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Effects of microelements on soil nematode assemblages seven years after contaminating an agricultural field
Science of the Total Environment 320 (2004) 131–143
Effects of microelements on soil nematode assemblages seven years after contaminating an agricultural field ´ Nagya,*, Gabor ´ ´ ´ c, Miklos ´ Fabian ´ ´ a, Istvan ´ Kissa Peter Bakonyia, Tom Bongersb, Imre Kadar a ´ University, Godollo, ¨ ¨ ¨ Pater ´ Department of Zoology and Ecology, Szent Istvan K. u. 1 H-2103, Hungary Laboratory for Nematology, Wageningen University, P.O. Box 8123, Wageningen 6700 ES, The Netherlands c Research Institute for Soil Science and Agricultural Chemistry of the Hungarian Academy of Sciences, Herman O. u. 15, Budapest H-1025, Hungary b
Received 28 January 2003; accepted 13 August 2003
Abstract Long-term effects of Cd, Cr, Cu, Se and Zn were studied 7 years after artificially contaminating plots of an agricultural field on a calcareous chernozem soil. Effects of three to four different contamination levels (originally 10, 30, 90 and 270 mg kgy1) were studied. Nematode density was significantly reduced by 90 and 270 mg kgy1 Se as well as by 270 mg kgy1 Cr, while 90 and 270 mg kgy1 Se also reduced nematode generic richness. Maturity Index values (calculated for c-p 2–5 nematodes) consistently decreased with increasing Cr and Se concentration and to a lesser extent in Zn plots as well. Structure Index showed decreasing trends in increasing Cr, Se and (to a lesser extent) in Zn treatments, while in Cd it shows a moderate increase. Distribution of c-p groups was negatively affected by the increasing Cr and Se concentration, while in Zn plots, this decrease was not significant. Response of feeding groups to pollutions was similar to other parameters: Cr and Se caused significant changes toward the loss of variability. The proportion of the most sensitive omnivorous and predatory nematodes decreased clearly as a consequence of Cr and Se treatments. Zn pollution also resulted in a slight decrease in this group, while Cd caused an increase. Nematode diversity profiles showed a significant decrease in the plots of increased Cr and Se concentrations, while increased concentrations of Cu and Zn resulted in ambiguous effects. Besides providing evidence on the harmful effects of Cr and Se on a soil nematode assemblage, our results suggest that simultaneous analysis of Maturity Index, Structure Index and diversity profiles provide a promising tool in nematological indication of soil pollution. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Soil pollution; Microelement concentration gradients; Nematodes; Maturity index; Structure index; Diversity profiles; Heavy metals
1. Introduction Increasing anthropogenic pollution of ecosystems by heavy metals and microelements means *Corresponding author. Fax: q36-28-410804. E-mail address: [email protected] (P. Nagy).
a growing hazard to living organisms, including mankind. In spite of this fact, data on long-term effects of heavy metals on soil biota are scarce to find. A unique long-term field experiment in Hungary has been providing opportunities since 1991 to study long-term effects of plant micronutrients,
0048-9697/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2003.08.006
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including heavy metals on soil biota, crops and ´ ´ 1994). their consumers (Kadar, Various crops grown at this site in previous years gave different responses to the contamination ´ ´ 1995). As a general trend, high Se expo(Kadar, sure had a strong phytotoxic effect on all grown crop species (maize, carrot, potato, peas, beet, spinach, winter wheat and sunflower) and this effect did not disappear since the start of the experiment. Deleterious effects of Se on nodulation, nitrogen-fixation and arbuscular endomycorrhizal colonisation were also detected 4 years after the application (Biro´ et al., 1998). Cr proved to be toxic for plants in the first 6 years of the experiment. Thereafter, no toxic effect was found. Cd was not toxic during the first 4 years, but significantly decreased plant production from the 5th year of the experiment until 2002. It is not clear whether this result is due to the increased toxicity of Cd or to the planting of more Cdsensitive plant species during these years. Cu and Zn caused no phytotoxic effects at all. Nematodes are often used as indicators of environmental disturbances, including heavy metal pollution (e.g.: Zullini and Peretti, 1986; Samoiloff, 1987; Bongers, 1990; Korthals et al., 1996a). There are, however, only a limited number of studies on plant microelements under conditions at least partly comparable to the present study regarding contamination effects on nematode assemblages. In a microcosm study, Parmelee et al. (1993) found Cu to decrease nematode density in concentrations above 200 mg kgy1. In a microplot experiment, no remarkable reduction of nematode density was found in soils polluted with 12 various microelements, except in the V plot, where the density was 25% of the control (D. Sturhan, personal communication). More recently, Korthals et al. (1996b) carried out an experiment on the short-term toxic effects of Cd, Cu, Ni and Zn on nematode assemblages in an acid sandy soil collected from a cultivated field. In their experiment, increased heavy metal concentrations resulted in a significant decrease in density and Maturity Index (MI 2–5). This effect was significant at the concentration level of 200 mg kgy1, which is comparable to the maximum initial concentrations used in the present experi-
ment. Cd applied in a concentration of 160 mg kgy1 as a maximum had no significant effect on nematode density. In another paper, Korthals et al. (1996c) reported effects of an aged Cu pollution and pH levels on nematode assemblage of an experimental field polluted 10 years earlier. Cu effects on the Maturity Index (MI 2–5) showed a clear stepwise trend along a concentration gradient of 250, 500, 750 kg hay1 and were largely enhanced by decreasing soil pH within a range of 3.9–5.5. In a field site contaminated long before sampling, Weiss and Larink (1991) found adverse effects of a mixture of heavy metals (among which Cd, Cr, Cu and Zn were also involved in this study) and sewage sludge. In terms of density, nematodes gave a positive reaction to this intervention, due to the high numbers of enrichment opportunists. However, contamination markedly decreased omnivorous nematodes. In another study on application of sewage sludge contaminated with various heavy metals, Georgieva et al. (2002) found Cu and Zn to have a negative effect on various parameters of a nematode assemblage on a sandy loam in England. Cu, Zn and ZnqCu decreased taxon richness and MI, affected c-p group and feeding group distribution. In our previous study (Nagy, 1999), certain elements were found to affect nematode assemblages of the same experimental field. Cr and especially Se had a negative influence while Zn appeared to stimulate nematodes in terms of density, generic richness and Maturity Index 6 years after the contamination. No effects were found, however, for Al, As, Ba, Cu, Hg, Mo, Ni, Pb and Sr. Regarding long-term observations (Bakonyi et al., 2003), Cr and especially Se showed harmful effects even 10 years after the contamination, while the advantageous effects of Zn disappeared. However, remarkable fluctuations have been found in the composition of nematode assemblage among the studied years. The aim of our study was to evaluate whether different doses of Cd, Cr, Cu, Se and Zn had recognisable effects on the structure of the nematode assemblages in the calcareous chernozem soil of an agricultural field 7 years after application. Particular emphasis was given to applying coen-
P. Nagy et al. / Science of the Total Environment 320 (2004) 131–143
ological methods in order to see whether various nematological indicators, such as taxon richness, MI, SI, c-p group distribution and feeding dominance lead to similar conclusions regarding the given disturbance. 2. Materials and methods Soil samples were collected from the experimental field of Research Institute for Soil Science and Agricultural Chemistry (RISSAC) of the Hun¨ ¨ Hungarian Academy of Sciences at Nagyhorcsok, gary (UTM-code: CT 00). Soil characteristics and ´ ´ (1994, experimental design are detailed by Kadar 1995). Therefore, only some of the most important soil characteristics are mentioned here. The soil of the experimental plots is a calcareous loamy chernozem with medium to deep humus layer formed on loess. Exchangeable cations comprise of 80% Ca, 16% Mg, 3% K, 1% Na. Ss40 meqy100 g, water soluble saltss1 meqy100 g, pH(KCl)s7.4. Particle distribution in percentage of mineral content was as follows. Coarse sand: 0.8%, fine sand: 15.7%, silt: 60.4%, clay: 23.1%. Soil organic matter content was approximately 3%, with a C:N ratio of 8–8.5. Experimental plots were polluted with single microelements given as CdSO4, K2CrO4, CuSO4, Na2SeO3 and ZnSO4, respectively. Contaminants were ploughed into the soil of the plots in April 1991. Initial total element doses were 90, 270 and 810 kg hay1. These doses were roughly equal to 30 mg kgy1, 90 mg kgy1 and 270 mg kgy1, respectively, based on the typical soil density value of 1.5 and the average plough layer of 0.2 m. In the case of Cd and Se, an additional treatment of 10 mg kgy1 in total concentration was also applied. Fertilisers were given at concentrations of Ns100 kg hay1 yeary1 (as NH4NO3), P2O5s100 kg hay1 yeary1, Ks100 kg hay1 yeary1. P, K and half of the N dose were applied before the autumn tillage, while the second half of N was applied in early spring. The above treatments are aimed to assess the effects of microelements on crops grown under conditions of regular cropping systems. No pesticides were applied, weed control was performed by manual cultivation. Experimental units were arranged in a split-plot design encompassing an area of 21 m2
