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Uptake and partitioning of sludge-borne copper in field-grown maize (Zea mays L.)
European Journal of Agronomy ELSEVIER
European Journal of Agronomy 5 (1996) 259-271
Uptake and partitioning of sludge-borne copper in field-grown maize (Zea mays L.) B. Jarausch-Wehrheim,
B. Mocquot, M. Mench *
INRA, Unit&d’ Agronomie, Centre de Recherches de Bordeaux, BP 81, F-33883 Villenave d’ Ornon Cedex, France
Abstract Accumulation of sludge-borne copper (Cu) by field-grown maize and its distribution between the different plant organs was studied in detail in a long-term sewage sludge field trial. Since 1974, field plots on a coarse sandy soil have been amended each year with farmyard manure (FYM) at a rate of 10 t dry matter (DM) ha-’ year-’ and with sewage sludge at the two levels of 10 t DM ha-’ year- 1 (SS 10) and 100 t DM ha-’ per 2 years (SS 100). All field plots have been cropped annually with maize. In 1993, five replicate plants per treatment were examined at six different growth stages from seedling to grain maturity. Each plant was separated into at least 12 different parts and the Cu content of each was determined. Regarding growth parameters, no visible deleterious effects on plant development due to the different soil treatments could be observed, although the dry matter yield of roots and stalks of SS loo-treated plants was significantly reduced. Significantly increased Cu concentrations of up to 60 mg Cu kg-’ DM in the roots of young SS lOO-grown maize plants and of up to 20 mg Cu kg -’ DM in the upper leaves at silage stage were found. No critical Cu amounts were reached in the grains until harvest. Keywords: Copper; Foodstuff quality; Maize; Metal uptake; Sewage sludge
1. Introduction The agricultural use of sewage sludge has been increasingly favoured as a means of inexpensive waste disposal and beneficial re-use (Sauerbeck, 1987; Yingming and Corey, 1993). This land application of sewage sludge could even be enhanced as, for example in France, landfilling will soon be banned and, thus, sludge incineration and soil application will be the only alternatives. Sewage sludge is added to agricultural soils primarily for its fertilizer value in terms of N and * Corresponding author. Tel.: +33 05 56 843042; fax: f33 05 56 843054;e-mail: [email protected].
P (Tiller
and Merry, 198 1; Juste and Mench, 1992), as a source of organic matter, trace elements (e.g., Cu, Mn, Zn) and sometimes as an alkaline soil amendment. However, besides the beneficial effects, the application of sewage sludges as well as other organic wastes may lead to a considerable accumulation of trace metals such as Cu, Cd, Ni, Pb and Zn in the topsoil (Juste and Mench, 1992). This could produce phytotoxic effects or unwanted accumulation of metals in the food chain (Heckman et al., 1987). Sewage sludges are generally rich in Cu, Cd and Zn compared to soils and plants (Juste et al., 1995). Although Cu is required as an essential nutrient element for plant development and normal growth (Clijsters and
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Van Assche, 1985; Lou& 1993), even a small increase in the amounts of Cu in the soil or growth medium may be detrimental to the plants (Lin and Wu, 1994) and excessive Cu concentrations are highly toxic (Woolhouse, 1983; Macnicol and Beckett, 1985; Verkleij and Schat, 1990). High soil Cu concentrations may inhibit the normal plant growth or may operate as a physiological stress factor in plant metabolism (Ouzounidou et al., 1995). Besides the direct toxic effects, Cu can also indirectly decrease primary production through its inhibitory effects on the processes responsible for nutrient cycling in soils and waters (Fernandes and Henriques, 1991). While toxicity to higher plants is of major concern, soil biological activity may be much more sensitive to sludge Cu inputs (McGrath et al., 1995). Cu contamination in fodder, such as maize, may increase the metal transfer into the food chain even if no direct decrease in crop yield due to metal phytotoxicity occurs. Damages to water, soil and food quality may be found in particular in sandy soils which have a low buffer capacity. These soils are very abundant in the south-west of France. In this area, maize is at present one of the main cultivated crops and in some cases the pioneer crop on former vineyards. The treatment of grapevines with Bordeaux mixture (solution of copper sulfate neutralized with hydrated lime) (Scheinberg, 1991) has already caused a considerable input of Cu and other metals into the soil. The phytotoxic Cu effects on grapes and maize were first reported by Delas (1963). Therefore, the further Cu input into soil via sludge application is questionable in these areas. To examine long-term effects of sewage sludge application on continuous maize cropping under natural cultivation conditions, a field trial was established at Bordeaux in 1974. Numerous reports on this long-term experiment have been published concerning the accumulation and distribution of metals in sludge-treated soils and plants (Gomez et al., 1992; Juste and Mench, 1992; Weissenhorn et al., 1995). Mench et al. (1994) examined the distribution of sludge-borne Cd, Cu, Fe, Mn and Ni among roots, stalks, leaves and grains of fieldgrown maize at tasseling and found that Cu was uniformly distributed among the different plant
Journal of Agronomy 5 (1996) 259-271
parts. However, treatment with highly metal polluted sludges caused an accumulation of Cu, Fe and Ni in maize grains. Weissenhorn et al. (1995) compared shoot and root metal concentrations of sludge-borne Cd, Cu, Mn, Ni, Pb and Zn at three different developmental stages of field-grown maize. Concentrations of Cu were always greater in roots than in shoots, and increased Cu contents could be related to the highest sludge application rate. The Cu concentrations in both organs were highest at the six-leaf stage and decreased until maturity. The results showed that sludge-borne Cu inputs in the soil resulted in an increased Cu content in the whole maize plant. Furthermore, the accumulation and distribution of Cu between the different plant organs was highly dependent on the growth state. Although detailed observations have been reported for wheat (Loneragan et al., 1980), few data are available for maize (Karlen et al., 1988), and the pathway and mechanism of Cu translocation and storage are poorly understood. The objective of this study was to characterize the uptake and distribution of sludge-borne Cu in different plant parts of field-grown maize and to localize Cu concentration and storage sites within the maize plant.
2. Materials and methods 2.1. Sampling site and soil treatment The field site was a long-term sewage-sludge trial on an acid sandy soil (Arenic Udifluvent, US classification, pH 5.5) at the INRA experimental site of Couhins, Bordeaux, France. The field trials were established in 1974 and received an anaerobically-digested dehydrated sludge at rates of 10 t DM ha-’ year-’ (SS 10) and 100 t DM ha-’ per 2 years (SS loo), respectively. Plots treated only with farmyard manure (FYM) at a rate of 10 t DM ha-’ year-’ served as controls. Each treatment was replicated five times. The pH of the soil, measured in 1993, was 6.61, 6.79 and 6.04 for FYM, SS 10 and SS 100 treatments, respectively. Fertilization and other practices as well as soil and sludge characteristics were previously described by
261
B. Jarausch- Wehrheim et aLlEuropean Journal of Agronomy 5 (1996) 259-271
Gomez et al. (1992) and Weissenhorn et al. (1995). Cumulative sludge inputs since 1974 led to a total Cu input until 1993 of 5.62 kg ha-’ Cu for FYM, 45kgha-‘CuforSS lOand227.5kghaK’Cufor SS 100 treatment. Since 1974, all field plots have been annually cropped with maize (Zea muys L. cv. INRA 260) at a density of 75 000 plants ha-‘. These field plots (3 m wide, 6 m long) were used for the present study. To avoid the modification of the long-term monitoring of data based on a standard number of plants, all field plots were planted in addition to Zea muys L. cv. INRA 260 with Zea muys L. cv. Volga, leading to a total density of 85 000 plants ha- ’ in the study year of 1993. Maize was planted using an 0.8 x 0.165 m spacing in a randomized block design. The only plant protection treatment was an application of Atrazine. Due to inadequate rainfall, 300-400 mm of water was applied by sprinkler irrigation from June to September.
leaf level 4
stalk level 3
stalk level 1
2.2. Plant sampling, fractionation and growth parameters Corresponding to the main developmental stages, whole maize plants were sampled on six dates and then subdivided into different plant fractions depending on the growth stage (Table 1 and Fig. 1). Among leaves and stalks, we distinguished between four different levels (Fig. 1). In our study, each leaf level was defined as a batch of four fully developed leaves, grouped adjacent
Fig. 1. Scheme for the fractionation of the whole maize plant into different organs and levels. Levels are defined as: LL 1 (SL l)=leaf 1-4; LL 2 (SL 2) =leaf 5-8; LL 3 (SL 3) =leaf 9-12; and LL 4 (SL 4)=leaf 13-16.
to each other on the stem. Stalk levels corresponded to leaf levels. Levels were counted from the bottom to the apex: levels 1 and 2 corresponded
Table 1 Harvest dates and fractionation of whole maize plant into its organs and into four leaf and stalk levels, respectively Days after sowing (sowing date: 5 May 1993)
Development stage
Subdivision of plant
41 56 14 95 118 152
6-leaf stage 12-leaf stage silking l&leaf stage silage maturity
root, root, root, root, root, root,
LL LL LL LL LL LL
1, LL 1, LL 1, LL 1, LL 1, LL 1, LL
2, 2, 2, 2, 2, 2,
SL LL LL LL LL LL
No. of fractions per plant 1 3, 3, 3, 3, 3,
SLl LL 4, LL 4, LL 4, LL 4,
SLl, SL 2, SL 3, SL 4, whole ear SL 1, SL 2, SL 3, SL 4, husks, cob, grains SL 1, SL 2, SL 3, SL 4, husks, cob, grains SLl, SL 2, SL 3, SL 4, husks, cob, grains
4 5 10 12 12 12
A level is defined as a batch of four fully developed leaves, grouped adjacent to each other on the stem (see Fig. 1). LL, leaf level; SL, stalk level. Levels are defined as: LL 1 (SL 1) =leaf l-4; LL 2 (SL 2) =leaf 5-8; LL 3 (SL 3) =leaf 9-12; and LL 4 (SL 4)= leaf 13-16.
