Measurement of symbiotic nitrogen-fixation in leguminous host-plants grown in heavy metal-contaminated soils amended with sewage sludge

Environmental Pollution 111 (2001) 311±320

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Measurement of symbiotic nitrogen-®xation in leguminous host-plants grown in heavy metal-contaminated soils amended with sewage sludge J.P. Obbard *, K.C. Jones Environmental Science Division, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK Received 17 March 1999; accepted 15 January 2000

``Capsule'': White clover, broad bean and pea plants responded di€erently to metals in soils from sewage sludge applications. Abstract Rates of nitrogen ®xation by Rhizobium in symbiosis with leguminous host-plants including white clover, broad bean and peas have been established in soils that have been amended experimentally with heavy metal-contaminated sewage sludges. Results from 15Ndilution experiments for the measurement of N2 ®xation have shown that adverse heavy metal e€ects are apparent on symbiotic N2 ®xation rates for white clover grown in inter-speci®c competition with ryegrass under mixed sward conditions, compared to white clover grown in pure sward. Further experiments on broad bean and pea indicated a signi®cant, but minor-inhibitory metal-related e€ect on the rate of N2 ®xation compared to untreated soils and soils amended with a relatively uncontaminated sludge. The implications of the results with respect to sludge utilisation in agriculture are discussed. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Nitrogen ®xation; Rhizobium; Metal contamination; Soils; Sewage sludge

1. Introduction The addition of sewage sludge to agricultural soil improves physical characteristics and acts as a valuable source of nutrients to growing crops (Smith, 1996). However, there is concern over the use of sludge that has been contaminated with potentially toxic elements (PTEs) from both industrial and domestic sources, and it is the concentration of PTEs in sludge that is often the main determinant in restricting its application to agricultural soils. The level of PTEs in sewage sludgeamended soils in the UK is controlled by The Sludge (Use in Agriculture) Regulations, 1989 (as amended) (UK Statutory Snstrument, 1989; DoE, 1995) which implement the European Community Directive 86/278/ EEC (CEC, 1986). Maximum permissible limits of PTEs in soil are expressed in terms of total metal concentra* Corresponding author. Present address: National University of Singapore, Department of Chemical Engineering, 10 Kent Ridge Crescent, 119260 Singapore; tel.: +65-874-2884; fax: +65-779-1936. E-mail address: [email protected] (J.P. Obbard).

tions (as mg/kg dry soil) for individual metals in the soil. Table 1 presents PTE concentrations permitted in soil under the CEC (1986) Directive and under the UK Statutory Instrument (1989, as amended) Regulations. Soil pH is an important factor controlling the availability of PTEs to plants in soils (Logan and Chaney, 1983), where total permissible limits account for the increased availability of metals at lower soil pH. Current debate on metal accumulation in soils as a result of sewage sludge applications is focussed on acceptable loading levels, clari®cation and interpretation of environmental risk (McGrath et al., 1994; Witter, 1996; Dahlin et al., 1997; Werner and Warnusz, 1997; Giller et al., 1998). Heavy metals have been implicated in impairing a range of microbial characteristics and processes in sewage sludge amended soils, including non-symbiotic and symbiotic N2 ®xation (Brookes et al., 1986; McGrath et al., 1988; Giller et al., 1989; Martensson and Witter, 1990; Lorenz et al., 1992; Chaudri et al., 1993; Barakah et al, 1996; Martensson and Torstensson 1996; Obbard and Jones, 1993; Obbard et al., 1993). Microbial

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J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320

Table 1 Comparison of Directive 86/278/EEC and UK limit values for total concentrations of heavy metals in sludge-amended soil (mg/kg dry soil) Directive 86/ 278/EECa

Element Zn Cu Ni Cd Pb Crc Hg

Soil pH 6±7 150±300 50±140 30±75 1±3 50±300 ± 1±1.5

UK Statutory Instrument (1989) Regulations 5.0<5.5

5.5<6.0

6.0<7.0

>7.0

b

b

b

300b 200 110 3 300 400d 1

200 80 50 3 300 400d 1

200 100 60 3 300 400d 1

200 135 75 3 300 400d 1

a For soil with a pH of 6±7. Limit values may be reduced on soil with a pH<6 to account for increased mobility and availability to crops. Higher limits are permitted on soil with a pH>7 and >5% calcium carbonate, but these must not exceed the adopted pH 6±7 values by more than 50%. b As amended (DoE, 1995). c No statutory limits have been set for Cr; proposed amendment to the Directive has been removed from the legislative process. d Cr limits are provisional.

