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003%0717(94)00179-0
Soil Bid. Bmchen~.
Vol.
27. No. 3. 287-296. pp. 1995
Copyright (’ 1995 Elsevier Science
Lid
Printed in Great Britain. All rights reserved
0038-0717/95 $9.50 + 0.00
BIOAVAILABILITY OF HEAVY METALS AND
ARBUSCULAR MYCORRHIZA IN A
SEWAGE-SLUDGE-AMENDED SANDY SOIL
I. WEISSENHORN,‘* M. MENCH’ and C. LEYVAL’
‘Centre
de Pkdologie Biologique. CNRS. Laboratoire Associk i I’Universiti de Nancy I, BP 5, 54501
Vandoeuvre-l&-Nancy Cedex, France and ‘INRA. Unite Agronomic Centre de Recherches de Bordeaux,
BP 81. 33883 Villenave d’Ornon Cedex, France
(Accepted 9
August 1994)
Summary-The bioavailability of metals (Cd, Ni. Zn. Cu, Pb and Mn) and abundance of arbuscular
mycorrhiza were studied in a long-term sewage-sludge field trial on an acid sandy soil, at INRA-Bordeaux,
France. Zn/Mn-(El) and Cd/Ni-(E2)contaminated sludge had been applied at two rates (10 t DM ha- ’
Y ’ and 100 t DM ha
’
2y _
’
)
for 18 and 5 y, respectively. Inorganic fertilizer and farm yard manure
treatments served as unpolluted controls. Soil extraction with EDTA-NH,OAc and Ca(NO,), and plant
(Zeu WKIJX L.) uptake demonstrated an unusually high Zn
(El), Cd and
Ni (E2) availability in the
sludge-amended plots. The spore density of arbuscular mycorrhizal fungi ranged from 16 to 67 spores
50 g-
’
dry soil, and root colonization between O&33%. No relationship between mycorrhizal abundance
and degree of metal exposure in soil or inside plant roots could be established, but root colonization across
the different treatments correlated well with plant P status. The results suggest a better tolerance of the
indigenous population of arbuscular mycorrhizal fungi to elevated metal than to high P concentrations.
INTRODUCTION
Application
of sewage sludge to agricultural land is
a widely practised means of inexpensive waste dis-
posal and improvement of soil physical properties
and nutrient status (Sauerbeck, 1987). Sludges can,
nevertheless, contain considerable amounts of heavy
metals that persist in the soil long after application
(Juste and Mench, 1992). At present in the European
Community (EC). 50-70% of sewage solids are dis-
posed of on land. This represents average application
rates of 50-100 kg dry matter ha.-’ of agricultural
soil y-l, and the trend is rising (Brookes and
Verstraete, 1989). Thus, the contribution of sewage-
sludge to the overall input of metals to the
environment is considerable.
Current EC regulations for maximum metal inputs
into agricultural soils (CEC, 1986) and the maximum
admissible metal concentrations of food and food-
stuffs (Ewers, 1991) are based on numerous studies
concerned with metal uptake by plants, metal phyto-
toxicity and zootoxicity. Recent studies have, how-
ever, shown adverse effects of metals on soil microbial
populations and their activities at concentrations
close to EC limits for sludge application
(Brookes
et al., 1986; McGrath et
a/..
1988: Chaudri
et a/..
1993).
Arbuscular mycorrhizal (AM) fungi form symbi-
otic associations with the roots of most agricultural
and horticultural plants, enhancing uptake of P, Zn
and Cu (Tinker and Gildon, 1983) under conditions
of deficiency. However, few and variable results exist
on the significance of AM in soils contaminated with
metals derived from sewage-sludge application. Boyle
and Paul (1988) reported a negative correlation
between Zn concentrations in a soil treated with
urban-industrial sludge and AM colonization of
barley. Koomen
et al.
(1990) observed suppression of
AM in pot cultures with soil from a long-term sludge
trial, while under field conditions the amount of
colonization in the same soil was not different from
that of the farmyard manure control treatment.
Arnold and Kapustka (1987) found no effect of the
addition of metal-containing sludge on AM develop-
ment in field plots and pot culture, whereas Leyval
et al.
(1991) detected a lower sensitivity to heavy
metals in indigenous AM fungi from a sludge-pol-
luted site as compared to a reference isolate from an
unpolluted soil. These authors also observed that
shoot accumulation of Zn, Cu and Pb decreased with
mycorrhizal colonization for the highest amounts of
metal pollution, but not for the lower levels. Similar
observations were made by Schiiepp
et al.
(1987) for
Zn, whereas shoot uptake of Cd was always less in
mycorrhizal plants. independent of the amount of
pollution.
Our aim was to assess the relation between metal
availability, mycorrhizal abundance and plant metal
*Author
for correspondence.
accumulation in soil polluted by long-term appli-
cation of sewage-sludge. Metal availability and hence
287
288 1. Weissenhorn et
al
potential toxicity was estimated by chemical extrac-
tions of soil at one harvest time. Plant metal concen-
trations and mycorrhizal indices (spore density, root
colonization) were surveyed throughout one growing
season.
