Effects of microelements on soil nematode assemblages seven ...

Science <strong>of</strong> the Total Environment 320 (2004) 131–143
<strong>Effects</strong> <strong>of</strong> <strong>microelements</strong> on soil nematode assemblages seven
years after contaminating an agricultural field
a, a b c a a
Peter ´ Nagy *, Gabor ´ Bakonyi , Tom Bongers , Imre Kadar ´ ´ , Miklos ´ Fabian ´ ´ , Istvan ´ Kiss
a Department <strong>of</strong> Zoology and Ecology, Szent Istvan ´ University, Godollo, ¨ ¨ ¨ Pater ´ K. u. 1 H-2103, Hungary
bLaboratory
for Nematology, Wageningen University, P.O. Box 8123, Wageningen 6700 ES, The Netherlands
Research Institute for Soil Science and Agricultural Chemistry <strong>of</strong> the Hungarian Academy <strong>of</strong> Sciences, Herman O. u. 15,
Budapest H-1025, Hungary
c
Abstract
Received 28 January 2003; accepted 13 August 2003
Long-term effects <strong>of</strong> Cd, Cr, Cu, Se and Zn were studied 7 years after artificially contaminating plots <strong>of</strong> an
agricultural field on a calcareous chernozem soil.<strong>Effects</strong> <strong>of</strong> three to four different contamination levels (originally
y1 y1
10, 30, 90 and 270 mg kg ) were studied.Nematode density was significantly reduced by 90 and 270 mg kg Se
y1 y1
as well as by 270 mg kg Cr, while 90 and 270 mg kg 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 <strong>of</strong> c-p groups was negatively affected
by the increasing Cr and Se concentration, while in Zn plots, this decrease was not significant.Response <strong>of</strong> feeding
groups to pollutions was similar to other parameters: Cr and Se caused significant changes toward the loss <strong>of</strong>
variability.The proportion <strong>of</strong> the most sensitive omnivorous and predatory nematodes decreased clearly as a
consequence <strong>of</strong> Cr and Se treatments.Zn pollution also resulted in a slight decrease in this group, while Cd caused
an increase.Nematode diversity pr<strong>of</strong>iles showed a significant decrease in the plots <strong>of</strong> increased Cr and Se
concentrations, while increased concentrations <strong>of</strong> Cu and Zn resulted in ambiguous effects.Besides providing evidence
on the harmful effects <strong>of</strong> Cr and Se on a soil nematode assemblage, our results suggest that simultaneous analysis <strong>of</strong>
Maturity Index, Structure Index and diversity pr<strong>of</strong>iles provide a promising tool in nematological indication <strong>of</strong> soil
pollution.
2003 Elsevier B.V. All rights reserved.
Keywords: Soil pollution; Microelement concentration gradients; Nematodes; Maturity index; Structure index; Diversity pr<strong>of</strong>iles;
Heavy metals
1. Introduction
Increasing anthropogenic pollution <strong>of</strong> ecosystems
by heavy metals and <strong>microelements</strong> means
*Corresponding author.Fax: q36-28-410804.
E-mail address: nagypeterdr@freemail.hu (P.Nagy).
0048-9697/04/$ - see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2003.08.006
a growing hazard to living organisms, including
mankind.In spite <strong>of</strong> this fact, data on long-term
effects <strong>of</strong> 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 <strong>of</strong> plant micronutrients,

132 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
including heavy metals on soil biota, crops and
their consumers (Kadar, ´ ´ 1994).
Various crops grown at this site in previous
years gave different responses to the contamination
(Kadar, ´ ´ 1995).As a general trend, high Se exposure
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 <strong>of</strong> the
experiment.Deleterious effects <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> the experiment until 2002.It is not
clear whether this result is due to the increased
toxicity <strong>of</strong> Cd or to the planting <strong>of</strong> more Cdsensitive
plant species during these years.Cu and
Zn caused no phytotoxic effects at all.
Nematodes are <strong>of</strong>ten used as indicators <strong>of</strong> environmental
disturbances, including heavy metal pollution
(e.g.: Zullini and Peretti, 1986; Samoil<strong>of</strong>f,
1987; Bongers, 1990; Korthals et al., 1996a).
