Plant accumulation of potentially toxic elements in sewage sludge as affected by soil organic matter level and mycorrhizal fungi

Plant accumulation of potentially toxic elements in sewage sludge as affected by soil organic matter level and mycorrhizal fungi

Environmental Pollution 116 (2002) 293–300 www.elsevier.com/locate/envpol Plant accumulation of potentially toxic elements in sewage sludge as affecte...

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Environmental Pollution 116 (2002) 293–300 www.elsevier.com/locate/envpol

Plant accumulation of potentially toxic elements in sewage sludge as affected by soil organic matter level and mycorrhizal fungi M. Oudeh a, M. Khan b, J. Scullion c,* a Faculty of Agriculture, Al-Baath University, PO Box 77, Homs, Syria World Wide Fund for Nature Pakistan, PO Box 5180, Ferozepur Road Lahore-54600, Pakistan c Soil Science Unit, University of Wales, Penglais, Aberystwyth SY23 3DE, UK

b

Received 28 September 2000; accepted 24 February 2001

‘‘Capsule’’: Mycorrhizal activity and soil organic matter levels have the potential to modify the risks for plants from metals in sludges. Abstract Leek (Allium ameloprasum) was grown in pot trials in two clay loams of contrasting organic contents, with and without indigenous mycorrhizal propagules. Sewage sludges containing varying levels of Cd, Cu and Zn were added. Extractable soil metals, plant growth, major nutrient content and accumulation of metals, and soil microbial indices were investigated. The aim was to establish whether soil organic content and mycorrhizal status affected plant and microbial exposure to these metals. Extractable metals were higher and responses to inputs more pronounced in the arable, lower organic matter soil, although only Cd showed a soil difference in the CaCl2 fraction. There were no metal toxic effects on plants and some evidence to suggest that they promoted growth. Uptake of each metal was higher in the larger plants of the grassland, higher organic matter soil. Inoculation with arbuscular mycorrhizal fungi increased root Cd and Zn concentrations. With the exception of Cd (roots) and Zn (shoots), higher inputs of sludge metals did not increase plant metals. Zn and Cu, but not Cd, concentrations were higher in roots than in shoots. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Plants; Organic matter; Mycorrhizas; Sewage metals

1. Introduction The application of sewage sludge to land offers an environmentally acceptable and agronomically favourable means of waste disposal. Sludge contains large amounts of nitrogen and phosphorus; it can also replenish organic matter levels with beneficial effects on physical and biological conditions (Hall et al., 1986). However, it has long been recognised that potentially toxic elements (PTEs) in sludge may pose a risk to plant and human health. Many countries (Sheppard et al., 1992) have established guidelines limiting the loading of PTEs to soils. PTEs can be classified (Alloway, 1995) into elements posing a threat mainly to human health (e.g. cadmium [Cd], lead [Pb] and mercury [Hg]) and those mainly of concern as phytotoxins (e.g. boron [B], copper [Cu], * Corresponding author. Tel.: +44-1970-622303/04; fax: +441970-622307. E-mail address: [email protected] (J. Scullion).

nickel [Ni] and zinc [Zn]). Intake of PTEs in food is a potential human exposure pathway (Webber, 1972), particularly where much food is locally sourced or home grown on contaminated soils. Many factors affect plant availability of PTEs. Soil pH has an important influence on their solubility. For example, Martinez and Motto (1999) identified pH thresholds for Zn (6.2) and Cu (5.5) below which the solubility of these metals increased markedly. Richards et al. (2000) found that Zn concentrations in percolates were higher from soils at pH 5 than at pH 7; Cu solubility was less sensitive to pH than Zn, and Cd responses varied with soil type and sewage sludge type. Another important factor affecting PTE availability is the sorption capacity of soil since the concentration of PTEs in solution is controlled by equilibrium phase partitioning between the aqueous and solid phases (Berrow and Burridge, 1980). To varying degrees, this sorption capacity is determined by soil clay and organic matter contents (Alloway, 1995). Although clay content is an inherent feature of soils, their organic levels are

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directly influenced by agronomic factors such as tillage frequency (Douglas and Goss, 1982). Many PTEs (e.g. Cu, Ni and Zn) are important trace nutrients for plant growth. At higher pH, uptake of these elements is promoted by vesicular arbuscular mycorrhizal fungi (AMF; Khothari et al., 1991). The extent of mycorrhizal infection (Eason et al., 1999) and its effectiveness in nutrient uptake is affected by agronomic practices. Crops in more intensive farming systems are likely to have lower rates of root infection than those in less intensive systems (Douds et al., 1995; Eason et al., 1999). An experiment was established to determine whether two factors, soil organic matter content and mycorrhizal infection, had a marked effect on plant accumulation of metals applied at different rates in sewage sludge. It was suggested that PTEs would have lower plant availability in soil with higher organic contents and that VA mycorrhizal fungi would promote metal accumulation by plants. In addition to improving the understanding of metal behaviour in soil–plant systems, results were intended to inform discussion on variations in plant responses under differing conditions to metal inputs from sludge.