MATERIALS AND METHODS
Sampling site and soil characteristics
The field site was a long-term sewage-sludge trial
on an acid sandy soil (Arenic Udifluvent, pH 5.5) at
the INRA (Institut National de la Recherche
Agronomique) experimental site of Couhins (Bor-
deaux, southwest France). Two experiments, in the
same field, corresponded to two different sludge types
(Ambares and Louis-Fargue) with the following four
treatments (five replicate plots): C = control with
inorganic fertilizer only; F = farmyard manure at a
rate of 10 t DM ha-’ y-‘; Sl =sewage sludge at a
rate of 10 t DM ha- ’ ye ‘; S2 = sewage sludge at a
rate of IOOt DM haa’ 2~~‘.
Experiment I (El) was established in 1974 and has
since received an anaerobically-digested, mechani-
cally-dehydrated sludge from the sewage treatment
plant of a small residential area (Ambares). This
sludge is characterized by high Zn and Mn concen-
trations (Juste and Mench, 1992). Up to the time of
our study (1991) a total of 180 and 900 t DM of
sludge had been applied ha
’
for treatments Sl and
S2, respectively. Mineral fertilization of the plots was
adjusted to the same level for the treatments C, F and
Sl, i.e. 200 kg of N, P at the Sl level (275 kg in 1991).
166 kg of K and 50 kg of Mg ha
’ y
’ .
assuming that
50% of the N added with the organic materials
(manure or sludge) is mineralized during the same
year.
Experiment 2 (E2) was started in 1976 and has
received sludge from a sewage treatment plant
(Louis-Fargue) in the Bordeaux district. This is a
solid, anaerobically-digested and heat-dehydrated
sludge with high Cd and Ni contents, from wastes
discharged by a battery manufacturer. Sludge appli-
cation ceased after 1980, when a strong phytotoxic
effect was observed on maize [50% yield decrease on
S2 plots; Juste and Mench (1992)]. These plots offered
the possibility to study the long-lasting effects of
metals due to a total sludge application of 50 (Sl) and
300 t DM ha
’
(S2). Since 1981, all plots have been
fertilized annually with 200 kg N, 87 kg P, 166 kg K
and 50 kg Mg ha
’
as ammonium nitrate, potassium
sulphate, superphosphate and magnesium sulphate.
All field plots have been annually cropped with
maize
(Zea mays
L. cv. INRA 260) at a seed rate of
75,000 ha
’
The only plant protection treatment has
been application of the herbicide Atrazine. Usually,
only parts of plants have been removed for element
analysis and grains for yield determination, crop
residues being chopped and ploughed in every year at
the end of winter. Table 1 presents the soil character-
istics of the plots concerned by the present study,
reflecting the accumulation of organic matter. P and
heavy metals as well as an evolution of the pH
depending on the treatment.
Soil sampling and preparation
Soil was sampled in April (before sowing) and
September (at harvest) in 1991. Five subsamples were
collected at random points in the middle of each plot
Table I. PropertIes of soil from the plots of the Bordeaux experimental site’
El (“Ambar&“) E? (“Louis-Fargue”)
Treatment! C F SI S? C F SI s2
Plot number: 20 IO 4 I6 33 39 35 30
Clay
k
kg
7
45 57 35
Silt (g kg- ‘) 88 134 104
Sand (g kg ‘) 867 809 861
PH (H,D) 5.4 6.4 6.2
Organic matter (g kg ‘) 21.3 34.9 20.6
Organic C (g kg ‘) 12.4 20.3 12.0
Total N (g kg ‘) I.1 1.7 I.1
P Olsen (mg kg ‘) 72 129 80
P Dyer (mg kg
‘)d
304 499 426
CEC (cm01 kg ‘) 3.3 5.3 3.2
E.uchungecrhL
ccrrions (mg kg
’ )
Cd
634 I298 748
K 50 95 53
Mg 20 83 38
Na 9 20 9
Total’ mrrd concentration (mg kg ‘)
Cd 0.3 0.5 I
CU I4 23 I9
Zn I9 51 199
Pb IX 43 45
M” 33 X6 312
Ni 2.4 3.2 6.4
63
126
811
5.8
42.7
24.8
2.7
146
2360
8.5
I586 396 704 746 1486
90 46 31 37 65
59 I7 33 29 28
I5 7 8 9 09
5.7
67
1074
I89
I789
31
1.3 2.9 28
4.5 4.x I6
8.1 I2 46
II I4 22
23 25 33
3.6 7.6 74
96
46
I55
44
69
247
26 22 21
I29 I04 96
x4.5 x74 883
ix
5.9 6.8
12.7 20.3 16.7
7.4 I I.8 9.7
0.5 0.9 0.7
31 44 70
I35 I52 263
1.7 2.9 2.6
29
I21
850
7.1
22.2
12.9
0.9
87
793
2.8
“Data refer to dry matter (105 C) of surface soil (G20 cm) sieved at <2 mm (Coma ef <I/.. 1992).
%I = conlrol, inorganic fertilizer; F = farmyard manure; SI = sewage-sludge level I; S2 = sewage-sludge level 2.
‘Extraction with 0.5 M NaHCO, (Olsen CI al.. 1954).
dExtraclion with 2% citric acid (Afnor, 1991).
‘Aqua regicc digestion (Gomez CI rrl., 1992).
