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The effect of soil moisture content on nitrogen transformation using OECD test guideline 216
Applied Soil Ecology 46 (2010) 478–482
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
The effect of soil moisture content on nitrogen transformation using OECD test guideline 216 Gary Roberts a,∗ , Alison Penwell a , Fabrice Peurou b , Alan Sharpe a a b
AstraZeneca Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon TQ5 8BA, UK Jasco France, Zone de la Bouvre, 17 rue Gutenberg, 44340 Bouguenais, Nantes, France
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 11 August 2010 Accepted 6 September 2010 Keywords: Nitrification Nitrate Ammonification Ammonium Nitrapyrin Terrestrial ecotoxicity
a b s t r a c t One of the most important microbial processes in soil is the mineralisation of nitrogen in organic matter and this is vital for the maintenance of soil fertility. When performing a risk assessment for a substance that may enter the soil environment it is important to consider the potential effects the substance may have on the soil’s microbial activity. The standardised test produced by the OECD (Organisation for Economic Co-operation and Development) (OECD, 2000) to address this requires that the soil moisture content should be maintained at between 40% and 60% of the maximum water holding capacity (MHC) with a range of ±5%. However, our investigations have suggested that the soil moisture content may adversely affect the performance of the test. The current research aimed to identify the moisture content range, within which the measured concentration of nitrate-N was sufficiently above background levels in soil. This moisture content range, which will be different for soils of different texture types, focused on a sandy loam soil fulfilling the criteria of the OECD 216 guideline (OECD, 2000). The nitrate-N yield and the EC50 (the concentration causing 50% inhibition of nitrogen transformation) of the reference substance, nitrapyrin (2-chloro-6-(trichloromethyl)-pyridine), were determined at varying soil moisture contents. The results indicate that the current OECD recommended upper limit for the soil moisture content (60% MHC) may be too high. The yield of nitrate-N and the EC50 for nitrapyrin, were relatively constant between 20 and 40% MHC. Other reported data (Roberts et al., 2003) suggests that 50% MHC produced similar results. Therefore, we suggest that the OECD test be conducted between 20 and 50% MHC. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The mineralisation of organic nitrogen by soil microbial communities is vital for the maintenance of soil fertility. Any long-term interference with these biochemical processes could potentially affect nutrient cycling and this could alter soil fertility. Although the microbial communities will differ from soil to soil, the pathways of transformation are similar. In order to protect soil microorganisms, when performing a risk assessment for substances which may be released directly or indirectly into the soil, it is important to consider their potential effects on soil microbial activity. The OECD (Organisation for Economic Co-operation and Development) test guideline 216 (OECD, 2000) and ISO (International Organisation for Standardisation) test method 14238 (ISO, 1997) both describe laboratory test methods to investigate potential adverse effects of a substance on nitrogen transformation activity of soil microorganisms.
∗ Corresponding author. Tel.: +44 01803 882882; fax: +44 01803 882974. E-mail address: [email protected] (G. Roberts). 0929-1393/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2010.09.003
The OECD 216 guideline recommends using nitrapyrin (2chloro-6-(trichloromethyl)-pyridine) as a reference substance, known to inhibit nitrification. Roberts et al. (2003) report an EC50 (the concentration causing 50% inhibition of nitrogen transformation) for nitrapyrin of 3.1 mg kg−1 dry soil at 50% MHC (maximum water holding capacity). Both the OECD and ISO guidelines require that the soil moisture content should be maintained at between 40% and 60% of the MHC with a range of ±5%. Moisture content may influence the yield of nitrate as a result of the formation of anoxic regions. As ammonium is converted to nitrate only in aerated habitats (Alexander, 1977), in anoxic regions, organic nitrogen may be converted to ammonium by ammonification but the further oxidation of ammonium to nitrite and nitrate is hindered, reducing nitrate yield and thereby affecting the precision and accuracy of results from tests run under anoxic conditions. Ammonification
Organic N
−→
NH4 +
2-Step nitrification
−→
NO3 −
Observations made during previous work in our laboratory (data not published) suggested that the upper guideline moisture requirements may be too high for a sandy loam soil fulfilling the
G. Roberts et al. / Applied Soil Ecology 46 (2010) 478–482
criteria of the OECD 216 guideline. Therefore, the effect of soil moisture content on the production of ammonium-N, nitrite-N and nitrate-N and on the observed EC50 of the reference substance, nitrapyrin, was investigated. 2. Materials and methods 2.1. Soil A soil, fulfilling the criteria specified by the OECD 216 guideline, has the following characteristics: • • • •
sand content; not less than 50% and not greater than 75%. pH: 5.5–7.5. organic carbon content: 0.5–1.5%. the microbial biomass should be measured and its carbon content should be at least 1% of the total soil organic carbon.
A Dystric Cambisol sandy loam soil with these characteristics was collected from a set-aside field in the Royal Leamington Spa area, UK (OS map reference: SP 3289 6926) from a depth of 5–20 cm. The soil pH was 6.5, the organic carbon content 1.2% and the microbial biomass was determined to be 188 g C g−1 dry weight soil using the method described by Jenkinson and Powlson (1976); this was equivalent to 1.57% of total soil organic carbon. The soil was sieved to a particle size of ≤2 mm and was stored under refrigeration at a nominal temperature of 4 ◦ C until used in the test, within 6 weeks of collection. The particle size distribution was 74% sand (63 m–2 mm), 12% silt (2–63 m) and 14% clay (<2 m).
