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Home , Earthworm, Isothiocyanate

european journal of 45 (2009) 176–183

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Original article collection from agricultural fields: Comparisons of selected expellants in presence/absence of hand-sorting

Ce´line Pelosia,*, Michel Bertrandb, Yvan Capowiezc, Hubert Boizardd, Jean Roger-Estradea aAgroParisTech, UMR 211 Agronomie, F-78850 Thiverval-Grignon, France bINRA, UMR 211 Agronomie, INRA/AgroParisTech, F-78850 Thiverval-Grignon, France cINRA, UR 1114, Plantes et Syste`mes Horticoles, F-84914 Avignon cedex 9, France dINRA, UR 1158 Agronomie, F-80200 Estre´es-Mons, France article info abstract

Article history: The role play in is under increasing scientific scrutiny, especially Received 8 June 2008 in light of the fact that farmers are seeking to reduce soil tillage. However, there are many Received in revised form discrepancies in earthworm sampling methods. The aim of this study is to compare the 22 September 2008 efficiency of three chemical expellants (, commercial ‘hot’ and allyl Accepted 29 September 2008 , abbreviated AITC), with two sampling methods: (i) a simple method that Published online 26 October 2008 consisted in spreading an expellant solution on the soil and retrieving earthworms that emerged at the soil surface, and (ii) a combined method that consisted in applying Keywords: a chemical to expel earthworms and then hand-sorting the remaining earthworms from Earthworms the block of soil. Sampling efficiency was measured in terms of earthworm density and Hand-sorting , for endogeic and anecic earthworms. Formaldehyde With the simple method, a higher density of earthworms was sampled using formaldehyde and AITC than with mustard. Formaldehyde, AITC and mustard expelled not significantly Commercial ‘hot’ different biomasses of 47.7, 31.9 and 20.5 g m2, respectively, on average over the three Mustard plots. The combined method did not yield a significantly different density or biomass with Sampling method the different chemicals. Agricultural fields Formaldehyde is toxic and commercial ‘hot’ mustard is difficult to standardise and ineffi- cient when used without hand-sorting. Accounting for the accuracy of the sampling methods as well as the toxicity of the chemicals to users and soil organisms, AITC appears to be a reliable and promising chemical expellant whether or not in combination with hand- sorting. Its use would be a step towards standardizing earthworm sampling methods. ª 2008 Elsevier Masson SAS. All rights reserved.

1. Introduction structure [37], and they are central to the ecological func- tioning of agroecosystems [9]. Earthworm sampling methods The role earthworms play in soil fertility is under increasing are the means to obtain accurate data on the number and scientific scrutiny, especially in light of the fact that farmers biomass of individuals m2 in addition to the and are seeking to reduce soil tillage. Earthworms are known to ecological group composition of a given community. However, positively influence dynamics and soil there are discrepancies in earthworm sampling methods.

* Corresponding author. þ33 1 30815347; fax: þ33 1 30815425. E-mail address: [email protected] (C. Pelosi). 1164-5563/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2008.09.013 european journal of 45 (2009) 176–183 177