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per plot. Each plot was surrounded by paths of 1 m width. The two replicates were set up as two adjacent blocks of plots. Both replicates of each treatment and two control plots were sampled on 30 June 1998, 7 years after the contamination. The NH4-acetateqEDTA soluble (mobile) fraction of element was determined according to Lakanen and Ervio¨ (1971). These analyses were performed in 1991, 1992, 1994 and 1997. Sunflower (Helianthus annuus L.) was grown in the experimental field in 1998. By the date of sampling it was close to flowering. For nematological analysis composite samples (20 subsampleyplot) of approximately 300–400 g soil were taken using a soil corer of 2 cm in diameter. The top 10 cm of soil was sampled. Nematodes were extracted from soil using Cobb’s decanting and sieving method (modified according to s’Jacob and van Bezooijen, 1984), and enumerated (typically approx. 20% of the obtained suspension). Then after fixing with 80–90 8C hot formalin (cc. 8%), samples were stored until further processing, during which at least 150 specimens per sample were identified, possibly to genus level. This process resulted in nematode taxon richness values (i.e. number of nematode taxa) and coenological data for each sample. (In cases of the maximum concentration Se treatment where this amount of nematodes was not available, samples were processed totally.) As a result, c-p group distribution patterns and Maturity Index values were calculated for nematode assemblages in each sample according to Bongers (1990). Korthals et al. (1996a) indicated that omitting c-p 1 nematodes that results in a ‘MI (2–5)’ value gives a much better response to disturbances than the MI (including c-p 1). The reason for this is that taxa in c-p 1 group react to soil organic enrichment as well. When calculating c-p distribution, intermediate taxa of c-p 3 were assigned to the conglomerate of c-p 4–5 (persister nematodes). However, representatives of the c-p 3 group occurred in only 27% of the samples where their average proportion was as low as 0.75%. Structure Index (SI) values (Ferris et al., 2001) were also computed. SI is a measure of the stabile and structured status of a soil nematode community. It is based on the proportion of feeding types combined with their
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appropriate guild weightings. High value shows a more structured community, while low SI is a sign of ‘basal conditions’, i.e. previous disturbance. Nematode taxa were assigned to feeding types sensu Yeates et al. (1993). Data were analysed using one-way ANOVA to discover significant differences in nematode density as well as nematode generic richness values. For density values, correlation was also calculated along the concentration gradient. The significant differences in c-p group distributions were tested with standard Pearson x2 test. Most of the classical diversity indices have the serious disadvantage of varying in sensitivity to the density of the common and rare species. To overcome this problem, Patil and Taillie (1979) suggested the use of the community diversity profile. The community diversity profile outlines scale-dependent diversity of a community. Some values of the diversity profile are related to classical diversity indexes, namely at as0 equal to lgS, where Ssnumber of species in the community, at as1 equal to the value of the Shannon ´ ´ ´ index etc. Tothmeresz (1993) developed new diversity function families and the computer program package DivOrd. In this study, DivOrd 1.50 was used to calculate diversity profiles. Diversity profiles between scale parameters 0–40 of all plots were generated. The low values refer to the rare taxa, while the higher scale parameters depict the status of the more frequent ones. Above as4, the curves were more or less parallel to each other, therefore, diversity profiles between as0 and 4 are presented. Significant differences can be found among 2 curves when these do not cross each ´ other and Renyi diversity values for the points of the curves differ at Ps5%. In this study, curves have been compared with an increment of 0.5 along the range of as0–4. 3. Results The mobile fractions of the target element in the studied plots are shown in Table 1. These data show that the actual available values differed (often remarkably) from the originally added total values (depending on the pollutant). However, a trend of increasing concentrations over the dose
gradient was still well recognisable 6 years after the contamination. In general, increased microelement concentrations decreased nematode density in all cases except Cu. Nematode density was significantly lower than the control in the Se treatments at 90 and 270 mg kgy1 concentration and in the plots of the highest Cr contamination, 270 mg kgy1 (Table 2). There were significant correlations between microelement concentration and nematode density in Cr, Se and Zn treatments (P-0.05). Nematode taxon richness values were decreased significantly by the 90 and 270 mg kgy1 Se treatment (Table 3). The increasing concentrations of the other elements did not correlate with this parameter. However, this could be due to the relatively low richness and high S.D. values in certain cases (Cr, Cu). Nematode MI (2–5) values (Table 4) were consistently lower in the Cr treatments of the 270 mg kgy1 concentration and in the Se treatments from 30 mg kgy1 onwards, according to a clear dose-related pattern. The low number of nematodes extracted from the 270 mg kgy1 Se-plots made it impossible to calculate MI for this treatment. For Zn, a slight decrease appeared with the increasing concentration, while in Cd samples there was a little increase in MI toward the highest treatment investigated. No trend could be observed in Cutreated samples. The c-p group distribution of samples was also significantly affected by Cr and Se pollution levels (Table 5). Cu contamination induced no consistent response. This parameter was not sensitive to Cd and Zn treatment. The c-p 3–5 nematodes occurred in relatively high proportion in 270 mg kgy1 Cd and Cu samples. Structure Index (SI) values are displayed in Table 6. This parameter shows clear decreasing trends in Cr, Se and (to a lesser extent) in Zn treatments, while in Cd it shows a moderate increase. In the values of the Cu treatments there is no obvious trend. Feeding group dominance data are shown in Table 7. Regarding bacterial feeders, Cr and Se more or less increased, Cd, Cu and Zn decreased this parameter compared to the control. Fungal feeders were enhanced by all treatments, except
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Table 1 The NH4-acetateqEDTA soluble (mobile) concentration values along the microelement gradients as measured in the subsequent analyses Element, year
Control
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Cd 1991 1992 1994 1997 2000
– – – – –
14.0 0 0 0 0
27.0 18.0 14.0 26.8 14.0
96.0 62.0 44.0 84.7 44.0
270.0 228.0 164.0 190.0 124.0
Cr 1991 1992 1994 1997 2000
0 0 0 0.2 0
– – – – –
1.0 2.0 1.0 0.45 0.4
3.0 5.0 2.0 0.77 0.9
9.0 10.0 4.0 1.4 1.6
Cu 1991 1992 1994 1997 2000
9.0 4.0 4.0 6.0 4.0
– – – – –
29.0 34.0 23.0 19.4 20.0
47.0 94.0 65.0 54.1 44.0
200.0 270.0 192.0 133.0 128.0
Se 1991 1992 1994 1997 2000
– – – – –
1.0 0 0 0.5 0
6.0 7.0 8.0 1.56 2.0
34.0 66.0 33.0 9.32 4.0
84.0 81.0 89.0 36.0 11.0
Zn 1991 1992 1994 1997 2000
1.0 3.0 1.0 2.0 2.0
– – – – –
22.0 29.0 19.0 22.0 16.0
66.0 68.0 44.0 53.0 37.0
120.0 213.0 147.0 143.0 85.0
–: no such treatment.
Table 2 Effects of pollutant concentration gradients on nematode density; average specimenU100 g soily1 ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
1275.0 ("1104) – x x – x
– x – – 1160.0 ("587) –
– 1105.0 ("824) 1055.0 ("754) 981.0 ("501) 954.0 ("861) 1150.0 ("1033)
– 937.5 ("735) 834.5 ("724) 1184.0 ("945) 422.5 ("370)* 1021.0 ("648)
– 815.0 ("613) 484.5 ("267)* 1063.0 ("987) 20.0 ("3.5)** 882.5 ("628)
–: no such treatment, x: not sampled, *: significant decrease in respective values, P-0.05, **: significant decrease in respective value, P-0.01.
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Table 3 Effects of pollutant concentration gradients on nematode taxon richness; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
19.0 ("2.8) – x x – x
– x – – 16.5 ("2.1) –
– 22.5 20.5 21.0 16.0 18.5
– 17.0 16.0 15.5 12.5 18.5
– 18.0 (0) 15.0 ("2.8) 17.5 ("5.0) 3.0 ("1.4)* 18.5 ("2.1)
("2.1) ("0.7) ("1.4) ("2.8) ("2.1)
("y) (0) ("0.7) ("2.1)* ("0.7)
–: no such treatment, x: not sampled, y: no data available, *: significant decrease in respective values, P-5%.
the maximum Se level. As for predators and omnivores, this group was very sensitive to high Cr and Se levels, while in other treatments showed ratios comparable to the control. Bacterial feeders and fungal feeders being differently sensitive to heavy metal and microelement pollution, BFyFF ratio was displayed separately in Table 8. Based on this parameter, bacterial feeders became dominant under higher Se concentrations (90 and 270 mg kgy1), while increasing Cd and Cu levels favoured fungal feeders. Diversity-profiles for nematode samples were obviously affected by some of the treatments. Cd pollution did not result in a clear trend of diversity profiles (Fig. 1). The lowest level of Cr seemed to have slightly stimulated nematode diversity while the two highest ones resulted in a significant decrease, though there was an adverse trend between these latter: the highest level of contaminant was more depressive for the rare taxa than for the most common ones (Fig. 2). In the Cu treated plots, the highest level treatment resulted in lower diversity values than the others (except for the rare taxa) and there was an overall trend of decrease with increasing concentration (Fig. 3).