479
400 mL glass beakers to give the equivalent of 100 g dry soil in each beaker. The glass beakers were incubated in polythene boxes fitted with gas-tight lids (9 beakers per box). At each moisture content, two boxes were connected in series with butyl rubber tubing, so that all replicate beakers were exposed to similar air-flow (Fig. 1). A stream of humidified air was passed through the boxes during the study, to reduce evaporative losses but allow gas exchange thereby preventing the development of anaerobic conditions which could cause losses of nitrogen through denitrification. The containers were incubated at 20 ± 2 ◦ C, in the dark, for 28 days. The moisture content of the soils was checked at weekly intervals and was maintained at the specified level ±5% by readjustment gravimetrically with deionised water as necessary. 2.3. Determination of ammonium-N, nitrite-N and nitrate-N On day 0 the background concentrations of ammonium-N, nitrite-N and nitrate-N were determined in duplicate nominal 20 g dry soil equivalent sub-samples of control soil. On day 28 of the incubation, nominal 20 g dry soil equivalent aliquots of soil were sampled from each of the beakers in triplicate. Each soil sub-sample was extracted with 100 mL of 2 M KCl on a wrist action shaker (Stuart Scientific, Model SF1) at 150 rpm for 1 h and the resultant extracts were filtered using a Millipore filtration system (StericupTM and SteritopTM filtration system, 0.22 m GV DuraporeTM membrane) to sterilise the extracts and prevent further microbial activity. These filter-sterilised extracts were stored at 4 ◦ C until they were analysed. Day 28 nitrate-N analyses were determined within 4 days of sampling and all other analyses were completed within 1 month of sampling.
2.2. Experimental design 2.4. Ammonium-N Sub-samples of the soil were adjusted to 60, 40, 30, 20 and 14% of the MHC with deionised water, and were pre-incubated at that MHC for between 6 and 12 days. MHC was determined using the method described by ISO (1997). A 2.5 kg dry weight equivalent sample of soil, at each moisture content, was amended with lucerne meal (Dengie Crops Limited, Essex, UK), an organic substrate with a C:N ratio of 12.2:1, at a rate of 5 g kg−1 dry soil and thoroughly mixed. Stock solutions of 4 and 40 g L−1 nitrapyrin (Sigma–Aldrich, Dorset, UK. 99%) in ethanol were prepared. Appropriate volumes of these stock solutions were added to 4 g aliquots of fine quartz sand, the ethanol was evaporated and the sand subsequently mixed with 400 g dry weight equivalent soil samples to give concentrations of 1.0, 3.2, 10, 32 and 100 mg nitrapyrin kg−1 dry soil, at each of the five soil moisture contents. Control soil samples were prepared, containing 4 g of fine quartz sand per 400 g soil. Triplicate sub-samples of each soil treatment including the controls were weighed into
The 2 M KCl extracts were analysed for ammonium-N using Palintest® ammonium reagents (Palintest® , Gateshead, UK) according to the supplier’s instructions. This method was based on the formation of indophenol from the reaction of phenol, hypochlorite and ammonium, in the presence of catalytic quantities of sodium nitroprusside. Ammonium-N was determined by measuring indophenol at 640 nm spectrophotometrically (Uvikon 930, NorthStar Scientific, Bedfordshire, UK). A calibration curve was constructed using prepared standards of ammonium chloride in the concentration range 0.1–1.0 mg ammonium-N L−1 . 2.5. Nitrite-N and nitrate-N Each 2 M KCl extract was analysed for total oxidised nitrogen and nitrite-N using the Palintest® kit (Nitratest and Nitricol,
Air flow
Humidifier
9 Beakers of soil incubated in each of 2 boxes connected in series
Fig. 1. Schematic of the apparatus used for incubation of soils at a range of moisture contents and nitrapyrin concentrations.
480
G. Roberts et al. / Applied Soil Ecology 46 (2010) 478–482
Palintest® , Gateshead, UK) according to the supplier’s instructions. In this method, nitrate-N in the extract was first reduced to nitrite-N and the resulting nitrite-N was then determined by reaction with sulphanilic acid in the presence of N-(1-naphthyl)ethylene diamine to form a reddish dye (Montgomery and Dymock, 1961). The quantities of total oxidised nitrogen in each reacted extract were then determined at 545 nm spectrophotometrically (Uvikon 930, NorthStar Scientific, Bedfordshire, UK). A calibration curve was constructed using prepared standards of potassium nitrate and sodium nitrite in the concentration range 0.1–1.0 mg nitrate/nitrite-N L−1 . The nitrite-N concentration in each extract was also determined by an analogous procedure omitting the reduction step. The levels of nitrate-N were determined by subtraction of nitrite-N from the measured total oxidised nitrogen values.
Nitrate-N
Yield (mg N kg-1)
60
Ammonium-N * Below LOD
50 40 30 20 10
*
*
*
*
*
0 14
20
30
40
60
Soil moisture content (% of MHC) Fig. 2. Mean ammonium-N, nitrite-N and nitrate-N yield (with standard errors) at different soil moisture contents. Nitrite-N was not detected at any soil moisture content, so has been omitted from the figure. Limit of detection (LOD) for ammonium-N was 6.4 mg kg−1 . Limit of detection (LOD) for nitrite-N was 0.6 mg kg−1 . Limit of detection (LOD) for nitrate-N was 0.6 mg kg−1 .