2. Methods Leek (Allium ameloprasum L. Porrum Group) was grown in two soils with widely differing organic matter contents to which Cd, Zn and Cu contaminated sludge was added. Leek is responsive to mycorrhizal infection (Eason et al., 1999) and an important horticultural crop. Cd was included as a metal which is a potent toxin to livestock and humans (WHO/FAO, 1998) but which has relatively low toxicity to plants (Wong and Bradshaw, 1982). Cu and Zn were selected as examples of trace nutrients having the potential at higher concentrations to damage plants (Wong and Bradshaw, 1982). These three elements vary somewhat in their behaviour within soils and plants, with the organic fraction of soils playing a less significant role for Cd than for Zn (Balabane et al., 1999) or particularly Cu (Moreno et al., 1996). Partitioning within the plant also differs and affects the potential risk to food quality. Cu

(Marschner, 1995) and Cd (Fergusson, 1990) tend to accumulate in the roots whereas Zn is more likely to be transferred into foliage (White et al., 1979). 2.1. Experimental details Experimental variables included all combinations of two different growing media, control and mycorrhizal plants, and three sludges with varying concentrations of metals. This arrangement gave a total of 12 treatment combinations, each replicated five times, in a completely randomised design. The two growing media were slightly calcareous clay loams (Evesham series) of almost neutral pH (Table 1) obtained from 0 to 10 cm depth under adjacent grassland and 2nd year cereal in a grass–arable rotation. Organic matter content, extractable phosphorus (P) and extractable potassium (K) (MAFF, 1986) were markedly higher under grassland. Soil metal contents were generally low, with the exception of Cd, and similar, with the exception of total and ethylenediaminetetraacetic acid (EDTA)-Zn, which were slightly higher under grass. Soils were sieved, air dried, then g irradiated (20k Gy - Isotron, Swindon). AMF inoculation used spores and root fragments from the original soils. Spores and fresh roots cut into small pieces were mixed with the soil prior to placement in growth tubes. Spores were extracted following the method of Brundrett et al. (1996). Nonmycorrhizal controls received spore washings and sterilised roots only, respectively to re-inoculate the sterilised soils with micro-organisms and to compensate for any effects of root materials. Metal inputs were guided by European Commission Directives (CEC, 1986) on the use of sludges on agricultural land. The low metal sludge used in the experiment was obtained from Aberystwyth sewage works which serves a generally rural area. On a dry solids basis, the sludge contained 27.8–29.5% organic carbon and 2.4–2.6% total nitrogen (Khan, 1999). Three sludge metal treatments were included, each combining a single level of Cd, Cu and Zn. The low treatment contained indigenous sludge metals only (Table 1 ). For the medium (enriched to approximately 75% of EC maxima for each metal) and high (enriched to approximately EC

Table 1 Metal contents of soils prior to sludge application in relation to European Commission limitsa Soil

Cd mg kg

Cu

Zn

Total Grass Cereal a

3.8a 2.3a

Cd

Cu

Zn

Cd

Cu

Zn

0.5a 0.5a

1.6a 1.5a

1

EDTA 18.9a 16.1a

64.0a 50.1b

1.2a 1.2a

CaCl2 3.5a 5.3a

5.8a 3.4b

Comparing soils, means with a common letter suffix do not differ at a 5% level of probability (LSD test).