For Se treatments, a slight to moderate pollution (up to 90 mg kgy1) kept diversity profiles around the control level and only 270 mg kgy1 caused a massively significant decrease in diversity (Fig. 4). The Zn levels resulted in a remarkable pattern: no significant differences were found for the whole diversity profile for most treatments, but the highest concentration slightly increased diversity compared to the two lowest ones for rare taxa, while the frequent taxa appeared to become much less diverse (Fig. 5). 4. Discussion Effects of Cd on soil nematodes were not significant in terms of most parameters. Only BFy FF ratio was decreased by the increasing concentration and the diversity profile seemed to benefit from the 30 mg kgy1 treatment, i.e. LOEC (Lowest Observable Effect Concentration) value of Cd under present conditions should be above the theoretical level of 270 mg kgy1 applied as maximum rate (In available concentration it was equal to approx. 190 mg kgy1). This finding is in agreement with Kammenga et al. (1994) who
Table 4 Effects of pollutant concentration gradients on Maturity Index (2–5); average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
2.45 ("0.11) – x x – x
– x – – 2.52 ("0.06) –
– 2.25 2.33 2.34 2.19 2.42
– 2.3 ("y) 2.22 ("0.18) 2.11 ("0.06) 2.05 ("0.01) 2.39 ("0.04)
– 2.57 2.03 2.42 n.c. 2.33
("0.13) ("0.13) ("0.1) ("0.08) ("0.05)
–: no such treatment, x: not sampled, y: no data available, n.c.: not calculable due to the too low nematode density.
("0.09) ("0.04) ("0.18) ("0.13)
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Table 5 Effects of pollutant concentration gradients on the c-p group distribution patterns of non-plant feeding nematodes; average values Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control c-p 1(%) c-p 2(%) c-p 3–5(%)
3.5 79.7 16.9
– – –
– – –
– – –
– – –
Cd c-p 1(%) c-p 2(%) c-p 3–5(%) P-
– – –
x x x
4.0 85.8 10.1 n.s.
2.4 87.2 10.4 n.s.
1.1 79.2 19.9 n.s.
Cr c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
2.0 86.0 12.3 n.s.
0.7 90.0 9.0 0.01
0.7 98.8 0.8 0.001
Cu c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
1.7 85.9 12.2 n.s.
1.3 94.8 4.3 0.001
0.9 83.0 16.1 n.s.
Se c-p 1(%) c-p 2(%) c-p 3–5(%) P-
– – –
0.8 81.2 18.2 n.s.
1.3 92.6 5.9 0.01
6.0 92.6 1.5 0.001
n.c. n.c. n.c.
Zn c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
0.9 83.9 15.2 n.s.
1.4 84.6 14.2 n.s.
1.6 86.3 12.0 n.s.
–: no such treatment, x: not sampled, n.c.: not calculable due to the too low nematode density, P-significance level compared to the control resulting from the x2 test, n.s.: non-significant.
found nematodes to be relatively insensitive to Cd, compared to other soil animals. Though the cited study was carried out in vitro with a short-term
aspect, it included 12 nematode species of various feeding and life-strategy groups, which made its conclusions more realistic than usual single-species
Table 6 Effects of pollutant concentration gradients on SI; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
52.69("7.8) – x x – x
– x – – 56.93("3.6) –
– 35.04 42.64 47.24 28.58 50.60
– 40.59 ("y) 31.01 ("20.7) 26.52 ("6.0) 9.12 ("0.8) 48.49 ("2.0)
– 59.42 10.12 49.58 n.c. 43.16
("13.3) ("11.3) ("10.3) ("9.4) ("3.7)
–: no such treatment, x: not sampled, y: no data available, n.c. not calculable due to the too low nematode density.
("4.2) (0) ("13.3) ("10.8)
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Table 7 Effects of pollutant concentration gradients on feeding group ratios; average percentage values Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control BF FF PqO PF
34.4 21.9 9.1 34.6
– – – –
– – – –
– – – –
– – – –
– – – –
x x x x
42.5 30.3 6.5 20.7 0.01
36.0 29.1 6.3 28.6 n.s.
28.7 35.2 13.8 22.3 0.01
x x x x
– – – –
32.8 34.3 7.2 25.7 0.05
47.2 36.9 5.0 10.9 0.001
43.2 41.1 0.6 15.0 0.001
x x x
– – – –
34.2 30.0 7.6 28.2 n.s.
29.1 32.5 2.7 35.7 0.05
22.5 33.9 7.2 36.3 0.05
Cd BF FF PqO PF PCr BF FF PqO PF PCu BF FF PqO PF PSe BF FF PqO PF P-
– – – –
32.3 27.1 12.1 28.4 n.s.
39.8 30.6 4.1 25.5 0.05
56.2 30.6 1.0 12.2 0.001
n.c. n.c. n.c. n.c.
Zn BF FF PqO PF P-
x x x x
– – – –
21.2 31.8 7.2 39.7 0.05
26.8 31.7 7.9 33.7 n.s.
20.0 31.1 6.0 42.9 0.01
BF: bacterial feeders, FF: fungal feeders, PqO: predatorsqomnivores, PF: plant feeders, –: no such treatment, x: not sampled, n.c.: not calculable due to the too low nematode density, P-significance level compared to the control resulting from the x2 test, n.s.: non-significant.
laboratory tests. Korthals et al. (1996b) also found nematode fauna uneffected by Cd up to 160 mg kgy1 (though in a short-term study). Effects of Cr on nematode assemblages have so far been paid little attention to. Yeates et al. (1995) presented results on mixed effects of As, Cr and Cu but their data are hard to compare with ours due to the different approach (multi- vs. singleelement treatment). In his study D. Sturhan (per-
sonal communication) showed an MI-decrease for Cr. In our present experiment, Cr had still a negative impact on nematode assemblages, similarly to earlier results (Nagy, 1999). Increasing concentration of this element affected most of the parameters applied to indicate disturbance on nematode assemblage. Density, taxon richness, MI, SI, PqO ratio and proportion of c-p 3–5 nematodes all clearly decreased along the concentration gra-
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Table 8 Effects of pollutant concentration gradients on bacterial feederyfungal feeder ratios; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
1.90 ("1.4) – x x – x
– x – – 1.19 ("0.46) –
– 1.52 1.00 1.14 1.36 0.68
– 1.24 ("y) 1.29 ("0.2) 0.92 ("0.3) 1.95("0.6) 0.85 ("0.2)
– 0.82 1.14 0.66 n.c. 0.64
("0.6) ("0.3) ("0.2) ("0.2) ("0.1)
("0.1) ("0.5) ("0.0) ("0.0)
–: no such treatment, x: not sampled, y: no data available, n.c. not calculable due to the too low nematode density, BF: bacterial feeders, FF: fungal feeders.
Fig. 1. Diversity profiles for the Cd treatment. Cd 2:30 mg kgy1, Cd 3:90 mg kgy1, Cd 4:270 mg kgy1.
Fig. 2. Diversity profiles for the Cr treatment. Cr 2:30 mg kgy1, Cr 3:90 mg kgy1, Cr 4:270 mg kgy1.
Fig. 3. Diversity profiles for the Cu treatment. Cu 2:30 mg kgy1, Cu 3:90 mg kgy1, Cu 4:270 mg kgy1.
dient. Moreover, the diversity profiles also showed a significant decrease as a consequence of the 90 mg kgy1 level. Therefore, it can be concluded that LOEC value for Cr is approximately 0.5 mg kgy1 available concentration (expressed in NH4acetateqEDTA soluble). In the higher treatments (90 and 270 mg kgy1), where the remaining available Cr in 1997 was 0.77 mg kgy1 and 1.4 mg kgy1, respectively, all parameters showed a considerable depression. It is difficult, however, to quantify what proportion of available Cr acts in the soil as the more toxic Cr(VI) form. In case of the Cu gradients, no obvious response of nematodes could be observed based on the most parameters. Only diversity profiles showed a more or less consistent stepwise difference suggesting that 90 and 270 mg kgy1 concentrations might
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Fig. 4. Diversity profiles for the Se treatment. Se 1:10 mg kgy1, Se 2:30 mg kgy1, Se 3:90 mg kgy1, Se 4:270 mg kgy1.
have an unfavourable effect on nematode assemblages. Korthals et al. (1996c) observed several clear effects on nematode trophic structure of a field contaminated with a Cu concentration gradient 10 years prior to sampling. In the most comparable case, where actual pH was 5.5 (originally 6.1) total numbers were similar along the whole concentration gradient. Bacterial feeders slightly, fungal feeders remarkably increased with higher Cu levels. Plant nematodes showed no consistent reaction. Omnivorous and carnivorous nematodes decreased along the concentration gradient. These
Fig. 5. Diversity profiles for the Zn treatment. Zn 2:30 mg kgy1, Zn 3:90 mg kgy1, Zn 4:270 mg kgy1.