2.6. Statistical analysis To assess the effect of soil moisture content on nitrogen transformation, the control soil concentrations of ammonium-N, nitrite-N and nitrate-N, at each of the moisture contents, were compared using one-way analysis of variance, followed by a series of Tukey t-tests (Zar, 1999). To determine whether soil moisture content had an effect upon the inhibition of nitrogen transformation by nitrapyrin, two-way analysis of variance (Zar, 1999) was performed, with soil moisture and nitrapyrin concentration as the main variables. Subsequently, one-way analysis of variance followed by Dunnett’s test (Dunnett, 1955) was undertaken on each of the nitrapyrin-exposed soils to determine the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC). Additionally, a Weibull model (Rawlings and Cure, 1985) was fitted to the data to determine the EC50 and 95% confidence interval for nitrapyrin ecotoxicity.
centration in the soil was between 4.7 and 6.3 mg kg−1 . There was no obvious relationship between the day 0 total oxidised nitrogen concentration and soil moisture content (data not shown). This concentration represented approximately 10% of the highest analysed levels of total oxidised nitrogen in the soil on day 28 (Table 1). Therefore, these values were considered to be adequately low background levels to allow the nitrogen transformation activity of the soil to be assessed. Ammonium-N, nitrite-N and nitrate-N concentrations were determined in control soil incubated at different soil moisture contents after 28 days of incubation. The data (Fig. 2) show that the soil moisture content affected the yield of ammonium-N and nitrate-N. Nitrite-N was not detected above the limit of detection (0.6 mg kg−1 ) at any time during the experiments. Therefore, total oxidised nitrogen was equivalent to nitrate-N. Ammonium-N was only detected at the lowest soil moisture content tested (14% MHC) which suggests that at this soil moisture content the first step of nitrification was impeded. This was also suggested by the lower yield of nitrate observed at this soil moisture content compare to soils incubated at higher soil moisture contents (Fig. 2). Ammonium-N yields were below the limit of detection at the higher soil moisture contents, suggesting that nitrification was converting any ammonium-N to nitrate-N (via nitrite-N). However, at 60% MHC the yield of nitrate-N was also below the limit of detection (0.6 mg kg−1 ), suggesting that at this soil moisture con-
3. Results and discussion 3.1. The effect of soil moisture content on the yield of ammonium-N, nitrite-N and nitrate-N Background concentrations of ammonium-N and total oxidised nitrogen were determined on day 0. The ammonium-N concentration in the soil on day 0 was between <6.4 mg kg−1 (the limit of detection) and 15.2 mg kg−1 , which was approximately twice the limit of detection. These analyses were sufficient to set a baseline and to detect an increase in ammonium-N above the background. At day 0 the total oxidised nitrogen (nitrite-N plus nitrate-N) con-
Table 1 Yield of ammonium-N, nitrite-N and nitrate-N after 28 days of exposure to a range of soil moisture contents and nitrapyrin concentrations. Nitrapyrin concentration (mg kg soil−1 )
Soil moisture content (% MHC) Nitrate-N (mg kg soil−1 ) 14
0 (control) 1 3.2 10 32 100
11.2 7.9 6.3 4.9# 5.6# 5.9#
20
Ammonium-N (mg kg soil−1 ) 30
+
43.9 53.2 38.4 6.2# 7.0# 6.1#
40 +
44.8 54.1 40.2# 4.5# 4.0# 4.9#
+
53.7 56.8 33.7# 3.8# 3.8# 4.5#
60
14
20
30
40
60
<0.6 <0.6 <0.6 <0.6 <0.6 <0.6
14.9 16.2 13.6 11.5 20.7 16
<6.4 <6.4 9.7 22.3 33.6 56.2
<6.4 <6.4 7.1 18.2 22.8 33.5
<6.4 <6.4 18.6 40.2 42.8 44.5
<6.4 <6.4 <6.4 22.5 16.6 23.3
As ammonium-N yields were below the limit of detection, reliable statistical analysis could not be performed. All day 28 control nitrite-N data were below 0.6 mg kg−1 (the limit of detection), therefore total oxidised nitrogen was equivalent to nitrate-N. Values represent the mean of individual analyses from triplicate soil sub-samples. + Significant increase (p < 0.05) in control nitrate-N production compared to soil with a moisture content of 14% of MHC. # Significant reduction (p < 0.05) in nitrate-N production compared to the control for the relevant moisture content.