Authors reviewing earthworm sampling methods have (FAO classification), sampling was carried out in a yearly distinguished between ethological methods and hand-sorting ploughed plot (30 cm depth), labelled plot 1, and in a plot that [1,6,25]. Ethological methods, the efficiency of which depends on had been superficially tilled since 2001, labelled plot 2 (Table 1). earthworm activity, include the use of an electrical, i.e., octet In April 2008 sampling was carried out in another yearly method, or chemical methods to expel earthworms from the ploughed plot (30 cm depth), labelled plot 3, at the Thiverval- soil. Octet method efficiency has been discussed [7,18,35] and Grignon INRA experimental centre (4851’ N, 155’E, elevation different chemicals have been evaluated. Hand-sorting, which is 125 m), near Paris, which has orthic luvisol (FAO classifi- very time-consuming and labour intensive [8,38], entails a direct cation) (Table 1). The three plots were chosen in order to assess sampling of earthworms by excavating blocks of soil. Several sampling efficiency in cropping systems under different soil authors have reported the efficiency of a method combining the tillage regimes and thus with differing infiltration rates and application of a chemical expellant with hand-sorting [2,6]. depths [39]. The cropping systems are ‘‘intensive’’ ones, in Generally speaking, they found that hand-sorting is more suit- terms of high yield and the amount of pesticides and fertilisers. able than an expellant solution for small earthworms [26] such as endogeics and juvenile anecics. However, when this method 2.2. Experimental design and earthworm is used alone, the number of larger earthworms such as anecics sampling methods is often under-estimated because these earthworms can easily move into deeper soil. Similarly, expellants may give better Earthworms were collected in May 2006 during their results for large anecic earthworms [8,10,42] but smaller earth- maximum period of activity under temperate climatic condi- may be under-estimated because their low mobility tions [4,16] to limit the effect of dormancy. In April 2008, might prevent them reaching the surface. earthworms were collected during a cold week (mean air The chemical expellants commonly used in experimental temperature was near 7 C). Soil water content was 17.9%, sampling are formaldehyde [31], commercial hot mustard 14.8% and 21.0% in plots 1, 2 and 3, respectively. Selected [19,27] and, more recently, allyl isothiocyanate (AITC) [43]. weather conditions during the four weeks before the 2006 and Formaldehyde is the most commonly used one [8,33,34] but it 2008 samplings are presented in Fig. 1. has been recently recognised as a cancer-producing chemical Sampling was undertaken using a metal frame (40 40 cm) [23]. It is also toxic to earthworms, plants and their environ- which was placed on the soil and driven into the ground to ment [17,20]. Despite these findings, it is still recommended by a depth of 1 cm to retain the chemical on the sampling site. the ISO/DIS 23611-1, 2007-02 [14]. The expellant efficiency of Chemicals were applied twice at 10-min intervals. Three litres commercially available mustard paste or mustard powder has of expellant were applied for each experiment. Emerging been questioned [2]. Moreover, because its composition is earthworms were retrieved during a 20-min period after the generally a well-kept manufacturing secret [43], protocols first expellant application. Then, a 40 cm 40 cm 30 cm- cannot be standardised. Allyl isothiocyanate, or AITC, is deep block of soil was excavated from the same place and a natural breakdown product of formed in many remaining earthworms were extracted by hand in the labo- Cruciferae; it is the irritant molecule in commercial ‘hot’ ratory to obtain the results for the combined method. In each mustard. It is not noxious to humans [22] and is easily degraded plot, four replicates were made for each of the three chemicals in soil [3]. The isopropanol, used to dilute AITC in water, is also in a randomised design. Earthworms, preserved in 4% not harmful to human health [12] and rapidly biodegrades in formalin solution, were fresh weighed (without emptying gut water [41]. Only two studies have tested AITC as an earthworm contents), counted and identified at the species level accord- expellant [11,43] and it is still rarely used in field samplings. ing to the identification key established by Sims and Gerard Given these discrepancies in earthworm sampling methods, [36]. The development stage of each earthworm was noted. there is a need to move towards standardization. This study Earthworms are considered sub-adult if they have a full compares the efficiency of three chemical expellants, which have tubercula pubertatis but no clitellum and adult if they are never been evaluated in a single study (formaldehyde, commer- clitellate [36]. They are considered juvenile if they have cial ‘hot’ mustard and AITC) with two sampling methods: (i) neither tubercula pubertatis nor clitellum. a simple method that consisted in spreading an expellant solu- tion on the soil and retrieving earthworms that emerged at the soil surface, and (ii) a combined method that consisted in 2.3. Chemicals applying a chemical to expel earthworms and then hand-sorting the remaining earthworms from the block of soil. Sampling effi- Formaldehyde solution (VWR, France; 36%; density 1.09) was ciency was measured in terms of earthworm density and diluted with water to 2 g l1, according to Bouche´ and Aliaga biomass, for endogeic and anecic earthworms. [5] and Gunn [20]. Following Zaborski [43], AITC was first diluted with isopropanol (propan-2-ol) to give a 5 g l1 solu- tion. Then this solution was diluted with water to reach 2. Materials and methods a concentration of 0.1 g l1, which is considered optimal [43]. Commercial ‘hot’ mustard (store-brand mustard) was thor- 2.1. Sites and cropping systems oughly mixed with water to obtain a solution at a concentra- tion of 15 g l1 [10,20]. Both the mustard solution and AITC Field data were collected in two locations in northern France. In stock solution with isopropanol were prepared in the labora- May 2006, at the Estre´es-Mons INRA experimental centre tory shortly before the experiment because the molecule loses (4952’ N, 300’ E, elevation 85 m), which has haplic luvisol soils its irritating activity after a certain time in water [27] due to its 178 european journal of soil biology 45 (2009) 176–183