results are partly in line with our findings: fungal feeders seemed to slightly benefit from the increasing Cu levels and the BFyFF ratio decreased. Also, plant feeders fluctuated around basically the same percentage value. However, in our study there was no systematic decrease in the number of omnivorous and carnivorous nematodes and bacterial feeders rather decreased than increased in their proportion. The probable main reasons for the partly dissimilar findings can be the different pH values and soil type. Under present conditions, nematodes did not appear to be particularly sensitive to Cu up to an available concentration level of approximately 130 mg kgy1. The relatively high proportion of c-p 3–5 nematodes in 270 mg kgy1 Cd and Cu samples may be due to the insensitivity of this group of persister nematodes to the given metals or an increase in algal growth in the upper millimetres of the soil, where the heavy metals had been washed out. The most expressed effects on target parameters in this experiment were observed in the Se treatments. The first concentration level (10 mg kgy1) slightly increased nematode MI, while the two higher concentrations (30 and 90 mg kgy1) resulted in a clearly detectable decrease in all the studied nematode parameters. In the plots of highest dose (270 mg kgy1) there were not enough nematodes to calculate indices. It should also be pointed out, however, that the remarkable negative effects of Se might partly be due to the almost complete lack of vegetation in the 270 mg kgy1 plots. This assumption is supported by the observations that terrestrial nematodes are without respect to their feeding types, closely connected to vegetation of a field (Freckman and Caswell, 1985; Yeates, 1987). It requires further studies to discriminate between direct and indirect (plant-mediated) toxic effects of high Se loads. Zn has apparently lost its previously shown (Nagy, 1999) favourable character for the soil nematode assemblage by the time of the present experiment. None of the concentration levels had any significant effect on any of the parameters of the studied soil invertebrate group. The only indication of its earlier positive effect was that the diversity of the rare taxa was higher in plots of high than of low Zn levels. Diversity of the
P. Nagy et al. / Science of the Total Environment 320 (2004) 131–143
frequent taxa showed, however, an opposite trend. Nematode density, MI, SI values showed, though not significantly, a decrease with the increasing concentrations of Zn. These findings appear to be in line with the results of Korthals et al. (1996b) who found Zn to depress nematodes in a concentration of 200 mg kgy1 during a short-term experiment. In our long-term study performed under more favourable soil conditions, Zn doses of approximately 140 ppm available concentration in plots of the maximum treatment gave the first signs of destruction for the nematode fauna. In a recent paper, Georgieva et al. (2002) reported several negative effects of Zn, Cu and ZnqCu treatments on nematodes in an English agroecosystem on a sandy loam treated with sewage sludge. The metal levels applied by them extended to a higher level (e.g. for Zn: 160–600 mg kgy1 in total concentration) than in our case. It should be pointed out, however, that repeated sewage sludge application means a more realistic, but less controllable treatment than ploughing in a given amount of metal salt at the start of the experiment; the latter is easily washed out and biologically more available. Density decreased significantly by Cr and Se concentration gradient, while in Cd and Zn plots, it showed a non-significant decline. The former can be attributed to a severe destruction, since pure nematode density is a rather insensitive parameter (Bongers, 1990). Richness of nematode taxa is a more sensitive parameter of activity of nematofauna as well as soil nitrogen status and decomposition function (Ekschmitt et al., 1999). In our study, richness was slightly decreased by increasing Cr and remarkably decreased by the subsequent steps of Se treatment. The other pollutants did not generate any clear effect. The clear stepwise response of Maturity Index and Structure Index to Cr and Se treatments also underlines the destructive character of these pollutants. The c-p distribution of samples shows very clearly the sensitivity of the ‘persisters’ to Cr and Se pollution. The c-p 3 nematodes being very rare, the apparent trends in this parameter can be attributed to the sensitivity of the K-strategist nematodes (in this case belonging mostly to the order Dory-
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laimida). This is in accordance with Bongers (1990, 1999). Zullini and Peretti (1986) found a similar phenomenon for lead (Pb) pollution. Regarding distribution of feeding groups: the percentage dominance bacterial feeders (chiefly Acrobeloides, Chiloplacus and Heterocephalobus) clearly increase along the concentration gradients of Cr and Se. In the light of other parameters, this indicates the relative insensitivity of this group to the given disturbances. It is remarkable how dominant Chiloplacus is in the 90 and 270 ppm Seplots. This indicates a considerable tolerance for Se, at least compared to other nematode taxa that occur in the studied area. This phenomenon has also been observed earlier, except the stepwise response (Nagy, 1999). Proportion of fungal feeding nematodes remains stable (Zn) or slightly increases (Cd, Cr, Cu), but even in case of Se, it decreases strongly only in the highest treatment level. In the latter case, the low value is based on the (probably random) occurrence of a single Tylencholaimus. This shows that fungivorous nematodes (dominated by Aphelenchus, Aphelenchoides and Ditylenchus) were quite insensitive to most pollutions. This is in agreement with D. Sturhan (personal communication) and Korthals et al. (1996c), who found Aphelenchoides to tolerate heavy metal (including Cu) stress. The proportion of omnivorous and predatory nematodes decreased clearly as a consequence of Cr and Se treatments. Zn also resulted in a slight decrease, while to Cd this group reacted with an increase. Since dominant representatives of this group were in this study basically the same as the c-p 4–5 nematodes, these results underline the harmful effects of Cr and Se demonstrated by the above mentioned parameters as well. In general, MI (and the closely related c-p distribution), SI values and diversity profiles gave basically similar indications of the effects of the studied elements. While for Cr and Se, a clear depression could be demonstrated and for Zn a weak depression appears, Cd generated no sensitivity and Cu treatments showed no obvious patterns. The fact that the above parameters of different theoretical nature led to comparable consequences shows the robustness of the present results. The combined use of these tools may be a
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relevant method in field toxicity studies, offering several advantages compared to single indices. Such advantages of diversity ordering are the firm background built on various diversity indices and the suitability of clear graphic interpretation. However, the Maturity Index and Structure Index are easy-to-calculate measures of highly relevant nematological basis (Bongers and Bongers, 1998; Bongers, 1999; Bongers and Ferris, 1999; Ferris et al., 2001). In conclusion, both favourable soil conditions and time elapsed (over 7 years) since the contamination as well as not very high available element concentrations may explain the less pronounced effects of certain elements (Cd, Cu, Zn). However, Se and Cr still show significant long-term effects on terrestrial nematodes, an important component of the soil biota involved in a crop production system. Acknowledgments This study was supported by a HungarianFlemish Intergovernmental R&D Co-operative Agreement (Contract No. B15y97) and by the Ministry of Culture and Education (Contract No. FKFP 0280y1999). References ´ ´ I. Long-term effects of heavy Bakonyi G, Nagy P, Kadar metals and microelements on nematode assemblage. Toxicol Lett 2003;140–141:391 –401. ¨ ´ ¨ ¨ I, Kadar ´ ´ I. Toxicity of some Biro´ B, Koves-Pechy K, Voros field applied heavy metal salts to the rhizobial and fungal ´ ´ microsymbionts of alfalfa and red clover. Agrokemia es Talajtan 1998;47:265 –276. Bongers T. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 1990;83:14 –19. Bongers T. The maturity index, the evolution of nematode life history traits, adaptive radiation and c-p scaling. Plant Soil 1999;212:13 –22. Bongers T, Bongers M. Functional diversity of nematodes’. Appl Soil Ecol 1998;10:239 –251. Bongers T, Ferris H. Nematode community structure as a bioindicator in environmental monitoring. TREE 1999;14:224 –228. ¨ S, Ekschmitt K, Bakonyi G, Bongers M, Bongers T, Bostrom Dogan H, Harrison A, Kallimanis A, Nagy P, O’Donnel AG, Papatheodoru EM, Sohlenius B, Stamou GP, Wolters V. Nematode community structure as indicator of soil
functioning in European grassland soils. Eur J Soil Biol 1999;37:263 –268. Ferris H, Bongers T, deGoede RGM. A framework for soil food web diagnostics: extension of the nematode faunal analysis concept. Appl Soil Ecol 2001;18:13 –29. Freckman DW, Caswell EP. The ecology of nematodes in agroecosystems. Annu Rev Phytopathol 1985;23:275 –296. Georgieva SS, McGrath SP, Hooper DJ, Chambers BS. Nematode communities under stress: the long-term effects of heavy metals in soil treated with sewage sludge. Appl Soil Ecol 2002;20:27 –42. ´ ´ I. Importance of long-term field experiments in sustainKadar ´ ´ Talajtan able agriculture for Hungary. Agrokemia es 1994;43:291 –304. ´ ´ I. A talaj-noveny-allat-ember ¨ ´ ´ ´ ´ ´ ´ szennyezo˜ Kadar taplaleklanc wContamination of ´ kemiai ´ ´ dese elemekkel Magyarorszagon. the soil-plant-animal-human food-chain by chemicals in Hungary.x MTA-TAKI, Budapest, 1995, p. 388 (in Hungarian). Kammenga JE, Van Gestel CAM, Bakker J. Patterns of sensitivity to cadmium and pentachlorophenol among nematode species from different taxonomic and ecological groups. Arch Environ Con Tox 1994;27:88 –94. Korthals GW, deGoede RGM, Kammenga JE, Bongers T. The maturity index as an instrument for risk assessment of soil pollution. In: vanStraalen NM, Krivolutsky DA, editors. Bioindicator systems for soil pollutionKluwer, 1996a. p. 85 –93. Korthals GW, van der Ende A, van Megen H, Lexmond TM, Kammenga JE, Bongers T. Short-term effects of cadmium, copper, nickel and zinc on soil nematodes from different feeding and life-history strategy groups. Appl Soil Ecol 1996b;4:107 –117. Korthals GW, Alexiev AD, Lexmond TM, Kammenga JE, Bongers T. Long-term effects of copper and pH on the nematode community in an agroecosystem. Environ Toxicol Chem 1996c;15:979 –985. Lakanen E, Ervio¨ R. A comparison of eight extractants for the determination of plant available micronutrients in soil. Acta Agr Finn 1971;123:223 –232. Nagy P. Effects of an artificial metal pollution on nematode assemblage of a calcareous loamy chernozem soil. Plant Soil 1999;212:35 –47. Parmelee RW, Wentsel RS, Philips CT, Simini M, Checkai RT. Soil microcosm for testing the effects of chemical pollutants on soil fauna communities and trophic structure. Environ Toxicol Chem 1993;12:1477 –1486. Patil GP, Taillie C. An overwiev of diversity. In: Grassle JF, Patil GP, Smith W, Taillie C, editors. Ecological diversity in theory and practice. Fairland, Maryland: International Cooperative Publishing House, 1979. p. 3 –27. Samoiloff MR. Nematodes as indicators of toxic environmental contaminants. In: Veech JA, Dickson DW, editors. Vistas on nematology. Florida: E.O. Painter Printer Co., DeLeon Springs, 1987. p. 433 –439. s’Jacob JJ, van Bezooijen J. A manual for practical work in nematologyWageningen Agricultural University Department of Nematology, 1984. p. 77
P. Nagy et al. / Science of the Total Environment 320 (2004) 131–143 ´ ´ ´ B. DivOrd 1.50. A program for diversity ordering. Tothmeresz Tiscia 1993; 27: 33–44. Weiss B, Larink O. Influence of sewage sludge and heavy metals on nematodes in an arable soil. Biol Fert Soils 1991;12:5 –9. Yeates GW. How plants affect nematodes. Adv Ecol Res 1987;17:61 –113. Yeates GW, Bongers T, de Goede RGM, Freckman DW, Georgieva SS. Feeding habits in soil nematode families and
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genera: an outline for soil ecologists. J Nematol 1993;25:315 –331. Yeates GW, Orchard VA, Speir TW. Reduction in faunal populations and decomposition following pasture contamination by a Cu–Cr–As based timber preservative. Acta Zoologica Fennica 1995;196:297 –300. Zullini A, Peretti E. Lead pollution and moss-inhabiting nematodes of an industrial area. Water Air Soil Poll 1986;27:403 –410.