G. Roberts et al. / Applied Soil Ecology 46 (2010) 478–482
481
Table 2 Ecotoxicity endpoints (EC50 , NOEC and LOEC) of nitrapyrin (expressed as mg nitrapyrin kg soil−1 ) determined at different soil moisture contents. Soil moisture content (% MHC)
EC50 (mg kg−1 )
Lower 95% confidence limit(mg kg−1 )
Upper 95% confidence limit (mg kg−1 )
NOEC (mg kg−1 )
LOEC (mg kg−1 )
14 20 30 40
36 5.7 5.6 4
8 2.5 3.4 2.2
64 9 7.9 5.8
3.2 3.2
10 10 1 1
a a
tent ammonification (the formation of ammonium-N from organic nitrogen) was being inhibited. As ammonium-N yields were below the limit of detection, reliable statistical analysis could not be performed. Comparison of the control data (no added nitrapyrin) from each soil moisture content indicated that the 14% MHC and 60% MHC soil moisture content showed significantly lower (p < 0.01) nitrogen transformation than the 20, 30 and 40% MHC soils (Table 1). Data from control soils amended with lucerne meal at 50% MHC from our laboratory (data not shown) have a mean nitrateN concentration of 49.0 mg kg−1 (Std. dev. 13.3, n = 4), which is similar to the concentrations observed between 20 and 40% MHC in this study and consistent with the levels reported previously by Roberts et al. (2003). Therefore, for a sandy loam soil, of the type specified by the OECD 216 guideline, these data suggest that a more appropriate soil moisture content range would be between 20 and 50% MHC to ensure sufficient formation of nitrate-N to enable an inhibitory effect to be determined. The data (Table 1) suggest that nitrapyrin inhibited the first step of nitrification as, at 20–40% MHC, the ammonium-N increased with nitrapyrin concentration, whilst the nitrite-N stayed below the LOD and low concentrations of nitrate-N were observed. It was not possible to determine if ammonification was inhibited, but as ammonium-N was observed at high concentrations of nitrapyrin, but not at low concentrations or in the controls, it suggests that ammonification was not sensitive to nitrapyrin. This is consistent with published data, which shows that the mode of action of nitrapyrin is to inhibit the first step of nitrification, ammonium oxidation to nitrite (Goring, 1962; Campbell and Aleem, 1965). 3.2. Effect of soil moisture content on ecotoxicity endpoints (EC50 , NOEC and LOEC) of nitrapyrin To assess if the accuracy of the ecotoxicity endpoints (EC50 , NOEC and LOEC) were affected by the moisture content of the soil, soil was incubated at different soil moisture contents in the presence of lucerne (the source of organic nitrogen) and exposed to a series of concentrations of a known nitrification inhibitor, nitrapyrin (Goring, 1962). The EC50 for nitrapyrin varied between 4.0 and 36.0 mg kg−1 (Fig. 3). The two-way analysis of variance indicated that the 14% MHC soil resulted in a significantly different dose response to nitrapyrin than the other soil moisture concentrations, possibly due to the overall effect of low moisture on nitrogen transformation, irrespective of the presence of nitrapyrin. The NOEC and LOEC values, in addition to the EC50 values with confidence intervals, derived from each of the nitrapyrin exposures at different soil moisture contents are given in Table 2. At 20, 30 and 40% MHC there was not a significant difference in the EC50 value (5.7, 5.6 and 4.0 mg kg−1 (p = 0.05)) and these were consistent with the previously reported EC50 value (3.1 mg kg−1 ) for nitrapyrin determined at 50% MHC (Roberts et al., 2003).
Nitrapryrin EC50 (mg nitrapyrin kg soil-1)
EC50 values were determined using the Weibull model using analysed nitrate-N concentrations. It was not possible to determine the EC50 at 60% MHC as the nitrate-N was below the limit of detection (0.6 mg kg−1 ). a NOEC was not obtained since the LOEC was the lowest tested concentration.
70 60 50 40 30 20 10 0 10
20
30
40
Soil moisture content (% of MHC) Fig. 3. Observed nitrapyrin EC50 (with 95% confidence interval) at different soil moisture contents. It was not possible to determine the EC50 at 60% MHC as the nitrate-N was below the limit of detection (0.6 mg kg−1 ).
4. Conclusion The results presented here indicate that the current OECD 216 guideline recommended upper limit for the soil moisture content of the test soil (60% MHC) may be too high, which can lead to inhibition of the nitrification process and results in low yields of nitrate-N. Consequently, at 60% MHC, it was not possible to determine an EC50 for nitrapyrin, a known inhibitor of nitrification in soil. However, the yield of nitrate-N was relatively constant between 20 and 40% MHC and produced consistent EC50 values for nitrapyrin. Other reported data (Roberts et al., 2003) suggested that 50% MHC produced similar results. Therefore, we suggest that the OECD 216 test be conducted between 20 and 50% MHC. Acknowledgements The authors gratefully acknowledge the skilled technical assistance given by M Daniel and J Good, AstraZeneca Brixham Environmental Laboratory. References Alexander, M., 1977. Introduction to Soil Microbiology, second ed. J Wiley and Sons Inc, 253. Campbell, N.E.R., Aleem, M.I.H., 1965. The effect of 2-chloro-6(trichloromethyl)pyridine on the chemoautotrophic metabolism of nitrifying bacteria. I. Ammonia and hydroxylamine oxidation by Nitrosomonas. Antonie van Leeuwenhoek 31, 124–136. Dunnett, C.W., 1955. A multivariant comparison procedure for comparing several treatments with a control. American statistical association journal 50, 1096–1119. Goring, C.A.I., 1962. Control of nitrification of ammonium fertilizers and urea by 2-chloro, 6-trichloromethyl pyridine. Soil Sci. 93, 431–439. ISO 14238, 1997. Soil Quality–Biological Methods–Determination of Nitrogen Mineralisation and Nitrification in Soils and the Influence of Chemicals on these Processes.
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Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biology & Biochemistry 8, 209–213. Montgomery, H.A.C., Dymock, J.F., 1961. The determination of nitrite in water. Analyst (London) 86, 414–416. OECD, 2000. Guideline for the Testing of Chemicals No. 216. In: Soil Microorganisms: Nitrogen Transformation Test. Organisation of Economic Cooperation and Development, Paris.
Rawlings, S.O., Cure, W.W., 1985. The Weibull function as a dose-response model to describe ozone effects on crop yields. Crop Science 25, 807–814. Roberts, G.C., Peurou, F., Penwell, A.J., 2003. Assessing the toxic effect of nitrapyrin on nitrogen transformation in soil. Soil Biology & Biochemistry 35 (3), 479–481. Zar, J.H., 1999. Statistical Analysis, 4th ed. Prentice Hall, New Jersey.