processes within soil ecosystems play a vital role in determining the quantities of plant-available nitrogen via metabolic transformation reactions (Paul and Clark 1996; Sylvia et al., 1998). Studies on symbiotic nitrogen ®xation have been largely focussed on the host plant white clover (Trifolium repens L.) in association with Rhizobium leguminosarum biovar trifolii, due to the agronomic importance of this plant in mixed-grass swards. Under favourable conditions, N2 ®xation rates of up to 200 kg N haÿ1 yearÿ1 for T. repens have been recorded (Robson et al., 1989). The possibility of a long-term impairment in the N2 ®xation capacity of agricultural soils, due to contamination, therefore warrants concern. For the purposes of this study, quantitative estimates of N2 ®xation were measured using the 15N-dilution technique on the host-plants white clover (T. repens) with the symbiont R. leguminosarum biovar trifolii, and broad bean (Vicea faba) and pea (Pisum sativum) with the symbiontR. leguminosarum biovarvicea. In a study by McGrath et al. (1988), 15N-dilution was only detected in T. repens when e€ective R. leguminosarum biovar trifolii cells isolated from a soil amended with farmyard manure were inoculated into a metal-contaminated sludge-amended plot. Isolates from the sludged soil were later found to be genetically ine€ective at ®xing nitrogen (Giller et al., 1989; Hirsch et al., 1993). In the investigation reported here indigenous strains of e€ective bacteria were tested to determine N2 ®xation rates in soils that had been previously amended with sewage sludge. Competition for nutrients and other envir-

onmentally limiting factors between T. repens and grass species in the ®eld sward are known to a€ect the rate of N2 ®xation (Haynes, 1980). The implications of this have been also studied by conducting 15N-dilution experiments on T. repens growing in pure and mixed swards with ryegrass (Lolium perenne) in sludge amended soils. The 15N-dilution investigation was extended to broad bean and pea plants because of the commercial importance of these legumes in agriculture and the opportunity to study e€ects on a di€erent variety of Rhizobium i.e. R. leguminosarum biovar viceae. Previous studies on soybean in soils freshly amended with sewage sludge have indicated that sludge amendment in agriculture may be an acceptable practice with respect to growth, nodulation and nitrogen ®xation characteristics of soybean (Angle et al., 1992). 2. Materials and methods 2.1. Soils used for experimentation 2.1.1. Luddington experimental site, UK The trial was established by the UK Agricultural Development and Advisory Service in collaboration with the Macaulay Institute for Soil Research in 1968. Soil treatments included untreated control plots; plots with single sewage sludge applications of 125 t dry matter (DM)/ha and plots with four successive annual application of 31 t DM/ha. A local relatively uncontaminated sludge was used to dilute four metalcontaminated UK sludges selected on the basis of their high concentrations of either Zn, Cu, Ni, or Cr, to achieve target concentrations of 16000, 8000, 4000, and 8800 mg/kg of theses elements, respectively, in the dry sludges, or half of these values. Sixteen soil treatments were replicated in four blocks, and horticultural crops were grown during the period 1968±72, after which pasture grasses were sown. 2.1.2. 15N-dilution experiments Quantitative estimates of N2 ®xation rates were determined using the 15N-dilution technique in conjunction with combustion mass spectrometry of plant material grown on soil samples from the Luddington experimental plots. The 15N-dilution technique, when used for the quantitative assessment of symbiotic N2 ®xation, is dependent on isotopic composition di€erences between a test legume and a non-®xing reference plant caused by the isotopic dilution of root assimilated 15 N by the ®xation of atmospheric 14N2 (McAuli€e et al., 1958). The use of the stable, non-radioactive 15N isotope in studies of plant nutrition and the calculation and interpretation of data are described in an International Atomic Energy Agency report (IAEA, 1983), and

J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320

the subject has also been reviewed extensively by Chalk (1985) and Danso (1986). Variations in sludge-related soil properties (apart from heavy metal concentrations) were controlled as much as possible by the use of soil from the Luddington experimental plots where adequate controls (i.e. sludged soil of low metal concentrations) were present. The determination as to whether e€ects on N2 ®xation were induced by an e€ect on the micro-symbiont, or by a direct phytotoxic e€ect on the host-plant itself, was ascertained, as far as possible, by conducting growth trials with added nitrogen. If nitrogen additions compensated for any yield reduction then it could be deduced that metal e€ects on the symbiosis were non-phytotoxic. Host plants tested included T. repens grown in pure and mixed sward (with ryegrass L. perenne), broad bean and pea. Reference plants used were ryegrass for white clover, and barley for broad bean and pea. 2.1.3. Soil preparation Homogenized moist soil (<2 mm) from the Luddington experimental site was labelled by mixing in a 15 N solution, containing 15N as 15NH4 15NO3 (99 or 66% atom excess 15N), at the rate of 37.5 mg 15N per kg/dry soil (McGrath et al., 1988). The moisture content of the soil was determined and adjusted to 20% (dry weight basis). The soil was again thoroughly homogenised and left in darkness for 6 weeks with daily mixing. After this period soil sample replicates were placed into 15-cm-diameter plant pots for the growth trial. Moisture content was re-checked and any necessary adjustments were made. Soil (350 g; dry weight equivalent) for each of four replicates for each soil sample was used. The plant pots were placed on individual pot trays and the total weight of the replicate pot taken. Pre-germinated seedlings of the various plants used in the assay were then planted into the soil. The number of plants grown in each pot was species dependent and selected on the basis of pre-experimental trials to determine the optimum number of plants per pot. Plant density for the mixed sward of white clover/ryegrass was selected to reduce interspecies competition for light and nutrients. Plant densities per pot were as follows: 1. white clover and ryegrass (pure sward): 40 plants per pot; 2. white clover and ryegrass (mixed sward): 20 plants per pot; and 3. broad bean, pea and barley (pure sward): two plants per pot. Plants were grown for 8 weeks in a random block design in a Fisons growth cabinet (20 C, 16/14 h light/ dark cycle), and maintained at 20% moisture content by daily watering with sterile distilled±deionised water to weight. The soil surface of each pot was covered with 50 g of high-density polyethylene beads to reduce eva-