There are, however, only a limited number <strong>of</strong>
studies on plant <strong>microelements</strong> 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
y1
concentrations above 200 mg kg .In a microplot
experiment, no remarkable reduction <strong>of</strong> nematode
density was found in soils polluted with 12 various
<strong>microelements</strong>, except in the V plot, where the
density was 25% <strong>of</strong> the control (D.Sturhan,
personal communication).
More recently, Korthals et al. (1996b) carried
out an experiment on the short-term toxic effects
<strong>of</strong> 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 <strong>of</strong> 200
y1 mg kg , which is comparable to the maximum
initial concentrations used in the present experi-
ment.Cd applied in a concentration <strong>of</strong> 160 mg
y1 kg as a maximum had no significant effect on
nematode density.In another paper, Korthals et al.
(1996c) reported effects <strong>of</strong> an aged Cu pollution
and pH levels on nematode assemblage <strong>of</strong> 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
y1
<strong>of</strong> 250, 500, 750 kg ha and were largely
enhanced by decreasing soil pH within a range <strong>of</strong>
3.9–5.5.
In a field site contaminated long before sampling,
Weiss and Larink (1991) found adverse
effects <strong>of</strong> a mixture <strong>of</strong> heavy metals (among which
Cd, Cr, Cu and Zn were also involved in this
study) and sewage sludge.In terms <strong>of</strong> density,
nematodes gave a positive reaction to this intervention,
due to the high numbers <strong>of</strong> enrichment
opportunists.However, contamination markedly
decreased omnivorous nematodes.In another study
on application <strong>of</strong> sewage sludge contaminated with
various heavy metals, Georgieva et al. (2002)
found Cu and Zn to have a negative effect on
various parameters <strong>of</strong> 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
<strong>of</strong> the same experimental field.Cr and
especially Se had a negative influence while Zn
appeared to stimulate nematodes in terms <strong>of</strong> 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 <strong>of</strong> Zn disappeared.
However, remarkable fluctuations have been found
in the composition <strong>of</strong> nematode assemblage among
the studied years.
The aim <strong>of</strong> our study was to evaluate whether
different doses <strong>of</strong> Cd, Cr, Cu, Se and Zn had
recognisable effects on the structure <strong>of</strong> the nematode
assemblages in the calcareous chernozem soil
<strong>of</strong> an agricultural field 7 years after application.
Particular emphasis was given to applying coen-

P. Nagy et al. / Science <strong>of</strong> 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 <strong>of</strong> Research Institute for Soil Science
and Agricultural Chemistry (RISSAC) <strong>of</strong> the Hungarian
Academy <strong>of</strong> Sciences at Nagyhorcsok, ¨ ¨ Hungary
(UTM-code: CT 00).Soil characteristics and
experimental design are detailed by Kadar ´ ´ (1994,
1995).Therefore, only some <strong>of</strong> the most important
soil characteristics are mentioned here.The soil <strong>of</strong>
the experimental plots is a calcareous loamy chernozem
with medium to deep humus layer formed
on loess.Exchangeable cations comprise <strong>of</strong> 80%
Ca, 16% Mg, 3% K, 1% Na.Ss40 meqy100 g,
water soluble saltss1 meqy100 g, pH s7.4.
KCl
( )
Particle distribution in percentage <strong>of</strong> 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 <strong>of</strong> 8–8.5. Experimental plots were polluted
with single <strong>microelements</strong> given as CdSO ,
4
K CrO , CuSO , Na SeO and ZnSO , respective-
2 4 4 2 3 4
ly.Contaminants were ploughed into the soil <strong>of</strong>
the plots in April 1991.Initial total element doses
y1
were 90, 270 and 810 kg ha .These doses were
y1 y1
roughly equal to 30 mg kg , 90 mg kg and
y1
270 mg kg , respectively, based on the typical
soil density value <strong>of</strong> 1.5 and the average plough
layer <strong>of</strong> 0.2 m. In the case <strong>of</strong> Cd and Se, an
y1
additional treatment <strong>of</strong> 10 mg kg in total con-
centration was also applied.Fertilisers were given
y1 y1
at concentrations <strong>of</strong> Ns100 kg ha year (as
y1 y1
NH4NO 3), P2O5s100 kg ha year , Ks100
y1 y1
kg ha year .P, K and half <strong>of</strong> the N dose were
applied before the autumn tillage, while the second
half <strong>of</strong> N was applied in early spring.The above
treatments are aimed to assess the effects <strong>of</strong>
<strong>microelements</strong> on crops grown under conditions
<strong>of</strong> 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 <strong>of</strong> 21 m2 133
per plot.Each plot was surrounded by paths <strong>of</strong> 1
m width.The two replicates were set up as two
adjacent blocks <strong>of</strong> plots.Both replicates <strong>of</strong> each
treatment and two control plots were sampled on
30 June 1998, 7 years after the contamination.
The NH4-acetateqEDTA soluble (mobile) fraction
<strong>of</strong> element was determined according to Lak-
anen 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 <strong>of</strong> sampling
it was close to flowering.