0.2a 0.3a

M. Oudeh et al. / Environmental Pollution 116 (2002) 293–300

maxima+50% for each metal) treatments, metal chlorides were added to sludges 2 weeks prior to their application to soils. A single rate (15 g dry solids kg 1 soil) of dewatered sludge was physically mixed with the growing media prior to its placement in pots. Several studies have indicated that metals added to sludges may be more ‘plant available’ than indigenous sludge metals, and that the fractionation of metals in sludges may change over periods of years (Berrow and Burridge, 1980; Adams and Sanders, 1984). However, Adams and Sanders (1984) found that the CaCl2extractable concentrations of metals in sludges were similarly low for unamended and metal-enriched sludges above critical pH thresholds. The pH of the sludge (7.1) used was markedly higher than the threshold pH values for the metals used in our study. Furthermore, preliminary experiments (Khan, 1999) indicated that the proportions of total metals extractable by CaCl2 in our sludge were similarly low for added or indigenous sludge Cd, Cu and Zn. Since our study was short term, it was considered that this CaCl2 metal fraction, rather than other less easily extractable metal pools, was likely to be an appropriate indicator of potential metal toxicity. Plants were grown under standard environmental conditions (20–25 C), a photoperiod of 16 h and moisture conditions which were unlikely to limit plant growth. There were no pest or disease problems during the experiment. Plants were grown in 4010 cm diameter tubes cut at 30 cm to allow separation of the upper growing medium from a sand drainage layer underneath, with a disc of Typar (Dupont) placed at soil-sand boundary to act as a root barrier.

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and stained (Phillips and Hayman, 1970). The percentage of AMF infection was measured under a dissecting microscope using the line intersect method (Giovannetti and Mosse, 1980). Previous work using similar sludge metal rates had indicated microbial responses to these inputs (Khan and Scullion, 2000). For this reason, biomass C was measured as an indicator of gross microbial effects. The fumigation-extraction method was used for samples taken following plant harvest, with a standard conversion factor for transforming extractable to biomass C (Vance et al., 1987). Organic carbon in extracts was measured by high temperature catalytic combustion (Kaplin, 1993) using a Shimadzu Total Organic Carbon Analyser (TOC-5050). For ‘total’ metals and nutrient concentrations, soil and plant materials were digested for 20 h in hot, concentrated perchloric-nitric acid digests (MAFF, 1986). For extractable metals (McGrath and Cegara, 1992), soils were shaken (Griffen reciprocal shaker) for 1 h in 0.5 M EDTA or for 16 h in 0.1 M CaCl2. All metal concentrations in extracts were measured (MAFF, 1986) by atomic absorption spectroscopy (Pye-Unicam SP9). Soil samples for extraction were taken 20 days after sludge application. Total foliar nitrogen (Kjeldal digestion and titration), phosphorus (by spectrophotometry as a phospho-vanado-molybdate complex) and potassium (by flame photometry) concentrations were also determined (MAFF, 1986). Results from the experiment were analysed (Statgraphics, 1993) using three-way analysis of variance.

3. Results 2.2. Measurements Treatment responses were assessed in terms of plant growth, foliar nutrient content, foliar and root metals, and microbial indices including mycorrhizal roots and biomass carbon. Microbial indices were determined in order to detect potential effects of treatments on nutrient cycling and uptake. Shoot fresh and dry weights were recorded following a final destructive harvest after 4 months growth. Roots were washed free of soil; sub-samples were then cleared

Table 2 Metal contents of sludges added to soils in relation to European Commission limits Treatment

Cd (mg kg 1)

Cu (mg kg 1)

Zn (mg kg 1)

EC Limits

40

1750

4000

Metal rates Low Medium High

10 30 60

143 1300 2600

317 3000 6000

Observations during the experiment and at harvest did not indicate any symptoms of toxicity or deficiency relating to either of the trace nutrient (Cu and Zn) treatments. Initially, plants grown in the cereal soil grew markedly better than those in the grassland soil. Since soils were sampled 20 days after the mycorrhizal inoculation treatment the absence of any inoculation effect on ectractable metal concentrations was expected (Table 3). EDTA extractable values were consistently higher in the cereal soil for all metals; CaCl2 extractable Cu and Cd were higher in the cereal soil. Adding metals to sludges resulted in highly significant (P < 0.001) progressive increases in EDTA extractable metals. Effects on CaCl2 extractable values were less consistent, with no metal input response for Cu or Zn; the medium and high inputs showed a similar increase over the low treatment for Cd. There were highly significant (P < 0.001) soilmetal input interaction effects for EDTA extractable levels of all three metals, but for CaCl2 this interaction was significant for extractable Cd only. These interactions followed a similar pattern in all cases, as illustrated by data

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for EDTA–Cu (Fig. 1). In cereal soils, higher inputs of metals caused a progressive increase in extractable metals but, in the grassland soil, increases were less pronounced with little difference between medium and high input rates. Plant growth (Table 4) was markedly higher in grassland soils, but AMF inoculation had little effect. Both sludge metal enrichment treatments increased growth by a similar amount compared with unamended sludge. Main treatment responses for foliar N concentrations were non-significant, but foliar P and K were higher in grassland soils. All nutrient uptakes were significantly greater in high growth grassland soils; there were no other significant treatment responses. Foliar K concentrations were lower for the high metal compared with lower metal treatments.

