Chemosphere 57 (2004) 215–224

www.elsevier.com/locate/chemosphere

Contrasting effects of manure and compost on soil pH,

heavy metal availability and growth
of Chenopodium album L. in a soil
contaminated by pyritic mine waste
David J. Walker, Rafael Clemente, M. Pilar Bernal *

Department of Soil and Water Conservation and Organic Waste Management, Centro de Edafolog
ıa y Biolog
ıa
Aplicada del Segura, CSIC, Apartado 164, 30100 Espinardo, Murcia, Spain
Received 12 September 2003; received in revised form 6 May 2004; accepted 19 May 2004

Abstract

Chenopodium album L. was found to be one of the initial plant species colonising a heavy metal-contaminated site,
polluted by pyritic (sulphide-rich) waste from the Aznalc
ollar mine spill (South-western Spain). This indicates its
importance in the re-vegetation of this soil. In a pot experiment, C. album was sown in soil collected from the con-
taminated site, either non-amended or amended with cow manure or compost produced from olive leaves and olive mill
wastewater, in order to study the effect on heavy metal bioavailability and soil pH. In non-amended and compost-
amended soils, soil acidification, probably resulting from oxidation and hydrolysis of sulphide, led to increases in the
concentrations of soluble sulphate and plant-available Cu, Zn and Mn in the soil (extractable with 0.1 M CaCl2 ). Under
these conditions, shoot growth of C. album was negligible and shoot concentrations of Zn (2420–5585 lg g
1 ) and Mn
(5513–8994 lg g
1 ) were phytotoxic. Manure application greatly increased shoot growth and reduced the shoot con-
centrations of Cu, Zn, and Mn, and their plant-available concentrations in the soil. These effects appeared to be related
to an increase of soil pH, due to an inhibition of sulphide oxidation/hydrolysis, relative to the non-amended soil. For
metal sulphides-contaminated soil, liable to acidification, manure application appears to be able to enhance the initial
stages of re-vegetation, by species such as C. album.

2004 Elsevier Ltd. All rights reserved.

Keywords: Bioavailability; Chenopodium album L.; Heavy metals; Organic amendments; Remediation; Soil contamination

1. Introduction Spain). The toxic sludge covered 40 km2 of the adjoining

The rupture of a dam wall enclosing pyritic mine
ollar in April 1998 released 5 million m3
slurry at Aznalc