For nematological analysis composite samples
(20 subsampleyplot) <strong>of</strong> approximately 300–400 g
soil were taken using a soil corer <strong>of</strong> 2 cm in
diameter.The top 10 cm <strong>of</strong> 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% <strong>of</strong> 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 <strong>of</strong> nematode taxa)
and coenological data for each sample. (In cases
<strong>of</strong> the maximum concentration Se treatment where
this amount <strong>of</strong> 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 <strong>of</strong> c-p 3 were assigned to the conglomerate
<strong>of</strong> c-p 4–5 (persister nematodes).However,
representatives <strong>of</strong> the c-p 3 group occurred in only
27% <strong>of</strong> 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 <strong>of</strong> the stabile and structured status <strong>of</strong> a
soil nematode community.It is based on the
proportion <strong>of</strong> feeding types combined with their

134 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
appropriate guild weightings.High value shows a
more structured community, while low SI is a sign
<strong>of</strong> ‘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
2
with standard Pearson x test.
Most <strong>of</strong> the classical diversity indices have the
serious disadvantage <strong>of</strong> varying in sensitivity to
the density <strong>of</strong> the common and rare species.To
overcome this problem, Patil and Taillie (1979)
suggested the use <strong>of</strong> the community diversity
pr<strong>of</strong>ile.The community diversity pr<strong>of</strong>ile outlines
scale-dependent diversity <strong>of</strong> a community.Some
values <strong>of</strong> the diversity pr<strong>of</strong>ile are related to classical
diversity indexes, namely at as0 equal to
lgS, where Ssnumber <strong>of</strong> species in the community,
at as1 equal to the value <strong>of</strong> 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 pr<strong>of</strong>iles.Diversity
pr<strong>of</strong>iles between scale parameters 0–40 <strong>of</strong> all plots
were generated.The low values refer to the rare
taxa, while the higher scale parameters depict the
status <strong>of</strong> the more frequent ones.Above as4, the
curves were more or less parallel to each other,
therefore, diversity pr<strong>of</strong>iles 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 <strong>of</strong>
the curves differ at Ps5%.In this study, curves
have been compared with an increment <strong>of</strong> 0.5
along the range <strong>of</strong> as0–4.
3. Results
The mobile fractions <strong>of</strong> the target element in
the studied plots are shown in Table 1.These data
show that the actual available values differed
(<strong>of</strong>ten remarkably) from the originally added total
values (depending on the pollutant).However, a
trend <strong>of</strong> 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
y1
and 270 mg kg concentration and in the plots
<strong>of</strong> 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
y1
significantly by the 90 and 270 mg kg Se
treatment (Table 3).The increasing concentrations
<strong>of</strong> 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 <strong>of</strong> the 270
y1 mg kg concentration and in the Se treatments
y1
from 30 mg kg onwards, according to a clear
dose-related pattern.The low number <strong>of</strong> nematodes
y1
extracted from the 270 mg kg 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 <strong>of</strong> 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
y1
in relatively high proportion in 270 mg kg 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 <strong>of</strong> 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

P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Table 1
The NH4-acetateqEDTA soluble (mobile) concentration values along the microelement gradients as measured in the subsequent
analyses
Element, year Control 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Cd
1991 – 14.0 27.0 96.0 270.0
1992 – 0 18.0 62.0 228.0
1994 – 0 14.0 44.0 164.0
1997 – 0 26.8 84.7 190.0
2000
Cr
– 0 14.0 44.0 124.0
1991 0 – 1.0 3.0 9.0
1992 0 – 2.0 5.0 10.0
1994 0 – 1.0 2.0 4.0
1997 0.2 – 0.45 0.77 1.4
2000
Cu
0 – 0.4 0.9 1.6
1991 9.0 – 29.0 47.0 200.0
1992 4.0 – 34.0 94.0 270.0
1994 4.0 – 23.0 65.0 192.0
1997 6.0 – 19.4 54.1 133.0
2000
Se
4.0 – 20.0 44.0 128.0
1991 – 1.0 6.0 34.0 84.0
1992 – 0 7.0 66.0 81.0
1994 – 0 8.0 33.0 89.0
1997 – 0.5 1.56 9.32 36.0
2000
Zn
– 0 2.0 4.0 11.0
1991 1.0 – 22.0 66.0 120.0
1992 3.0 – 29.0 68.0 213.0
1994 1.0 – 19.0 44.0 147.0
1997 2.0 – 22.0 53.0 143.0
2000 2.0 – 16.0 37.0 85.0
–: no such treatment.