313

poration and the growth of cyanobacteria, and to prevent the cross-contamination of Rhizobium strains. For each treatment, three additional replicates had nitrogen added upon watering (0.05% KNO3 solution) in order to determine whether growth e€ects were induced by a nitrogen de®ciency or a direct phytotoxic e€ect of heavy metals on the host plant. After the growth period, plants were harvested and yield determined (dry weight at 105 C, 48 h). 2.1.4. Analysis of 15N-labelled plant material Dried plant shoot material was ground in a liquid nitrogen freezer mill (Spex Industries, USA). Root material was not analysed to avoid the risk of analysing soil particulates adhering to root tissue and, in the case of the mixed sward, to avoid mixing of clover and ryegrass root material. Low temperature guarantees rapid grinding of the plant material to a ®ne powder without loss of temperature-labile material. Homogeneity of the sample is particularly important in this method as the weight taken for analysis is only 5±10 mg. Plant material was added to pre-weighed tin capsules that were dried (105 C, 20 min), sealed, cooled, re-weighed and analysed using a Dumas-type continuous ¯ow CHN analyser (Roboprep) coupled to a tracer-mass isotope-ratio mass spectrometer (Europa Scienti®c limited, Crewe, UK). The analytical technique was based on the method of Harris and Paul (1989), where samples were combusted directly and results obtained for both total nitrogen and the atom % 15N excess of plant material and soils. The proportion of nitrogen in the test plant as a function of N2 ®xation was calculated using the following equation: atom % excess of 15 N in the test plant †  100 atom % excess of 15 N in the reference plant † 

Minus natural abundae of

15

…1†

N (0.366%)M.

3. Results and discussion 3.1. Soil properties and

15

N-dilution results

Results are divided into two sections. The ®rst section is further sub-divided into results for white clover grown in pure and mixed sward (with ryegrass), and the second section results for broad bean and pea. 3.1.1. White clover Ð pure sward Soil chemical data for the Luddington experimental soils used for this experiment are given in Table 2. Soil pH in the untreated control was 5.1, with sludged soils ranging between pH 4.4 and 5.2. Organic matter content in the untreated control was 4.7%, with sludged soils ranging from 4.8 to 6.7%. Heavy metals concentrations exceeded soil limit values for Zn (i.e. 200 mg/kg) in the

314

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Table 2 Soil physiochemical characteristics of Luddington soil samples used for the estimation of symbiotic N2 ®xation for white clover grown in pure swarda Sample

pH

Organic matter (%)

Totalb Zn

Total Cu

Total Ni

Total Cr

Soil total N (%)

Soil 15N atom %c

Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

5.1 4.4 4.8 4.9 4.8 4.8 5.1 5.2 5.2

4.7 5.6 5.1 4.8 7.9 5.9 6.7 5.7 5.7

146 110 366 442 664 206 270 255 200

101 53 91 42 44 193 283 52 43

32 28 28 33 79 32 35 117 36

162 153 192 186 264 356 220 253 347

0.16 0.15 0.18 0.17 0.21 0.18 0.22 0.18 0.22

0.031 0.022 0.020 0.020 0.027 0.019 0.023 0.021 0.023

a Figures underlined indicate that the UK limit values are exceeded. Classi®cation is `ungrazed grassland'. Metal concentrations in mg/kg of dry soil. Soil was labelled with 66% 15N excess (15NH415NO3) at 37.5 mg/kg dry soil. b Metal concentrations in mg/kg dry soil. c At 5% atom excess analytical standard deviation is <1%.