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to the older (lower) organs; and levels 3 and 4 to the younger (upper) parts. Before fractionation, the following physiological and morphological parameters were recorded per plant: plant height; number of leaves; number of dry leaves; number of ears; shoot diameter (at a height of 15 cm from the soil surface); total leaf area (determined from the length L and the width W of each leaf using the formula S=O.75 x LW (Bonhomme et al., 1982). The mean chlorophyll content of leaves 6, 10 and 13 was directly measured on the field with a portable chlorophyll meter (Minolta SPAD 502). The calibration and verification of the values obtained was done by extraction of chlorophyll with N,N-dimethylformamide (DMF) and its quantification by spectrophotometry (Inskeep and Bloom, 1985). 2.3. Preparation and analysis of plant material Adhering particles were removed by several washings using first de-ionized water with 1% (v/v) Triton X-100 and 1% (v/v) pure nitric acid ( 14 M HNO,), second de-ionized water and then distilled water. Washed plant material was oven-dried at 80°C to constant weight, weighed, cut into small pieces, and subsampled. Roots and above-ground plant parts were milled in a zirconium oxide grinder (PM 4, Retsch, Dusseldorf, Germany); grains were ground in a stainless steel mill using a 0.375 mm sieve. Aliquots of plant material were also oven-dried at 105°C to determine dry matter (DM) content. Plant samples (0.5 g DM) were wet digested overnight in 14 M HN03 (5 ml) and 30% (v/v) H,O, (10 ml). Digestion was completed in a heating system (Gerhardt, Bonn, Germany) under reflux in four steps, at 50°C for 30 min, 100°C for 30 min, 150°C for 30 min and 220°C for 2 h. After cooling for 35 min, the digest was made up to 100 ml with distilled water. Two certified reference samples (rye-grass BCR 281 and sea lettuce BCR 279 from the EU Community Bureau of References) and controls of reagents without plant material were analysed along with plant samples in every batch. Cu concentrations were determined either by flame air-acetylene (Varian Spectra A 20) or graphite furnace (Varian Spectra
A300/A400 with Zeeman background correction) atomic absorption spectrophotometry or by inductively coupled plasma atomic emission spectrometry (Varian Liberty 200) depending on the metal concentration. Each solution was analysed in triplicate using standards in a similar matrix. European standard values for the Cu concentration in the certified reference samples are 9.65_+ 0.38 mg kg-’ for rye-grass and 13.14kO.37 mg kg-’ for sea lettuce. In our conditions, we determined mean Cu values of 9.92 +0.17 and 11.46kO.46 mg kg-’ and thus recovery rates were 102.82 and 87.2% for rye-grass and sea lettuce (n = 6), respectively. The digestion quality for each plant part was tested by repeated preparation and determination of three replicates. The standard deviation of the results for each plant part was less than 6%. The working value was examined by the Cu determination in the control without plant material. The mean Cu concentration in 20 control replicates was less than 2 ng Cu ml-‘. 2.4. Data analysis and statistics All concentrations refer to plant DM determined at 105°C. Means and standard errors were calculated for three replicate values. All data were statistically evaluated by analysis of variance; means and standard errors were compared for error variance and significant differences by a multiple range test (PcO.05) (Keuls, 1952). The relationship between Cu concentration and plant DM was fitted by non-linear regression using an exponential model. The coefficient r2 refers to the fraction of variance explained by the correlation.
3. Results and discussion 3.1. Morphological observations
Sludge application at two different concentrations (SS 10 and SS 100) did not significantly affect plant height or total leaf area compared to the control treatment with farmyard manure (FYM) (data not shown). At maximum growth stage (day 118), the mean plant height was
263
B. Jarausch- Wehrheim et aLlEuropean Journal of Agronomy 5 (1996) 259-271
3.2. Copper uptake kinetics
250+20 cm and the total plant leaf area was 0.7 +0.05 m2 for all treatments. Although no visual morphological treatment effects could be observed, biomass production was strongly affected in some plant parts. Significant DM yield reduction was found especially in the roots of SS loo-treated plants and in different stalk levels of plants grown in SS lo- and SS loo-treated plots (Table 2). In contrast, grains of SS lOOgrown plants had a significantly increased DM at silage stage compared to FYM-treated plants. In general, chlorophyll contents were greater in leaf 10 and 13 of SS lo- and SS loo-treated plants (data not shown) than in the FYM controls. As chlorophyll contents varied considerably between individual plants, a significant treatment effect could only be confirmed for leaf 10 of SS loo-treated plants at day 95. This was associated with a raised Cu concentration (Table 3) and a significant DM reduction (Table 2) in leaf level 3 at day 95. Therefore, deleterious effects of high Cu concentrated sludge application on the plant metabolism cannot be excluded.
The soil Cu supply significantly affected the temporal change in Cu concentration in the different plant parts (Fig. 2(B)). Regarding the whole plant, the SS 100 treatment increased Cu concentrations at the earliest growth stages followed by a progressive decrease during the vegetation cycle. The subdivision of the whole plant into above-ground parts and roots shows that the initially raised Cu concentrations in SS 100 plants were mainly due to the Cu concentration in the roots. These raised Cu concentrations in the roots were maintained until day 95. They were up to 5-fold higher than mean concentrations of all above-ground parts (Fig. 2(B)). However, at maturity (day 152) the root Cu concentration in SS 100 plants was below that of FYM and SS 10 plants. Two main phases of decrease in the root Cu concentration can be distinguished: the first decrease, between day 41 and day 56 (Fig. 2(B)), was correlated with an increase in DM (Fig. 2(A)) and may thus be due to a dilution effect. In
Table 2 Dry matter of the different plant parts of FYM, SS IO- and SS loo-amended maize plants at six sampling dates during the growth cycle expressed in g DM per plant and significant treatment-dependent variances classified by the letters a, b and c Days after sowing
D 41
D 56
D 74
D 95
D 118
D 152
Treatment
Plant fraction: DM (g per plant) Roots
FYM ss 10 ss 100 FYM SSlO ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
3.8 a 3.9 a 2.8 b 8.5 a 9.3 a 8.9 a 28.7 a 25.1 ab 19.7 b 36.2 a 29.1 a 33.0 a 38.3 a 30.9 b 29.4 b 39.3 a 31.4 a 38.5 a
Leaf levels Ll
L2
4.3 a 4.1 a 3.2 a 2.4 a 3.7a 3.1 a 5.0 a 3.3 a 5.0 a 3.0 b 2.8 b 6.5 a 3.0 a 3.0 a 3.7 a
7.1 a 7.3 a 7.0 a 12.1 a 11.4a 12.7 a 16.9 a 15.5 a 15.5 a 14.7 b 14.8 b 19.7 a 16.6 a 16.0 a 16.9 a 14.5 a 13.3 a 17.0 a
Stalk levels L3
18.9 a 16.6a 16.9 a 18.5 a 14.5 a 14.2 a 20.8 a 18.4 b 18.9 b 21.5 a 19.8 a 19.7 a 17.1 a 18.8 a 16.5 a
Ear
L4
Sl
s2
s3
S4
Total
9.6 a 11.5 a 9.8 a 11.9 a 9.2 a 9.1 a 12.6 a 9.3 a 10.9 a 11.5 a 9.2 b 9.5 b
7.0 a 6.0 b 5.3 c 30.6 a 26.3 b 27.6 b 35.3 a 30.5 a 25.7 b 42.9 a 43.9 a 49.2 a 47.6 a 41.9 b 27.5 c 58.3 a 33.3 b 41.1 b
47.3 a 43.1 b 30.7 c 65.2 a 49.8 b 49.8 b 65.1 a 52.3 b 44.8 c 65.5 a 58.8 a 63.1 a
24.7 a 24.5 a 23.6 a 45.0 a 34.7 b 35.9 b 44.3 a 34.2 b 35.8 b 46.4 a 41.7 a 47.5 a
11.4 a 10.8 a 8.2 b 14.9 a 10.8 b 10.7 b 12.2 a 8.7 b 10.5 b 10.6 a 11.0 a 10.4 a
5.9 a 11.3 a 7.0 a
Ear Husks
Cob
Grains
28.1 a 26.6 a 28.6 a 23.0 a 22.2 a 22.0 a 23.6 a 21.6 a 22.5 a
32.3 a 36.3 b 46.3 c 40.5 a 39.6 a 44.6 a 46.9 a 41.8 a 49.2 a
21.7 a 23.8 a 25.6 a 150.6 b 161.6 ab 190.4 a 282.1 a 263.4 a 287.4 a
B. Jarawch- Wehrheim er al./European Journal of Agronomy 5 (1996) 259-271
264
Table 3 Cu concentration in the different plant parts of FYM, SS lo- and SS loo-amended maize plants at six sampling dates during the growth cycle, expressed in mg Cu kg-’ DM and significant treatment-dependent variances classified by the letters a, b and c Days after sowing
D 41
D 56
D 74
D 95
D 118
D 152
Treatment
Plant fraction: mg Cu kg-’ DM Roots
FYM ss 10 SSlOO FYM ss 10 SSlOO FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
16.8 b 22.5 b 60.5 a 20.9 a 16.5 a 32.5 a 7.3 c 12.2 b 29.8 a 11.4 b 13.5 b 30.7 a 4.0 a 15.0 a 10.2 a 7.3 a 17.8 a 1.9 a
Leaf levels Ll
L2
10.3 a 12.0 a 13.7 a 7.9 b 9.2 a 13.7 a 4.8 b 6.4 b 10.1 a 10.6 a 9.5 a 8.3 a 7.8 b 11.9 a 12.0 a
9.7 a 8.3 a 11.2 a 8.0 a 10.9 a 12.5 a 5.1 b 6.4 b 11.2 a 5.2 b 5.6 b 8.0 a 5.3 b 5.9 b 8.6 a 6.0 a 6.8 a 8.0a
Stalk levels L3
5.8 b 7.3 b 10.3 a 5.6 a 6.8 a 9.4 a 5.2 a 6.5 a 8.7 a 5.8 b 9.2 b 14.2 a 9.1 a 9.9 a 11.8a
Ear
L4
Sl
s2
s3
s4
total
4.2 b 7.7 a 8.5 a 4.8 b 6.7 a 7.6 a 8.4 c 15.8 b 20.7 a 8.0 b 14.6 a 18.8 a
5.8 a 6.1 a 6.3 a 2.8 b 3.5 a 4.8 a 2.0 b 2.4 b 5.4 a 1.3 a 1.3 a 1.9 a 1.2 a 1.1 a 1.8 a 1.4 b 1.4b 3.5 a
1.9 b 2.2 b 4.3 a 1.7 a 1.6 a 2.7 a 1.2 b 1.4 b 6.7 a 2.7 a 3.4a 2.1 a
4.2 a 3.5 a 5.4 a 2.7 b 3.6 a 5.1 a 3.0 a 4.5 a 5.1 a 6.1 a 6.0a 5.7 a
2.5 b 3.3 b 5.7 a 3.4 a 3.0 a 4.0 a 3.2 a 4.3 a 5.6 a 4.8 a 4.6a 5.7 a
4.6 a 3.1 a 5.2 a
contrast, the second decrease step, between days 95 and 118, cannot be explained by a simple dilution as no further increase of the biomass occurred. Thus, the root bound Cu was translocated to the upper plant parts during plant development. To examine this Cu translocation from the roots to the above-ground organs, we subdivided the above-ground parts into their main organs: leaves, stalks and ear. A comparison of the kinetics of Cu accumulation between lower and upper leaves is shown in Fig. 3. The Cu concentration of leaves varied with their position on the stem, their age and the soil Cu supply. In leaves as well as in stalks (Table 3), the highest concentrations were found in the lower levels (1 and 2) on the first harvest dates, followed by a progressive decrease until maturity. The decrease in the lower levels was correlated with raised Cu concentrations in the upper levels at the end of the vegetation period, indicating a Cu translocation from the lower to the upper parts during plant development (Table 3 and Fig. 3).