Table 1 – Sampling dates, site locations and soil characteristics of the three plots used to compare earthworm sampling methods. Plot number 1 2 3

Sampling date May 2006 May 2006 April 2008

Site location Estre´es-Mons Estre´es-Mons Thiverval-Grignon 4952 N, 300 E, elevation 85m 4952 N, 300 E, elevation 85m 4851 N, 155 E, elevation 125m

Soil tillage Ploughing Direct-drilling Ploughing

Crop Flax Flax None

Last ploughing date November 2005 – January 2008

Clay (g kg1) 210 180 230 Silt (g kg1) 726 724 770 Sand (g kg1)63 61 80 Organic matter (g kg1) 17.9 15.8 33.0 1 CaCO3 (kg ) 12.0 7.0 1.2 C/N ratio 9.6 8.0 10.8 pH 7.8 7.0 7.9 Rotation Flax/wheat/pea/wheat Flax/wheat/pea/wheat Maize/winter barley/wheat/rape

short life-span. The diluted commercial ‘hot’ mustard was Density of individuals in the age class in the simple and the vigorously agitated to prevent the phases from separating out. combined methods is presented in Table 3. Juveniles were dominant in plots 1 and 3 but more sub-adults and adults were retrieved in plot 2. Chemical expellants did not influence 2.4. Statistical analysis the proportion of individuals in each age class. In the simple method, on average for the three plots, the proportion of Sample data were first transformed to record earthworm juveniles varied between 59% (with AITC) and 63% (with 2 number and biomass m . Simple and combined methods formaldehyde and mustard). In the combined method, were analyzed without and with inclusion of the hand sorting between 60% (with formaldehyde and mustard) and 63% (with data, respectively. When the homoscedasticity of variance AITC) of individuals were juveniles. was not respected, data were transformed by f(x) ¼ ln(x þ 1). The results were compared by an analysis of variance (ANOVA) using the statistical program R (version 2.6.1, 2007) 3.2. Comparison of expellant effect in the simple method [24]. For each sampling method, plot and chemical effects were tested, as well as the effect of their interaction. When On average, combining data from the three plots, formalde- treatments were significantly different in the ANOVA anal- hyde and AITC were significantly more efficient than mustard ysis, a Tukey test was used to pinpoint which treatments were for sampling endogeic species (Table 4). The density of anecic different. species as well as the total density was higher with formal- dehyde and AITC than with mustard although differences were not always statistically significant. However, the biomass of anecic earthworms and total biomass were not 3. Results significantly different depending on the chemical used, although lower biomasses were sampled with mustard than 3.1. Species and age class composition of with the two other chemicals. Endogeic biomass was signifi- the earthworm community cantly higher with AITC and formaldehyde than with mustard. Density of the species found in the three sampling plots in the When we considered the three plots separately, the same simple and the combined methods are presented in Table 2. trend was observed since density and biomass using mustard Densities of chlorotica and giardi were lower compared to data obtained with the two other were low, whatever the site location and the chemical. The chemicals, but the differences were not always significant. sensitivity to chemical expellants was not always the same for Differences were never significant for plot 3, for example. the species of a given ecological group. Aporrectodea icterica did With diluted commercial ‘hot’ mustard the total earth- not respond to vermifuge applications and individuals were density found in the plots was between 1.6 (plot 3) and only found with hand-sorting. AITC and formaldehyde 5.7 (plot 2) times lower than with formaldehyde, and between appeared more efficient than mustard for Aporrectodea cal- 2.0 (plot 3) and 5.2 (plot 2) times lower than with AITC (Table 4). iginosa and , although the major part of them The total biomass obtained using formaldehyde was between was retrieved by hand. It was the opposite for ter- 1.8 (plot 2) and 14.0 (plot 1) times higher than with mustard. restris since between 64 and 100% of the individuals were The total biomass retrieved using AITC was between 1.1 (plot retrieved with AITC or formaldehyde applications. 2) and 14.9 (plot 3) times higher than with mustard. No european journal of soil biology 45 (2009) 176–183 179

rainfall (mm) soil temperature (°C)

32 16

28 14 Soil temperature (°C) 24 12 20 10 16 8 12 6

Rainfall (mm) 8 4 4 2 0 0 week -4 week -3 week -2 week -1 week -4 week -3 week -2 week -1