of heavy metal-rich mud and acidic water into the river
Guadiamar, in the province of Seville (Andaluc
ıa,
*
Corresponding author. Tel.: +34-968-396343; fax: +34-968-
396213.
E-mail address: pbernal@cebas.csic.es (M.P. Bernal).
0045-6535/$ - see front matter
2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.05.020
arable land, with the main contaminants being As, Cd,
Cu, Pb, Sb, Tl and Zn (Sim
on et al., 1999, 2001; Del R
ıo
et al., 2002). Although much of the sludge was physically
removed from the land, traces remain. When this con-
taminated soil is moist and exposed to air, oxidation and
hydrolysis of sulphides (S2
) in the sludge leads to the
formation of sulphuric acid, thus lowering soil pH and
greatly increasing heavy metal solubility (Williamson
and Johnson, 1981; Sim
on et al., 1999, 2001; Clemente
et al., 2003). This, and the fact that in much of the
216 D.J. Walker et al. / Chemosphere 57 (2004) 215–224
affected area the total heavy metal levels exceed Euro-
pean Union maximum permitted values for agricultural
soils (Council of the European Communities, 1986),
make the land unusable for cultivation of crops.
Addition of organic matter (OM) to mine waste-
contaminated soils has been employed to aid their re-
vegetation, by improving fertility and structure and
decreasing the plant-availability of heavy metals, al-
though this may require the co-application of lime
(Williamson and Johnson, 1981; Tordoff et al., 2000;
Abbott et al., 2001). Organic amendments can decrease
heavy metal bioavailability, shifting them from ‘‘plant-
available’’ forms, extractable with water or solutions of
neutral salts such as CaCl2 , to fractions associated with
OM, carbonates or metal oxides. This depends upon the
particular metal and soil type involved, and also upon
the characteristics of the OM, particularly with respect to
its degree of humification, content of heavy metals and
salts and its effect on soil pH (Ross, 1994; Narwal and
Singh, 1998; Alm as et al., 1999; Shuman, 1999; Walker
et al., 2003). Application of fresh manure can increase
heavy metal solubility, due to the provision of soluble
organic compounds which form complexes with the
metals (Alm as et al., 1999; Shuman, 1999). But the humic
substances, which constitute a major part of the OM of
compost, can reduce metal solubility by formation of
stable metal chelates (Ross, 1994). Differences with re-
spect to effects on metal availability between amendment
treatments can be related to differences not only in the
OM (humification), but also in the mineral fractions (salt
content, pH) and cation exchange capacity (CEC), as
well as changes in the redox conditions of the soil (Ross,
1994; Shuman, 1999; Walker et al., 2003).
The location of El Vicario (longitude W 06 130 1000 ,
latitude N 37 260 1700 ), 10 km downstream of the rup-
tured dam at Aznalc
ollar, has been designated by the
regional government of Andaluc
ıa (Junta de Andaluc
ıa)
for field experimentation into remediation of the con-
taminated soil. The aim of this is to re-establish the
natural vegetation. One part of the remediation research
involves the addition of soil amendments, such as OM.
At El Vicario, the annual weed species Chenopodium
album L. (Chenopodiaceae) was one of the most abun-
dant plants in the summer of 2000 and one of the first
plant species to colonise this land. This indicates possi-
ble heavy metal tolerance, although Del R
ıo et al. (2002)
found that C. album sampled from other sites contami-
nated by the Aznalc
ollar spill did not have elevated
tissue levels of heavy metals. The aim of the current
study was to determine the ability of two organic
amendments, compost and manure, to enhance growth
of C. album in a non-calcareous soil from El Vicario.
This species was considered an appropriate test species
since its abundance indicates that it will play an
important role in the initial re-vegetation of this site.
The two organic materials chosen have been shown to
decrease plant-availability of heavy metals in calcareous
soils (Walker et al., 2003).
2. Materials and methods
2.1. Collection and analysis of field samples of soil and C.
album plants
In May 2000, seven samples of soil and plants of C.
album were taken within the experimental site at El
Vicario (20 · 80 m). Plants were cut just above the soil
surface and samples of the top 20 cm of soil around the
plant roots were collected. The plants were transported
in refrigerated boxes to the laboratory, washed with tap
water and then with deionised water. The plants were
then separated into stems and leaves, which were oven-
dried (70 C for 72 h), milled, subjected to a nitric:per-
chloric acid (2:1) digestion and analysed for Cu, Pb, Zn,
Mn and Fe by atomic absorption spectrometry (AAS)
(SOLAAR 969; Unicam, Cambridge, UK).
The soil at the experimental site, El Vicario, is a non-
calcareous (<0.5% CaCO3 ) loam Typic Xerofluvent (Soil
Survey Staff, 1987). Soil pH was determined for satu-
rated soil pastes (30 ml deionised water per 100 g soil).
Total heavy metals were extracted by nitric acid–per-
chloric acid digestion (Abrisqueta and Romero, 1969).
Metals were extracted also with 5 mM diethylenetri-
aminepentaacetic acid/10 mM CaCl2 /100 mM trietha-
nolamine (pH 7.3) (DTPA), according to Lindsay and
Norvell (1969). Metal concentrations in each extract,
measured by AAS, were adjusted to values for oven-
dried (12 h at 105 C) soil.
2.2. Characterisation of organic amendments and soil used
in the pot experiment
For the pot experiment, soil from the top 20 cm,
adjacent to soil sample 1 (Table 1), was collected in
February 2001, mixed, air-dried for 1 week, and sieved
to 2 mm. The pH of this soil differed from that of sample
1 due to the acidification process of the sulphides which
occurred from May 2000 to February 2001 and the
spatial heterogeneity of the soil contamination (Cle-
mente et al., 2003). Two organic amendments were used
(Table 2), a compost (prepared from a mixture of olive
leaves and the solid fraction of olive-mill wastewater in a
pilot plant, as described by Garc
ıa-Gomez et al. (2003))
and a fresh cow manure. The organic amendments were
air-dried (1 week) and ground before analysis and use.
The manure was rich in soluble organic compounds (40
g kg
1 of water-soluble organic-C), had 113 g kg
1 of
NaOH-extractable organic-C, and a humification ratio
of 14.7% (100 · extractable-C/total organic-C). In con-
trast, the compost had low water-soluble organic-C
(22.5 g kg
1 ), and higher proportions of NaOH-
 D.J. Walker et al. / Chemosphere 57 (2004) 215–224 217