Effects of microelements on soil nematode assemblages seven years after contaminating an agricultural field ´ Nagya,*, Gabor ´ ´ ´ c, Miklos ´ Fabian ´ ´ a, Istvan ´ Kissa Peter Bakonyia, Tom Bongersb, Imre Kadar a ´ University, Godollo, ¨ ¨ ¨ Pater ´ Department of Zoology and Ecology, Szent Istvan K. u. 1 H-2103, Hungary Laboratory for Nematology, Wageningen University, P.O. Box 8123, Wageningen 6700 ES, The Netherlands c Research Institute for Soil Science and Agricultural Chemistry of the Hungarian Academy of Sciences, Herman O. u. 15, Budapest H-1025, Hungary b
Received 28 January 2003; accepted 13 August 2003
Abstract Long-term effects of Cd, Cr, Cu, Se and Zn were studied 7 years after artificially contaminating plots of an agricultural field on a calcareous chernozem soil. Effects of three to four different contamination levels (originally 10, 30, 90 and 270 mg kgy1) were studied. Nematode density was significantly reduced by 90 and 270 mg kgy1 Se as well as by 270 mg kgy1 Cr, while 90 and 270 mg kgy1 Se also reduced nematode generic richness. Maturity Index values (calculated for c-p 2–5 nematodes) consistently decreased with increasing Cr and Se concentration and to a lesser extent in Zn plots as well. Structure Index showed decreasing trends in increasing Cr, Se and (to a lesser extent) in Zn treatments, while in Cd it shows a moderate increase. Distribution of c-p groups was negatively affected by the increasing Cr and Se concentration, while in Zn plots, this decrease was not significant. Response of feeding groups to pollutions was similar to other parameters: Cr and Se caused significant changes toward the loss of variability. The proportion of the most sensitive omnivorous and predatory nematodes decreased clearly as a consequence of Cr and Se treatments. Zn pollution also resulted in a slight decrease in this group, while Cd caused an increase. Nematode diversity profiles showed a significant decrease in the plots of increased Cr and Se concentrations, while increased concentrations of Cu and Zn resulted in ambiguous effects. Besides providing evidence on the harmful effects of Cr and Se on a soil nematode assemblage, our results suggest that simultaneous analysis of Maturity Index, Structure Index and diversity profiles provide a promising tool in nematological indication of soil pollution. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Soil pollution; Microelement concentration gradients; Nematodes; Maturity index; Structure index; Diversity profiles; Heavy metals
1. Introduction Increasing anthropogenic pollution of ecosystems by heavy metals and microelements means *Corresponding author. Fax: q36-28-410804. E-mail address: [email protected] (P. Nagy).
a growing hazard to living organisms, including mankind. In spite of this fact, data on long-term effects of heavy metals on soil biota are scarce to find. A unique long-term field experiment in Hungary has been providing opportunities since 1991 to study long-term effects of plant micronutrients,
0048-9697/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2003.08.006
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including heavy metals on soil biota, crops and ´ ´ 1994). their consumers (Kadar, Various crops grown at this site in previous years gave different responses to the contamination ´ ´ 1995). As a general trend, high Se expo(Kadar, sure had a strong phytotoxic effect on all grown crop species (maize, carrot, potato, peas, beet, spinach, winter wheat and sunflower) and this effect did not disappear since the start of the experiment. Deleterious effects of Se on nodulation, nitrogen-fixation and arbuscular endomycorrhizal colonisation were also detected 4 years after the application (Biro´ et al., 1998). Cr proved to be toxic for plants in the first 6 years of the experiment. Thereafter, no toxic effect was found. Cd was not toxic during the first 4 years, but significantly decreased plant production from the 5th year of the experiment until 2002. It is not clear whether this result is due to the increased toxicity of Cd or to the planting of more Cdsensitive plant species during these years. Cu and Zn caused no phytotoxic effects at all. Nematodes are often used as indicators of environmental disturbances, including heavy metal pollution (e.g.: Zullini and Peretti, 1986; Samoiloff, 1987; Bongers, 1990; Korthals et al., 1996a). There are, however, only a limited number of studies on plant microelements under conditions at least partly comparable to the present study regarding contamination effects on nematode assemblages. In a microcosm study, Parmelee et al. (1993) found Cu to decrease nematode density in concentrations above 200 mg kgy1. In a microplot experiment, no remarkable reduction of nematode density was found in soils polluted with 12 various microelements, except in the V plot, where the density was 25% of the control (D. Sturhan, personal communication). More recently, Korthals et al. (1996b) carried out an experiment on the short-term toxic effects of Cd, Cu, Ni and Zn on nematode assemblages in an acid sandy soil collected from a cultivated field. In their experiment, increased heavy metal concentrations resulted in a significant decrease in density and Maturity Index (MI 2–5). This effect was significant at the concentration level of 200 mg kgy1, which is comparable to the maximum initial concentrations used in the present experi-
ment. Cd applied in a concentration of 160 mg kgy1 as a maximum had no significant effect on nematode density. In another paper, Korthals et al. (1996c) reported effects of an aged Cu pollution and pH levels on nematode assemblage of an experimental field polluted 10 years earlier. Cu effects on the Maturity Index (MI 2–5) showed a clear stepwise trend along a concentration gradient of 250, 500, 750 kg hay1 and were largely enhanced by decreasing soil pH within a range of 3.9–5.5. In a field site contaminated long before sampling, Weiss and Larink (1991) found adverse effects of a mixture of heavy metals (among which Cd, Cr, Cu and Zn were also involved in this study) and sewage sludge. In terms of density, nematodes gave a positive reaction to this intervention, due to the high numbers of enrichment opportunists. However, contamination markedly decreased omnivorous nematodes. In another study on application of sewage sludge contaminated with various heavy metals, Georgieva et al. (2002) found Cu and Zn to have a negative effect on various parameters of a nematode assemblage on a sandy loam in England. Cu, Zn and ZnqCu decreased taxon richness and MI, affected c-p group and feeding group distribution. In our previous study (Nagy, 1999), certain elements were found to affect nematode assemblages of the same experimental field. Cr and especially Se had a negative influence while Zn appeared to stimulate nematodes in terms of density, generic richness and Maturity Index 6 years after the contamination. No effects were found, however, for Al, As, Ba, Cu, Hg, Mo, Ni, Pb and Sr. Regarding long-term observations (Bakonyi et al., 2003), Cr and especially Se showed harmful effects even 10 years after the contamination, while the advantageous effects of Zn disappeared. However, remarkable fluctuations have been found in the composition of nematode assemblage among the studied years. The aim of our study was to evaluate whether different doses of Cd, Cr, Cu, Se and Zn had recognisable effects on the structure of the nematode assemblages in the calcareous chernozem soil of an agricultural field 7 years after application. Particular emphasis was given to applying coen-
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ological methods in order to see whether various nematological indicators, such as taxon richness, MI, SI, c-p group distribution and feeding dominance lead to similar conclusions regarding the given disturbance. 2. Materials and methods Soil samples were collected from the experimental field of Research Institute for Soil Science and Agricultural Chemistry (RISSAC) of the Hun¨ ¨ Hungarian Academy of Sciences at Nagyhorcsok, gary (UTM-code: CT 00). Soil characteristics and ´ ´ (1994, experimental design are detailed by Kadar 1995). Therefore, only some of the most important soil characteristics are mentioned here. The soil of the experimental plots is a calcareous loamy chernozem with medium to deep humus layer formed on loess. Exchangeable cations comprise of 80% Ca, 16% Mg, 3% K, 1% Na. Ss40 meqy100 g, water soluble saltss1 meqy100 g, pH(KCl)s7.4. Particle distribution in percentage of mineral content was as follows. Coarse sand: 0.8%, fine sand: 15.7%, silt: 60.4%, clay: 23.1%. Soil organic matter content was approximately 3%, with a C:N ratio of 8–8.5. Experimental plots were polluted with single microelements given as CdSO4, K2CrO4, CuSO4, Na2SeO3 and ZnSO4, respectively. Contaminants were ploughed into the soil of the plots in April 1991. Initial total element doses were 90, 270 and 810 kg hay1. These doses were roughly equal to 30 mg kgy1, 90 mg kgy1 and 270 mg kgy1, respectively, based on the typical soil density value of 1.5 and the average plough layer of 0.2 m. In the case of Cd and Se, an additional treatment of 10 mg kgy1 in total concentration was also applied. Fertilisers were given at concentrations of Ns100 kg hay1 yeary1 (as NH4NO3), P2O5s100 kg hay1 yeary1, Ks100 kg hay1 yeary1. P, K and half of the N dose were applied before the autumn tillage, while the second half of N was applied in early spring. The above treatments are aimed to assess the effects of microelements on crops grown under conditions of regular cropping systems. No pesticides were applied, weed control was performed by manual cultivation. Experimental units were arranged in a split-plot design encompassing an area of 21 m2