Ear Husks
Cob
Grains
2.6 a 2.4 a 2.6 a 1.9 a 2.3 a 3.0 a 2.5 a 3.0 a 3.6 a
3.1 a 2.9 a 3.1a 1.6a 2.5a 2.6a 2.7 a 3.3a 2.6 a
0.7 b 2.1 a 2.6a 0.8a 1.2a 1.4a 1.2 a 1.4a 1.3 a
Significantly raised Cu concentrations were observed in the upper leaf levels of SS lo- and SS loo-treated plants from the silage stage to maturity and in different stalk levels during the whole vegetation period (Table 3). However, while Cu concentrations in the stalks never reached critical values, potentially toxic Cu concentrations up to 20 mg kg-’ were found in leaf level 4 at the silage stage (Table 3). The ear was subdivided into its three main organs: husks, cobs and grains. The Cu concentrations in these plant parts were determined at the latest harvest dates. The Cu concentrations in the ear components were always lower than in leaves and stalks. Besides the Cu concentration in grains at day 95, no significant treatment effect was observed (Table 3). The Cu concentrations in the grains at harvest were even lower than in the other ear organs. As Cu concentration sites they are thus of negligible importance, and they may safely be used as animal feeds. Our results confirm data from previous studies. Weissenhom et al. (1995) determined the highest
B. Jarausch- Wehrheim et al./Eurapean Journal of Agronomy 5 (19961259-271
___
Wholoplants
P 1 p
100
lbovs-ground plant part
Total
SO 600
L
t 0
400
0
0 P
1,
55
74
9s
119
II
(52 25
Whole plrnts
_I
8
59
71
95
119
Total ~bovo-bond
152
plant parts
FYM = SSlO
(00
so
A SSlOO
0 i
ss
A 5s
10 100
Ic
---2mo
Wholm roots
0
8
1h
1
FYM SSlO
n
A SSlOO
Whole plants
. 2000
=
,500.
(SO0
E l E
WOO500
=
FYM ss 10
A
ss
0
2000
8
n
200
l
1
I
0 FYM
200
23
‘=
Wholo roots
SO
0.
3 k f: g
A
265
100
.
i?
I
04 0
41 55
74
9s
II9
152
drrs &tot sowing
200
,rr.r
1,
I
55
I4
9s
119
152
drrs after sowing
200
0
41 I9
74
95
119
IS2
IO
dart aftor sowing
Fig. 2. Comparison of the dry matter development (A), the Cu concentration expressed in mg Cu g-’ DM (B) and the total Cu content expressed in g DM per plant (C) in whole plants, total above-ground parts and whole roots during the whole growth cycle.
Cu concentrations in the roots of the young maize plant and a general decrease with plant age. Root metal concentrations were less strongly correlated with soil metal concentrations at later growth stages. Weissenhorn et al. also showed a general decrease in shoot Cu concentration between sixleaf stage and grain maturity of field-~0~ maize. Loneragan et al. (1980) found progressively decreasing Cu concentrations in whole shoots of wheat (Triticum aestivum) at adequate and ‘luxury’ external Cu supply from the first harvest date to maturity. They found that young organs had higher Cu concentrations than older plant parts and that the Cu concentration in all young organs of wheat decreased with age. Thus, the Cu accumulation seems to be related to the plant metabolism
and occurs in the metabolically most active plant parts. 3.3. Critical copper concentrations In our studies, average Cu concentrations in the whole plant or in the above-ground parts did not reach critical values for plant damage (Fig. 2(B)). However, we found Cu concentrations of up to 60 mg kg-’ DM Cu in the young roots and of up to 20 mg kg-’ DM Cu in the upper leaves of SS loo-treated plants at silage stage. Macnicol and Beckett (1985) determined an upper critical level of 20 mg kg- ’ DM Cu for the occurrence of yield reduction of 10% in maize plant tissues. Using the measurement of guaiacol-~roxidase capacity
B. Jarausch- Wehrheim et al./European Journal of Agronomy 5 (1996) 259-271
266
Lower r
T
a
Leaves
lular level which could be revealed by molecular metal stress biomarkers in these plant parts.
IL1 +L21
1
l n
i
3.4. Copper accumulation andpartitioning
FYM SSlO
2
E 6 0
!
III
0
41
56
74
days
40 35
a i
2
I
Upper
I
I
I
95
116
152
after
Leaves
200
sowing
jL3+L41
I
30 25 20 15 10
n
5
SSlO
A SSlOO II,
0 0
41
I 56
74
days
95
after
1 116
152
200
sowing
Fig. 3. Comparison of the Cu concentration in the lower leaf levels (Ll + L2) and in the upper leaf levels (L3 + L4) during the whole growth cycle of the maize plant, expressed in mg Cu g-’ DM.
Mocquot et al. (1996) found a threshold value of 26 mg kg- ’ DM Cu for the appearance of physiological damage in the roots of growth chamber cultivated maize plants. The raised Cu concentrations in roots of SS loo-treated plants were correlated with significant DM yield reductions of up to 31% (Table 2 and Table 3). In contrast, the high Cu concentrations in the upper leaves did not significantly affect leaf biomass production (Tables 2 and 3). These observations provide evidence for the presence of direct toxic effects on the young roots. However, the high Cu concentrations in leaves may indicate damage at a subcel-
To follow the total Cu mass flow in the whole plant, we determined the total Cu content and its distribution in the different plant organs during the whole growth cycle. Table 4 shows the total Cu content calculated by multiplication of the Cu concentration (Table 3) by the DM of the different organs (Table 2). The correlation between DM yield, Cu concentration and total Cu content is shown in Fig. 2. Despite a gradual decrease in the Cu concentration, the increase of the Cu content was due to a great biomass production during the entire growing season in the whole plant and in the aboveground plant parts of FYM and SS lo-treated plants. In contrast, the Cu amount of whole SS loo-treated plants reached a plateau between day 95 and grain harvest. The luxury soil Cu supply in SS 100 plots caused an excess of Cu in the young roots. However, a net loss of Cu from the roots occurred between days 95 and 118, which was correlated with an increase of the Cu amount in the above-ground parts. This indicates Cu translocation from the roots to the upper plant organs during plant development. The subdivision of the whole plant into its different organs made possible an analysis of the Cu uptake and partitioning among the different vegetative and reproductive organs. Because of the large variability in biomass, the analysis of the percentage Cu distribution gives a better comparison between the differently treated maize plants. Fig. 4 shows the percentage Cu accumulation and distribution among the main organs of maize during the whole plant development. The following aspects are evident: for all treatments, the Cu mass flow occurred from the roots to the above-ground vegetative plant parts, leaves and stalks, and least to the grains. This indicates that the different soil treatments had no negative effect on the normal pathway of essential micronutrients such as Cu. The soil Cu supply influenced Cu distribution and storage in the different plant parts. Thus, Cu retention in the young roots of SS loo-treated
FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
D 41
D 152
D 118
D 95
D 74
D 56
Treatment
Days after sowing
63.3 b 86.8 b 171.2 a 177.9 b 154.1 b 288.6 a 209.2 b 305.7 ab 585.6 a 413.0 b 393.3 b 1012.2 a 153.1 a 463.5 a 299.6 a 287.1 a 558.4 a 304.4 a
Roots
90.0 a 94.8 a 132.1 a 110.8 a 79.6 a 134.4 a 78.1 b 73.3 b 300.2 a 176.8 a 199.8 a 132.5 a
40.2 b 88.2 a 83.1 a 57.3 a 61.6 a 68.9 a 105.8 b 147.4 b 226.3 a 92.2 b 133.9 ab 179.2 a
40.5 a 36.4 a 33.1 a 85.8 a 92.2 a 132.3 a 70.7 a 73.2 a 139.0 a 55.8 b 57.1 b 93.5 a 57.1 a 46.1 a 49.5 a 81.6 a 46.6 a 144.0 a
68.5 b 60.5 a 78.6 a 96.4 a 124.5 a 158.8 a 96.4 b 99.1 b 173.3 a 76.4 b 82.9 b 157.4 a 88.1 b 94.2 ab 145.3 a 86.8 b 90.3 b 136.0 a
44.5 a 49.4 a 44.1 a 18.7 b 33.8 a 42.1 a 24.1 b 20.8 b 50.8 a 31.8 b 26.6 b 54.0 a 23.2 a 35.3 a 44.0 a 109.5 b 121.3 b 174.2 a 103.8 a 98.3 a 133.7 a 108.2 a 119.6 a 164.2 a 124.5 b 181.9 ab 280.2 a 155.3 a 186.1 a 194.7 a
s2
Sl
L4
L2
Ll L3
Stalk levels
Leaf levels
Plant fraction: Cu content (mg per plant)
103.6 a 85.6 a 127.5 a 121.5 b 124.9 b 182.9 a 132.9 a 153.9 a 182.4 a 283.2 a 250.0 a 270.8 a
s3
28.6 a 35.5 a 47.0 a 50.7 a 32.4 a 42.9 a 38.9 a 37.3 a 58.9 a 50.9 a 50.5 a 59.1 a
s4
26.9 a 41.8 a 36.2 a
total
Ear
73.0 a 63.8 a 74.4 a 43.8 a 51.1 a 66.1 a 59.1 a 64.9 a 80.9 a
Husks
100.1 a 105.3 a 143.6 a 64.9 a 98.9 a 116.0 a 126.7 a 138.0 a 127.9 a
Cob
Ear
15.2 b 49.9 a 66.6 a 120.5 b 193.9 ab 266.6 a 338.5 a 368.7 a 373.6 a
Grains
Table 4 Cu accumulation in the different plant parts of FYM, SS lo- and SS loo-amended maize plants at six sampling dates during the growth cycle expressed in mg Cu per plant and significant treatment-dependent variances classified by the letters a, b and c
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Journal
of Agronomy 5 (1996) 259-271
FYM
41
41
74
56
74
56
118
95
74
I52
95
118
152
9s
118
L-52
t (days) _._.~_
Fig. 4. Percentage
distribution
@ Roots ill Stalks
a Leaves
of the total Cu content
among
10 Husks m Ear - _..___ -__ _-. the different
plants exceeded 50% of the total Cu uptake, while in FYM and SS 10 plants most Cu was retained in the developing leaves. While the Cu accumulation in the roots of FYM and SS loo-treated plants decreased at the end of the vegetation cycle, the Cu amount in the roots of SS 10 plants was
organs
i.1 Cobs
fi Grains
of FYM, SS lo- and SS loo-treated
maize plants.