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
The effect of soil moisture content on nitrogen transformation using OECD test guideline 216 Gary Roberts a,∗ , Alison Penwell a , Fabrice Peurou b , Alan Sharpe a a b
AstraZeneca Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon TQ5 8BA, UK Jasco France, Zone de la Bouvre, 17 rue Gutenberg, 44340 Bouguenais, Nantes, France
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 11 August 2010 Accepted 6 September 2010 Keywords: Nitrification Nitrate Ammonification Ammonium Nitrapyrin Terrestrial ecotoxicity
a b s t r a c t One of the most important microbial processes in soil is the mineralisation of nitrogen in organic matter and this is vital for the maintenance of soil fertility. When performing a risk assessment for a substance that may enter the soil environment it is important to consider the potential effects the substance may have on the soil’s microbial activity. The standardised test produced by the OECD (Organisation for Economic Co-operation and Development) (OECD, 2000) to address this requires that the soil moisture content should be maintained at between 40% and 60% of the maximum water holding capacity (MHC) with a range of ±5%. However, our investigations have suggested that the soil moisture content may adversely affect the performance of the test. The current research aimed to identify the moisture content range, within which the measured concentration of nitrate-N was sufficiently above background levels in soil. This moisture content range, which will be different for soils of different texture types, focused on a sandy loam soil fulfilling the criteria of the OECD 216 guideline (OECD, 2000). The nitrate-N yield and the EC50 (the concentration causing 50% inhibition of nitrogen transformation) of the reference substance, nitrapyrin (2-chloro-6-(trichloromethyl)-pyridine), were determined at varying soil moisture contents. The results indicate that the current OECD recommended upper limit for the soil moisture content (60% MHC) may be too high. The yield of nitrate-N and the EC50 for nitrapyrin, were relatively constant between 20 and 40% MHC. Other reported data (Roberts et al., 2003) suggests that 50% MHC produced similar results. Therefore, we suggest that the OECD test be conducted between 20 and 50% MHC. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The mineralisation of organic nitrogen by soil microbial communities is vital for the maintenance of soil fertility. Any long-term interference with these biochemical processes could potentially affect nutrient cycling and this could alter soil fertility. Although the microbial communities will differ from soil to soil, the pathways of transformation are similar. In order to protect soil microorganisms, when performing a risk assessment for substances which may be released directly or indirectly into the soil, it is important to consider their potential effects on soil microbial activity. The OECD (Organisation for Economic Co-operation and Development) test guideline 216 (OECD, 2000) and ISO (International Organisation for Standardisation) test method 14238 (ISO, 1997) both describe laboratory test methods to investigate potential adverse effects of a substance on nitrogen transformation activity of soil microorganisms.
∗ Corresponding author. Tel.: +44 01803 882882; fax: +44 01803 882974. E-mail address: [email protected] (G. Roberts). 0929-1393/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2010.09.003
The OECD 216 guideline recommends using nitrapyrin (2chloro-6-(trichloromethyl)-pyridine) as a reference substance, known to inhibit nitrification. Roberts et al. (2003) report an EC50 (the concentration causing 50% inhibition of nitrogen transformation) for nitrapyrin of 3.1 mg kg−1 dry soil at 50% MHC (maximum water holding capacity). Both the OECD and ISO guidelines require that the soil moisture content should be maintained at between 40% and 60% of the MHC with a range of ±5%. Moisture content may influence the yield of nitrate as a result of the formation of anoxic regions. As ammonium is converted to nitrate only in aerated habitats (Alexander, 1977), in anoxic regions, organic nitrogen may be converted to ammonium by ammonification but the further oxidation of ammonium to nitrite and nitrate is hindered, reducing nitrate yield and thereby affecting the precision and accuracy of results from tests run under anoxic conditions. Ammonification
Organic N
−→
NH4 +
2-Step nitrification
−→
NO3 −
Observations made during previous work in our laboratory (data not published) suggested that the upper guideline moisture requirements may be too high for a sandy loam soil fulfilling the
G. Roberts et al. / Applied Soil Ecology 46 (2010) 478–482
criteria of the OECD 216 guideline. Therefore, the effect of soil moisture content on the production of ammonium-N, nitrite-N and nitrate-N and on the observed EC50 of the reference substance, nitrapyrin, was investigated. 2. Materials and methods 2.1. Soil A soil, fulfilling the criteria specified by the OECD 216 guideline, has the following characteristics: • • • •
sand content; not less than 50% and not greater than 75%. pH: 5.5–7.5. organic carbon content: 0.5–1.5%. the microbial biomass should be measured and its carbon content should be at least 1% of the total soil organic carbon.
A Dystric Cambisol sandy loam soil with these characteristics was collected from a set-aside field in the Royal Leamington Spa area, UK (OS map reference: SP 3289 6926) from a depth of 5–20 cm. The soil pH was 6.5, the organic carbon content 1.2% and the microbial biomass was determined to be 188 g C g−1 dry weight soil using the method described by Jenkinson and Powlson (1976); this was equivalent to 1.57% of total soil organic carbon. The soil was sieved to a particle size of ≤2 mm and was stored under refrigeration at a nominal temperature of 4 ◦ C until used in the test, within 6 weeks of collection. The particle size distribution was 74% sand (63 m–2 mm), 12% silt (2–63 m) and 14% clay (<2 m).