Table 2
y1
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on nematode density; average specimenU100 g soil ("S.D.)
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control 1275.0 ("1104) – – – –
Cd – x 1105.0 ("824) 937.5 ("735) 815.0 ("613)
Cr x – 1055.0 ("754) 834.5 ("724) 484.5 ("267)*
Cu x – 981.0 ("501) 1184.0 ("945) 1063.0 ("987)
Se – 1160.0 ("587) 954.0 ("861) 422.5 ("370)* 20.0 ("3.5)**
Zn x – 1150.0 ("1033) 1021.0 ("648) 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.
135

136 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Table 3
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on nematode taxon richness; average values ("S.D.)
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control 19.0 ("2.8) – – – –
Cd – x 22.5 ("2.1) 17.0 ("y) 18.0 (0)
Cr x – 20.5 ("0.7) 16.0 (0) 15.0 ("2.8)
Cu x – 21.0 ("1.4) 15.5 ("0.7) 17.5 ("5.0)
Se – 16.5 ("2.1) 16.0 ("2.8) 12.5 ("2.1)* 3. 0 ("1.4)*
Zn x – 18.5 ("2.1) 18.5 ("0.7) 18.5 ("2.1)
–: 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
y1
(90 and 270 mg kg ), while increasing Cd and
Cu levels favoured fungal feeders.
Diversity-pr<strong>of</strong>iles for nematode samples were
obviously affected by some <strong>of</strong> the treatments.Cd
pollution did not result in a clear trend <strong>of</strong> diversity
pr<strong>of</strong>iles (Fig.1).The lowest level <strong>of</strong> 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 <strong>of</strong> 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
<strong>of</strong> decrease with increasing concentration (Fig.3).
Table 4
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on Maturity Index (2–5); average values ("S.D.)
For Se treatments, a slight to moderate pollution
y1
(up to 90 mg kg ) kept diversity pr<strong>of</strong>iles around
y1
the control level and only 270 mg kg 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 pr<strong>of</strong>ile 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
<strong>Effects</strong> <strong>of</strong> Cd on soil nematodes were not
significant in terms <strong>of</strong> most parameters.Only BFy
FF ratio was decreased by the increasing concentration
and the diversity pr<strong>of</strong>ile seemed to benefit
y1
from the 30 mg kg treatment, i.e. LOEC (Low-
est Observable Effect Concentration) value <strong>of</strong> Cd
under present conditions should be above the
y1
theoretical level <strong>of</strong> 270 mg kg applied as max-
imum rate (In available concentration it was equal
y1
to approx.190 mg kg ).This finding is in
agreement with Kammenga et al. (1994) who
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control 2.45 ("0.11) – – – –
Cd – x 2.25 ("0.13) 2.3 ("y) 2.57 ("0.09)
Cr x – 2.33 ("0.13) 2.22 ("0.18) 2.03 ("0.04)
Cu x – 2.34 ("0.1) 2.11 ("0.06) 2.42 ("0.18)
Se – 2.52 ("0.06) 2.19 ("0.08) 2.05 ("0.01) n.c.
Zn x – 2.42 ("0.05) 2.39 ("0.04) 2.33 ("0.13)
–: no such treatment, x: not sampled, y: no data available, n.c.: not calculable due to the too low nematode density.