Sampling Sampling Estrées-Mons, 2006 date Thiverval-Grignon, 2008 date

Fig. 1 – Sum of rainfall (mm) and mean values of soil temperature (8C) at 10 cm depth at Estre´es-Mons and Thiverval-Grignon four weeks before sampling. Vertical bars are standard deviations.

significant differences were found between the efficiency of to react to the expellant applications. These findings are in formaldehyde and AITC (Table 4). accordance with other studies [2,27,40] in which the latter species was not found. In the same way, they found L. terrestris 3.3. Comparison of expellant effect in the combined to be sensitive to chemical expellants [27,40], as in our case, method but A. giardi was not found in their case. Although the chemicals did not work with the same effi- As shown in Table 5, when we used the combined chemical/ ciency, neither of the two methods showed bias in the hand sorting method to sample earthworms, no significant proportion of juveniles and sub-adults/adults collected. The differences in density and biomass were found in terms of the higher proportion of sub-adults and adults in plot 2 compared ecological group in different plots, except in one case. More to the two others may be explained by the absence of soil endogeic earthworms were collected in plot 2 using mustard tillage which may have reduced the mortality of larger but this value was not significantly different from the value earthworms. using AITC. Temperature conditions as well as the lack of rain during Compared to the combined method (if we assume 100% of the weeks before the sampling in plot 3 may explain the lower earthworms were sampled with this method), with the simple density found in this plot (Table 4) because earthworm pop- method using formaldehyde and AITC, 76% and 63%, respec- ulation dynamics vary greatly with temperature and soil tively, of the overall anecic density was retrieved, while water content [16,30,42]. Earthworms might have been in commercial ‘hot’ mustard only forced 27% of this ecological or in the deeper layer of soil during the sampling group to the surface, on average for the three plots (Tables 4, period and thus may have been less affected by the irritating 5). The same trend was observed in the case of endogeics for effect of the chemicals than when fully active. Moreover, the three expellants. Only approximately 3%, 14% and 15% of efficiency of expellants might have been diminished by dry endogeics were expelled with commercial ‘hot’ mustard, soil conditions in plot 3 since chemical infiltration depends on formaldehyde and AITC, respectively, on average for the three soil moisture [5]. plots (Tables 4, 5). The findings of this study, in agreement with others [2,26], confirm that earthworm populations are greatly under-esti- mated without hand-sorting. Following treatment with 4. Discussion mustard, formaldehyde and AITC, hand-sorting showed the overall efficiency to be 94%, 75% and 79% of the total density, The sampling of earthworm density and biomass was carried respectively. This means that only 6% of the earthworms out under three different pedoclimatic conditions. The would have been collected by applying commercial mustard expellant efficiency of formaldehyde and AITC was not without hand-sorting. Bartlett et al. [2] found that when using significantly different and was better than mustard when a mustard solution, 36% of total earthworms were expelled. used with the simple method. In the combined method, no They worked in a permanent pasture, with an earthworm significant differences were found between the three chem- community including epigeic species which are small earth- icals, either for density or biomass. This might be due to the worms living in the surface litter. This earthworm ecological fact that all the earthworms were retrieved when using the group was absent in our study plots. Moreover, combined method, including the anecic earthworms that and root mats are known to play a role in expellant efficiency, were not expelled by the chemical application. influencing the extent and the speed of the chemical infiltra- A. caliginosa, A. rosea and A. chlorotica presented quite the tion in the soil [32]. Thus, soil tillage and the slightly developed same sensitivity to chemicals while A. icterica did not appear root system of our sampled plots may account for differences. 180 european journal of soil biology 45 (2009) 176–183

Although it is time consuming, labour intensive [8,38] and physically destructive, hand-sorting appears necessary for an accurate sampling of the total earthworm community, in particular for endogeics and small anecics. Previous studies reported that the efficiency of methods using chemical

the simple expellants depends on solution concentrations, temperature and humidity conditions, and soil tillage, as well as earth- worm size and age. However, most large earthworms can be