following soils: `low Zn' (by 166 mg/kg), `mid Zn' (by 242 mg/kg), `high Zn' (by 464 mg/kg), `low Cu' (by 6 mg/kg), `high Cu' (by 70 mg/kg) and `high Ni' (by 55 mg/kg). The Cu limit (i.e. 80 mg/kg) was exceeded in the following soils: untreated control (by 23 mg/kg) `low Cu' (by 113 mg/kg) and `high Cu' (by 203 mg/kg). The Ni limit (i.e. 50 mg/kg) was exceeded in the `high Ni' soil (by 67 mg/kg). Therefore, contaminated soils exceeded UK limit values for one or more elements. Cadmium was not analysed in this set of soils, but was assumed to be below the UK limit value on the basis of previous analysis (unpublished data). It must also be noted that The Code of Practice for Agricultural Use of Sewage Sludge (DoE, 1989) recommends caution when applying sludge to soils with a pH<5.2, as found in the Luddington soils. The experimental soils therefore represent a rather extreme case with respect to current sludge utilisation practices in the UK, i.e. soils of low pH with elevated metal levels above UK limit values. Total nitrogen levels (%) ranged from 0.16 to 0.22 % in the test soils, with an excess 15N (atom %) of 0.019±

0.031% achieved by the addition of 66% 15N excess (15NH415NO3) at 37.5 mg 15N /kg dry soil. 15 N atom % excess data of test and reference (i.e. ryegrass) plant shoot material are given in Table 3, together with N2 ®xation estimates (expressed as the percentage of total shoot nitrogen) for white clover. Eq. (1), with the mean 15N-atom % excess of reference material, was used to calculate N2 ®xation (%) rates for white clover. Mean plant yield and total nitrogen (%) content of shoot material are given for test and reference plants in Table 4 together with calculated mean plant nitrogen yields. Yield data from a separate growth trial, where plants with and without supplemented nitrogen (0.05% KNO3 w/v) are presented in Table 5. N2 ®xation estimates for contaminated sludge treatments varied between 38.2 and 60.9% compared to the untreated control at 48.1% and the sludged control at 46.7%. Although there was variation in the chemical properties between soil treatments, N2 ®xation estimates were not signi®cantly correlated to these. N2 ®xation estimates did not di€er between samples from the untreated

Table 3 Atom % 15N excess in reference (ryegrass) and test plants (white clover) and N2 ®xation estimates for white clover grown on Luddington experimental soils Ð pure sward Sample

Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

Reference plant (ryegrass)

Test plant (white clover)

Mean atom % excess of 15N

S.D.

Mean atom % excess of 15N

S.D.

Mean N2 ®xation rate (%)

S.D.

1.553 1.269 1.305 1.549 1.763 1.502 1.396 2.115 1.371

0.124 0.055 0.070 0.180 0.148 0.147 0.072 0.108 0.197

0.806 0.678 0.822 0.867 1.007 0.649 0.716 0.827 0.822

0.056 0.066 0.106 0.326 0.115 0.026 0.040 0.057 0.114

48.1 46.7 38.2 52.8 42.9 56.8 48.7 60.9 44.0

3.63 5.17 7.53 14.10 6.53 1.70 2.83 2.71 3.39

J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320 Table 4 Plant yields, total nitrogen content (%) and calculated mean plant nitrogen yield for plants grown on the Luddington experimental samples Ð pure sward Sample

Mean yield (mg)

S.D.

Total N%

S.D.

Mean plant N yield (mg)

White clover Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

22 22 22 26 26 28 23 27 27

5.6 2.1 4.3 6.2 4.6 2.1 4.4 5.1 3.0

3.6 3.6 3.4 3.7 3.2 3.6 3.7 3.9 3.6

0.34 0.46 0.34 0.11 0.24 0.23 0.29 0.73 0.13

0.79 0.78 0.77 0.97 0.83 0.85 0.84 1.07 0.98

Ryegrass Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

38 40 42 46 44 41 31 52 38

± 7.4 7.6 6.4 11.0 9.2 4.5 10.0 0.2

1.28 1.27 1.42 1.34 1.28 1.48 1.84 1.23 1.21

0.19 0.22 0.21 0.18 0.23 0.23 0.07 0.15 0.13

0.48 0.51 0.59 0.62 0.56 0.60 0.56 0.63 0.46

Table 5 Yields of plants grown in soil from the Luddington experimental trial with and without supplemented nitrogen (0.05% KNO3 w/v) Ð pure sward Sample

Mean yield (mg) minus N

S.D.

Mean yield (mg) plus N

S.D.