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per plot. Each plot was surrounded by paths of 1 m width. The two replicates were set up as two adjacent blocks of plots. Both replicates of each treatment and two control plots were sampled on 30 June 1998, 7 years after the contamination. The NH4-acetateqEDTA soluble (mobile) fraction of element was determined according to Lakanen and Ervio¨ (1971). These analyses were performed in 1991, 1992, 1994 and 1997. Sunflower (Helianthus annuus L.) was grown in the experimental field in 1998. By the date of sampling it was close to flowering. For nematological analysis composite samples (20 subsampleyplot) of approximately 300–400 g soil were taken using a soil corer of 2 cm in diameter. The top 10 cm of soil was sampled. Nematodes were extracted from soil using Cobb’s decanting and sieving method (modified according to s’Jacob and van Bezooijen, 1984), and enumerated (typically approx. 20% of the obtained suspension). Then after fixing with 80–90 8C hot formalin (cc. 8%), samples were stored until further processing, during which at least 150 specimens per sample were identified, possibly to genus level. This process resulted in nematode taxon richness values (i.e. number of nematode taxa) and coenological data for each sample. (In cases of the maximum concentration Se treatment where this amount of nematodes was not available, samples were processed totally.) As a result, c-p group distribution patterns and Maturity Index values were calculated for nematode assemblages in each sample according to Bongers (1990). Korthals et al. (1996a) indicated that omitting c-p 1 nematodes that results in a ‘MI (2–5)’ value gives a much better response to disturbances than the MI (including c-p 1). The reason for this is that taxa in c-p 1 group react to soil organic enrichment as well. When calculating c-p distribution, intermediate taxa of c-p 3 were assigned to the conglomerate of c-p 4–5 (persister nematodes). However, representatives of the c-p 3 group occurred in only 27% of the samples where their average proportion was as low as 0.75%. Structure Index (SI) values (Ferris et al., 2001) were also computed. SI is a measure of the stabile and structured status of a soil nematode community. It is based on the proportion of feeding types combined with their
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appropriate guild weightings. High value shows a more structured community, while low SI is a sign of ‘basal conditions’, i.e. previous disturbance. Nematode taxa were assigned to feeding types sensu Yeates et al. (1993). Data were analysed using one-way ANOVA to discover significant differences in nematode density as well as nematode generic richness values. For density values, correlation was also calculated along the concentration gradient. The significant differences in c-p group distributions were tested with standard Pearson x2 test. Most of the classical diversity indices have the serious disadvantage of varying in sensitivity to the density of the common and rare species. To overcome this problem, Patil and Taillie (1979) suggested the use of the community diversity profile. The community diversity profile outlines scale-dependent diversity of a community. Some values of the diversity profile are related to classical diversity indexes, namely at as0 equal to lgS, where Ssnumber of species in the community, at as1 equal to the value of the Shannon ´ ´ ´ index etc. Tothmeresz (1993) developed new diversity function families and the computer program package DivOrd. In this study, DivOrd 1.50 was used to calculate diversity profiles. Diversity profiles between scale parameters 0–40 of all plots were generated. The low values refer to the rare taxa, while the higher scale parameters depict the status of the more frequent ones. Above as4, the curves were more or less parallel to each other, therefore, diversity profiles between as0 and 4 are presented. Significant differences can be found among 2 curves when these do not cross each ´ other and Renyi diversity values for the points of the curves differ at Ps5%. In this study, curves have been compared with an increment of 0.5 along the range of as0–4. 3. Results The mobile fractions of the target element in the studied plots are shown in Table 1. These data show that the actual available values differed (often remarkably) from the originally added total values (depending on the pollutant). However, a trend of increasing concentrations over the dose
gradient was still well recognisable 6 years after the contamination. In general, increased microelement concentrations decreased nematode density in all cases except Cu. Nematode density was significantly lower than the control in the Se treatments at 90 and 270 mg kgy1 concentration and in the plots of the highest Cr contamination, 270 mg kgy1 (Table 2). There were significant correlations between microelement concentration and nematode density in Cr, Se and Zn treatments (P-0.05). Nematode taxon richness values were decreased significantly by the 90 and 270 mg kgy1 Se treatment (Table 3). The increasing concentrations of the other elements did not correlate with this parameter. However, this could be due to the relatively low richness and high S.D. values in certain cases (Cr, Cu). Nematode MI (2–5) values (Table 4) were consistently lower in the Cr treatments of the 270 mg kgy1 concentration and in the Se treatments from 30 mg kgy1 onwards, according to a clear dose-related pattern. The low number of nematodes extracted from the 270 mg kgy1 Se-plots made it impossible to calculate MI for this treatment. For Zn, a slight decrease appeared with the increasing concentration, while in Cd samples there was a little increase in MI toward the highest treatment investigated. No trend could be observed in Cutreated samples. The c-p group distribution of samples was also significantly affected by Cr and Se pollution levels (Table 5). Cu contamination induced no consistent response. This parameter was not sensitive to Cd and Zn treatment. The c-p 3–5 nematodes occurred in relatively high proportion in 270 mg kgy1 Cd and Cu samples. Structure Index (SI) values are displayed in Table 6. This parameter shows clear decreasing trends in Cr, Se and (to a lesser extent) in Zn treatments, while in Cd it shows a moderate increase. In the values of the Cu treatments there is no obvious trend. Feeding group dominance data are shown in Table 7. Regarding bacterial feeders, Cr and Se more or less increased, Cd, Cu and Zn decreased this parameter compared to the control. Fungal feeders were enhanced by all treatments, except
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Table 1 The NH4-acetateqEDTA soluble (mobile) concentration values along the microelement gradients as measured in the subsequent analyses Element, year
Control
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Cd 1991 1992 1994 1997 2000
– – – – –
14.0 0 0 0 0
27.0 18.0 14.0 26.8 14.0
96.0 62.0 44.0 84.7 44.0
270.0 228.0 164.0 190.0 124.0
Cr 1991 1992 1994 1997 2000
0 0 0 0.2 0
– – – – –
1.0 2.0 1.0 0.45 0.4
3.0 5.0 2.0 0.77 0.9
9.0 10.0 4.0 1.4 1.6
Cu 1991 1992 1994 1997 2000
9.0 4.0 4.0 6.0 4.0
– – – – –
29.0 34.0 23.0 19.4 20.0
47.0 94.0 65.0 54.1 44.0
200.0 270.0 192.0 133.0 128.0
Se 1991 1992 1994 1997 2000
– – – – –
1.0 0 0 0.5 0
6.0 7.0 8.0 1.56 2.0
34.0 66.0 33.0 9.32 4.0
84.0 81.0 89.0 36.0 11.0
Zn 1991 1992 1994 1997 2000
1.0 3.0 1.0 2.0 2.0
– – – – –
22.0 29.0 19.0 22.0 16.0
66.0 68.0 44.0 53.0 37.0
120.0 213.0 147.0 143.0 85.0
–: no such treatment.
Table 2 Effects of pollutant concentration gradients on nematode density; average specimenU100 g soily1 ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
1275.0 ("1104) – x x – x
– x – – 1160.0 ("587) –
– 1105.0 ("824) 1055.0 ("754) 981.0 ("501) 954.0 ("861) 1150.0 ("1033)
– 937.5 ("735) 834.5 ("724) 1184.0 ("945) 422.5 ("370)* 1021.0 ("648)
– 815.0 ("613) 484.5 ("267)* 1063.0 ("987) 20.0 ("3.5)** 882.5 ("628)
–: no such treatment, x: not sampled, *: significant decrease in respective values, P-0.05, **: significant decrease in respective value, P-0.01.