P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Table 5
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on the c-p group distribution patterns <strong>of</strong> non-plant feeding nematodes; average values
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control
c-p 1(%) 3. 5 – – – –
c-p 2(%) 79.7 – – – –
c-p 3–5(%)
Cd
16.9 – – – –
c-p 1(%) – x 4.0 2.4 1.1
c-p 2(%) – x 85.8 87.2 79.2
c-p 3–5(%) – x 10.1 10.4 19.9
P-
Cr
n.s. n.s. n.s.
c-p 1(%) x – 2.0 0.7 0.7
c-p 2(%) x – 86.0 90.0 98.8
c-p 3–5(%) x – 12.3 9.0 0.8
P-
Cu
n.s. 0.01 0.001
c-p 1(%) x – 1.7 1.3 0.9
c-p 2(%) x – 85.9 94.8 83.0
c-p 3–5(%) x – 12.2 4.3 16.1
P-
Se
n.s. 0.001 n.s.
c-p 1(%) – 0.8 1.3 6.0 n.c.
c-p 2(%) – 81.2 92.6 92.6 n.c.
c-p 3–5(%) – 18.2 5.9 1.5 n.c.
P-
Zn
n.s. 0.01 0.001
c-p 1(%) x – 0.9 1.4 1.6
c-p 2(%) x – 83.9 84.6 86.3
c-p 3–5(%) x – 15.2 14.2 12.0
P- n.s. n.s. n.s.
–: no such treatment, x: not sampled, n.c.: not calculable due to the too low nematode density, P-significance level compared
2
to the control resulting from the x 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
Table 6
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on SI; average values ("S.D.)
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control 52.69("7.8) – – – –
Cd – x 35.04 ("13.3) 40.59 ("y) 59.42 ("4.2)
Cr x – 42.64 ("11.3) 31.01 ("20.7) 10.12 (0)
Cu x – 47.24 ("10.3) 26.52 ("6.0) 49.58 ("13.3)
Se – 56.93("3.6) 28.58 ("9.4) 9.12 ("0.8) n.c.
Zn x – 50.60 ("3.7) 48.49 ("2.0) 43.16 ("10.8)
–: no such treatment, x: not sampled, y: no data available, n.c. not calculable due to the too low nematode density.
137
aspect, it included 12 nematode species <strong>of</strong> various
feeding and life-strategy groups, which made its
conclusions more realistic than usual single-species

138 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Table 7
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on feeding group ratios; average percentage values
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control
BF 34.4 – – – –
FF 21.9 – – – –
PqO 9. 1 – – – –
PF
Cd
34.6 – – – –
BF – x 42.5 36.0 28.7
FF – x 30.3 29.1 35.2
PqO – x 6.5 6.3 13.8
PF – x 20.7 28.6 22.3
P- 0.01 n.s. 0.01
Cr x –
BF x – 32.8 47.2 43.2
FF x – 34.3 36.9 41.1
PqO x – 7.2 5.0 0.6
PF 25.7 10.9 15.0
P-
Cu
0.05 0.001 0.001
BF – 34.2 29.1 22.5
FF x – 30.0 32.5 33.9
PqO x – 7.6 2.7 7.2
PF x – 28.2 35.7 36.3
P-
Se
n.s. 0.05 0.05
BF – 32.3 39.8 56.2 n.c.
FF – 27.1 30.6 30.6 n.c.
PqO – 12.1 4.1 1.0 n.c.
PF – 28.4 25.5 12.2 n.c.
P-
Zn
n.s. 0.05 0.001
BF x – 21.2 26.8 20.0
FF x – 31.8 31.7 31.1
PqO x – 7.2 7.9 6.0
PF x – 39.7 33.7 42.9
P- 0.05 n.s. 0.01
BF: bacterial feeders, FF: fungal feeders, PqO: predatorsqomnivores, PF: plant feeders, –: no such treatment, x: not sampled,
2
n.c.: not calculable due to the too low nematode density, P-significance level compared to the control resulting from the x test,
n.s.: non-significant.
laboratory tests.Korthals et al.(1996b) also found
nematode fauna uneffected by Cd up to 160 mg
y1 kg (though in a short-term study).
<strong>Effects</strong> <strong>of</strong> Cr on nematode assemblages have so
far been paid little attention to.Yeates et al.(1995)
presented results on mixed effects <strong>of</strong> 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 <strong>of</strong> this element affected most <strong>of</strong> the
parameters applied to indicate disturbance on nematode
assemblage.Density, taxon richness, MI, SI,
PqO ratio and proportion <strong>of</strong> c-p 3–5 nematodes
all clearly decreased along the concentration gra-

P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Table 8
<strong>Effects</strong> <strong>of</strong> pollutant concentration gradients on bacterial feederyfungal feeder ratios; average values ("S.D.)