maintained at a relatively constant proportion of about 30% during the whole growing season. This observation could be explained by a stronger root mycorrhization in SS 10 plots, as observed by Weissenhorn et al. (1995). They assumed that the 0.1 in the rhizosphere of SS 10 plants was addition-
B. Jarausch- Wehrheim et al./European Journal of Agronomy 5 (1996) 259-271
ally bound by the mycorrhiza and thus less available to the plants. This is supported by the lower percentage of Cu content in the leaves and stalks of SS 10 plants at the end of the vegetation period compared to FYM and SS 100 plants. Thus at the silage stage (day 118), more than 60% of the total Cu amount in FYM and SS loo-treated plants was retained in the shoot, equally distributed in leaves and stalks, while in SS 10 plants the shoot accounted only for 45% of the total Cu content. In FYM and SS loo-treated plants, the decline in the root Cu concentration between days 95 and 152 was correlated with a simultaneous Cu increase, primarily in the leaves and in the grains, at maturity, indicating a direct translocation from the Cu storage in the roots to the grains with an intermediate Cu storage in the upper leaves. In contrast, the Cu demand during grainfill of SS 10 plants was uniformly supplied by the Cu in the upper shoot parts, the husks and the cob. Among the ear components, the grains represented the main Cu storage organs, accounting for 20% of the total Cu content at maturity for all treatments. Regarding the absolute Cu contents (Table 4), the large amount of Cu in the stalks at silage stage was mainly due to their high DM yield (Table 2), Cu concentrations being relatively low (Table 3). In contrast, the Cu accumulation in the leaves was due to raised Cu concentrations mainly in the upper leaves of SS 100 plants (Fig. 3; Table 3). Thus, due to their high biomass at silage stage, the shoots appeared as the main Cu storage sites with an equal distribution between the leaf and stalk fractions. 3.5. Copper-biomass relationship The results illustrate the influence of the total biomass on Cu accumulation during the plant growth cycle. To describe the relationship between Cu concentration and plant DM, a model was fitted to the data, using non-linear regression. As shown in Fig. 5, we obtained an inverse relationship, following an exponential curve, for all treatments in the case of above-ground plant organs. However, no satisfactory fit was found in the case of roots, as indicated by the low regression coefficients (Table 5). This observation suggests a
269
16 14
00 0
100
200
Above-ground
300
400
plant
l
FYM
w
ss
A
ss
500
biomass
10 100
600
700
(g DM)
Fig. 5. Relationship between Cu concentration (mg Cu kg-’ DM) and biomass of above-ground parts using an exponential model of non-linear regression presented as mean values of three replicates per treatment with standard deviations. Table 5 Values for the parameters a and b and coefficient of determination rz for the relationship between Cu concentration and plant DM in aerial plant parts and roots using the exponential model y=axb Treatment
FYM ss 10 ss 100
Above-ground plant parts
Roots
a
b
r2
a
b
r2
24.84 22.72 20.55
-0.38 -0.34 -0.24
0.983 0.967 0.919
90.21 90.17 91.38
-0.79 -0.78 -0.83
0.213 0.161 0.637
common Cu accumulation and translocation system in all above-ground organs and, in contrast, the existence of different concentration-dependent uptake and transport mechanisms in the roots.
4. Conclusions The aim of this study was to examine the influence of sludge applications on the Cu uptake and distribution among the different organs of field-grown maize. The most important result was the localization of critical Cu concentration and storage sites within the plant. It was shown that, although no visual morphological treatment effects on the normal plant growth were observed, the
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Cu concentration in individual organs reached critical values at certain dates during the growth cycle. As demonstrated by DM yield reduction, these high Cu concentrations may have produced harmful effects at a subcellular level. Such effects deserve more attention in future studies. With respect to food quality, two points should be emphasized: first sewage sludge application did not result in critical Cu concentrations in the grains at harvest, thus indicating that they could be used safely for animal feeding. Even though critical Cu values were not reached, sludges contain other metals which could have toxic effects. In particular, the effects of Cd and Zn are currently under investigation in our laboratory and their content in grains in relation to food quality is being studied. Second, more attention should be paid to the use of above-ground parts as fodder. We found that the highest Cu concentrations in the upper leaves appeared at silage stage. As the microbial biomass is more sensitive to raised Cu concentrations than higher plants (McGrath et al., 1995), microbial processes during ensilage may be affected. Furthermore, raised Cu amounts in silage taken as fodder may be harmful to animals and humans (Scheinberg, 199 1). Thus, a regular analysis of the Cu concentration in those plant parts taken as fodder is recommended to detect possible detrimental effects in the food chain. Long-term effects of sewage sludge application should be taken into consideration as sludge decays in the soil for a long time after its application (Juste and Mench, 1992). Even if there are no risks at present, the long-lasting effects of sewage sludge application and the reuse of polluted plant material for soil treatments should be further studied.
The authors thank P. Solda and for skilful technical assistance, P. LERMAVE for their help in metal W. Jarausch and Dr. D. Plenet discussions.
S. Buss&es Masson and analysis, Dr. for helpful
References Bonhomme, R., Ruget F., Derieux, M. and Vincourt, P., 1982. Relations entre production de mat&e s&he drienne et energie interceptee chez differems genotypes de rndis. CR Acad. Sci., 294: 3933398. Clijsters, H. and Van Assche, F., 1985. Inhibition of photosynthesis by heavy metals. Photosynth. Res., 7: 31-40. Delas, J., 1963. La toxicite du cuivre accumule dans les ~01s. Agrochimica, VII: 257-288. Fernandes, J.C. and Henriques, F.S., 1991. Biochemi~l, physiological, and structural effects of excess copper in plants. Bot. Rev., 57: 246-273. Gomez, A., Solda, P., Lambrot, C., Wilbert, J, and Juste, C. (1992). Bilan des elements traces metalliques transfer&s dans un sol sableux apres 16 anntes d’apports continus et connus de boues de station d’tpuration et de fumier de ferme en monoculture irriguee de ma&.. Convention de recherche no. 89-256, Neuilly sur Seine: Ministtre de 1’Environnement. Heckman, J.R., Angle, J.S. and Chaney, R.L., 1987. Residual effects of sewage sludge on soybean. I. A~umulation of heavy metals. J. Environ. Qual., 16: 113-I 17. Inskeep, W.P. and Bloom, P.R., 1985. Extinction coefficients of chlorophyll a and b in N,N-dimethylformamid and 80% acetone. Plant Physiol., 71: 483-85. Juste, C. and Mench, M., 1992. Long-term application of sewage sludge and its effects on metal uptake by crops. In: D.C. Adrian0 (Editor), Biogeo~h~ist~ of Trace Metals: Advances in Trace Substances Research. Lewis Publishers, Boca Raton, FL, pp. 159-193. Juste, C., Chassin, P., Gomez, A., Lineres, M. and Mocquot, B, Feix, I. and Wiart, J, 1995. Les micro-polluants metalliques dans les boues residuaires des stations d’epuration urbaines. ADEME, Angers, and INRA, Bordeaux. Karlen, D.L., Flannery, R.L. and Sadler, E.J., 1988. Aerial accumulation and partitioning of nutrients by corn. Agron. J., 80: 232-242. Keuls, M., 1952. The use of the ‘student&l’ range in connection with an analysis of variance. Euphytica, 1: 112-122. Lin, S-L. and Wu, L., 1994. Effects of copper concentration on mineral nutrient uptake and copper accumulation in protein of doper-tolerant and nontolerant Lotus pursh~anus L. Ecotoxicol. Environ. Safety, 29: 214-228. Loneragan, J.F., Snowball, K. and Robson, A.D., 1980. Copper supply in relation to content and redistribution of copper among organs of the wheat plant. Ann. Bot., 45: 621-632. Lou&, A., 1993. Le cuivre. In: A. Lotte (Editor), Oligo-elements en Agriculture. Editions SCPA and Nathan, Paris, pp. 155-177. Macnicol, R.D. and Beckett, P.H.T., 1985. Critical tissue concentrations of potentially toxic elements. Plant Soil, 85: 107-129. McGrath, S.P., Chaudri, A.M. and Giller, K.E., 1995. Longterm effects of metals in sewage sludge on soils, microorganisms and plants. J. Indust. Microbial., 14: 94-104. Mench, M.J., Martin, E. and Solda, P., 1994. After effects of
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metals derived from a highly metal-polluted sludge on maize (Zea mays L.). Water Air Soil Pollut., 75: 277-291. Mocquot, B., Vangronsveld, .I., Clijsters, H. and Mench, M., 1996. Copper toxicity in young maize (Zea mays L.) plants: effects on growth, mineral and chlorophyll contents, and enzyme activities. Plant Soil (in press). Ouzounidou, G., Ciamporova, M., Moustakas, M. and Karataglis, S., 1995. Responses of maize (Zea mays L.) plants to copper stress. I. Growth, mineral content and ultrastructure of roots. Environ. Exp. Bot., 35: 167-76. Sauerbeck, D.R., 1987. Effects of agricultural practices on the physical, chemical and biological properties of soils. Part II: Use of sewage sludge and agricultural wastes. In: H. Barth and P.L. L’Hermite (Editors), Scientific Basis for Soil Protection in the European Community. Elsevier Applied Science, London, pp. 181-210. Scheinberg, H., 1991. Copper. In: E. Merian (Editor), Metals and their Compounds in the Environment. VCH, Weinheim, pp. 893-907.