White clover Untreated Sludged control 1. High Zn 2. High Cu 3. High Ni 4. High Cr

26 29 21 24 22 32

2.96 3.24 2.19 3.14 1.55 4.51

35 36 31 38 28 51

4.49 9.04 4.90 9.31 6.89 8.19

Ryegrass Untreated Sludged control 1. High Zn 2. High Cu 3. High Ni 4. High Cr

28 24 30 26 43 42

1.79 4.71 2.30 1.96 4.86 7.80

57 58 69 50 64 54

5.29 4.07 4.39 4.16 6.54 4.90

and contaminated soil treatments, except for the high Ni sample (60.9%, P<0.003) and the `low Cu' sample (56.8%, P<0.020) where N2 ®xation was signi®cantly greater. The mean N2 ®xation estimate for the sludged control treatment was also less (46.7%) than the `low Cu' sample (56.8%, P<0.24). N2 ®xation in the `high Ni' and the `low Cu' samples was also signi®cantly higher than other contaminated treatments, but no other di€erences were determined between contaminated treatments. Despite these samples exceeding UK limit values for Ni

315

(117 mg/kg) and Cu (193 mg/kg), respectively, mean N2 estimates were actually higher than the controls and other treatments, thereby indicating that no adverse metal e€ect on the N2 ®xing process was occurring. One-way analysis of variance (ANOVA) tests revealed no signi®cant inter-treatment di€erences in white clover yields, and only small di€erences in ryegrass yields (Table 4). The supplemented N-growth trial demonstrated that plant yields were increased by between 33 and 59% for white clover and 30 and 147% for ryegrass, thereby indicating that heavy metals alone were not limiting plant growth in the soils tested. Overall, data have shown that no adverse e€ects on the rate of N2 ®xation were occurring for white clover growing in pure sward on contaminated sludged soils of low pH which exceeded UK limit values for either Zn, Cu, or Ni compared to control samples. 3.1.2. White clover Ð mixed sward Soil chemical data for the Luddington experimental soils used for this experiment are given in Table 6. Soil pH in the sludged control was pH 5.1, with sludged soils ranging between pH 5.0 and 5.7. Organic matter content in the sludged control was 5.1%, with sludged soils ranging from 3.6 to 6.7%. Heavy metals concentrations exceeded soil limit values for Zn (i.e. 200 mg/kg) in the following soils: `low Zn' (by 97 mg/kg), `high Zn' (by 342 mg/kg), `low Cu' (by 54 mg/kg), `high Cu' (by 49 mg/kg) and `high Ni' (by 30 mg/kg). The Cu limit (i.e. 80 mg/kg) was exceeded in the following soils: `low Cu' (by 67 mg/ kg) and `high Cu' (by 155 mg/kg). The Ni limit (i.e. 50 mg/ kg) was exceeded in the `low Ni' (by 13 mg/kg), `high Ni' soil (by 103 mg/kg). Total nitrogen levels (%) ranged from 0.16 to 0.27% in the test soils, with an excess 15N (atom %) of 0.272±0.574% achieved by the addition of 99% 15N excess (15NH415NO3) at 37.5 mg 15N /kg dry soil. 15 N atom % excess data of test and reference (i.e. ryegrass) plant shoot material are given in Table 7, together with combined plant yield and N2 ®xation estimates (expressed as the percentage of total shoot N) for white clover. ANOVA (one-way) tests on N2 ®xation data proved that signi®cant di€erences existed between treatments (F=12.98, P<0.001) and the intertreatment variation contrasted sharply with that for white clover growing in pure sward. N2 ®xation of white clover in the sludged control soil (40.1%) was signi®cantly greater than for the `low Zn' sample (15.2%, P<0.001), the `high Zn' sample (16.0%, P<0.023), and the `high Cu' sample (8.2%, P<0.034). `Low Ni' (70.7%, P<0.07) was the only sample to have a signi®cantly higher N2 ®xation estimate than the sludged control. The 'high Cr' and 'low Cr' samples showed no signi®cant di€erence in N2 ®xation estimates to the control. It is interesting to note that these three soils were the only samples not to exceed the UK limit values for heavy metals in sludged soils.

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J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320

Table 6 Characteristics of Luddington soil samples used for the estimation of N2 ®xation for white clover grown in mixed sward with ryegrassa Soil

pH

Organic matter (%)

Totalb Zn

Total Cu

Total Ni

Total Cr

Soil total N (%)

Soil atom % 15 N excessc

Sludged control 1. Low Zn 2. High Zn 3. Low Cu 4. High Cu 5. Low Ni 6. High Ni 7. Low Cr 8. High Cr

5.2 5.0 5.0 5.1 5.0 5.2 5.4 5.7 5.6

5.1 5.4 4.5 5.0 6.7 5.9 5.3 3.8 3.6

112 297 542 254 249 121 230 99 143

33 32 34 147 235 30 35 32 74

48 31 48 30 35 63 153 25 31

78 81 117 135 160 144 168 230 331

0.16 0.18 0.19 0.20 0.27 0.20 0.19 0.16 0.16

0.518 0.502 0.574 0.312 0.437 0.272 0.450 0.467 0.552

a Figures underlined indicate that the UK limit values are exceeded. Classi®cation is `ungrazed grassland'. Metal concentrations in mg/kg of dry soil. Soil was labelled with 99% 15N excess (15NH415NO3) at 37.5 mg/kg dry soil. b Metal concentrations in mg/kg dry soil. c At 5% atom excess analytical standard deviation is <1%.