Element 0mgkg y1 10 mg kg y1 30 mg kg y1 90 mg kg y1 270 mg kg y1
Control 1.90 ("1.4) – – – –
Cd – x 1.52 ("0.6) 1.24 ("y) 0.82 ("0.1)
Cr x – 1.00 ("0.3) 1.29 ("0.2) 1.14 ("0.5)
Cu x – 1.14 ("0.2) 0.92 ("0.3) 0.66 ("0.0)
Se – 1.19 ("0.46) 1.36 ("0.2) 1.95("0.6) n.c.
Zn x – 0.68 ("0.1) 0.85 ("0.2) 0.64 ("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 pr<strong>of</strong>iles for the Cd treatment.Cd 2:30 mg
y1 y1 y1
kg , Cd 3:90 mg kg , Cd 4:270 mg kg .
Fig.2.Diversity pr<strong>of</strong>iles for the Cr treatment.Cr 2:30 mg
y1 y1 y1
kg , Cr 3:90 mg kg , Cr 4:270 mg kg .
139
Fig.3.Diversity pr<strong>of</strong>iles for the Cu treatment.Cu 2:30 mg
y1 y1 y1
kg , Cu 3:90 mg kg , Cu 4:270 mg kg .
dient.Moreover, the diversity pr<strong>of</strong>iles also showed
a significant decrease as a consequence <strong>of</strong> the 90
y1 mg kg level.Therefore, it can be concluded that
LOEC value for Cr is approximately 0.5 mg
y1 kg available concentration (expressed in NH4- acetateqEDTA soluble).In the higher treatments
y1
(90 and 270 mg kg ), where the remaining
y1
available Cr in 1997 was 0.77 mg kg and 1.4
y1 mg kg , respectively, all parameters showed a
considerable depression.It is difficult, however, to
quantify what proportion <strong>of</strong> available Cr acts in
the soil as the more toxic Cr(VI) form.
In case <strong>of</strong> the Cu gradients, no obvious response
<strong>of</strong> nematodes could be observed based on the most
parameters.Only diversity pr<strong>of</strong>iles showed a more
or less consistent stepwise difference suggesting
y1
that 90 and 270 mg kg concentrations might

140 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
Fig.4.Diversity pr<strong>of</strong>iles for the Se treatment.Se 1:10 mg
y1 y1 y1
kg , Se 2:30 mg kg , Se 3:90 mg kg , Se 4:270 mg
kg . y1
have an unfavourable effect on nematode
assemblages.
Korthals et al. (1996c) observed several clear
effects on nematode trophic structure <strong>of</strong> 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 pr<strong>of</strong>iles for the Zn treatment.Zn 2:30 mg
y1 y1 y1
kg , Zn 3:90 mg kg , Zn 4:270 mg kg .
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 <strong>of</strong> 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
<strong>of</strong> approximately 130 mg kg . y1
The relatively high proportion <strong>of</strong> c-p 3–5 nem-
y1
atodes in 270 mg kg Cd and Cu samples may
be due to the insensitivity <strong>of</strong> this group <strong>of</strong> persister
nematodes to the given metals or an increase in
algal growth in the upper millimetres <strong>of</strong> 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 treat-
y1
ments.The first concentration level (10 mg kg )
slightly increased nematode MI, while the two
y1
higher concentrations (30 and 90 mg kg ) result-
ed in a clearly detectable decrease in all the studied
nematode parameters.In the plots <strong>of</strong> highest dose
y1
(270 mg kg ) there were not enough nematodes
to calculate indices.It should also be pointed out,
however, that the remarkable negative effects <strong>of</strong>
Se might partly be due to the almost complete
y1
lack <strong>of</strong> vegetation in the 270 mg kg plots.This
assumption is supported by the observations that
terrestrial nematodes are without respect to their
feeding types, closely connected to vegetation <strong>of</strong>
a field (Freckman and Caswell, 1985; Yeates,
1987).It requires further studies to discriminate
between direct and indirect (plant-mediated) toxic
effects <strong>of</strong> high Se loads.