479
400 mL glass beakers to give the equivalent of 100 g dry soil in each beaker. The glass beakers were incubated in polythene boxes fitted with gas-tight lids (9 beakers per box). At each moisture content, two boxes were connected in series with butyl rubber tubing, so that all replicate beakers were exposed to similar air-flow (Fig. 1). A stream of humidified air was passed through the boxes during the study, to reduce evaporative losses but allow gas exchange thereby preventing the development of anaerobic conditions which could cause losses of nitrogen through denitrification. The containers were incubated at 20 ± 2 ◦ C, in the dark, for 28 days. The moisture content of the soils was checked at weekly intervals and was maintained at the specified level ±5% by readjustment gravimetrically with deionised water as necessary. 2.3. Determination of ammonium-N, nitrite-N and nitrate-N On day 0 the background concentrations of ammonium-N, nitrite-N and nitrate-N were determined in duplicate nominal 20 g dry soil equivalent sub-samples of control soil. On day 28 of the incubation, nominal 20 g dry soil equivalent aliquots of soil were sampled from each of the beakers in triplicate. Each soil sub-sample was extracted with 100 mL of 2 M KCl on a wrist action shaker (Stuart Scientific, Model SF1) at 150 rpm for 1 h and the resultant extracts were filtered using a Millipore filtration system (StericupTM and SteritopTM filtration system, 0.22 m GV DuraporeTM membrane) to sterilise the extracts and prevent further microbial activity. These filter-sterilised extracts were stored at 4 ◦ C until they were analysed. Day 28 nitrate-N analyses were determined within 4 days of sampling and all other analyses were completed within 1 month of sampling.
2.2. Experimental design 2.4. Ammonium-N Sub-samples of the soil were adjusted to 60, 40, 30, 20 and 14% of the MHC with deionised water, and were pre-incubated at that MHC for between 6 and 12 days. MHC was determined using the method described by ISO (1997). A 2.5 kg dry weight equivalent sample of soil, at each moisture content, was amended with lucerne meal (Dengie Crops Limited, Essex, UK), an organic substrate with a C:N ratio of 12.2:1, at a rate of 5 g kg−1 dry soil and thoroughly mixed. Stock solutions of 4 and 40 g L−1 nitrapyrin (Sigma–Aldrich, Dorset, UK. 99%) in ethanol were prepared. Appropriate volumes of these stock solutions were added to 4 g aliquots of fine quartz sand, the ethanol was evaporated and the sand subsequently mixed with 400 g dry weight equivalent soil samples to give concentrations of 1.0, 3.2, 10, 32 and 100 mg nitrapyrin kg−1 dry soil, at each of the five soil moisture contents. Control soil samples were prepared, containing 4 g of fine quartz sand per 400 g soil. Triplicate sub-samples of each soil treatment including the controls were weighed into
The 2 M KCl extracts were analysed for ammonium-N using Palintest® ammonium reagents (Palintest® , Gateshead, UK) according to the supplier’s instructions. This method was based on the formation of indophenol from the reaction of phenol, hypochlorite and ammonium, in the presence of catalytic quantities of sodium nitroprusside. Ammonium-N was determined by measuring indophenol at 640 nm spectrophotometrically (Uvikon 930, NorthStar Scientific, Bedfordshire, UK). A calibration curve was constructed using prepared standards of ammonium chloride in the concentration range 0.1–1.0 mg ammonium-N L−1 . 2.5. Nitrite-N and nitrate-N Each 2 M KCl extract was analysed for total oxidised nitrogen and nitrite-N using the Palintest® kit (Nitratest and Nitricol,
Air flow
Humidifier
9 Beakers of soil incubated in each of 2 boxes connected in series
Fig. 1. Schematic of the apparatus used for incubation of soils at a range of moisture contents and nitrapyrin concentrations.
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Palintest® , Gateshead, UK) according to the supplier’s instructions. In this method, nitrate-N in the extract was first reduced to nitrite-N and the resulting nitrite-N was then determined by reaction with sulphanilic acid in the presence of N-(1-naphthyl)ethylene diamine to form a reddish dye (Montgomery and Dymock, 1961). The quantities of total oxidised nitrogen in each reacted extract were then determined at 545 nm spectrophotometrically (Uvikon 930, NorthStar Scientific, Bedfordshire, UK). A calibration curve was constructed using prepared standards of potassium nitrate and sodium nitrite in the concentration range 0.1–1.0 mg nitrate/nitrite-N L−1 . The nitrite-N concentration in each extract was also determined by an analogous procedure omitting the reduction step. The levels of nitrate-N were determined by subtraction of nitrite-N from the measured total oxidised nitrogen values.
Nitrate-N
Yield (mg N kg-1)
60
Ammonium-N * Below LOD
50 40 30 20 10
*
*
*
*
*
0 14
20
30
40
60
Soil moisture content (% of MHC) Fig. 2. Mean ammonium-N, nitrite-N and nitrate-N yield (with standard errors) at different soil moisture contents. Nitrite-N was not detected at any soil moisture content, so has been omitted from the figure. Limit of detection (LOD) for ammonium-N was 6.4 mg kg−1 . Limit of detection (LOD) for nitrite-N was 0.6 mg kg−1 . Limit of detection (LOD) for nitrate-N was 0.6 mg kg−1 .