Anecic species sampled more effectively by applying an irritant solution on the soil. For an accurate estimation of earthworm populations, a combined method should thus be used [5]. L. terrestris A. giardi Some previous studies have focused on the efficiency of 10.9 (46.9) – (–) 9.4 (21.9) 1.6 (6.3) 35.9 (35.9) 3.1 (6.3) 1.6 (4.7) – (7.8) 4.7 (6.3) – (3.1) 4.7 (6.3) 1.6 (7.8) hand-sorting versus a chemical expellant [8,13,15]. Others compared the efficiency of several chemical expellants without hand-sorting, including a comparison of mustard and formaldehyde. Chan and Munro [10] and Gunn [20] found that (6.3) (7.8) (9.4) (25.0)(4.7) 18.8 (26.6) – (–) (9.4) (–) a15mll1 English mustard solution retrieved more earth- worms than formalin at different concentrations. These authors, as well as others [7,18,21,28] used mustard powder, Unidentifiable which seems to have a different expellant efficiency than the – 4.7 (29.7) 29.7 (45.3) – (–) 3.1– (18.8) 32.8 (51.6) – (–) – – commercial mustard used in this present study. However, Grønstøl et al. [19], using commercially available mustard paste also found no significant differences between the proportion of juveniles, earthworm size and the number of emerging earthworms following mustard and formalin treat- ments. The discrepancy with our findings could possibly be – (–) – (–) – (–) explained by the fact that the AITC concentration in the commercial ‘hot’ mustard we used was much lower than those used in the aforementioned studies. This highlights just how difficult it is to standardise the diluted commercial ‘hot’ mustard method. Only two studies compared AITC and formaldehyde efficiency. Zaborski [43] and Coja et al. [11], found no significant differences when they compared AITC A. chlorotica A. icterica 1 1 Endogeic species (100 mg l ) with a 200 mg l [43] or a 0.2% [11] formalin solution. The results found in this study are consistent with these studies since AITC and 2 g l1 formaldehyde were found to have the same efficiency. A. rosea

L Table 3 – Density (number m 2) of individuals in the age class in the three sampling plots, using mustard, formaldehyde and AITC in the simple method, which involves spreading a chemical expellant on to the soil and retrieving earthworms emerging at the soil surface, and the combined method (between brackets), which follows the simple method with hand sorting A. caliginosa earthworms from the block of soil. 6.3 (207.8) 3.1 (40.6) – (3.1) – (–) 18.8 (137.5) 10.9 (23.4) 1.3 (6.3) – (–) 28.1 (173.8) 17.2 (35.9) 3.1 (7.8) – (–) – (71.9) – (115.6) – (–) 10.9 (48.4) 12.5 (70.3) – (–) 1.6 (71.9) – (3.1) 4.7 (76.6) – (7.8) – 14.1 (39.1) 23.4 (121.9) – (–) 3.1 (42.2) – (4.7) 4.7 (68.8) – (17.2) – 6.3 (65.6) – (20.3) 3.1 (118.8) – (21.9) –

) of the species found in the three sampling plots, using mustard, formaldehyde and AITC in the simple method, which involves spreading Plot Chemical expellant Age classes 2 L Juveniles Sub-adults þ adults

1 Mustard 20.3 (242.2) 0.0 (62.5) Formaldehyde 50.0 (187.5) 15.6 (54.7) AITC 70.3 (218.8) 14.1 (68.8)

2 Mustard 3.1 (92.2) 7.8 (131.3) Formaldehyde 31.3 (67.2) 31.3 (103.1) Formaldehyde AITC Formaldehyde AITC Formaldehyde AITC AITC 18.8 (104.7) 37.5 (107.8)

3 Mustard 4.7 (103.1) 3.1 (73.4) Formaldehyde 7.8 (95.3) 4.7 (56.3) Table 2 – Density (number m a chemical expellant on tomethod the with soil hand and sorting retrieving earthworms earthworms fromPlot emerging the at block the of soil soil. surface, and the combined method Chemical (between expellant brackets), which follows 1 Mustard –, No individual was found. Unidentifiable earthworms are endogeic juveniles. 2 Mustard 3 Mustard AITC 9.4 (150.0) 6.3 (90.6) european journal of soil biology 45 (2009) 176–183 181

L L Table 4 – Density (number m 2) and biomass (g m 2) of the two ecological groups and the total population for the three sampling plots, using mustard, formaldehyde and AITC in the simple method, which involves spreading a chemical expellant on to the soil and retrieving earthworms emerging at the soil surface. Plot Chemical expellant Density Biomass

Endogeics Anecics Total Endogeics Anecics Total

1 Mustard 9.4 a 10.9 a 20.3 a 0.6 a 1.6 a 2.2 a Formaldehyde 35.9 ab 29.7 a 65.6 ab 3.6 ab 27.2 b 30.8 b AITC 51.6 b 32.8 a 84.4 b 6.5 b 12.4 b 18.9 b