Table 7 Atom % 15N excess in reference (ryegrass) and test plants (white clover) and N2 ®xation estimates for white clover on soils from the Luddington experimental trial Ð mixed sward Sample

Sludged control 1. Low Zn 2. High Zn 3. Low Cu 4. High Cu 5. Low Ni 6. High Ni 7. Low Cr 8. High Cr

Reference plant (ryegrass)

Test plant (white clover)

Mean atom % excess of 15N

S.D.

Mean atom % excess of 15N

S.D.

Nitrogen ®xation (%)

S.D.

Total yield (mg)

Yield S.D.

5.950 5.088 5.105 5.544 5.312 5.252 5.801 5.294 6.141

0.580 0.339 0.442 0.425 0.076 0.595 0.081 0.612 0.818

3.563 4.316 4.289 3.669 4.877 1.542 4.455 2.409 3.644

1.067 0.294 0.332 0.120 0.088 0.241 0.292 0.462 0.834

40.1 15.2 16.0 33.8 8.2 70.7 23.2 54.5 40.7

17.94 5.78 6.31 2.16 1.66 4.57 5.03 8.73 13.58

863 1127 586 798 305 982 905 1100 773

210 142 110 269 66 175 285 48 85

For all samples tested, N2 ®xation was signi®cantly reduced where plants were grown in soils which had the higher concentration of a speci®c element compared to the lower, except for the `high' and `low' Zn samples. For these samples N2 ®xation rates were signi®cantly reduced compared to the sludged control, and were similar for both despite the di€erence in Zn concentration (`low Zn' 297 mg/kg 15.2% N2 ®xation, `high Zn' 542 mg/kg±16% N2 ®xation). Therefore, in general, the e€ect of an enhanced concentration of heavy metals is to signi®cantly reduce N2 ®xation rates of white clover when in inter-speci®c competition. The combination of elements present at di€erent relative concentrations between soils makes it particularly dicult to determine which elements are having the greatest e€ect on N2 ®xation. In contrast to plants grown in pure sward signi®cant total yield di€erences were found between treatments (F=9.73, P<0.001), where reductions occurred between the higher and lower concentrations of Zn, Cu, and Cr, but

not Ni. Reductions in yield may re¯ect the adverse e€ect of heavy metals on the plants when in inter-speci®c competition. Grass species are known to be at a competitive advantage when grown in mixed swards with clover due to allelochemical e€ects and their greater ability to extract soil mineral nutrients (Haynes, 1980; Frame and Newbould, 1986). Shading by grass foliage also limits photosynthesis by clover plants and hence their ability to compete e€ectively. 3.1.3. 15N-dilution results for broad bean and pea plants Physicochemical properties of soil samples used for experimentation are given in Table 2. 15N atom % excess for broad bean, pea and barley are given in Table 8 and N2 ®xation estimates are given in Table 9. Plant yields, total nitrogen (%) content and calculated mean plant nitrogen yields are given in Table 10. Broad bean yields when grown in soil with and without supplemented nitrogen (0.05% KNO3) are given in Table 11. Pea growth trials with and without nitrogen failed due

J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320 Table 8 Atom %

317

15

N excess in reference plants (barley) and N2 ®xing broad bean and pea plants in Luddington experimental soils

Sample

Reference plant (barley)

Test plants Broad bean

Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

Mean atom % excess of 15N

S.D.

Mean atom % excess of 15N

S.D.

Mean atom % excess of 15N

S.D.

2.373 2.274 2.270 1.572 2.811 2.696 2.317 2.983 2.700

0.197 0.180 0.342 0.082 0.266 0.240 0.515 0.192 0.15

0.275 0.309 0.441 0.409 ± 0.467 0.470 ± 0.428

0.063 0.052 0.100 0.100 ± 0.120 0.102 ± 0.056

0.938 0.488 0.983 0.834 ± 0.952 ± ± 0.761

0.267 0.131 0.177 0.133 ± 0.180 ± ± 0.187

Table 9 N2 ®xation (%) of broad bean and pea plants grown on Luddington experimental soils Sample

Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

Pea

Broad bean

Pea

Nitrogen ®xation (%)

S.D.

Nitrogen ®xation (%)

S.D.