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Table 3 Effects of pollutant concentration gradients on nematode taxon richness; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
19.0 ("2.8) – x x – x
– x – – 16.5 ("2.1) –
– 22.5 20.5 21.0 16.0 18.5
– 17.0 16.0 15.5 12.5 18.5
– 18.0 (0) 15.0 ("2.8) 17.5 ("5.0) 3.0 ("1.4)* 18.5 ("2.1)
("2.1) ("0.7) ("1.4) ("2.8) ("2.1)
("y) (0) ("0.7) ("2.1)* ("0.7)
–: no such treatment, x: not sampled, y: no data available, *: significant decrease in respective values, P-5%.
the maximum Se level. As for predators and omnivores, this group was very sensitive to high Cr and Se levels, while in other treatments showed ratios comparable to the control. Bacterial feeders and fungal feeders being differently sensitive to heavy metal and microelement pollution, BFyFF ratio was displayed separately in Table 8. Based on this parameter, bacterial feeders became dominant under higher Se concentrations (90 and 270 mg kgy1), while increasing Cd and Cu levels favoured fungal feeders. Diversity-profiles for nematode samples were obviously affected by some of the treatments. Cd pollution did not result in a clear trend of diversity profiles (Fig. 1). The lowest level of Cr seemed to have slightly stimulated nematode diversity while the two highest ones resulted in a significant decrease, though there was an adverse trend between these latter: the highest level of contaminant was more depressive for the rare taxa than for the most common ones (Fig. 2). In the Cu treated plots, the highest level treatment resulted in lower diversity values than the others (except for the rare taxa) and there was an overall trend of decrease with increasing concentration (Fig. 3).
For Se treatments, a slight to moderate pollution (up to 90 mg kgy1) kept diversity profiles around the control level and only 270 mg kgy1 caused a massively significant decrease in diversity (Fig. 4). The Zn levels resulted in a remarkable pattern: no significant differences were found for the whole diversity profile for most treatments, but the highest concentration slightly increased diversity compared to the two lowest ones for rare taxa, while the frequent taxa appeared to become much less diverse (Fig. 5). 4. Discussion Effects of Cd on soil nematodes were not significant in terms of most parameters. Only BFy FF ratio was decreased by the increasing concentration and the diversity profile seemed to benefit from the 30 mg kgy1 treatment, i.e. LOEC (Lowest Observable Effect Concentration) value of Cd under present conditions should be above the theoretical level of 270 mg kgy1 applied as maximum rate (In available concentration it was equal to approx. 190 mg kgy1). This finding is in agreement with Kammenga et al. (1994) who
Table 4 Effects of pollutant concentration gradients on Maturity Index (2–5); average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
2.45 ("0.11) – x x – x
– x – – 2.52 ("0.06) –
– 2.25 2.33 2.34 2.19 2.42
– 2.3 ("y) 2.22 ("0.18) 2.11 ("0.06) 2.05 ("0.01) 2.39 ("0.04)
– 2.57 2.03 2.42 n.c. 2.33
("0.13) ("0.13) ("0.1) ("0.08) ("0.05)
–: no such treatment, x: not sampled, y: no data available, n.c.: not calculable due to the too low nematode density.
("0.09) ("0.04) ("0.18) ("0.13)
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Table 5 Effects of pollutant concentration gradients on the c-p group distribution patterns of non-plant feeding nematodes; average values Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control c-p 1(%) c-p 2(%) c-p 3–5(%)
3.5 79.7 16.9
– – –
– – –
– – –
– – –
Cd c-p 1(%) c-p 2(%) c-p 3–5(%) P-
– – –
x x x
4.0 85.8 10.1 n.s.
2.4 87.2 10.4 n.s.
1.1 79.2 19.9 n.s.
Cr c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
2.0 86.0 12.3 n.s.
0.7 90.0 9.0 0.01
0.7 98.8 0.8 0.001
Cu c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
1.7 85.9 12.2 n.s.
1.3 94.8 4.3 0.001
0.9 83.0 16.1 n.s.
Se c-p 1(%) c-p 2(%) c-p 3–5(%) P-
– – –
0.8 81.2 18.2 n.s.
1.3 92.6 5.9 0.01
6.0 92.6 1.5 0.001
n.c. n.c. n.c.
Zn c-p 1(%) c-p 2(%) c-p 3–5(%) P-
x x x
– – –
0.9 83.9 15.2 n.s.
1.4 84.6 14.2 n.s.
1.6 86.3 12.0 n.s.
–: no such treatment, x: not sampled, n.c.: not calculable due to the too low nematode density, P-significance level compared to the control resulting from the x2 test, n.s.: non-significant.
found nematodes to be relatively insensitive to Cd, compared to other soil animals. Though the cited study was carried out in vitro with a short-term
aspect, it included 12 nematode species of various feeding and life-strategy groups, which made its conclusions more realistic than usual single-species
Table 6 Effects of pollutant concentration gradients on SI; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
52.69("7.8) – x x – x
– x – – 56.93("3.6) –
– 35.04 42.64 47.24 28.58 50.60
– 40.59 ("y) 31.01 ("20.7) 26.52 ("6.0) 9.12 ("0.8) 48.49 ("2.0)
– 59.42 10.12 49.58 n.c. 43.16
("13.3) ("11.3) ("10.3) ("9.4) ("3.7)
–: no such treatment, x: not sampled, y: no data available, n.c. not calculable due to the too low nematode density.
("4.2) (0) ("13.3) ("10.8)
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Table 7 Effects of pollutant concentration gradients on feeding group ratios; average percentage values Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control BF FF PqO PF
34.4 21.9 9.1 34.6
– – – –
– – – –
– – – –
– – – –
– – – –
x x x x
42.5 30.3 6.5 20.7 0.01
36.0 29.1 6.3 28.6 n.s.
28.7 35.2 13.8 22.3 0.01
x x x x
– – – –
32.8 34.3 7.2 25.7 0.05
47.2 36.9 5.0 10.9 0.001
43.2 41.1 0.6 15.0 0.001
x x x
– – – –
34.2 30.0 7.6 28.2 n.s.
29.1 32.5 2.7 35.7 0.05
22.5 33.9 7.2 36.3 0.05
Cd BF FF PqO PF PCr BF FF PqO PF PCu BF FF PqO PF PSe BF FF PqO PF P-
– – – –
32.3 27.1 12.1 28.4 n.s.
39.8 30.6 4.1 25.5 0.05
56.2 30.6 1.0 12.2 0.001
n.c. n.c. n.c. n.c.
Zn BF FF PqO PF P-
x x x x
– – – –
21.2 31.8 7.2 39.7 0.05
26.8 31.7 7.9 33.7 n.s.
20.0 31.1 6.0 42.9 0.01
BF: bacterial feeders, FF: fungal feeders, PqO: predatorsqomnivores, PF: plant feeders, –: no such treatment, x: not sampled, n.c.: not calculable due to the too low nematode density, P-significance level compared to the control resulting from the x2 test, n.s.: non-significant.
laboratory tests. Korthals et al. (1996b) also found nematode fauna uneffected by Cd up to 160 mg kgy1 (though in a short-term study). Effects of Cr on nematode assemblages have so far been paid little attention to. Yeates et al. (1995) presented results on mixed effects of As, Cr and Cu but their data are hard to compare with ours due to the different approach (multi- vs. singleelement treatment). In his study D. Sturhan (per-
sonal communication) showed an MI-decrease for Cr. In our present experiment, Cr had still a negative impact on nematode assemblages, similarly to earlier results (Nagy, 1999). Increasing concentration of this element affected most of the parameters applied to indicate disturbance on nematode assemblage. Density, taxon richness, MI, SI, PqO ratio and proportion of c-p 3–5 nematodes all clearly decreased along the concentration gra-
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Table 8 Effects of pollutant concentration gradients on bacterial feederyfungal feeder ratios; average values ("S.D.) Element
0 mg kgy1
10 mg kgy1
30 mg kgy1
90 mg kgy1
270 mg kgy1
Control Cd Cr Cu Se Zn
1.90 ("1.4) – x x – x
– x – – 1.19 ("0.46) –
– 1.52 1.00 1.14 1.36 0.68
– 1.24 ("y) 1.29 ("0.2) 0.92 ("0.3) 1.95("0.6) 0.85 ("0.2)
– 0.82 1.14 0.66 n.c. 0.64
("0.6) ("0.3) ("0.2) ("0.2) ("0.1)
("0.1) ("0.5) ("0.0) ("0.0)
–: no such treatment, x: not sampled, y: no data available, n.c. not calculable due to the too low nematode density, BF: bacterial feeders, FF: fungal feeders.
Fig. 1. Diversity profiles for the Cd treatment. Cd 2:30 mg kgy1, Cd 3:90 mg kgy1, Cd 4:270 mg kgy1.
Fig. 2. Diversity profiles for the Cr treatment. Cr 2:30 mg kgy1, Cr 3:90 mg kgy1, Cr 4:270 mg kgy1.