2 Mustard 0.0 a 10.9 a 10.9 a 0.0 a 58.2 a 58.2 a Formaldehyde 23.4 b 39.1 b 62.5 b 6.0 b 96.0 a 102.0 a AITC 37.5 b 18.8 ab 56.3 b 8.3 b 53.7 a 62.0 a

3 Mustard 6.2 a 1.6 a 7.8 a 0.9 a 0.1 a 1.0 a Formaldehyde 7.8 a 4.7 a 12.5 a 1.2 a 9.1 a 10.3 a AITC 9.4 a 6.2 a 15.6 a 1.3 a 13.6 a 14.9 a

3 Plots Mustard 5.2 a 7.8 a 13.0 a 0.5 a 20.0 a 20.5 a Formaldehyde 22.4 b 24.5 b 46.9 ab 3.6 b 44.1 a 47.7 a AITC 32.8 b 19.3 ab 52.1 b 5.4 b 26.6 a 31.9 a

For each variable in each plot, values followed by the same letter are not significantly different at a ¼ 0.05.

Although commercial ‘hot’ mustard was not very efficient 2007-02 [14], its use should be limited given that it is a health in the simple method, it cannot be dismissed considering hazard. The results of this study, in agreement with Zaborski results from other studies as well as ours that show it can be [43], show that AITC is an efficient chemical. Its half-life an acceptable expellant if it is followed by hand-sorting. ranges from 20 to 60 h in the soil [3] and it is less noxious than However, this chemical presents many drawbacks. Indeed, to formaldehyde for handlers, earthworms and the environment be soluble in water, its concentration cannot exceed 25 g l1. [11]. Moreover, AITC is economical and easy to standardise Increasing the concentration of commercial ‘hot’ mustard since it is a pure molecule. However, it is important to note solution leads to higher numbers of retrieved earthworms; that AITC can be used as a biopesticide [29], and therefore if however, because of the threshold for solubility, the optimal communities other than earthworms are under study, it is concentration for sampling earthworms remains unknown necessary to be cautious. [20]. Furthermore, the AITC concentration in commercial ‘hot’ This study is a step towards standardizing earthworm mustard is often not known because manufacturers keep it sampling methods, accounting for the accuracy of the a secret. Thus, this method cannot be easily compared or samples as well as the toxicity of the chemicals to users and standardised. soil organisms. Further research on AITC should be conducted Furthermore, even if formaldehyde is nearly as efficient as to test a wide range of chemical concentrations, soil types, AITC, and even if it is recommended by the ISO/DIS 23611-1, cropping systems, and climate conditions.

L L Table 5 – Density (number m 2) and biomass (g m 2) of the two ecological groups and the total population for the three sampling plots, using mustard, formaldehyde and AITC in the combined method, which involves spreading a chemical expellant on to the soil and retrieving earthworms emerging at the soil surface, and then hand sorting remaining earthworms from the block of soil. Plot Chemical expellant Endogeics Anecics Total Endogeics Anecics Total

1 Mustard 257.8 a 46.9 a 304.7 a 41.0 a 7.4 a 48.4 a Formaldehyde 196.9 a 45.3 a 242.2 a 28.7 a 29.6 a 58.3 a AITC 235.9 a 51.6 a 287.5 a 35.9 a 32.7 a 68.6 a

2 Mustard 195.3 b 28.1 a 223.4 a 36.8 a 83.7 a 120.5 a Formaldehyde 128.1 a 42.2 a 170.3 a 28.2 a 99.3 a 127.5 a AITC 185.9 ab 26.6 a 212.5 a 30.8 a 55.4 a 86.2 a

3 Mustard 164.1 a 12.5 a 176.6 a 29.7 a 9.6 a 39.3 a Formaldehyde 142.2 a 9.4 a 151.6 a 21.9 a 12.5 a 34.4 a AITC 226.5 a 14.1 a 240.6 a 33.6 a 24.6 a 58.2 a

3 Plots Mustard 205.7 a 29.2 a 234.9 a 35.8 a 33.6 a 69.4 a Formaldehyde 155.7 a 32.3 a 188.0 a 26.3 a 47.1 a 73.4 a AITC 216.1 a 30.8 a 246.9 a 33.4 a 37.6 a 71.0 a

For each variable in each plot, values followed by the same letter are not significantly different at a ¼ 0.05. 182 european journal of soil biology 45 (2009) 176–183

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