Arbuscular mycorrhiza
in a sewage-sludge-amended soil 289
(to avoid
edge effects) from the topsoil (O-20cm sieves) and centrifugation in 50% sucrose solution
depth). Samples were then bulked, sieved moist to (Walker et al., 1982). They were spread on filter paper
<4mm, and air-dried for soil extractions and esti- with grid lines in a Petri dish and intact spores
mation of mycorrhizal spore density. Subsamples counted under a dissecting microscope.
were oven-dried at 105°C to determine moisture For determination of mycorrhizal root
contents. colonization, subsamples of I g fresh lateral roots
Soil extractions
were randomly taken from
1
cm segments dispersed
in water, cleared and stained with acid glycerol
Single extractions with two different extractants- trypan blue according to Koske and Gemma (1989).
(a)
1 M
CH,CHOONH,-0.1
M
EDTA and (b) 0.1
N
Thirty stained root segments were mounted on slides
Ca(NO,),-were performed on the soil sampled in PVLG [polyvinyl alcohol-lacto-glycerol; Koske
in September. Triplicate aliquots of 5 g and Tessier (1983)J and examined under a compound
(NH,OAc-EDTA) or log [Ca(NO,),] of soil were microscope ( x 150). The percentage of mycorrhizal
shaken in plastic flasks with 50 ml of extractant for root cortex was estimated by rating the density of
2 h at 20°C. Extracts were filtered through ash-free infection using a five class system according to
paper, and the Ca(NO& extracts were acidified with Trouvelot
et al.
(1986).
I4
N
nitric acid
(1
ml) to prevent metal adsorption.
All solutions were kept at 4C until analysis. Concen-
Data analwis and presentation
trations of metals (Cd, Zn, Pb, Cu. Ni and Mn) were All concentrations refer to soil or plant dry matter
determined by either flame (Varian Spectra A20) or determined at 105 C. Means and standard errors
graphite furnace (Varian Spectra A300 with deu- were calculated for three (soil data) or five (plant
terium background correction. A400 with Zeeman data) replicate values. To describe the relationship
background correction) atomic absorption between plant P and mycorrhizal root colonization,
spectrophotometry, depending on the metal a curve was fitted by non-linear regression using an
concentration. Each solution was analysed in tripli- exponential model
(r’ =
corrected coefficient of deter-
cate using standards in a similar matrix. Blanks were mination). Means of shoot dry weights and plant P
analysed in the same way. The standard deviation concentrations were compared by Tukey’s multiple
was kept below 2%. range test (P < 0.05) following a significant
Plant sampling and preparation (P < 0.01)
one-way ANOVA.
Five maize plants per plot were sampled at three
growth stages: six-leaf (June 1991), tasselling (July
1991) and maturity (September 1991) corresponding,
respectively to 26, 57 and I I8 days after sowing.
Plants were divided into roots and shoots and care-
fully washed free of adhering soil particles using
deionized water. The surrounding soil was wet-sieved
to recover most of the fine root system. Washed plant
material was dried at 8O‘C, weighed, cut into small
pieces, subsampled and ground (~300 pm) in a
RESULTS
AND DISCUSSION
Bioal~ailahility of metals
E.xtractability.
According to the EC directive
(CEC, 1986) the total soil content of Zn in the E I -S2
plot, Cd in the E2-Sl and E2-S2 plots and Ni in the
E2-S2 plot largely exceeded the upper permitted
values for sewage-sludge application to arable soils
(Table I). Since there is usually no simple relationship
between the total amount of a metal in soil and its
zirconium oxide grinder (Retsch PM4). Aliquots were biological implications, the available metal fraction
oven-dried at 105°C to determine dry matter. in the soils was evaluated by two single chemical
Plant analysis
extractions (Birke and Werner, 1991): (1) an
EDTA-NH,OAc extraction to estimate the fraction
Plant samples (I g) were wet digested overnight in that can be mobilized from the soil solid phase by
14
N
HNO, (5 ml) and 30% H,Oz (IO ml). After desorption and decomplexing, thus simulating the
heating at 120°C under reflux for 2 h, the digest was effect of plant and microbial exudates; and (2)
made up to IOOml with distilled water. A certified extraction with unbuffered Ca(N0,)2 solution to
reference sample [ryegrass CRM 28
1,
EC Community estimate concentrations of metals in the soil solution
Bureau of References (BCR)] was included in the and readily-exchangeable fraction. Extracted metal
analysis as well as blanks of reagents. The digests concentrations and their proportion of the total
were analysed for metals in the same way as the soil amount of soil metal varied considerably with the
extracts. Phosphorus concentration was determined differences in metal load and other soil character-
by inductively-coupled plasma atomic emission spec- istics, particularly organic matter and pH (Tables I
trometry (Jobin Yvon 38 plus). and 2).