211
Tiller, K.G. and Merry, R.H., 1981. Copper pollution of agricultural soils. In: J.F. Loneragan, A.D. Robson, and R.D. Graham (Editors), Copper in Soils and Plants. Academic Press, Sydney, New York and London, pp. 119-137. Verkleij, J.C.A. and Schat, H., 1990. Mechanisms of metal tolerance in higher plants. In: A.J. Shaw (Editor), Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, FL, pp. 179-193. Weissenhorn, I., Mench, M. and Leyval, C., 1995. Bioavailability of heavy metals and arbuscular mycorrhiza in a sewage-sludge amended sandy soil. Soil Biol. Biochem., 27: 287-296. Woolhouse, H.W., 1983. Toxicity and tolerance in the responses of plants to metals. In: A. Pirson and M.H. Zimmermann (Editors), Encyclopedia of Plant Physiology. Springer Verlag, Berlin, pp. 245-300. Yingming, L. and Corey, R.B., 1993. Redistribution of sludgeborne cadmium, copper, and zinc in a cultivated plot. J. Environ. Qual., 22: l-8.
European Journal of Agronomy 5 (1996) 259-271
Uptake and partitioning of sludge-borne copper in field-grown maize (Zea mays L.) B. Jarausch-Wehrheim,
B. Mocquot, M. Mench *
INRA, Unit&d’ Agronomie, Centre de Recherches de Bordeaux, BP 81, F-33883 Villenave d’ Ornon Cedex, France
Abstract Accumulation of sludge-borne copper (Cu) by field-grown maize and its distribution between the different plant organs was studied in detail in a long-term sewage sludge field trial. Since 1974, field plots on a coarse sandy soil have been amended each year with farmyard manure (FYM) at a rate of 10 t dry matter (DM) ha-’ year-’ and with sewage sludge at the two levels of 10 t DM ha-’ year- 1 (SS 10) and 100 t DM ha-’ per 2 years (SS 100). All field plots have been cropped annually with maize. In 1993, five replicate plants per treatment were examined at six different growth stages from seedling to grain maturity. Each plant was separated into at least 12 different parts and the Cu content of each was determined. Regarding growth parameters, no visible deleterious effects on plant development due to the different soil treatments could be observed, although the dry matter yield of roots and stalks of SS loo-treated plants was significantly reduced. Significantly increased Cu concentrations of up to 60 mg Cu kg-’ DM in the roots of young SS lOO-grown maize plants and of up to 20 mg Cu kg -’ DM in the upper leaves at silage stage were found. No critical Cu amounts were reached in the grains until harvest. Keywords: Copper; Foodstuff quality; Maize; Metal uptake; Sewage sludge
1. Introduction The agricultural use of sewage sludge has been increasingly favoured as a means of inexpensive waste disposal and beneficial re-use (Sauerbeck, 1987; Yingming and Corey, 1993). This land application of sewage sludge could even be enhanced as, for example in France, landfilling will soon be banned and, thus, sludge incineration and soil application will be the only alternatives. Sewage sludge is added to agricultural soils primarily for its fertilizer value in terms of N and * Corresponding author. Tel.: +33 05 56 843042; fax: f33 05 56 843054;e-mail: [email protected].
P (Tiller
and Merry, 198 1; Juste and Mench, 1992), as a source of organic matter, trace elements (e.g., Cu, Mn, Zn) and sometimes as an alkaline soil amendment. However, besides the beneficial effects, the application of sewage sludges as well as other organic wastes may lead to a considerable accumulation of trace metals such as Cu, Cd, Ni, Pb and Zn in the topsoil (Juste and Mench, 1992). This could produce phytotoxic effects or unwanted accumulation of metals in the food chain (Heckman et al., 1987). Sewage sludges are generally rich in Cu, Cd and Zn compared to soils and plants (Juste et al., 1995). Although Cu is required as an essential nutrient element for plant development and normal growth (Clijsters and
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Van Assche, 1985; Lou& 1993), even a small increase in the amounts of Cu in the soil or growth medium may be detrimental to the plants (Lin and Wu, 1994) and excessive Cu concentrations are highly toxic (Woolhouse, 1983; Macnicol and Beckett, 1985; Verkleij and Schat, 1990). High soil Cu concentrations may inhibit the normal plant growth or may operate as a physiological stress factor in plant metabolism (Ouzounidou et al., 1995). Besides the direct toxic effects, Cu can also indirectly decrease primary production through its inhibitory effects on the processes responsible for nutrient cycling in soils and waters (Fernandes and Henriques, 1991). While toxicity to higher plants is of major concern, soil biological activity may be much more sensitive to sludge Cu inputs (McGrath et al., 1995). Cu contamination in fodder, such as maize, may increase the metal transfer into the food chain even if no direct decrease in crop yield due to metal phytotoxicity occurs. Damages to water, soil and food quality may be found in particular in sandy soils which have a low buffer capacity. These soils are very abundant in the south-west of France. In this area, maize is at present one of the main cultivated crops and in some cases the pioneer crop on former vineyards. The treatment of grapevines with Bordeaux mixture (solution of copper sulfate neutralized with hydrated lime) (Scheinberg, 1991) has already caused a considerable input of Cu and other metals into the soil. The phytotoxic Cu effects on grapes and maize were first reported by Delas (1963). Therefore, the further Cu input into soil via sludge application is questionable in these areas. To examine long-term effects of sewage sludge application on continuous maize cropping under natural cultivation conditions, a field trial was established at Bordeaux in 1974. Numerous reports on this long-term experiment have been published concerning the accumulation and distribution of metals in sludge-treated soils and plants (Gomez et al., 1992; Juste and Mench, 1992; Weissenhorn et al., 1995). Mench et al. (1994) examined the distribution of sludge-borne Cd, Cu, Fe, Mn and Ni among roots, stalks, leaves and grains of fieldgrown maize at tasseling and found that Cu was uniformly distributed among the different plant
Journal of Agronomy 5 (1996) 259-271
parts. However, treatment with highly metal polluted sludges caused an accumulation of Cu, Fe and Ni in maize grains. Weissenhorn et al. (1995) compared shoot and root metal concentrations of sludge-borne Cd, Cu, Mn, Ni, Pb and Zn at three different developmental stages of field-grown maize. Concentrations of Cu were always greater in roots than in shoots, and increased Cu contents could be related to the highest sludge application rate. The Cu concentrations in both organs were highest at the six-leaf stage and decreased until maturity. The results showed that sludge-borne Cu inputs in the soil resulted in an increased Cu content in the whole maize plant. Furthermore, the accumulation and distribution of Cu between the different plant organs was highly dependent on the growth state. Although detailed observations have been reported for wheat (Loneragan et al., 1980), few data are available for maize (Karlen et al., 1988), and the pathway and mechanism of Cu translocation and storage are poorly understood. The objective of this study was to characterize the uptake and distribution of sludge-borne Cu in different plant parts of field-grown maize and to localize Cu concentration and storage sites within the maize plant.
2. Materials and methods 2.1. Sampling site and soil treatment The field site was a long-term sewage-sludge trial on an acid sandy soil (Arenic Udifluvent, US classification, pH 5.5) at the INRA experimental site of Couhins, Bordeaux, France. The field trials were established in 1974 and received an anaerobically-digested dehydrated sludge at rates of 10 t DM ha-’ year-’ (SS 10) and 100 t DM ha-’ per 2 years (SS loo), respectively. Plots treated only with farmyard manure (FYM) at a rate of 10 t DM ha-’ year-’ served as controls. Each treatment was replicated five times. The pH of the soil, measured in 1993, was 6.61, 6.79 and 6.04 for FYM, SS 10 and SS 100 treatments, respectively. Fertilization and other practices as well as soil and sludge characteristics were previously described by
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Gomez et al. (1992) and Weissenhorn et al. (1995). Cumulative sludge inputs since 1974 led to a total Cu input until 1993 of 5.62 kg ha-’ Cu for FYM, 45kgha-‘CuforSS lOand227.5kghaK’Cufor SS 100 treatment. Since 1974, all field plots have been annually cropped with maize (Zea muys L. cv. INRA 260) at a density of 75 000 plants ha-‘. These field plots (3 m wide, 6 m long) were used for the present study. To avoid the modification of the long-term monitoring of data based on a standard number of plants, all field plots were planted in addition to Zea muys L. cv. INRA 260 with Zea muys L. cv. Volga, leading to a total density of 85 000 plants ha- ’ in the study year of 1993. Maize was planted using an 0.8 x 0.165 m spacing in a randomized block design. The only plant protection treatment was an application of Atrazine. Due to inadequate rainfall, 300-400 mm of water was applied by sprinkler irrigation from June to September.