Table 1
Concentrations of Cu, Pb and Zn measured in soil (total and DTPA-extractable) and in stem and leaf tissue of C. album for seven
sampled areas within the experimental site of El Vicario, in May 2000
Soil pH Cu Pb Zn
sample Soila Plantb Soil Plant Soil Plant
Total DTPA Stem Leaves Total DTPA Stem Leaves Total DTPA Stem Leaves
1 6.8 110 25.5 23.6 38.0 182 8.0 14.4 25.5 527 102 87.4 339
2 6.5 248 35.5 13.7 39.7 482 5.1 7.7 22.8 1128 202 43.1 340
3 6.4 275 35.0 9.7 27.1 699 ndc 15.5 1.0 1432 279 38.1 425
4 3.5 201 16.3 6.2 26.0 566 nd 10.1 8.7 895 197 93.7 444
5 4.7 139 23.1 9.8 17.8 251 nd 16.1 5.2 522 173 94.5 623
6 4.7 139 23.1 11.7 27.2 251 nd 8.1 1.1 522 173 215 1168
7 7.0 120 19.7 16.6 31.2 197 5.2 15.4 8.6 597 88 257 950
a
Metal concentrations in soil are expressed as lg g
1 dry soil.
b
Metal concentrations in plant tissue are expressed as lg g
1 DM.
c
nd, not detected (<0.5 lg g
1 ).

Table 2 determined by a colorimetric method based on Berthe-

Characteristics of the initial soil, compost and manure used for lot’s reaction (Sommer et al., 1992).
the pot experiment (dry weight basis) Soil texture (46.0% sand, 34.3% silt and 19.7% clay)
Parameter Soil Compost Manure was revealed by sieving and sedimentation, using the
pH 5.29 7.75 9.09 hydrometer method (Gee and Bauder, 1986). For deter-
EC (dS m
1 ) 2.28 1.95 7.27 mination of CaCO3 , CO2 released by addition of HCl was
CEC (cmolc kg
1 ) 14.0 132 nda measured with a calcimeter (US Salinity Laboratory Staff,
Buffer capacity 50.6 nd nd 1954). The total N (NT ) and organic C (Corg ) concentra-
(meq kg
1 pH tions of the soil and amendments were measured with a
unit
1 ) Carlo Erba automatic microanalyser (Navarro et al.,
OM (%) 1.72 88.3 76.1 1991). The OM content was calculated by multiplying
Corg (g kg
1 ) 10.0 468 423
Corg by 1.72, for soil. Total-P was determined colorimet-
NT (g kg
1 ) 0.9 30.2 28.5
NHþ
1 rically as molybdovanadate phosphoric acid (Kitson and
4 -N (mg kg ) nd 355 583
Total P (g kg
1 ) nd 1.3 12.8 Mellon, 1944). Soluble sulphate was determined in a 1:5
Total K (g kg
1 ) nd 14.3 46.6 water extract according to Abrisqueta et al. (1962). The
Total metals CEC of the soil and amendments was determined using
(mg kg
1 ) BaCl2 -triethanolamine (Carpena et al., 1972; Lax et al.,
Cu 133 (50) 167 23 1986). The buffer capacity was determined according to
Pb 220 (50) <0.05 <0.05 Hartikainen (1986). Electrical conductivity (EC) was
Zn 521 (150) 80 262 determined for 1:5 and 1:10 aqueous extracts of soil and
Fe 45 713 4023 946 amendments, respectively. Total metal contents and
Mn 1044 76 273
concentrations of metals extractable with 0.1 M CaCl2 (3
For the total Cu, Pb and Zn, the values in parentheses are the g of air-dried soil shaken with 20 ml of 0.1 M CaCl2 for 16
European Union limits for heavy metal concentrations in h) were determined by AAS. All soil parameters were
agricultural soils, at pH 6.0 (Council of the European Com-
adjusted to values for oven-dried soil.
munities, 1986).
a
nd ¼ not determined.
2.3. Pot experiment