88.9 86.4 81.5 74.0 ± 82.7 79.3 ± 84.2

2.54 2.31 3.81 6.35 ± 4.43 4.23 ± 2.08

63.7 78.5 56.7 46.9 ± 64.7 ± 49.2 71.8

13.25 5.76 7.82 8.50 ± 6.72 ± 6.72 6.92

to persistent problems with mildew. 15N-dilution estimates were only determined for plants grown on treatments where nitrogenase activity had been previously con®rmed (unpublished data), i.e. all treatments for broad bean except the `high Zn' and `high Ni' soils and all for pea except the `high Zn' and `high Cu' soils. 3.1.3.1. Broad bean. ANOVA indicated that N2 ®xation estimates di€ered signi®cantly between treatments (F = 11.23, P<0.001), but not plant yields (F ˆ 0:69, P<0.65). Further statistical analysis using `t-test' statistics indicated that N2 ®xation estimates for the untreated control (88.9%) were signi®cantly greater than the `low Zn' sample (81.5%, P<0.002), the `mid Zn' sample (74%, P<0.005), the 'low Cu' sample (82.7 %, P< 0. 012), the `high Cu' sample (79.3%, P<0.001), and the `high Cr' sample (84.2%, P<0.004). The N2 ®xation estimate for the sludged control (86.4%) was signi®cantly greater than for the `low Zn' and `mid Zn' estimates. Although no signi®cant di€erences were found between plant yields it is worth noting that the mean yields for unnodulated plants in the `high Zn' sample

Table 10 Plant yields, plant total nitrogen content and calculated mean plant nitrogen yields for barley, broad bean and pea plants grown on soils from Luddington experimental site Sample

Mean yield (mg)

S.D.

Total N%

S.D.

Total N yield (mg)

Barley Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

655 592 617 820 500 615 641 653 711

114 106 100 161 111 96 98 127 67

1.96 2.37 2.50 1.82 2.49 2.16 2.56 2.10 2.03

0.16 0.29 0.31 0.14 0.36 0.36 0.28 0.38 0.11

14.2 14.0 15.4 14.9 12.5 13.5 16.4 13.7 14.2

Broad bean Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

2048 2220 2091 2000 1947 2138 2067 1841 2176

386 424 320 300 304 420 349 277 321

3.57 3.69 3.03 2.84 ± 3.28 3.18 ± 3.82

0.34 0.33 0.13 0.26 ± 0.69 0.32 ± 0.26

73.1 82.1 83.4 56.0 ± 70.6 66.1 ± 82.7

Pea Untreated Sludged control 1. Low Zn 2. Mid Zn 3. High Zn 4. Low Cu 5. High Cu 6. High Ni 7. High Cr

575 524 390 590 570 598 472 679 818

220 229 122 85 111 119 149 90 304

3.58 4.16 3.05 3.51 ± 3.43 ± 2.89 3.80

0.50 0.42 0.25 0.21 ± 0.36 ± 0.25 0.35

20.7 22.0 12.1 20.7 ± 20.3 ± 17.0 31.1

and the `high Ni' sample were the lowest of all the treatments. Yields of broad bean supplemented with nitrogen were not signi®cantly di€erent from those without added nitrogen. As no di€erences were determined in plant

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Table 11 Yields for plants growing in Luddington experimental soils with and without supplemented nitrogen (0.05% KNO3 w/v) Sample Broad bean Untreated Sludged control 1. High Zn 2. High Cu 3. High Ni 4. High Cr

Mean yield (mg) minus N

S.D.

Mean yield (mg) plus N

S.D.

994 1128 937 1016 860 1050

307 99 189 364 327 341

1052 1191 809 976 996 998

169 264 138 143 225 217

yield between treatments in the 15N-dilution trial the conclusion is that unsupplemented plants were able to satisfy their nitrogen-requirement through N2 ®xation alone. Excluding the `high Zn' and `high Ni' samples, the greatest reduction in N2 ®xation estimates was between the sludged control and the `mid Zn' sample. This represents a reduction in N2 ®xation rates from 86.4 to 74% only. Therefore, heavy metal e€ects, where e€ective symbiosis with R. leguminosarum biovar viceae was present, were signi®cant, but not severe enough at the above heavy metal concentrations, to result in signi®cant di€erences in yield. However, higher concentrations of heavy metals (664 mg/kg of Zn and 117 mg/kg Ni) resulted in the complete failure of nodulation, and hence N2 ®xation. Therefore, there appears to be a distinct contrast between the e€ects of heavy metals on Rhizobium populations in the soil and the ability of e€ective Rhizobium strains to ®x N2 once in symbiosis with the host plant when growing in contaminated soils. The ability of the nodule to protect Rhizobium from toxic metal exposure has been speculated by Giller et al. (1989) and Smith (1990) and this investigation supports their hypothesis. The phenomenon is however, dependent on a viable population of e€ective Rhizobium being present in the soil in the ®rst instance. 3.1.3.2. Pea. As for broad bean, ANOVA (one-way) tests proved that N2 ®xation estimates di€ered between treatments (F ˆ 12:79, P<0.001), but plant yield (F ˆ 3:05, P ˆ 0:14) did not. Further `t-test' analysis showed that N2 ®xation estimates were greater for pea plants growing on uncontaminated sludged soil (78.5%) than for plants grown on the untreated soil (63.7%, P ˆ 0:027). N2 ®xation estimates were signi®cantly lower for samples grown on contaminated treatments than the sludged control than for the following soils `low Zn' (56.7%, P ˆ 0), `mid Zn' (46.9%, P ˆ 0), `low Cu' (64.7%, P<0.002), and `high Ni' (49.2%, P ˆ 0). As for broad bean, the highest N2 ®xation estimates for plant samples grown in contaminated soils were for the