leaf level 4
stalk level 3
stalk level 1
2.2. Plant sampling, fractionation and growth parameters Corresponding to the main developmental stages, whole maize plants were sampled on six dates and then subdivided into different plant fractions depending on the growth stage (Table 1 and Fig. 1). Among leaves and stalks, we distinguished between four different levels (Fig. 1). In our study, each leaf level was defined as a batch of four fully developed leaves, grouped adjacent
Fig. 1. Scheme for the fractionation of the whole maize plant into different organs and levels. Levels are defined as: LL 1 (SL l)=leaf 1-4; LL 2 (SL 2) =leaf 5-8; LL 3 (SL 3) =leaf 9-12; and LL 4 (SL 4)=leaf 13-16.
to each other on the stem. Stalk levels corresponded to leaf levels. Levels were counted from the bottom to the apex: levels 1 and 2 corresponded
Table 1 Harvest dates and fractionation of whole maize plant into its organs and into four leaf and stalk levels, respectively Days after sowing (sowing date: 5 May 1993)
Development stage
Subdivision of plant
41 56 14 95 118 152
6-leaf stage 12-leaf stage silking l&leaf stage silage maturity
root, root, root, root, root, root,
LL LL LL LL LL LL
1, LL 1, LL 1, LL 1, LL 1, LL 1, LL
2, 2, 2, 2, 2, 2,
SL LL LL LL LL LL
No. of fractions per plant 1 3, 3, 3, 3, 3,
SLl LL 4, LL 4, LL 4, LL 4,
SLl, SL 2, SL 3, SL 4, whole ear SL 1, SL 2, SL 3, SL 4, husks, cob, grains SL 1, SL 2, SL 3, SL 4, husks, cob, grains SLl, SL 2, SL 3, SL 4, husks, cob, grains
4 5 10 12 12 12
A level is defined as a batch of four fully developed leaves, grouped adjacent to each other on the stem (see Fig. 1). LL, leaf level; SL, stalk level. Levels are defined as: LL 1 (SL 1) =leaf l-4; LL 2 (SL 2) =leaf 5-8; LL 3 (SL 3) =leaf 9-12; and LL 4 (SL 4)= leaf 13-16.
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to the older (lower) organs; and levels 3 and 4 to the younger (upper) parts. Before fractionation, the following physiological and morphological parameters were recorded per plant: plant height; number of leaves; number of dry leaves; number of ears; shoot diameter (at a height of 15 cm from the soil surface); total leaf area (determined from the length L and the width W of each leaf using the formula S=O.75 x LW (Bonhomme et al., 1982). The mean chlorophyll content of leaves 6, 10 and 13 was directly measured on the field with a portable chlorophyll meter (Minolta SPAD 502). The calibration and verification of the values obtained was done by extraction of chlorophyll with N,N-dimethylformamide (DMF) and its quantification by spectrophotometry (Inskeep and Bloom, 1985). 2.3. Preparation and analysis of plant material Adhering particles were removed by several washings using first de-ionized water with 1% (v/v) Triton X-100 and 1% (v/v) pure nitric acid ( 14 M HNO,), second de-ionized water and then distilled water. Washed plant material was oven-dried at 80°C to constant weight, weighed, cut into small pieces, and subsampled. Roots and above-ground plant parts were milled in a zirconium oxide grinder (PM 4, Retsch, Dusseldorf, Germany); grains were ground in a stainless steel mill using a 0.375 mm sieve. Aliquots of plant material were also oven-dried at 105°C to determine dry matter (DM) content. Plant samples (0.5 g DM) were wet digested overnight in 14 M HN03 (5 ml) and 30% (v/v) H,O, (10 ml). Digestion was completed in a heating system (Gerhardt, Bonn, Germany) under reflux in four steps, at 50°C for 30 min, 100°C for 30 min, 150°C for 30 min and 220°C for 2 h. After cooling for 35 min, the digest was made up to 100 ml with distilled water. Two certified reference samples (rye-grass BCR 281 and sea lettuce BCR 279 from the EU Community Bureau of References) and controls of reagents without plant material were analysed along with plant samples in every batch. Cu concentrations were determined either by flame air-acetylene (Varian Spectra A 20) or graphite furnace (Varian Spectra
A300/A400 with Zeeman background correction) atomic absorption spectrophotometry or by inductively coupled plasma atomic emission spectrometry (Varian Liberty 200) depending on the metal concentration. Each solution was analysed in triplicate using standards in a similar matrix. European standard values for the Cu concentration in the certified reference samples are 9.65_+ 0.38 mg kg-’ for rye-grass and 13.14kO.37 mg kg-’ for sea lettuce. In our conditions, we determined mean Cu values of 9.92 +0.17 and 11.46kO.46 mg kg-’ and thus recovery rates were 102.82 and 87.2% for rye-grass and sea lettuce (n = 6), respectively. The digestion quality for each plant part was tested by repeated preparation and determination of three replicates. The standard deviation of the results for each plant part was less than 6%. The working value was examined by the Cu determination in the control without plant material. The mean Cu concentration in 20 control replicates was less than 2 ng Cu ml-‘. 2.4. Data analysis and statistics All concentrations refer to plant DM determined at 105°C. Means and standard errors were calculated for three replicate values. All data were statistically evaluated by analysis of variance; means and standard errors were compared for error variance and significant differences by a multiple range test (PcO.05) (Keuls, 1952). The relationship between Cu concentration and plant DM was fitted by non-linear regression using an exponential model. The coefficient r2 refers to the fraction of variance explained by the correlation.
3. Results and discussion 3.1. Morphological observations
Sludge application at two different concentrations (SS 10 and SS 100) did not significantly affect plant height or total leaf area compared to the control treatment with farmyard manure (FYM) (data not shown). At maximum growth stage (day 118), the mean plant height was
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3.2. Copper uptake kinetics
250+20 cm and the total plant leaf area was 0.7 +0.05 m2 for all treatments. Although no visual morphological treatment effects could be observed, biomass production was strongly affected in some plant parts. Significant DM yield reduction was found especially in the roots of SS loo-treated plants and in different stalk levels of plants grown in SS lo- and SS loo-treated plots (Table 2). In contrast, grains of SS lOOgrown plants had a significantly increased DM at silage stage compared to FYM-treated plants. In general, chlorophyll contents were greater in leaf 10 and 13 of SS lo- and SS loo-treated plants (data not shown) than in the FYM controls. As chlorophyll contents varied considerably between individual plants, a significant treatment effect could only be confirmed for leaf 10 of SS loo-treated plants at day 95. This was associated with a raised Cu concentration (Table 3) and a significant DM reduction (Table 2) in leaf level 3 at day 95. Therefore, deleterious effects of high Cu concentrated sludge application on the plant metabolism cannot be excluded.
The soil Cu supply significantly affected the temporal change in Cu concentration in the different plant parts (Fig. 2(B)). Regarding the whole plant, the SS 100 treatment increased Cu concentrations at the earliest growth stages followed by a progressive decrease during the vegetation cycle. The subdivision of the whole plant into above-ground parts and roots shows that the initially raised Cu concentrations in SS 100 plants were mainly due to the Cu concentration in the roots. These raised Cu concentrations in the roots were maintained until day 95. They were up to 5-fold higher than mean concentrations of all above-ground parts (Fig. 2(B)). However, at maturity (day 152) the root Cu concentration in SS 100 plants was below that of FYM and SS 10 plants. Two main phases of decrease in the root Cu concentration can be distinguished: the first decrease, between day 41 and day 56 (Fig. 2(B)), was correlated with an increase in DM (Fig. 2(A)) and may thus be due to a dilution effect. In