Table 3 Effects of soil, mycorrhizal inoculation and sludge metal input on extractable soil metal concentrations Treatment

Soil Grass Cereal AMF None Inoculated Metal Low Medium High

CaCl2 (mg kg 1)a

EDTA (mg kg 1)a

Cd

Cu

Zn

Cd

Cu

Zn

1.19b 1.37a

0.49b 0.64a

2.02a 2.26a

2.08b 3.33a

5.81b 14.51a

22.01b 35.08a

1.24a 1.31a

0.96b 1.45a 1.43a

0.53a 0.59a

0.53a 0.61a 0.57a

Foliar metal concentrations and accumulations are given in Table 5. Growth was markedly lower in the cereal soils and this response resulted in lower accumulations of Zn and Cu, in spite of the higher concentrations of the latter for plants grown in the cereal soils. Mycorrhizal inoculation did not affect average yield or metal indices, although there was a highly significant inoculation interaction effect with soil type (Fig. 2) for Cu. Inoculation caused a slight decrease in Cu concentration for plants grown in the grassland soil but a marked increase in the cereal soil. There was a progressive increase in Zn concentration and accumulation with higher levels of metal contamination in the added sludge. For the other two metals no such trend was obtained. Total root mass was not measured as there was some decomposition before all samples could be air dried; for this reason metal accumulation in roots was not calculated. Root Zn and Cu concentrations (Table 6) were much higher than those in foliage. For Cd, in contrast,

2.14a 2.13a

1.58a 2.29a 2.55a

2.77a 2.35a

1.22c 2.78b 4.12a

10.42a 9.89a

4.68c 11.05b 14.77a

29.25a 27.84a

10.50c 33.92b 41.19a

a Comparing main treatments, means with a common letter suffix do not differ at a 5% level of probability (Duncan’s multiple range test).

Table 4 Plant yields and foliar nutrient concentrations Treatment

Yield (g pot 1)

N (%)a

P (%)a

K (%)a

Soil Grass Cereal

4.99a 2.42b

4.00a 4.28a

0.459a 0.360b

3.36a 1.67b

AMF None Inoculated

3.50a 3.91a

4.18a 4.11a

0.398a 0.422a

2.57a 2.46a

Metals Low Medium High

2.86b 4.20a 4.05a

4.26a 4.19a 3.98a

0.436a 0.405a 0.398a

2.70a 2.62a 2.23b

a

Comparing main treatments, means with a common letter suffix do not differ at a 5% level of probability (Duncan’s multiple range test).

Fig. 1. Soil-metal input rate interaction (P <0.01) effect for EDTA extractable copper.

Fig. 2. Soil-AMF inoculation interaction effect (P<0.01) effect for foliar copper concentration.

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root concentrations were lower than foliar levels. Root Cd levels were lower in plants grown on the cereal soil. Mycorrhizal roots had higher concentrations of Zn and Cd. Concentrations of Cu and Cd, but not Zn, tended to increase with higher sludge contamination, but metal input level effects were mainly non-significant. With the expected exception of the AMF-inoculation treatment (Table 7), root infection was not significantly affected by main experimental factors. Although there was no evidence of metals inhibiting overall infection rates, these tended to be higher for metal-enriched sludges in the grassland soil but progressively lower with increased metal inputs in the cereal soil, giving a significant (P < 0.01) interaction (Fig. 3). Microbial biomass was markedly higher in the grassland than in the cereal soil, but other main treatment and interaction effects were non-significant. Table 5 Mean foliar metal concentrations and metal accumulation Treatment concentration (mg kg 1)