Mycorrhizal parameters
The lowest and highest Cd and Ni concentrations
differed by a factor of I-200. The EDTA-NH,OAc-
Spore density was assessed for soil samples of April extractable fractions amounted to 40-50% of the
and September. Spores were extracted from three total soil concentration for most of the metals and
replicates of 50 g soil by wet-sieving
(I
mm and 63 pm plots. Only Mn remained below 30%. whilst up to
290
I. Weissenhorn CI al
Table 2. Extractable fractions of soil metals expressed in mg kg
’
dry matter and as a percentage of the
total soil metal concentration (values in parentheses)
Plot Cd Ni Zn Mn CU Pb
El-C
El-F
El-SI
El-S2
E2-C
EZ-F
EZ-S I
EZ-S2
El-C
El-F
El-SI
El-S2
E2-C
E2-F
EZ-SI
E2-S2
0.3 (100)
0.4 (80)
0.8 (80)
3. I (54)
I .2 (92)
2.8 (97)
16.0 (57)
56.0 (58)
0.04(13)
0.05 (IO)
0.06 (6)
0.40 (7)
0.37 (28)
0.68 (23)
2.78 (IO)
3.52 (4)
0.3 (13)
0.4(13)
0.8 (13)
I.5 (5)
I.8 (50)
5.6 (74)
26.2 (35)
66.8 (27)
0.08 (3.3)
0.05 (I .6)
0.06 (0.9)
0.28 (0.9)
0.10 (2.8)
ND
5.93 (8.0)
10.0 (4.0)
EDTA-NH,OA<
8.4 (44) 7.0 (21)
32.0 (63) 24.0 (28)
76.0 (38) 45.0 (14)
390.0 (36) 80.0 (4)
3.1 (38) 3.7 (16)
7.5 (63) 6.6 (26)
20.0 (43) 5.5 (17)
63.0 (41) I.4 (2)
Co(NO,),
0.42 (2.2) 0.72 (2.2)
I .04 (2.0) 0.96(l.l)
I .88 (0.9) 2.28 (0.7)
34.47 (3.2) 6.66 (0.4)
0.58 (7.2) 0.86 (3.7)
0.94 (7.8) I .04 (4.2)
2.04 (4.4) I.18 (3.6)
1.53 (1.0) 0.55 (0.8)
6.8 (21)
I I .o (48)
9.9 (52)
32.5 (49)
I .8 (40)
2.8 (58)
5.9 (37)
18.9 (41)
0.04 (0.3)
0.02 (0. I
)
0.12 (0.6)
0.33 (0.5)
<DL
0.02 (0.4)
0.03 (0.2)
0.06 (0.1)
12.6 (72)
21.6(51)
30.0 (67)
x3.5 (44)
7.3 (66)
12.7 (93)
12.1 (55)
22.4 (50)
0.02 (0. I)
<DL
0.01 (0.021
0.03 io.ozj
0.01 (0.09)
0.01 (0.07)
0.01 (0.05)
0.01 (0.021
El = Experimental I; E2 = Experimental 2; C = Control. inorganic fertilizer: F = Farmyard manure;
Sl = Sewage-sludge level I; S2 = Sewage-sludge level 2; ND = not determined; DL = detection limit;
OAc = acetate.
100% of Cd was extracted. Concentrations in the
Ca(NO,), extracts were roughly one order of
magnitude smaller than those determined by
EDTA-NH,OAc extraction, except for Pb and Cu
concentrations which differed by two orders of
magnitude. These two elements are widely known to
form stable complexes with organic matter. Their
potential toxicity in the Bordeaux plots can therefore
be considered as negligible, especially since their total
soil concentrations do not exceed EC limits. On the
other hand, Zn in El-S2, as well as Cd and Ni in
E2-Sl and E2-S2, were extracted by EDTA-NH,OAc
at concentrations far above EDTA-extractable Zn,
Cd and Ni concentrations reported to cause a 50%
reduction in N,-fixation by clover in soil [165, 5.3 and
7.3 mg kg-‘, respectively (McGrath et
al.,
1988)].
This reflects the high total metal load accumulated in
the soil after long-term application of heavily con-
taminated sewage-sludge (Juste and Mench, 1992).
Even in the E2 control plots (C, F), the Cd concen-
trations appeared relatively high compared to the
corresponding plots of El. Since the plots are rela-
tively small (3 x 6 m), this can be attributed to a
transfer of Cd from the polluted S2 and Sl plots by
cultivation practices and erosion, possibly even by
lateral migration of this mobile element in the form
of ions or soluble complexes (Juste and Mench,
1992). However, the chemical extractions only show
relative differences between treatments and give an
indication of the order of magnitude of availability or
potential toxicity of the studied metals. The real
metal exposure of the living organisms is far from
being defined by chemical extraction of bulk soil
samples, since it will be affected by soil heterogeneity,
metal speciation, ionic interactions and microbial
activity (Babich and Stotzky, 1983; Brtimmer et al.,
1986).
Plant
uptake.
According to the soil metal concen-
trations, the metal concentrations in maize shoots
and roots differed with treatment by up to two orders
of magnitude (Tables 3 and 4). The skewed distri-
bution of data and the small number of data points
(n = 8) did not allow a statistical evaluation of the
relationships between the metal concentrations in the
plant tissue and the different soil fractions. However,
it can be concluded that for Cd, Ni and Zn the
concentrations in shoots and roots (Tables 3 and 4)
reflected well the soil concentrations, particularly in
the extractable soil fractions (Table 2). The high plant
availability of these metals is in agreement with
previous findings (Sauerbeck, 1991; Juste and Mench,
1992). Thus, Cd, Ni and Zn uptake by plants on the
metal-contaminated plots was markedly increased
above the control. On the El-S2 plot, shoot Zn
concentrations, particularly at the six-leaf stage,
exceeded the phytotoxic threshold value of 300mg
kg-
’
for maize proposed by Hinesly
et al.