Table 2 Dry matter of the different plant parts of FYM, SS IO- and SS loo-amended maize plants at six sampling dates during the growth cycle expressed in g DM per plant and significant treatment-dependent variances classified by the letters a, b and c Days after sowing
D 41
D 56
D 74
D 95
D 118
D 152
Treatment
Plant fraction: DM (g per plant) Roots
FYM ss 10 ss 100 FYM SSlO ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
3.8 a 3.9 a 2.8 b 8.5 a 9.3 a 8.9 a 28.7 a 25.1 ab 19.7 b 36.2 a 29.1 a 33.0 a 38.3 a 30.9 b 29.4 b 39.3 a 31.4 a 38.5 a
Leaf levels Ll
L2
4.3 a 4.1 a 3.2 a 2.4 a 3.7a 3.1 a 5.0 a 3.3 a 5.0 a 3.0 b 2.8 b 6.5 a 3.0 a 3.0 a 3.7 a
7.1 a 7.3 a 7.0 a 12.1 a 11.4a 12.7 a 16.9 a 15.5 a 15.5 a 14.7 b 14.8 b 19.7 a 16.6 a 16.0 a 16.9 a 14.5 a 13.3 a 17.0 a
Stalk levels L3
18.9 a 16.6a 16.9 a 18.5 a 14.5 a 14.2 a 20.8 a 18.4 b 18.9 b 21.5 a 19.8 a 19.7 a 17.1 a 18.8 a 16.5 a
Ear
L4
Sl
s2
s3
S4
Total
9.6 a 11.5 a 9.8 a 11.9 a 9.2 a 9.1 a 12.6 a 9.3 a 10.9 a 11.5 a 9.2 b 9.5 b
7.0 a 6.0 b 5.3 c 30.6 a 26.3 b 27.6 b 35.3 a 30.5 a 25.7 b 42.9 a 43.9 a 49.2 a 47.6 a 41.9 b 27.5 c 58.3 a 33.3 b 41.1 b
47.3 a 43.1 b 30.7 c 65.2 a 49.8 b 49.8 b 65.1 a 52.3 b 44.8 c 65.5 a 58.8 a 63.1 a
24.7 a 24.5 a 23.6 a 45.0 a 34.7 b 35.9 b 44.3 a 34.2 b 35.8 b 46.4 a 41.7 a 47.5 a
11.4 a 10.8 a 8.2 b 14.9 a 10.8 b 10.7 b 12.2 a 8.7 b 10.5 b 10.6 a 11.0 a 10.4 a
5.9 a 11.3 a 7.0 a
Ear Husks
Cob
Grains
28.1 a 26.6 a 28.6 a 23.0 a 22.2 a 22.0 a 23.6 a 21.6 a 22.5 a
32.3 a 36.3 b 46.3 c 40.5 a 39.6 a 44.6 a 46.9 a 41.8 a 49.2 a
21.7 a 23.8 a 25.6 a 150.6 b 161.6 ab 190.4 a 282.1 a 263.4 a 287.4 a
B. Jarawch- Wehrheim er al./European Journal of Agronomy 5 (1996) 259-271
264
Table 3 Cu concentration in the different plant parts of FYM, SS lo- and SS loo-amended maize plants at six sampling dates during the growth cycle, expressed in mg Cu kg-’ DM and significant treatment-dependent variances classified by the letters a, b and c Days after sowing
D 41
D 56
D 74
D 95
D 118
D 152
Treatment
Plant fraction: mg Cu kg-’ DM Roots
FYM ss 10 SSlOO FYM ss 10 SSlOO FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
16.8 b 22.5 b 60.5 a 20.9 a 16.5 a 32.5 a 7.3 c 12.2 b 29.8 a 11.4 b 13.5 b 30.7 a 4.0 a 15.0 a 10.2 a 7.3 a 17.8 a 1.9 a
Leaf levels Ll
L2
10.3 a 12.0 a 13.7 a 7.9 b 9.2 a 13.7 a 4.8 b 6.4 b 10.1 a 10.6 a 9.5 a 8.3 a 7.8 b 11.9 a 12.0 a
9.7 a 8.3 a 11.2 a 8.0 a 10.9 a 12.5 a 5.1 b 6.4 b 11.2 a 5.2 b 5.6 b 8.0 a 5.3 b 5.9 b 8.6 a 6.0 a 6.8 a 8.0a
Stalk levels L3
5.8 b 7.3 b 10.3 a 5.6 a 6.8 a 9.4 a 5.2 a 6.5 a 8.7 a 5.8 b 9.2 b 14.2 a 9.1 a 9.9 a 11.8a
Ear
L4
Sl
s2
s3
s4
total
4.2 b 7.7 a 8.5 a 4.8 b 6.7 a 7.6 a 8.4 c 15.8 b 20.7 a 8.0 b 14.6 a 18.8 a
5.8 a 6.1 a 6.3 a 2.8 b 3.5 a 4.8 a 2.0 b 2.4 b 5.4 a 1.3 a 1.3 a 1.9 a 1.2 a 1.1 a 1.8 a 1.4 b 1.4b 3.5 a
1.9 b 2.2 b 4.3 a 1.7 a 1.6 a 2.7 a 1.2 b 1.4 b 6.7 a 2.7 a 3.4a 2.1 a
4.2 a 3.5 a 5.4 a 2.7 b 3.6 a 5.1 a 3.0 a 4.5 a 5.1 a 6.1 a 6.0a 5.7 a
2.5 b 3.3 b 5.7 a 3.4 a 3.0 a 4.0 a 3.2 a 4.3 a 5.6 a 4.8 a 4.6a 5.7 a
4.6 a 3.1 a 5.2 a
contrast, the second decrease step, between days 95 and 118, cannot be explained by a simple dilution as no further increase of the biomass occurred. Thus, the root bound Cu was translocated to the upper plant parts during plant development. To examine this Cu translocation from the roots to the above-ground organs, we subdivided the above-ground parts into their main organs: leaves, stalks and ear. A comparison of the kinetics of Cu accumulation between lower and upper leaves is shown in Fig. 3. The Cu concentration of leaves varied with their position on the stem, their age and the soil Cu supply. In leaves as well as in stalks (Table 3), the highest concentrations were found in the lower levels (1 and 2) on the first harvest dates, followed by a progressive decrease until maturity. The decrease in the lower levels was correlated with raised Cu concentrations in the upper levels at the end of the vegetation period, indicating a Cu translocation from the lower to the upper parts during plant development (Table 3 and Fig. 3).
Ear Husks
Cob
Grains
2.6 a 2.4 a 2.6 a 1.9 a 2.3 a 3.0 a 2.5 a 3.0 a 3.6 a
3.1 a 2.9 a 3.1a 1.6a 2.5a 2.6a 2.7 a 3.3a 2.6 a
0.7 b 2.1 a 2.6a 0.8a 1.2a 1.4a 1.2 a 1.4a 1.3 a
Significantly raised Cu concentrations were observed in the upper leaf levels of SS lo- and SS loo-treated plants from the silage stage to maturity and in different stalk levels during the whole vegetation period (Table 3). However, while Cu concentrations in the stalks never reached critical values, potentially toxic Cu concentrations up to 20 mg kg-’ were found in leaf level 4 at the silage stage (Table 3). The ear was subdivided into its three main organs: husks, cobs and grains. The Cu concentrations in these plant parts were determined at the latest harvest dates. The Cu concentrations in the ear components were always lower than in leaves and stalks. Besides the Cu concentration in grains at day 95, no significant treatment effect was observed (Table 3). The Cu concentrations in the grains at harvest were even lower than in the other ear organs. As Cu concentration sites they are thus of negligible importance, and they may safely be used as animal feeds. Our results confirm data from previous studies. Weissenhom et al. (1995) determined the highest
B. Jarausch- Wehrheim et al./Eurapean Journal of Agronomy 5 (19961259-271
___
Wholoplants
P 1 p
100
lbovs-ground plant part
Total
SO 600
L
t 0
400
0
0 P
1,
55
74
9s
119
II
(52 25
Whole plrnts
_I
8
59
71
95
119
Total ~bovo-bond
152
plant parts
FYM = SSlO
(00
so
A SSlOO
0 i
ss
A 5s
10 100
Ic
---2mo
Wholm roots
0
8
1h
1
FYM SSlO
n
A SSlOO
Whole plants
. 2000
=
,500.
(SO0
E l E
WOO500
=
FYM ss 10
A
ss
0
2000
8
n
200
l
1
I
0 FYM
200
23
‘=
Wholo roots
SO
0.
3 k f: g
A
265
100
.
i?
I
04 0
41 55
74
9s
II9
152
drrs &tot sowing
200
,rr.r
1,
I
55
I4
9s
119
152
drrs after sowing
200
0
41 I9
74
95
119
IS2
IO
dart aftor sowing
Fig. 2. Comparison of the dry matter development (A), the Cu concentration expressed in mg Cu g-’ DM (B) and the total Cu content expressed in g DM per plant (C) in whole plants, total above-ground parts and whole roots during the whole growth cycle.
Cu concentrations in the roots of the young maize plant and a general decrease with plant age. Root metal concentrations were less strongly correlated with soil metal concentrations at later growth stages. Weissenhorn et al. also showed a general decrease in shoot Cu concentration between sixleaf stage and grain maturity of field-~0~ maize. Loneragan et al. (1980) found progressively decreasing Cu concentrations in whole shoots of wheat (Triticum aestivum) at adequate and ‘luxury’ external Cu supply from the first harvest date to maturity. They found that young organs had higher Cu concentrations than older plant parts and that the Cu concentration in all young organs of wheat decreased with age. Thus, the Cu accumulation seems to be related to the plant metabolism
and occurs in the metabolically most active plant parts. 3.3. Critical copper concentrations In our studies, average Cu concentrations in the whole plant or in the above-ground parts did not reach critical values for plant damage (Fig. 2(B)). However, we found Cu concentrations of up to 60 mg kg-’ DM Cu in the young roots and of up to 20 mg kg-’ DM Cu in the upper leaves of SS loo-treated plants at silage stage. Macnicol and Beckett (1985) determined an upper critical level of 20 mg kg- ’ DM Cu for the occurrence of yield reduction of 10% in maize plant tissues. Using the measurement of guaiacol-~roxidase capacity
B. Jarausch- Wehrheim et al./European Journal of Agronomy 5 (1996) 259-271
266
Lower r
T
a
Leaves
lular level which could be revealed by molecular metal stress biomarkers in these plant parts.
IL1 +L21
1
l n
i
3.4. Copper accumulation andpartitioning
FYM SSlO
2
E 6 0
!
III
0
41
56
74
days
40 35
a i
2
I
Upper
I
I
I
95
116
152
after
Leaves
200
sowing
jL3+L41
I
30 25 20 15 10
n
5
SSlO
A SSlOO II,
0 0
41
I 56
74
days
95
after
1 116
152
200
sowing
Fig. 3. Comparison of the Cu concentration in the lower leaf levels (Ll + L2) and in the upper leaf levels (L3 + L4) during the whole growth cycle of the maize plant, expressed in mg Cu g-’ DM.