extractable organic-C (143 g kg
1 ) and humic acid-like C
(92.4 g kg
1 ), giving a humification ratio of 30.6%, and a
humification index of 19.8% (100 · humic acid-C/total
organic-C). These properties, determined according to
Bernal et al. (1998), indicate that, relative to the manure,
the OM of the compost had a high degree of humifica-
tion and was more resistant to microbial degradation.
The pH of the amendments was measured in 1:10 water
extracts. The NHþ 4 -N was extracted with 2 M KCl and
Three treatments were set up on day 1: Non-amended
soil (which received no OM addition), soil + compost
(‘‘+C’’), and soil + manure (‘‘+M’’). The amendments
provided 1.0% Corg to the soil (27 and 26 g air-dry
compost and manure kg
1 dry soil, respectively). The
soil in the three treatments was mixed thoroughly and
watered with deionised water every 5–7 days, any
drainage water was collected under each pot and re-
turned to the soil the following day. On day 31, samples
218 D.J. Walker et al. / Chemosphere 57 (2004) 215–224
were air-dried for analysis and the soils were then
transferred into 1-kg pots and treated with N:P:K fer-
tiliser (15:15:15), at a rate equivalent to 400 kg ha
1 .
Seeds of C. album collected from El Vicario in Novem-
ber 2000 were sown in all treatments, at a rate of 1.0 g
pot
1 (1400 seeds). Seeds from a population of C.
album growing in non-contaminated soil (University of
Reading, UK) (‘‘Reading’’) were also sown in the non-
amended soil, at a rate of 0.5 g pot
1 (650 seeds). Seeds
of both populations (1.0 g pot
1 ) were also sown in 0.5-
kg pots of sand, to determine seedling emergence in a
non-contaminated medium. There were six replicates of
each substrate-seed combination, except in the +M
treatment, which had 12 replicates (six for each harvest
time). Pots were placed in a completely randomised
design, in a growth chamber, with a 16-h day, a daytime
photosynthetically active radiation of 350 lmol m
2 s
1 ,
temperature of 30/18 C (day/night), and a relative
humidity of 50/70% (day/night). They were watered
initially with 1.0 mM Ca(NO3 )2 , to maximise germina-
tion (Bouwmeester and Karssen, 1993) and subsequently
with deionised water. On day 59, seedling emergence was
recorded and seedlings were thinned to 10 pot
1 . On day
83, all pots of plants in non-amended and +C soil, and
six pots of +M plants, were harvested. The remaining
pots of +M plants were harvested on day 119. Aerial
parts were cut just above the soil surface, washed, oven-
dried, weighed, digested, and their concentrations of Cu,
Pb, Zn, Mn, and Fe determined by AAS. For the El
Vicario and Reading plants grown in non-amended soil,
material from the six replicates was pooled for analysis,
due to the very small plant size. At each harvest, soil
samples were taken for analysis.
2.4. Statistical analysis
The data were subjected to ANOVA. In all Figures
and Tables, mean values denoted by different letters are
significantly different ðP < 0:05Þ according to Tukey’s
range test. Percentage seedling emergence values were
only approximations and were not analysed statistically.
Pearson’s correlation coefficients, a measure of the linear
association between two variables, were determined for
plant metal concentration versus CaCl2 -extractable met-
als from soil ðn ¼ 18Þ, plant metal concentration versus
soil pH ðn ¼ 18Þ and CaCl2 -extractable metals from soil
versus soil pH ðn ¼ 38Þ. Statistical tests were performed
using SPSS Version 11.5 software (SPSS Inc., 2002).
3. Results and discussion
3.1. Field samples of soil and of C. album plants
In Table 1, the great variability in soil pH, total and
DTPA-extractable concentrations of Cu, Pb and Zn,
within an experimental site of 20 · 80 m, can be seen,
reflecting spatial variation with respect to the physical
removal of the pyrite-containing sludge and acidic mine
waste from the soil following the mine spill in April 1998