(1977).
Despite this high Zn accumulation, no yield
depression compared to the farmyard manure control
has been recorded since the beginning of the
experiment in 1974 (Juste and Mench, 1992). In E2,
the Cd and Ni contents of maize from the sludge-
amended plots (Sl. S2) strongly exceeded average
yield-relevant toxicity limits (Cd, 5-10 mg kg- ‘; Ni,
20-30 mg kg- ‘) reported in the literature (Sauerbeck,
1982). Since 1987, however, a significant yield de-
pression has been recorded only for the high-level
sludge treatment S2, and no symptoms of metal
toxicity were visible on the above-ground plant parts
(Juste and Mench, 1992). As known for maize and
many other plants (Jarvis et (11.. 1976; Mench et al.,
1989; Sauerbeck and Hein, 1991) Cd and Ni were
retained in the roots, where they reached concen-
trations up to 5 (Cd) and 50 (Ni) times higher than
in shoots.
Root and particularly shoot Mn concentrations of
plants from the high Mn plots of El (Sl, S2) were
hardly higher than the control values, reflecting the
Arbuscular mycorrhiza in a sewage-sludge-amended soil
Table 3. Shoot metal concenlrations (mg kg
’
dry matter) at three growth stages (means k SE; n = 5)
PI01 Cd Ni Zn Mn CU Pb
291
El-C
El-F
El-SI
El-S2
E2-C
E2-F
EZ-SI
E2-S2
El-C
El-F
El-S1
El-S?
E2-C
EZ-F
E2-Sl
EZ-S2
El-C
El-F
El-S1
El-S2
EZ-C
E2-F
EZ-S I
I .03 + 0.06
0.52 f 0.08
0.86 + 0.1
I
0.87 f 0.28
33.91 + 5.92
13.68 i 3.63
55.07 k 2.25
85.50 k 4.65
0.47 * 0. I3
0.29 * 0.03
0.23 i 0.02
1.02~0.16
3.66 + 0.98
4.60 + 0.91
31.46 + 2.99
36.72 ? 2.03
0.14? 0.04
0.13 + 0.03
0.14 + 0.02
0.25 * 0.05
I .25 F 0.50
2.
I
I * 0.03
9.36 + 0.96
0.77 f 0.07
I.11 f0.14
I
.42 ? 0.41
I .99 * 0.54
I .90 f 0.29
1.26f0.10
10.03 + 0.38
24.54 + 2.21
1.53 + 0.23
I .25 k 0.08
0.97 + 0.08
1.62kO.15
2.72 i 0.48
I .60 + 0.09
8.28 ? 0.82
13.91 * I.17
0.87~O.lO
I .30 F 0.23
1.68f0.18
I.11 +0.11
0.84 k 0.07
I .72 + 0.32
4.24 + 0.55
Six
-leaf
l38? IO
I38 ir I9
272 + I8
452 + 33
l27+9
95 + 6
104 + 20
18Ok20
Tomlling
67 + 5
87+
II
139 + I5
314+26
48 * 9
63 + 4
103 If: 7
92 + 3
Mafurity
56 + 28
52i: I3
68k IO
III +21
28 * 2
33 i4
46 + 3
88 * 4.7
34 5 4.0
85 * 19.7
96 k 6.8
71 f 8.5
33 f 5.3
20 + 2.2
20 * I .4
79 k 6.5
29 + 2.9
31 k 2.7
44 f 3.5
52 f 7.8
62 + 3.4
25 k 2.3
12kO.4
26 k 5.1
I3 k 2.3
I8 k 2.7
23 f 2.3
I8 + 2.6
I9 * 3.3
8 + 1.3
I I.2 + 0.9
9. I F 0.4
10.9 + 0.7
14.0 li_ 0.4
8.5 * 0.5
7.7 f I.5
8.4 k 0.2
13.4 * I.3
9.8 & 0.6
10.9 f 1.0
8.3 + 0.4
13.9 k 0.6
6.3 k 0.4
6.7 f 0.4
8.9 f 0.5
10.0 * 0.4
3.9 * 0.3
4.4 i_ 0.4
4.9 + 0.3
5. I f 0.4
3.1 * 0.2
2.8 * 0.2
3.3 + 0.3
1.05&0.12
1.35+0.11
I
.90 i 0.42
3.67 f I.15
0.90 * 0.03
I .08 + 0. I3
1.07 + 0.1 I
I .65 k 0.39
0.76 k 0.07
0.91 f 0.06
0.79 k 0.06
0.93 * 0.07
0.69 & 0.05
0.80 k 0.08
0.79 * 0.07
0.8 I * 0.05
I. I I * 0.07
0.95 * 0.07
1.39&0.15
I.41 kO.15
I .35 + 0.04
I.41 k 0.06
1.06+0.13
E2-S2 12.54 + 2.48 5.87 IO.44 43+4 710.7 4.9 IO.3 1.31 IO.14
Abbreviations as in Table 2.
low
Mn
availability
seen in the low recovery rates by
extraction
(Table
2). In E2, Mn concentrations of
plants from the sludge-treated plots were markedly
lower than controls and close to deficiency threshold
values (Juste, 1988). This could be due to the higher
pH or to competition with Cd or Ni.