Zn has apparently lost its previously shown
(Nagy, 1999) favourable character for the soil
nematode assemblage by the time <strong>of</strong> the present
experiment.None <strong>of</strong> the concentration levels had
any significant effect on any <strong>of</strong> the parameters <strong>of</strong>
the studied soil invertebrate group.The only indication
<strong>of</strong> its earlier positive effect was that the
diversity <strong>of</strong> the rare taxa was higher in plots <strong>of</strong>
high than <strong>of</strong> low Zn levels.Diversity <strong>of</strong> the

P. Nagy et al. / Science <strong>of</strong> 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 <strong>of</strong> Zn.These findings appear to be
in line with the results <strong>of</strong> Korthals et al. (1996b)
who found Zn to depress nematodes in a concen-
y1tration
<strong>of</strong> 200 mg kg during a short-term exper-
iment.In our long-term study performed under
more favourable soil conditions, Zn doses <strong>of</strong>
approximately 140 ppm available concentration in
plots <strong>of</strong> the maximum treatment gave the first
signs <strong>of</strong> destruction for the nematode fauna.In a
recent paper, Georgieva et al. (2002) reported
several negative effects <strong>of</strong> 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 kg y1
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 <strong>of</strong> metal salt at the start <strong>of</strong> 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 <strong>of</strong> nematode taxa is a more sensitive
parameter <strong>of</strong> activity <strong>of</strong> nemat<strong>of</strong>auna 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 <strong>of</strong>
Se treatment.The other pollutants did not generate
any clear effect.
The clear stepwise response <strong>of</strong> Maturity Index
and Structure Index to Cr and Se treatments also
underlines the destructive character <strong>of</strong> these pollutants.The
c-p distribution <strong>of</strong> samples shows very
clearly the sensitivity <strong>of</strong> 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 <strong>of</strong> the K-strategist nematodes
(in this case belonging mostly to the order Dory-
141
laimida).This is in accordance with Bongers
(1990, 1999).Zullini and Peretti (1986) found a
similar phenomenon for lead (Pb) pollution.
Regarding distribution <strong>of</strong> feeding groups: the
percentage dominance bacterial feeders (chiefly
Acrobeloides, Chiloplacus and Heterocephalobus)
clearly increase along the concentration gradients
<strong>of</strong> Cr and Se.In the light <strong>of</strong> other parameters, this
indicates the relative insensitivity <strong>of</strong> 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 <strong>of</strong> fungal feeding
nematodes remains stable (Zn) or slightly
increases (Cd, Cr, Cu), but even in case <strong>of</strong> Se, it
decreases strongly only in the highest treatment
level.In the latter case, the low value is based on
the (probably random) occurrence <strong>of</strong> 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
<strong>of</strong> omnivorous and predatory nematodes decreased
clearly as a consequence <strong>of</strong> Cr and Se treatments.
Zn also resulted in a slight decrease, while to Cd
this group reacted with an increase.Since dominant
representatives <strong>of</strong> this group were in this study
basically the same as the c-p 4–5 nematodes, these
results underline the harmful effects <strong>of</strong> 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 pr<strong>of</strong>iles gave
basically similar indications <strong>of</strong> the effects <strong>of</strong> 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 <strong>of</strong>
different theoretical nature led to comparable consequences
shows the robustness <strong>of</strong> the present
results.The combined use <strong>of</strong> these tools may be a

142 P. Nagy et al. / Science <strong>of</strong> the Total Environment 320 (2004) 131–143
relevant method in field toxicity studies, <strong>of</strong>fering
several advantages compared to single indices.
Such advantages <strong>of</strong> diversity ordering are the firm
background built on various diversity indices and
the suitability <strong>of</strong> clear graphic interpretation.However,
the Maturity Index and Structure Index are
easy-to-calculate measures <strong>of</strong> 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 <strong>of</strong> certain elements (Cd, Cu, Zn).However,
Se and Cr still show significant long-term effects
on terrestrial nematodes, an important component
<strong>of</strong> the soil biota involved in a crop production
system.
Acknowledgments
This study was supported by a Hungarian-
Flemish Intergovernmental R&D Co-operative
Agreement (Contract No.B15y97) and by the
Ministry <strong>of</strong> Culture and Education (Contract No.
FKFP 0280y1999).
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