Fig. 3. Diversity profiles for the Cu treatment. Cu 2:30 mg kgy1, Cu 3:90 mg kgy1, Cu 4:270 mg kgy1.
dient. Moreover, the diversity profiles also showed a significant decrease as a consequence of the 90 mg kgy1 level. Therefore, it can be concluded that LOEC value for Cr is approximately 0.5 mg kgy1 available concentration (expressed in NH4acetateqEDTA soluble). In the higher treatments (90 and 270 mg kgy1), where the remaining available Cr in 1997 was 0.77 mg kgy1 and 1.4 mg kgy1, respectively, all parameters showed a considerable depression. It is difficult, however, to quantify what proportion of available Cr acts in the soil as the more toxic Cr(VI) form. In case of the Cu gradients, no obvious response of nematodes could be observed based on the most parameters. Only diversity profiles showed a more or less consistent stepwise difference suggesting that 90 and 270 mg kgy1 concentrations might
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Fig. 4. Diversity profiles for the Se treatment. Se 1:10 mg kgy1, Se 2:30 mg kgy1, Se 3:90 mg kgy1, Se 4:270 mg kgy1.
have an unfavourable effect on nematode assemblages. Korthals et al. (1996c) observed several clear effects on nematode trophic structure of a field contaminated with a Cu concentration gradient 10 years prior to sampling. In the most comparable case, where actual pH was 5.5 (originally 6.1) total numbers were similar along the whole concentration gradient. Bacterial feeders slightly, fungal feeders remarkably increased with higher Cu levels. Plant nematodes showed no consistent reaction. Omnivorous and carnivorous nematodes decreased along the concentration gradient. These
Fig. 5. Diversity profiles for the Zn treatment. Zn 2:30 mg kgy1, Zn 3:90 mg kgy1, Zn 4:270 mg kgy1.
results are partly in line with our findings: fungal feeders seemed to slightly benefit from the increasing Cu levels and the BFyFF ratio decreased. Also, plant feeders fluctuated around basically the same percentage value. However, in our study there was no systematic decrease in the number of omnivorous and carnivorous nematodes and bacterial feeders rather decreased than increased in their proportion. The probable main reasons for the partly dissimilar findings can be the different pH values and soil type. Under present conditions, nematodes did not appear to be particularly sensitive to Cu up to an available concentration level of approximately 130 mg kgy1. The relatively high proportion of c-p 3–5 nematodes in 270 mg kgy1 Cd and Cu samples may be due to the insensitivity of this group of persister nematodes to the given metals or an increase in algal growth in the upper millimetres of the soil, where the heavy metals had been washed out. The most expressed effects on target parameters in this experiment were observed in the Se treatments. The first concentration level (10 mg kgy1) slightly increased nematode MI, while the two higher concentrations (30 and 90 mg kgy1) resulted in a clearly detectable decrease in all the studied nematode parameters. In the plots of highest dose (270 mg kgy1) there were not enough nematodes to calculate indices. It should also be pointed out, however, that the remarkable negative effects of Se might partly be due to the almost complete lack of vegetation in the 270 mg kgy1 plots. This assumption is supported by the observations that terrestrial nematodes are without respect to their feeding types, closely connected to vegetation of a field (Freckman and Caswell, 1985; Yeates, 1987). It requires further studies to discriminate between direct and indirect (plant-mediated) toxic effects of high Se loads. Zn has apparently lost its previously shown (Nagy, 1999) favourable character for the soil nematode assemblage by the time of the present experiment. None of the concentration levels had any significant effect on any of the parameters of the studied soil invertebrate group. The only indication of its earlier positive effect was that the diversity of the rare taxa was higher in plots of high than of low Zn levels. Diversity of the
P. Nagy et al. / Science of the Total Environment 320 (2004) 131–143
frequent taxa showed, however, an opposite trend. Nematode density, MI, SI values showed, though not significantly, a decrease with the increasing concentrations of Zn. These findings appear to be in line with the results of Korthals et al. (1996b) who found Zn to depress nematodes in a concentration of 200 mg kgy1 during a short-term experiment. In our long-term study performed under more favourable soil conditions, Zn doses of approximately 140 ppm available concentration in plots of the maximum treatment gave the first signs of destruction for the nematode fauna. In a recent paper, Georgieva et al. (2002) reported several negative effects of Zn, Cu and ZnqCu treatments on nematodes in an English agroecosystem on a sandy loam treated with sewage sludge. The metal levels applied by them extended to a higher level (e.g. for Zn: 160–600 mg kgy1 in total concentration) than in our case. It should be pointed out, however, that repeated sewage sludge application means a more realistic, but less controllable treatment than ploughing in a given amount of metal salt at the start of the experiment; the latter is easily washed out and biologically more available. Density decreased significantly by Cr and Se concentration gradient, while in Cd and Zn plots, it showed a non-significant decline. The former can be attributed to a severe destruction, since pure nematode density is a rather insensitive parameter (Bongers, 1990). Richness of nematode taxa is a more sensitive parameter of activity of nematofauna as well as soil nitrogen status and decomposition function (Ekschmitt et al., 1999). In our study, richness was slightly decreased by increasing Cr and remarkably decreased by the subsequent steps of Se treatment. The other pollutants did not generate any clear effect. The clear stepwise response of Maturity Index and Structure Index to Cr and Se treatments also underlines the destructive character of these pollutants. The c-p distribution of samples shows very clearly the sensitivity of the ‘persisters’ to Cr and Se pollution. The c-p 3 nematodes being very rare, the apparent trends in this parameter can be attributed to the sensitivity of the K-strategist nematodes (in this case belonging mostly to the order Dory-
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laimida). This is in accordance with Bongers (1990, 1999). Zullini and Peretti (1986) found a similar phenomenon for lead (Pb) pollution. Regarding distribution of feeding groups: the percentage dominance bacterial feeders (chiefly Acrobeloides, Chiloplacus and Heterocephalobus) clearly increase along the concentration gradients of Cr and Se. In the light of other parameters, this indicates the relative insensitivity of this group to the given disturbances. It is remarkable how dominant Chiloplacus is in the 90 and 270 ppm Seplots. This indicates a considerable tolerance for Se, at least compared to other nematode taxa that occur in the studied area. This phenomenon has also been observed earlier, except the stepwise response (Nagy, 1999). Proportion of fungal feeding nematodes remains stable (Zn) or slightly increases (Cd, Cr, Cu), but even in case of Se, it decreases strongly only in the highest treatment level. In the latter case, the low value is based on the (probably random) occurrence of a single Tylencholaimus. This shows that fungivorous nematodes (dominated by Aphelenchus, Aphelenchoides and Ditylenchus) were quite insensitive to most pollutions. This is in agreement with D. Sturhan (personal communication) and Korthals et al. (1996c), who found Aphelenchoides to tolerate heavy metal (including Cu) stress. The proportion of omnivorous and predatory nematodes decreased clearly as a consequence of Cr and Se treatments. Zn also resulted in a slight decrease, while to Cd this group reacted with an increase. Since dominant representatives of this group were in this study basically the same as the c-p 4–5 nematodes, these results underline the harmful effects of Cr and Se demonstrated by the above mentioned parameters as well. In general, MI (and the closely related c-p distribution), SI values and diversity profiles gave basically similar indications of the effects of the studied elements. While for Cr and Se, a clear depression could be demonstrated and for Zn a weak depression appears, Cd generated no sensitivity and Cu treatments showed no obvious patterns. The fact that the above parameters of different theoretical nature led to comparable consequences shows the robustness of the present results. The combined use of these tools may be a
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relevant method in field toxicity studies, offering several advantages compared to single indices. Such advantages of diversity ordering are the firm background built on various diversity indices and the suitability of clear graphic interpretation. However, the Maturity Index and Structure Index are easy-to-calculate measures of highly relevant nematological basis (Bongers and Bongers, 1998; Bongers, 1999; Bongers and Ferris, 1999; Ferris et al., 2001). In conclusion, both favourable soil conditions and time elapsed (over 7 years) since the contamination as well as not very high available element concentrations may explain the less pronounced effects of certain elements (Cd, Cu, Zn). However, Se and Cr still show significant long-term effects on terrestrial nematodes, an important component of the soil biota involved in a crop production system. Acknowledgments This study was supported by a HungarianFlemish Intergovernmental R&D Co-operative Agreement (Contract No. B15y97) and by the Ministry of Culture and Education (Contract No. FKFP 0280y1999). References ´ ´ I. Long-term effects of heavy Bakonyi G, Nagy P, Kadar metals and microelements on nematode assemblage. Toxicol Lett 2003;140–141:391 –401. ¨ ´ ¨ ¨ I, Kadar ´ ´ I. Toxicity of some Biro´ B, Koves-Pechy K, Voros field applied heavy metal salts to the rhizobial and fungal ´ ´ microsymbionts of alfalfa and red clover. Agrokemia es Talajtan 1998;47:265 –276. Bongers T. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 1990;83:14 –19. Bongers T. The maturity index, the evolution of nematode life history traits, adaptive radiation and c-p scaling. Plant Soil 1999;212:13 –22. Bongers T, Bongers M. Functional diversity of nematodes’. Appl Soil Ecol 1998;10:239 –251. Bongers T, Ferris H. Nematode community structure as a bioindicator in environmental monitoring. TREE 1999;14:224 –228. ¨ S, Ekschmitt K, Bakonyi G, Bongers M, Bongers T, Bostrom Dogan H, Harrison A, Kallimanis A, Nagy P, O’Donnel AG, Papatheodoru EM, Sohlenius B, Stamou GP, Wolters V. Nematode community structure as indicator of soil
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