Plant Cu and Pb concentrations were better
correlated with the total and EDTA-extractable soil
fractions than with the Ca(NO&extractable
fraction. This agrees with the values for their ex-
tractability, which indicates the predominance of less
mobile species. However, plant Cu was about 10
times higher than plant Pb, in spite of similar concen-
trations of both metals in the soil extract (Table 2).
This illustrates that the relevance of extractability to
bioavailability is specific for each metal. Shoot Cu
and Pb concentrations were below average toxicity
limits (Sauerbeck, 1982) in all plots, corresponding to
the relatively low total concentration in the soil (Cu)
or to the low availability (Pb).
Table 4. Root metal concentrations (mg kg
’
dry matter) at three growth stages (means k SE; n = 5)
Plot Cd Ni Zn Mn CU Pb
Si.r -leaf
El-C 4.9 * 0.5 5.3 + 0.7 107 f 7 73* I2 58 ? 6 5.8 k 0.6
El-F 2.3 i 0.3 3.3 * I.1 69+ I4 17+3 38 + 7 3.9 f 0.5
El-SI 5.2 f 0.7 5.3 f 0.9 323 f 91 54 It 5 53 & 9 8.6 * 1.3
El-S2 8.9 f 0.7 13.2 f 2.3 505 k 65 II428 177*9 15.0 * I.0
E2-C 124.9 + 13.2 50.9 + 9.9 75+ I4 76 k 5 4117 5.8 i_ 1.3
EZ-F 39.1 + 6.9 19.2 k 3.9 86k IO 35* II 14*2 5.3 & 0.5
EZ-SI l25.3? 19.5 557.7 * 51.1 173 * 34 13+2 so+ IO 4.3 0.5 f
E2-S2 198.7 It 24.6 959.5 ? 68.3 128+8 14+ I 99 * 9 6.0 k 0.5
Tmscliing
El-C 1.4+0.1 I.5 fO.l 58 f 5 27 k I 101 I 3.4 0.5 *
El-F 0.7 ? 0.1 2.3 ?I 0.5 912 II 15+2 14+ I 2.5 0.1 *
El-SI 2.0 rt 0.3 2.4 f 0.2 132i4 55 + 7 22 3
i_ 6.2 & 0.8
El-S2 I.8 i 0.2 3.6 k 0.3 123 f I8 64* IO 35+4 6.1 2 0.8
EZ-C 53.7 f 6.8 II.1 *2.5 43f IO 39* I3 9+1 5.0 0.5 &
EZ-F 27.6 f 4.2 7.1 + I.5 42 + 7 I4 + 2 5 0.3
+ 2.8 * 0.2
E2-S I 78.6 t 10.0 236.8 + 28.8 60 + 4 9*1 l7+2 4.2 0.2 li_
EZ-S2 73.9 * 3.0 413.0 + 36.4 71 +5 II * I 30 3 It 5.2 0.3 *
Mrrruritj
El-C l.o*o.I l.SiO.2 34* II II * I 6+l 2.6 + 0.2
El-F I.1 +0.1 I.5 +0.3 32 f 4 II * I 7+1 7.1 0.7 i_
El-SI I.5 + 0.2 I .9 * 0.2 I35 + I2 54* I9 22 5 + 3.0 +0.1
El-S2 0.3 F0.I 3.4 * 0.2 77* IO 54 k 6 21 +2 2.8 0.3 f
E2-C 13.2 t I.3 16.1 k 3.7 22 * 2 IOk2 x+2 2.6 0.3 k
E2-F 4.x * I.3 4.7 * 0.5 ISi I 5*1 4 0.3 i: ND
EZ-S I 37.3 + 5.7 249.5 k 25.2 30 5 + 5*1 13 2 + 2.3 0.3 _+
E2-S2 50.1 f 4.7 298.9 f 35.5 28 k I 4 + 0.3 19+3 2.4 0.4 f
Abbreviations as in Table 2.
292
1. Weissenhorn et
al.
50
Experiment 1 Experiment 2
-0-C
5 7
c40
s
.B
30
5
8
~ 20
.!
g s 10
E”
0
20 40 60 60 100 120 ‘20 40 60 100 1
days after sowing
C
??
before sowing
??
a1 harvest
T
treatment
Fig. I. Mycorrhizal root colonization (A, B) and spore counts (C, D)
in the field plots during one growing
season (1991).
Means + SE (mycorrhizal colonization, n = 5; spore counts, n = 3). Abbreviations
as in
Table 2.
Plant metal concentrations generally decreased
with plant growth and, root metal concentrations in
particular, were less correlated with soil metal
concentrations at later growth stages. This indicates
a relatively higher metal uptake by the young plant
with translocation and dilution of metals during
growth, as reported for micronutrients Cu, Zn and
Mn in maize by Lubet and Juste (1985).