`high Cr' sample (71.8%) which did not exceed UK limit values for heavy metals in sludge amended soils. Intra-sample variation in N2 ®xation estimates and yields for pea plants (Table 10) were greater than for broad bean due to persistent problems with mildew during growth trials. This was not treated because of the possibility of causing interference with the symbiosis. Trials for pea plants with additional nitrogen failed due to persistent problems with mildew on the plants. Greater signi®cant inter-sample variation was also detected for pea plants (78.5% sludged control to 46.9% for `mid Zn' soil), but this was not sucient to result in yield di€erences between treatments. As for the white clover pure sward growth trial, it was dicult to ascertain whether reduced N2 estimates were the result of a direct e€ect on the plant or the Rhizobium, or due to an interactive e€ect. 4. Conclusion The main conclusions from the 15N-dilution data for white clover when grown in pure sward is that rates of N2 ®xation indicated that no adverse heavy metal e€ects were occurring for indigenous strains of Rhizobium in symbiosis with the host plant. However, growth of clover in mixed sward with rye grass resulted in a severe reduction of N2 ®xation for most the most contaminated soils when compared to the control. This was re¯ected in reduced yields for plants, where reductions in N2 ®xation were related to the concentration of heavy metals present. Signi®cant reductions in N2 ®xation were recorded in some soils which did not exceed UK limit values for heavy metal concentrations and reductions were large in soils which exceeded limit values for Cu, Zn and Ni. The reason for this e€ect under mixed sward conditions was that clover was presumed to be at a competitive disadvantage for nutrients and light with ryegrass, resulting in a lowered rate of photosynthesis and nitrogenase activity. The use of mixed clover/grass swards in agriculture is important in terms of providing nutritional grazing and enhancing nitrogen-reserves. Therefore, even though investigations have shown that there are no adverse e€ects on symbiotic N2 ®xation per se, competition between clover and other sward species on the ®eld may be detrimental to soil fertility in contaminated soils under conditions of inter-speci®c competition. However, it must be noted that the Luddington soils may represent an extreme case with respect to current sludge utilisation practices in the UK, as metal concentrations in the majority of samples used were well in excess of UK limit values and the soil was of a low pH. Indeed, The Code of Practice for Agricultural Use of Sewage Sludge (DoE, 1989) recommends caution in the application of sludge to soils at pH<5.2, as is the case for most of the Luddington samples studied.

J.P. Obbard, K.C. Jones / Environmental Pollution 111 (2001) 311±320

The main conclusion from the 15N-dilution data for broad bean and pea plants is that a reduction in N2 ®xation for plants grown in contaminated soil was apparent compared to the controls. However, these differences did not result in a signi®cant di€erence in yield where reductions were relatively slight, particularly for broad bean, even in soils well in excess of UK limit values for Zn, Cu and Ni. The e€ect, therefore, may not present a problem at lower metal concentrations. It appears that where e€ective Rhizobium are present in the soil and can achieve symbiosis with the host plant, then e€ects on N2 ®xation are not severe even where metal concentrations are in excess of UK limit values. The importance of any e€ect will be clearly dependent on the quantity of nitrogen-fertilisers applied under agronomic conditions to sludge-amended soils in the long term. Also, the coating of legume seed with e€ective strains of Rhizobium is an established practice (Vincent, 1981; Havelka et al., 1982) and this may circumvent the problem of low indigenous populations of e€ective cells in the soil and the establishment of e€ective symbiosis. Whilst important relationships between N2 ®xation rates and soil properties in sewage sludge-amended soils have been noted in this investigation, further studies are required to establish which metals (and concentrations) are having an adverse e€ect on symbiotic N2 ®xation under a range of agronomic conditions. Acknowledgements The research fund awarded by the Water Research Centre, UK, is gratefully acknowledged. The authors are also grateful to Mr. C. Quarmby of the Institute of Terrestrial Ecology, Grange-over-Sands, Cumbria, UK, for undertaking 15N analysis.

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