(Sim
on et al., 1999; Del R
ıo et al., 2002; Clemente et al.,
2003). In all cases, except for Cu and Pb in soil samples 1
and 7, the total levels of Cu, Pb, and Zn (Table 1) ex-
ceeded the European Union maximum permitted values
for agricultural soils at pH 6–7; 50–140, 50–300, and
150–300 lg g
1 for Cu, Pb, and Zn, respectively (Council
of the European Communities, 1986). The DTPA-
extractable concentrations were generally above the
minimum phytotoxic values for Cu (6–32 lg g
1 ) and Zn
(5–10 lg g
1 ) (Pichtel and Salt, 1998; Fageria, 2001).
These values, and the relatively high leaf levels of Cu (all
samples) and Zn (samples 5–7) suggest a possible ele-
vated tolerance of this population of C. album towards
Cu and Zn (Marschner, 1995; Chino et al., 1997). With
respect to metal levels determined for other plant species
at the El Vicario site (Del R
ıo et al., 2002), tissue Cu and
Pb values for C. album are similar or lower, whereas Zn
accumulation (up to 1168 lg g
1 ) is greater in C. album.
Close relationships have been demonstrated between
concentrations of heavy metals extractable with DTPA
and plant tissue heavy metal levels (Narwal and Singh,
1998; Walker et al., 2003). However, when Pearson
correlation coefficients were calculated for Cu, Pb and
Zn, none of the relationships were statistically significant
(P P 0:05, data not shown), reflecting the observation of
McLaughlin et al. (2000) that DTPA can over-estimate
heavy metal availability.
3.2. Pot experiment
Seedling emergence in the non-amended soil for the
El Vicario population was only about one-twentieth of
that in sand, whereas for the Reading population it was
about one-eighth of the sand value (Table 3). At day 83,
shoot growth of the El Vicario population was very
similar to that of the Reading population (Table 3).
These results indicate that the population of C. album
from the contaminated El Vicario site does not possess a
greater degree of heavy metal tolerance. Plants grown in
non-amended or +C soil were stunted (no more than
three pairs of leaves per plant) and unlikely to grow
more, whereas the +M plants were much larger with a
healthy appearance. By day 119, the dry weight of +M
plants had virtually doubled (2780 mg pot
1 ) with re-
spect to day 83, and they showed no visible signs of
metal toxicity. Although all the soil treatments received
N:P:K fertiliser, the macronutrients supplied by the
manure (Table 2) and improved soil conditions (Stewart
et al., 2000) may have contributed also to the vast in-
crease in growth caused by this treatment. The most
important benefits of manure addition to soil are related
to the increased OM content and biological activity. The
 D.J. Walker et al. / Chemosphere 57 (2004) 215–224
Table 3
Mean percentage seedling emergence, 28 days after sowing, and shoot dry weight (mg DM per pot of 10 plants), 52 days after sowing
( ¼ 83 days after mixing soils, with or without organic amendments), for C. album (from the contaminated El Vicario site and the non-
contaminated Reading site), sown in sand or in El Vicario soil. The soil was non-amended or was amended with compost (+C) or
manure (+M)
Population Treatment
El Vicario Sand
Non-amended soil
Soil + C
Soil + M
Reading Sand
Non-amended soil
a
Values denoted by different letters are significantly different ðP < 0:05Þ according to Tukey’s range test.
b
nd ¼ not determined.
OM from manures acts as a nutrient pool, improves
nutrient cycling, increases CEC and buffer capacity, re-
duces compaction, and improves soil physical properties
such as aggregation, friability, density, root penetration,
water holding capacity and water infiltration (Stewart
et al., 2000). The use of manure and compost is in
accordance with European Union policy on the appli-
cation of agro-industrial wastes to land for agricultural
or ecological benefit (Commission of the European
Communities, 1996).