Mocquot et al. (1996) found a threshold value of 26 mg kg- ’ DM Cu for the appearance of physiological damage in the roots of growth chamber cultivated maize plants. The raised Cu concentrations in roots of SS loo-treated plants were correlated with significant DM yield reductions of up to 31% (Table 2 and Table 3). In contrast, the high Cu concentrations in the upper leaves did not significantly affect leaf biomass production (Tables 2 and 3). These observations provide evidence for the presence of direct toxic effects on the young roots. However, the high Cu concentrations in leaves may indicate damage at a subcel-
To follow the total Cu mass flow in the whole plant, we determined the total Cu content and its distribution in the different plant organs during the whole growth cycle. Table 4 shows the total Cu content calculated by multiplication of the Cu concentration (Table 3) by the DM of the different organs (Table 2). The correlation between DM yield, Cu concentration and total Cu content is shown in Fig. 2. Despite a gradual decrease in the Cu concentration, the increase of the Cu content was due to a great biomass production during the entire growing season in the whole plant and in the aboveground plant parts of FYM and SS lo-treated plants. In contrast, the Cu amount of whole SS loo-treated plants reached a plateau between day 95 and grain harvest. The luxury soil Cu supply in SS 100 plots caused an excess of Cu in the young roots. However, a net loss of Cu from the roots occurred between days 95 and 118, which was correlated with an increase of the Cu amount in the above-ground parts. This indicates Cu translocation from the roots to the upper plant organs during plant development. The subdivision of the whole plant into its different organs made possible an analysis of the Cu uptake and partitioning among the different vegetative and reproductive organs. Because of the large variability in biomass, the analysis of the percentage Cu distribution gives a better comparison between the differently treated maize plants. Fig. 4 shows the percentage Cu accumulation and distribution among the main organs of maize during the whole plant development. The following aspects are evident: for all treatments, the Cu mass flow occurred from the roots to the above-ground vegetative plant parts, leaves and stalks, and least to the grains. This indicates that the different soil treatments had no negative effect on the normal pathway of essential micronutrients such as Cu. The soil Cu supply influenced Cu distribution and storage in the different plant parts. Thus, Cu retention in the young roots of SS loo-treated
FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100 FYM ss 10 ss 100
D 41
D 152
D 118
D 95
D 74
D 56
Treatment
Days after sowing
63.3 b 86.8 b 171.2 a 177.9 b 154.1 b 288.6 a 209.2 b 305.7 ab 585.6 a 413.0 b 393.3 b 1012.2 a 153.1 a 463.5 a 299.6 a 287.1 a 558.4 a 304.4 a
Roots
90.0 a 94.8 a 132.1 a 110.8 a 79.6 a 134.4 a 78.1 b 73.3 b 300.2 a 176.8 a 199.8 a 132.5 a
40.2 b 88.2 a 83.1 a 57.3 a 61.6 a 68.9 a 105.8 b 147.4 b 226.3 a 92.2 b 133.9 ab 179.2 a
40.5 a 36.4 a 33.1 a 85.8 a 92.2 a 132.3 a 70.7 a 73.2 a 139.0 a 55.8 b 57.1 b 93.5 a 57.1 a 46.1 a 49.5 a 81.6 a 46.6 a 144.0 a
68.5 b 60.5 a 78.6 a 96.4 a 124.5 a 158.8 a 96.4 b 99.1 b 173.3 a 76.4 b 82.9 b 157.4 a 88.1 b 94.2 ab 145.3 a 86.8 b 90.3 b 136.0 a
44.5 a 49.4 a 44.1 a 18.7 b 33.8 a 42.1 a 24.1 b 20.8 b 50.8 a 31.8 b 26.6 b 54.0 a 23.2 a 35.3 a 44.0 a 109.5 b 121.3 b 174.2 a 103.8 a 98.3 a 133.7 a 108.2 a 119.6 a 164.2 a 124.5 b 181.9 ab 280.2 a 155.3 a 186.1 a 194.7 a
s2
Sl
L4
L2
Ll L3
Stalk levels
Leaf levels
Plant fraction: Cu content (mg per plant)
103.6 a 85.6 a 127.5 a 121.5 b 124.9 b 182.9 a 132.9 a 153.9 a 182.4 a 283.2 a 250.0 a 270.8 a
s3
28.6 a 35.5 a 47.0 a 50.7 a 32.4 a 42.9 a 38.9 a 37.3 a 58.9 a 50.9 a 50.5 a 59.1 a
s4
26.9 a 41.8 a 36.2 a
total
Ear
73.0 a 63.8 a 74.4 a 43.8 a 51.1 a 66.1 a 59.1 a 64.9 a 80.9 a
Husks
100.1 a 105.3 a 143.6 a 64.9 a 98.9 a 116.0 a 126.7 a 138.0 a 127.9 a
Cob
Ear
15.2 b 49.9 a 66.6 a 120.5 b 193.9 ab 266.6 a 338.5 a 368.7 a 373.6 a
Grains
Table 4 Cu accumulation in the different plant parts of FYM, SS lo- and SS loo-amended maize plants at six sampling dates during the growth cycle expressed in mg Cu per plant and significant treatment-dependent variances classified by the letters a, b and c
268
B. Jarausch- Wehrheim et al/European
Journal
of Agronomy 5 (1996) 259-271
FYM
41
41
74
56
74
56
118
95
74
I52
95
118
152
9s
118
L-52
t (days) _._.~_
Fig. 4. Percentage
distribution
@ Roots ill Stalks
a Leaves
of the total Cu content
among
10 Husks m Ear - _..___ -__ _-. the different
plants exceeded 50% of the total Cu uptake, while in FYM and SS 10 plants most Cu was retained in the developing leaves. While the Cu accumulation in the roots of FYM and SS loo-treated plants decreased at the end of the vegetation cycle, the Cu amount in the roots of SS 10 plants was
organs
i.1 Cobs
fi Grains
of FYM, SS lo- and SS loo-treated
maize plants.
maintained at a relatively constant proportion of about 30% during the whole growing season. This observation could be explained by a stronger root mycorrhization in SS 10 plots, as observed by Weissenhorn et al. (1995). They assumed that the 0.1 in the rhizosphere of SS 10 plants was addition-
B. Jarausch- Wehrheim et al./European Journal of Agronomy 5 (1996) 259-271
ally bound by the mycorrhiza and thus less available to the plants. This is supported by the lower percentage of Cu content in the leaves and stalks of SS 10 plants at the end of the vegetation period compared to FYM and SS 100 plants. Thus at the silage stage (day 118), more than 60% of the total Cu amount in FYM and SS loo-treated plants was retained in the shoot, equally distributed in leaves and stalks, while in SS 10 plants the shoot accounted only for 45% of the total Cu content. In FYM and SS loo-treated plants, the decline in the root Cu concentration between days 95 and 152 was correlated with a simultaneous Cu increase, primarily in the leaves and in the grains, at maturity, indicating a direct translocation from the Cu storage in the roots to the grains with an intermediate Cu storage in the upper leaves. In contrast, the Cu demand during grainfill of SS 10 plants was uniformly supplied by the Cu in the upper shoot parts, the husks and the cob. Among the ear components, the grains represented the main Cu storage organs, accounting for 20% of the total Cu content at maturity for all treatments. Regarding the absolute Cu contents (Table 4), the large amount of Cu in the stalks at silage stage was mainly due to their high DM yield (Table 2), Cu concentrations being relatively low (Table 3). In contrast, the Cu accumulation in the leaves was due to raised Cu concentrations mainly in the upper leaves of SS 100 plants (Fig. 3; Table 3). Thus, due to their high biomass at silage stage, the shoots appeared as the main Cu storage sites with an equal distribution between the leaf and stalk fractions. 3.5. Copper-biomass relationship The results illustrate the influence of the total biomass on Cu accumulation during the plant growth cycle. To describe the relationship between Cu concentration and plant DM, a model was fitted to the data, using non-linear regression. As shown in Fig. 5, we obtained an inverse relationship, following an exponential curve, for all treatments in the case of above-ground plant organs. However, no satisfactory fit was found in the case of roots, as indicated by the low regression coefficients (Table 5). This observation suggests a
269
16 14
00 0
100
200
Above-ground
300
400
plant
l
FYM
w
ss
A
ss
500
biomass
10 100
600
700
(g DM)
Fig. 5. Relationship between Cu concentration (mg Cu kg-’ DM) and biomass of above-ground parts using an exponential model of non-linear regression presented as mean values of three replicates per treatment with standard deviations. Table 5 Values for the parameters a and b and coefficient of determination rz for the relationship between Cu concentration and plant DM in aerial plant parts and roots using the exponential model y=axb Treatment
FYM ss 10 ss 100
Above-ground plant parts
Roots
a
b
r2
a
b
r2
24.84 22.72 20.55
-0.38 -0.34 -0.24
0.983 0.967 0.919
90.21 90.17 91.38
-0.79 -0.78 -0.83
0.213 0.161 0.637
common Cu accumulation and translocation system in all above-ground organs and, in contrast, the existence of different concentration-dependent uptake and transport mechanisms in the roots.
4. Conclusions The aim of this study was to examine the influence of sludge applications on the Cu uptake and distribution among the different organs of field-grown maize. The most important result was the localization of critical Cu concentration and storage sites within the plant. It was shown that, although no visual morphological treatment effects on the normal plant growth were observed, the
270
B. Jarausch- Wehrheim et al./European Journal of Agronomy 5 (1996) 259-273
Cu concentration in individual organs reached critical values at certain dates during the growth cycle. As demonstrated by DM yield reduction, these high Cu concentrations may have produced harmful effects at a subcellular level. Such effects deserve more attention in future studies. With respect to food quality, two points should be emphasized: first sewage sludge application did not result in critical Cu concentrations in the grains at harvest, thus indicating that they could be used safely for animal feeding. Even though critical Cu values were not reached, sludges contain other metals which could have toxic effects. In particular, the effects of Cd and Zn are currently under investigation in our laboratory and their content in grains in relation to food quality is being studied. Second, more attention should be paid to the use of above-ground parts as fodder. We found that the highest Cu concentrations in the upper leaves appeared at silage stage. As the microbial biomass is more sensitive to raised Cu concentrations than higher plants (McGrath et al., 1995), microbial processes during ensilage may be affected. Furthermore, raised Cu amounts in silage taken as fodder may be harmful to animals and humans (Scheinberg, 199 1). Thus, a regular analysis of the Cu concentration in those plant parts taken as fodder is recommended to detect possible detrimental effects in the food chain. Long-term effects of sewage sludge application should be taken into consideration as sludge decays in the soil for a long time after its application (Juste and Mench, 1992). Even if there are no risks at present, the long-lasting effects of sewage sludge application and the reuse of polluted plant material for soil treatments should be further studied.
The authors thank P. Solda and for skilful technical assistance, P. LERMAVE for their help in metal W. Jarausch and Dr. D. Plenet discussions.
S. Buss&es Masson and analysis, Dr. for helpful
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Tiller, K.G. and Merry, R.H., 1981. Copper pollution of agricultural soils. In: J.F. Loneragan, A.D. Robson, and R.D. Graham (Editors), Copper in Soils and Plants. Academic Press, Sydney, New York and London, pp. 119-137. Verkleij, J.C.A. and Schat, H., 1990. Mechanisms of metal tolerance in higher plants. In: A.J. Shaw (Editor), Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, FL, pp. 179-193. Weissenhorn, I., Mench, M. and Leyval, C., 1995. Bioavailability of heavy metals and arbuscular mycorrhiza in a sewage-sludge amended sandy soil. Soil Biol. Biochem., 27: 287-296. Woolhouse, H.W., 1983. Toxicity and tolerance in the responses of plants to metals. In: A. Pirson and M.H. Zimmermann (Editors), Encyclopedia of Plant Physiology. Springer Verlag, Berlin, pp. 245-300. Yingming, L. and Corey, R.B., 1993. Redistribution of sludgeborne cadmium, copper, and zinc in a cultivated plot. J. Environ. Qual., 22: l-8.