2.6. Statistical analysis To assess the effect of soil moisture content on nitrogen transformation, the control soil concentrations of ammonium-N, nitrite-N and nitrate-N, at each of the moisture contents, were compared using one-way analysis of variance, followed by a series of Tukey t-tests (Zar, 1999). To determine whether soil moisture content had an effect upon the inhibition of nitrogen transformation by nitrapyrin, two-way analysis of variance (Zar, 1999) was performed, with soil moisture and nitrapyrin concentration as the main variables. Subsequently, one-way analysis of variance followed by Dunnett’s test (Dunnett, 1955) was undertaken on each of the nitrapyrin-exposed soils to determine the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC). Additionally, a Weibull model (Rawlings and Cure, 1985) was fitted to the data to determine the EC50 and 95% confidence interval for nitrapyrin ecotoxicity.
centration in the soil was between 4.7 and 6.3 mg kg−1 . There was no obvious relationship between the day 0 total oxidised nitrogen concentration and soil moisture content (data not shown). This concentration represented approximately 10% of the highest analysed levels of total oxidised nitrogen in the soil on day 28 (Table 1). Therefore, these values were considered to be adequately low background levels to allow the nitrogen transformation activity of the soil to be assessed. Ammonium-N, nitrite-N and nitrate-N concentrations were determined in control soil incubated at different soil moisture contents after 28 days of incubation. The data (Fig. 2) show that the soil moisture content affected the yield of ammonium-N and nitrate-N. Nitrite-N was not detected above the limit of detection (0.6 mg kg−1 ) at any time during the experiments. Therefore, total oxidised nitrogen was equivalent to nitrate-N. Ammonium-N was only detected at the lowest soil moisture content tested (14% MHC) which suggests that at this soil moisture content the first step of nitrification was impeded. This was also suggested by the lower yield of nitrate observed at this soil moisture content compare to soils incubated at higher soil moisture contents (Fig. 2). Ammonium-N yields were below the limit of detection at the higher soil moisture contents, suggesting that nitrification was converting any ammonium-N to nitrate-N (via nitrite-N). However, at 60% MHC the yield of nitrate-N was also below the limit of detection (0.6 mg kg−1 ), suggesting that at this soil moisture con-
3. Results and discussion 3.1. The effect of soil moisture content on the yield of ammonium-N, nitrite-N and nitrate-N Background concentrations of ammonium-N and total oxidised nitrogen were determined on day 0. The ammonium-N concentration in the soil on day 0 was between <6.4 mg kg−1 (the limit of detection) and 15.2 mg kg−1 , which was approximately twice the limit of detection. These analyses were sufficient to set a baseline and to detect an increase in ammonium-N above the background. At day 0 the total oxidised nitrogen (nitrite-N plus nitrate-N) con-
Table 1 Yield of ammonium-N, nitrite-N and nitrate-N after 28 days of exposure to a range of soil moisture contents and nitrapyrin concentrations. Nitrapyrin concentration (mg kg soil−1 )
Soil moisture content (% MHC) Nitrate-N (mg kg soil−1 ) 14
0 (control) 1 3.2 10 32 100
11.2 7.9 6.3 4.9# 5.6# 5.9#
20
Ammonium-N (mg kg soil−1 ) 30
+
43.9 53.2 38.4 6.2# 7.0# 6.1#
40 +
44.8 54.1 40.2# 4.5# 4.0# 4.9#
+
53.7 56.8 33.7# 3.8# 3.8# 4.5#
60
14
20
30
40
60
<0.6 <0.6 <0.6 <0.6 <0.6 <0.6
14.9 16.2 13.6 11.5 20.7 16
<6.4 <6.4 9.7 22.3 33.6 56.2
<6.4 <6.4 7.1 18.2 22.8 33.5
<6.4 <6.4 18.6 40.2 42.8 44.5
<6.4 <6.4 <6.4 22.5 16.6 23.3
As ammonium-N yields were below the limit of detection, reliable statistical analysis could not be performed. All day 28 control nitrite-N data were below 0.6 mg kg−1 (the limit of detection), therefore total oxidised nitrogen was equivalent to nitrate-N. Values represent the mean of individual analyses from triplicate soil sub-samples. + Significant increase (p < 0.05) in control nitrate-N production compared to soil with a moisture content of 14% of MHC. # Significant reduction (p < 0.05) in nitrate-N production compared to the control for the relevant moisture content.