Uptake (mg pot 1)a

Cd

Cu

Zn

Cd

Cu

Zn

1.59a 2.49a

5.53b 6.72a

45.3a 42.6a

7.86a 5.32a

27.1a 15.4b

AMF None 2.41a Inoculated 1.67a

5.87a 6.39a

38.7a 49.3a

6.98a 6.21a

Metals Low Medium High

6.60a 6.01a 5.77a

31.9b 43.7ab 56.3a

4.58a 8.61a 6.60a

Soil Grass Cereal

1.75a 2.81a 1.56a

4. Discussion The effects of sludge treatments on soil metals were largely as expected. Extractable metal concentrations were generally higher and showed a more pronounced increase with higher metal inputs in the cereal soil. These differences can probably be attributed the lower soil organic content under cereals, organic matter providing binding sites for metals, particularly Cu (Webber, 1972; Kiekens et al., 1984; Moreno et al., 1996). Compared with initial soil values, all sludge treatments caused increases in CaCl2-extractable Cd, but only enriched sludges had increased extractable Zn; Cu values were not affected by any treatment. McGrath and Cegarra (1992) found that the effectiveness of CaCl2 as an extractant decreased in order of Cd > Zn > Cu; they also found that a high proportion of Cd in sludges was extractable by CaCl2. There was no evidence, on the basis of CaCl2 data, of metals added to the sludge being more easily extractable than those Table 7 Mean mycorrhizal root infection and microbial biomass C Treatment

Root infection (%)a

Biomass-C (mg kg 1)a

238a 119b

Soil Grass Cereal

34.5a 39.5a

408a 191b

20.0a 22.6a

167a 190a

AMF None Inoculated

6.12b 67.9a

273a 327a

18.2a 22.9a 22.8a

96c 168b 271a

Metals Low Medium High

35.6a 42.5a 33.0a

313a 288a 299a

a Comparing main treatments, means with a common letter suffix do not differ at a 5% level of probability (Duncan’s multiple range test).

a Comparing main treatments, means with a common letter suffix do not differ at a 5% level of probability (Duncan’s multiple range test).

Table 6 Mean root metal concentrations Treatment

Cd (mg kg 1)a

Cu (mg kg 1)a

Zn (mg kg 1)a

Soil Grass Cereal

1.48a 0.91b

23.7a 24.5a

70.9a 83.3a

AMF None Inoculated

0.99b 1.41a

22.1a 26.1a

63.5b 90.7a

Metals Low Medium High

1.06b 1.11ab 1.42a

21.1a 23.5a 27.7a

80.6a 66.1a 84.6a

a

Comparing main treatments, means with a common letter suffix do not differ at a 5% level of probability (Duncan’s multiple range test).

Fig. 3. Soil-metal interaction (P <0.001) for percentage AMF root infection.

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already present in the non-enriched sludge; increases in extractable soil metal concentrations over initial values were proportional to the metal content of the added sludges. The generally small increases in the CaCl2 fraction of Cu and Zn may also be explained by the near neutral soil pH (Martinez and Motto, 1999; Richards et al., 2000). In contrast, CaCl2 extractable Cd showed a marked increase compared with initial values. Ge et al. (2000) found that Cd differed from Zn and Cu in soils with pH values similar to ours, with Cd being present mainly in ionic form rather than as organic or mineral complexes. Also, Cd may have less affinity for soil organic matter than Zn (Balabane et al., 1999) or Cu (Moreno et al., 1996), and the organic matter in sludge has been found not to limit Cd availability to microorganisms (Dar, 1996). Soil type provided the main differences in plant growth and nutrient concentration, with higher nutrient availability in grassland soil the probable cause. Mycorrhizal inoculation had little effect perhaps due to the relatively high availability of P in the growing media (Eason et al., 1999). Increases in growth were obtained from metal-enriched sludges, an effect which may be attributable in part to higher net mineralisation of nitrogen from these sludges (Khan and Scullion, 2000). The reduction in foliar K concentration at the highest metal input rate could not be explained by a growth dilution effect, suggesting interference in K uptake by sludge metals. Foliar Zn and Cu concentrations were well below suggested toxicity levels of 100–300 and 20–30 mg kg 1, respectively; indeed concentrations of these two metals were only slightly higher than suggested deficiency ranges of 15–20 and 1–5 mg kg 1, respectively (Marschner, 1995). For this reason, at least the absence of any visual symptoms of plant toxicity or growth limitation was to be expected. Foliar Cd concentrations for all sludge metal levels exceeded WHO/FAO (1998) draft maximum levels of 0.05 mg kg 1 for vegetables. Leafy vegetables such as leek are thought to be at particular risk of Cd uptake (Webber, 1972) and foliar Cd concentrations are similar to those found in previous studies of sludge treated soils (Moreno et al., 1996). The high foliar Cd concentrations were associated with 6– 12-fold increase in soil CaCl2–Cd concentrations compared with the initial soil. The influence of soil type, mainly organic content, on metal accumulation by plants varied between metals and portions of plants. Shoot metal accumulation was generally higher for the larger plants grown in grassland soil, but concentration of Cu in plants from the cereal soil exceeded those of plants in the grassland soil due largely to different mycorrhizal responses. The higher shoot concentrations of Cu on the cereal soil may be attributed to its lower organic content (Moreno et al.,