Mycorrhizal abundance
AM spore density and root colonization of maize
plants varied considerably between experiments and
treatments (Fig. I). In El, mycorrhiza development
was greatest in the Sl plot with more than 20%
colonized root cortex, 26 days after sowing, and more
than 30% at harvest [Fig. l(A)]. Only a little root
colonization was found in both unpolluted plots (C,
F), but spore counts in the C plot increased during
the growing season suggesting a
de nova
production
of spores [Fig. l(B)]. No root colonization was
detected in the El-S2 plot. The number of spores was
equal to that in the C plot, but did not increase
towards the end of the season. This might be linked
to the high Zn concentration in this plot (Tables
1
and 2), as has been suggested to explain the absence
of
Rhizobium
cells in this treatment in the same year
(A. Chaudri, pers. commun.), although an
investig;,ion of total microbial biomass and activity
revealed no deleterious effect of the sludge treatment
in this experiment (Lineres
et al., 1989).
Furthermore,
shoot biomass data (Table 5) gave no evidence of Zn
toxicity to the maize plants grown in this plot.
Factors other than Zn toxicity probably influence
mycorrhizal development in this experiment consider-
ing the equally weak root colonization in the two
unpolluted plots.
In E2, highest root colonization, around 30% from
the six-leaf stage on, was found in the S2 treatment
[Fig. l(C)], in spite of the high Cd and Ni concen-
trations in the Ca(NO,), extract (Table 2) and the
root tissue (Table 4). Thus, a toxic effect of the high
Cd or Ni concentration, as has been shown for
Rhizobium
(A. Chaudri, pers. commun.), could not be
detected for the development of mycorrhizal fungi
within roots. This agrees with results from other
studies demonstrating the presence of AM in soils
Table 5. Shoot dry matter (g plant ‘) of maze
plants at harvest (means + SE, n = 5)
Treatment El E2
C
315k32 a 259 k 24 a
F 234 k
I
I b l98+ II ab
SI 200 k IO b 219 k I8 ab
s2 229 F I7 b 149+25 b
Abbreviations as in Table 2. Means followed by
the same letter are not significantly different
(P CO.05).
Arbuscular mycorrhiza in a sewage-sludge-amended soil
contaminated with metals from sewage-sludge
application (Arnold and Kapustka, 1987; Koomen
et al.,
1990; Leyval
et al.,
1991). In these studies.
however, metal applications were never as high as in
the E2-S2 plot. Only Gildon and Tinker (1983)
reported considerable amounts of AM root coloniza-
tion in an extremely polluted metal mining area with
HCI-extractable Cd soil concentrations of more than
300 mg kg- ‘. In their work, an AM fungal isolate
from the polluted soil proved tolerant to heavy
metals, as compared to a strain from unpolluted soil.
The results from E2 also demonstrate tolerance of the
indigenous AM population to the high Cd and Ni
concentrations in the soil and inside the roots. How-
ever, metal detoxification by adsorption on the plant
cell walls and intracellular sequestration (Ernst
et al.,
1992) which lowers the actual metal exposure of the
fungus growing inside the root cells, must be taken
into account.
In contrast to root colonization, spore counts in
the E2-S2 plot were low and did not increase during
the growing season [Fig. l(D)]. Degradation of
harvest residues (roots etc.) is delayed in the E2-S2
plot (P. Solda, pers. commun.) compared to the other
plots. Thus mycorrhizal root colonization in this plot
seems mainly due to hyphae proliferating from root
pieces (Biermann and Linderman, 1983). Babich and
Stotzky (1977) also found that sporulation of certain
filamentous soil fungi was more sensitive to Cd than
mycelial growth.
In the three other treatments of E2 (Sl, F. C),
root colonization remained at a low level until the
tasselling stage and significantly increased only at
maturity (Fig. 1). On the E2-F plot, mycorrhizal
colonization was lower but spore density was
higher than in the other treatments. This is in con-
trast to El, where root colonization and spore den-
sity were better related, although these two indices
of fungal development are not necessarily correlated
(Hetrick and Bloom, 1986; Douds and Schenck.
1990).
Angle and Heckman (1986) found that the metal
content of sludges is not the only factor related to the
amount of mycorrhizal infection and concluded that
it was impossible to separate the effects of heavy
metals, organic matter and nutrient status of the
different sludges used in their study. The considerable
variation in physico-chemical soil factors between the
treatments of our study (Table I) also made it difficult
to clearly prove or rule out a toxic metal effect on
AM. The low mycorrhizal abundance in the control
treatments indicates that other soil properties may
have obscured the effect of high metal concentrations
by directly or indirectly affecting mycorrhizal
incidence (Kruckelmann. 1975). A strong inverse
relationship, following an exponential curve, was
found between mycorrhizal colonization and root
and shoot P concentrations [Fig. 2(A, B)]. However,
bicarbonate-soluble (Olsen) and citric-acid-extract-
able (Dyer) P concentrations (Table I). common
measures for the available fraction of soil P, did not
correlate with mycorrhizal colonization. The import-
ance of plant P in regulating mycorrhizal infection
rather than soil P has been emphasized by Sanders
et al. (1975). Menge
et al.
(1978) and Graham et
al.
(1981). No significant correlations between mycor-
rhizal indices (spore density, root colonization) and
other soil factors could be established. As for the
possible effect of metals, they might be masked by the
strong P effect. Soil pH was better related to root
(Y = -0.66) and shoot (Y = -0.62) P than to mycor-
rhizal colonization (r = 0.52) levels, indicating some
indirect influence on mycorrhiza via plant P status.