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Table 2 Ecotoxicity endpoints (EC50 , NOEC and LOEC) of nitrapyrin (expressed as mg nitrapyrin kg soil−1 ) determined at different soil moisture contents. Soil moisture content (% MHC)
EC50 (mg kg−1 )
Lower 95% confidence limit(mg kg−1 )
Upper 95% confidence limit (mg kg−1 )
NOEC (mg kg−1 )
LOEC (mg kg−1 )
14 20 30 40
36 5.7 5.6 4
8 2.5 3.4 2.2
64 9 7.9 5.8
3.2 3.2
10 10 1 1
a a
tent ammonification (the formation of ammonium-N from organic nitrogen) was being inhibited. As ammonium-N yields were below the limit of detection, reliable statistical analysis could not be performed. Comparison of the control data (no added nitrapyrin) from each soil moisture content indicated that the 14% MHC and 60% MHC soil moisture content showed significantly lower (p < 0.01) nitrogen transformation than the 20, 30 and 40% MHC soils (Table 1). Data from control soils amended with lucerne meal at 50% MHC from our laboratory (data not shown) have a mean nitrateN concentration of 49.0 mg kg−1 (Std. dev. 13.3, n = 4), which is similar to the concentrations observed between 20 and 40% MHC in this study and consistent with the levels reported previously by Roberts et al. (2003). Therefore, for a sandy loam soil, of the type specified by the OECD 216 guideline, these data suggest that a more appropriate soil moisture content range would be between 20 and 50% MHC to ensure sufficient formation of nitrate-N to enable an inhibitory effect to be determined. The data (Table 1) suggest that nitrapyrin inhibited the first step of nitrification as, at 20–40% MHC, the ammonium-N increased with nitrapyrin concentration, whilst the nitrite-N stayed below the LOD and low concentrations of nitrate-N were observed. It was not possible to determine if ammonification was inhibited, but as ammonium-N was observed at high concentrations of nitrapyrin, but not at low concentrations or in the controls, it suggests that ammonification was not sensitive to nitrapyrin. This is consistent with published data, which shows that the mode of action of nitrapyrin is to inhibit the first step of nitrification, ammonium oxidation to nitrite (Goring, 1962; Campbell and Aleem, 1965). 3.2. Effect of soil moisture content on ecotoxicity endpoints (EC50 , NOEC and LOEC) of nitrapyrin To assess if the accuracy of the ecotoxicity endpoints (EC50 , NOEC and LOEC) were affected by the moisture content of the soil, soil was incubated at different soil moisture contents in the presence of lucerne (the source of organic nitrogen) and exposed to a series of concentrations of a known nitrification inhibitor, nitrapyrin (Goring, 1962). The EC50 for nitrapyrin varied between 4.0 and 36.0 mg kg−1 (Fig. 3). The two-way analysis of variance indicated that the 14% MHC soil resulted in a significantly different dose response to nitrapyrin than the other soil moisture concentrations, possibly due to the overall effect of low moisture on nitrogen transformation, irrespective of the presence of nitrapyrin. The NOEC and LOEC values, in addition to the EC50 values with confidence intervals, derived from each of the nitrapyrin exposures at different soil moisture contents are given in Table 2. At 20, 30 and 40% MHC there was not a significant difference in the EC50 value (5.7, 5.6 and 4.0 mg kg−1 (p = 0.05)) and these were consistent with the previously reported EC50 value (3.1 mg kg−1 ) for nitrapyrin determined at 50% MHC (Roberts et al., 2003).
Nitrapryrin EC50 (mg nitrapyrin kg soil-1)
EC50 values were determined using the Weibull model using analysed nitrate-N concentrations. It was not possible to determine the EC50 at 60% MHC as the nitrate-N was below the limit of detection (0.6 mg kg−1 ). a NOEC was not obtained since the LOEC was the lowest tested concentration.
70 60 50 40 30 20 10 0 10
20
30
40
Soil moisture content (% of MHC) Fig. 3. Observed nitrapyrin EC50 (with 95% confidence interval) at different soil moisture contents. It was not possible to determine the EC50 at 60% MHC as the nitrate-N was below the limit of detection (0.6 mg kg−1 ).
4. Conclusion The results presented here indicate that the current OECD 216 guideline recommended upper limit for the soil moisture content of the test soil (60% MHC) may be too high, which can lead to inhibition of the nitrification process and results in low yields of nitrate-N. Consequently, at 60% MHC, it was not possible to determine an EC50 for nitrapyrin, a known inhibitor of nitrification in soil. However, the yield of nitrate-N was relatively constant between 20 and 40% MHC and produced consistent EC50 values for nitrapyrin. Other reported data (Roberts et al., 2003) suggested that 50% MHC produced similar results. Therefore, we suggest that the OECD 216 test be conducted between 20 and 50% MHC. Acknowledgements The authors gratefully acknowledge the skilled technical assistance given by M Daniel and J Good, AstraZeneca Brixham Environmental Laboratory. References Alexander, M., 1977. Introduction to Soil Microbiology, second ed. J Wiley and Sons Inc, 253. Campbell, N.E.R., Aleem, M.I.H., 1965. The effect of 2-chloro-6(trichloromethyl)pyridine on the chemoautotrophic metabolism of nitrifying bacteria. I. Ammonia and hydroxylamine oxidation by Nitrosomonas. Antonie van Leeuwenhoek 31, 124–136. Dunnett, C.W., 1955. A multivariant comparison procedure for comparing several treatments with a control. American statistical association journal 50, 1096–1119. Goring, C.A.I., 1962. Control of nitrification of ammonium fertilizers and urea by 2-chloro, 6-trichloromethyl pyridine. Soil Sci. 93, 431–439. ISO 14238, 1997. Soil Quality–Biological Methods–Determination of Nitrogen Mineralisation and Nitrification in Soils and the Influence of Chemicals on these Processes.
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Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biology & Biochemistry 8, 209–213. Montgomery, H.A.C., Dymock, J.F., 1961. The determination of nitrite in water. Analyst (London) 86, 414–416. OECD, 2000. Guideline for the Testing of Chemicals No. 216. In: Soil Microorganisms: Nitrogen Transformation Test. Organisation of Economic Cooperation and Development, Paris.
Rawlings, S.O., Cure, W.W., 1985. The Weibull function as a dose-response model to describe ozone effects on crop yields. Crop Science 25, 807–814. Roberts, G.C., Peurou, F., Penwell, A.J., 2003. Assessing the toxic effect of nitrapyrin on nitrogen transformation in soil. Soil Biology & Biochemistry 35 (3), 479–481. Zar, J.H., 1999. Statistical Analysis, 4th ed. Prentice Hall, New Jersey.