In non-amended soil, toxic shoot levels of Zn (5585
and 3941 lg g
1 for the El Vicario and Reading popu-
lations, respectively) and Mn (8994 and 6602 lg g
1 for
El Vicario and Reading, respectively) occurred (Pos-
chenrieder and Barcel
o, 1981; Marschner, 1995; Kitao
et al., 2001) (Fig. 1). Tissue Cu levels in non-amended or
+C soil were around or above the toxicity threshold of
20–40 lg g
1 (Marschner, 1995; Chino et al., 1997), al-
though shoot Pb levels were non-toxic (Trivedi and
Erdei, 1992) (Fig. 1). Compost lowered tissue concen-
trations of Pb, Zn, Fe and Mn, although Zn (2420
lg g
1 ) and Mn (5513 lg g
1 ) were still almost certainly
toxic. For plants growing in non-amended or +C soil,
leaves showed severe chlorosis and necrosis, typical of
Mn toxicity (Barcel
o and Poschenrieder, 1981; Kitao
et al., 2001). Manure application reduced shoot con-
centrations of all the metals measured, especially Zn and
Mn (476 and 195 lg g
1 , respectively, on day 83) (Fig.
1). Also, the effect of manure addition on the shoot
Fe:Mn ratio, raising it to 0.37 (day 83) and 0.52 (day
119), compared to the non-amended (0.03), Reading
(0.05), and +C plants (0.02), may have contributed to
the growth stimulation (Poschenrieder and Barcel
o,
1981).
The highly significant correlations between the shoot
and soil CaCl2 -extractable concentrations of Cu, Zn and
Mn (Table 4), which represent the soluble and easily-
exchangeable fractions in soil (Sanders et al., 1986), as
demonstrated for other species (Sanders et al., 1986),
suggest that C. album could serve as an ‘‘indicator’’ of
219
Emergence (%) Shoot dry weight (mg pot
1 )a
25 ndb
1 12.3c
3 32.4b
2 1450a
38 nd
5 13.1c
Cu, Zn and Mn, but not Fe or Pb, availability in the soil
(Baker, 1981). The shoot levels of Cu, Pb, Zn and Mn
were related inversely to soil pH (Table 4), for the range
of pH values measured (4.00–6.42) (Table 5), as ob-
served previously (Sanders et al., 1986; Chino et al.,
1997). This reflects the fact that pH is often the main
factor determining the plant-availability of these metals,
availability being greater at lower soil pH (Sanders,
1983; Ross, 1994; Yoo and James, 2002).
During exposure of the soil to air and water, oxida-
tion and hydrolysis reactions of pyritic material (metal
sulphides) remaining from the mining sludge probably
occurred, causing the observed soil acidification and
solubilisation of heavy metals (Williamson and Johnson,
1981; Sim
on et al., 1999, 2001). For the non-amended
soil, a decline in pH of 1.2 units from day 1, when the
soil was first mixed, to day 83, the first harvest, was
observed (Table 5). The pH-buffer capacity (Table 2)
was similar to that found in non-calcareous soils by
Hartikainen (1986), and the pH-instability may reflect
the absence of carbonate in the soil. The decline in pH
was offset by 0.4 units by addition of compost, whilst the
pH of +M soil was 1.6–2.0 units higher than that of the
control, remaining higher at the end of the experiment
(119 days). A similar effect of manure was reported by
Tsakelidou et al. (1999) for non-contaminated acid soils,
and other organic amendments have succeeded in raising
the pH of contaminated soils (Narwal and Singh, 1998;
Shuman, 1999). Differences in the pH values of the
compost (7.75) and manure (9.09) themselves (Table 3)
probably contributed to their differing effects on soil pH.
Also, the manure had a Kþ content more than three
times that of the compost (Table 2). An increase of soil
pH following organic matter addition could be largely
due to addition of basic cations and production of NH3
during decomposition, although adsorption of Hþ ions,
development of reducing conditions due to increased
microbial activity and displacement of hydroxyls from
sesquioxide surfaces by organic anions could contri-
bute also (Pocknee and Summer, 1997). The NHþ 4 -N
220 D.J. Walker et al. / Chemosphere 57 (2004) 215–224