1996) and are consistent with extractable soil Cu data. In contrast, higher extractable Cd concentrations in the cereal soil were associated with lower root concentrations. Exchangeable (CaCl2) and EDTA extractable levels did not provide a reliable prediction of plant Cd accumulation for different soils, a finding similar to that of other studies (Pichtel et al., 2000). Increasing metal input rates caused higher concentrations and accumulation of foliar Zn only. For roots only Cd showed a similar effect. Although metal input rates increased levels of all metals in the EDTA extractable pool, only Cd showed a significant increase in CaCl2 concentrations. Metal accumulation by plants in response to increased inputs is not consistent. Studies in alkaline soils have found no increase (Hernandez et al., 1991). Other work on soils similar to ours (Kiekens et al., 1984) indicated differences between metals broadly consistent with our findings; foliar accumulation of Cu, and of Zn at soil concentrations similar to those used in the present study, was unaffected by soil metal concentrations. In contrast, increases in Cd resulted in higher plant concentrations even at low soil concentrations. Most studies (Marschner, 1995) have concluded that Cu accumulates in roots rather than shoots when plants are exposed to high external supply. Although there may be some variation between species in the distribution of Zn and Cd (Dahmani-Muller et al., 2000; Pichtel et al., 2000), these metals are also generally found at higher concentrations in roots. However, shoot/root concentration ratios are usually less than those for Cu (White et al., 1979; Dahmani-Muller et al., 2000). Results reported here agree with these conclusions for Cu and Zn but not for Cd. Cd concentrations suggest little if any retention within the root system. However, our data show that foliar/root ratios were lowest at the highest Cd input rate. Soil and plant Cd concentrations in our study were low compared with those of other studies that found root accumulation of Cd (Guo et al., 1996; Dahmani-Muller et al., 2000) so the distribution of Cd within plants may be dependent on levels of exposure to this metal. Alternatively, the partitioning of Cd within leek may be characteristic of this species. Mycorrhizal acquisition may account for a high proportion of Cd, Cu and Zn in plant shoots (Guo et al., 1996). However, there is conflicting evidence as to the effects of mycorrhizal infection, with some studies (Symeonidis, 1990) indicating reduced tolerance of high metal concentrations whilst others (Weissenhorn et al., 1995) suggest that infection offers some protection against metal toxicity. Lambert and Weidensaul (1991) showed that mycorrhizal inoculation of soybean increased accumulation of Zn and Cu. In the present study, AMF inoculation generally increased metal concentration and accumulation although effects were significant only in roots. Increased metal inputs reduced root infection rates only in the cereal soil where CaCl2

M. Oudeh et al. / Environmental Pollution 116 (2002) 293–300

extractable metal concentrations were higher; this interaction complicated interpretation of our findings. Reductions in root infection rate with higher metal availability may provide a mechanism for limiting excess uptake of these metals through the hyphal network although this effect was not apparent for Cu. Several previous studies have found reduced infectivity in mycorrhizal propagules exposed to multiple (Leyval et al., 1995) or individual metals (for Cd, Cu and Zn — Gildon and Tinker, 1983). Microbial biomass responses were confined to soil differences, where the markedly higher biomass in the grassland soil was attributable to its greater organic content and potential for growth. The absence of any significant metal input effect was consistent with earlier studies (Khan and Scullion, 2000) on similar fine textured soils of neutral pH.

5. Conclusions Findings illustrate the complexity of soil–plant interactions with regard to metal accumulation. Although soil organic matter content had the expected effects on extractable metal concentrations, these differences were not always apparent in plant tissue. Mycorrhizal plants grown in low organic matter soil are more likely to take up excess metals. However, for the rates of application and the fine textured soils used in the present study the risk of an adverse effect on plant growth is low. In contrast, accumulation of Cd contamination was of greater significance. Soil variation, including mycorrhizal activity and organic matter levels, has the potential to modify the risks arising from metals in sludge.

Acknowledgements The first (British Council) and second (Pakistan Government) authors would like to acknowledge financial support received during the course of their work. We would also like to thank F. Tuffen for technical assistance during the experiment.

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