Thus, the higher mycorrhizal colonization of E 1-S I
and E2-S2 plants (Fig. I) could be due to the lower
plant P concentrations compared to the other treat-
ments (Fig. 3). The differences in plant P between
treatments in El can be explained by the adjustment
of P fertilization in the C and F plots to the level of
Sl by adding superphosphate, a highly available form
of P fertiliser compared to sludge-derived P. Arnold
and Kapustka (1987) also found reduced AM spore
A
- y = 1097.94
(-1.024~
293
B
-
y = 213.37 (-1.-W
r2 =
0.65
1 2 3 4 5
phosphorus concentration (g kg-l
DM)
Fig. 2. Relationship between shoot (A) and root (B) phosphorus concentration and mycorrhizal root
colonization of maize plants at the tasselling stage in the plots of El and E2 (n = 8).
294 1. Weissenhorn ef al.
numbers and root colonization in urea-phosphate
fertilized compared to sludge-amended plots. In E2,
where after 1980 sludge application was stopped and
every plot received the same amount of superphos-
phate (87 kg P ha- ’ y y
‘),
differences in plant P
concentrations were less pronounced (Fig. 3) which
is reflected by the mycorrhizal status (Fig. 1). How-
ever, the S2 plot differed significantly from the other
plots. This could be due to greater P complexation in
relation to the higher soil pH, organic matter and Ca
status in this treatment (Table 1). Although root
biomass was not assessed, there was visible evidence
of root stunting and a lower proportion of fresh
lateral roots compared to the other treatments.
Therefore, it is assumed that the decrease in P influx
might also be related to root damage by metal
toxicity. Thus, the high Ni and Cd contamination of
this plot indirectly supported a considerable mycor-
rhizal root colonization via P status of the plant.
fungal hyphae on one hand face the same situation of
competing for uptake of elements from the soil as do
plant roots themselves, and on the other hand their
development and functioning might be directly
impaired by toxic metals (Graham et
al.,
1986).
Conclusions
The lack of non-mycorrhizal controls under field
conditions and the high variability and confounda-
tion of factors between treatments made it difficult to
distinguish the mycorrhizal contribution to metal
uptake by plants. However, increased mycorrhizal
colonization was neither associated with a consistent
increase in plant metal concentration at low metal
availabilities, nor with a significant decrease at high
availabilities, as has been suggested by Schuepp er al.
(1987) and Leyval et al. (1991). The abundant root
colonization in the high Cd and Ni plot of E2 did not
provide an efficient protection of the host plant
against high metal accumulation and toxicity
(Tables 3 and 4). Plant Mn concentrations in the
same plot were at the deficiency limit (Tables 3 and
4) and plant P concentrations were smaller than in the
other treatments
[Fig. 3(B)]. This
is in contrast to the
mycorrhizal enhanced uptake of immobile nutrients
reported in literature, particularly for P, but also for
Mn (Krishna and Bagyaraj, 1984) and raises the
question of mycorrhizal efficiency in such an
extremely polluted environment. Absorbing external
We conclude that no correlation between mycor-
rhizal abundance and degree of metal exposure in soil
or inside plant roots could be established. By con-
trast, mycorrhizal root colonization across the differ-
ent treatments correlated well with plant P status.
The negative effect of high plant P on mycorrhizal
colonization might have obscured the effect of metal
concentration, especially in the case of the high-level
Zn plot. However, a considerable root colonization
(30%) was found in the E2-S2 plot compared to low
or no colonization in the unpolluted plots, in spite of
the high Cd and Ni exposure, which appeared toxic
to
Rhizobium
(A. Chaudri, pers. commun.) and maize
roots.
This suggests a better tolerance of the indigenous
AM population to elevated metal concentrations
than to P concentrations luxurious for plant growth.
In a different study we have observed that spore
germination of an AM fungal culture isolated from a
Cd-polluted loamy sand was less affected by Cd than
for a laboratory reference strain (Weissenhorn
et al.,
1993). However, more subtle metal effects on sporu-
Iation or shifts in population are not ruled out. They
may have severe ecological consequences such as the
absence of N, fixation due to the survival of only
ineffective
Rhizobium
strains in sludge-treated soil
(Giller
et al.,
1989, 1993).
The role of AM in plant metal-stress alleviation
and protection of the food chain should not be
overestimated. Considerations to introduce metal-
tolerant mycorrhizas into agricultural soils, in order
to facilitate sewage-based fertilization systems
(Moore, 1988) seem extremely dangerous, not only
with respect to metal uptake by plants but also to
Experiment
1
Experiment 2
E
l2
12
n H
shoot
7 10
10
??
root
2
38
8
2
8 6 6
z
e B4 4
P
5 2 2
E 0 0
C F Sl s2
treatment
Fig. 3. Root and shoot phosphorus concentrations of maize plants at tasselling stage in the experimental
plots. Means k SE (n = 5). Abbreviations as in Table
I.
Arbuscular mycorrhiza in a sewage-sludge-amended soil 295
other microbial populations and processes, that
might be more susceptible than
AM
fungi.
Acknowledgements-We
thank A. Gomez and V. Didier-
Sappin (INRA-Bordeaux) for helpful discussions, and P.
Solda, S. Fargues and P. Masson for technical assistance.
This work was financially supported by the French Ministry
of Research and Technology and by an EC sectorial grant.
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