Fig. 1. Metal concentrations in shoot DM for C. album plants grown in pots, harvested at day 83 or 119 after mixing soil (with
compost or manure) ( ¼ 52 or 88 days after sowing). Seeds from the Reading and El Vicario populations were both sown (on day 31) in
non-amended soil. The El Vicario population was also sown in soil amended with compost (+C) or manure (+M). Values are means of
6 replicates, except for the plants grown in the non-amended soil (Reading and El Vicario) which were not included in the ANOVA
since only single analyses of material pooled from all 6 replicates were made.

concentration in the manure was within the range de- (1400 mg kg
1 ), because the manure was air-dried before
scribed by Menzi (2002) for cow manure in Europe use. The concentration in the compost was typical of a
(300–3200 mg kg
1 ), but was lower than the average mature compost (<400 mg kg
1 ; Bernal et al., 1998).
 D.J. Walker et al. / Chemosphere 57 (2004) 215–224 221

Table 4
Pearson correlation coefficients (unilateral) calculated for the relationships between metal concentration extractable from soil with 0.1
M CaCl2 (lg g
1 dry soil) and soil pH, shoot metal concentration (lg g
1 DM) and soil pH, and shoot metal concentration and soil
metal concentration extractable with 0.1 M CaCl2
Metal Pearson correlation coefficienta
½Metal shoot
½Metal CaCl2 b ½Metal shoot
pHb ½Metal CaCl2
pHc
Cu 0.801

)0.949

)0.776

Pb 0.488 NS )0.856

)0.597

Zn 0.994

)0.978

)0.982

Fe 0.244 NS 0.205 NS )0.564

Mn 0.997

)0.972

)0.978

Significance levels: P < 0:001; NS, P P 0:05.

b
n ¼ 18.
c
n ¼ 38.

Table 5
Mean values of pH, electrical conductivity (EC), and soluble sulphate concentration for non-amended soil and soil amended with
compost or manure (used to grow the El Vicario or Reading populations of C. album) at days 1 and 31 (at sowing of C. album), and at
the first (day 83) and second (day 119) harvests of C. album
Treatment Day pHa EC (dS m
1 ) Sulphate (g kg
1 )
Non-amended (El Vicario) 1 5.29d 2.28d 7.80de
31 4.83e 2.56cd 8.37cd
83 4.05g 2.89ab 9.68a
Non-amended (Reading) 83 4.08g 3.01a 9.63a
+Compost 31 5.26d 2.59bc 8.35cd
83 4.44f 2.97a 9.77a
+Manure 31 6.41a 2.55cd 7.54e
83 6.04c 2.88ab 8.93bc
119 6.22b 3.10a 9.03b

ANOVAb

Treatment NS

Day

Treatment · day NS NS
a
Mean values denoted by different letters differ significantly ðP < 0:05Þ, according to Tukey’s range test.
b
Significance levels:

P < 0:001; NS, P P 0:05.

However, the decrease of soil pH, associated with
sulphide oxidation, during the experiment (Days 31–119,
Table 5) was only slight in +M soil. Thus, mineralisation
of the labile OM in the fresh cow manure (Ajwa and
Tabatabai, 1994) may have altered the redox state of the
soil, to less oxidising conditions, thus minimising the
oxidation of sulphide to sulphate and the consequent
soil acidification. In support of this, the concentration of
soluble sulphate, on all days of measurement, was lowest
in the +M soil (Table 5). In a field experiment at El
Vicario, Clemente et al. (2003) found a close linear
relationship between soil pH and sulphate concentra-
tion, the sulphate formation being reflected in an in-
crease in EC. Table 5 shows that, for all treatments,
there was an increase in soil EC with time. For the non-
amended and compost-treated soil, this may have been
due to the acidification and solubilisation of metals
discussed above, whilst for the manure-amended soil it
may have been caused by the relatively high EC of the
manure (Table 2), and also mineralisation of its OM and
the consequent formation of soluble salts.
The 0.1 M CaCl2 -extractable metal concentrations
were closely (inversely) related to soil pH (Table 4),
reflecting earlier findings (Williamson and Johnson,
1981; Sanders, 1983; Ross, 1994). The relationship was
closer for Zn and Mn than for Cu, perhaps because sol-
uble/exchangeable Cu levels in soils often are influenced
more by OM than by pH, due to the ease with which Cu
can be chelated by OM (Sanders, 1983; Sanders et al.,
1986; Ross, 1994). This may also explain why, despite
their differing effects upon soil pH (Table 5), the effects of
compost and manure upon the concentration of Cu
222 D.J. Walker et al. / Chemosphere 57 (2004) 215–224

extractable with 0.1 M CaCl2 were similar, compared to and Pb (Fig. 2), as found previously for cow manure/Zn
the findings for Zn and Mn (Fig. 2). in differing soil types (Narwal and Singh, 1998; Walker
Addition of OM, particularly from manure, de- et al., 2003). Raising the soil OM content can increase
creased the soluble/exchangeable levels of Cu, Zn, Mn soil CEC, a factor which may affect both soluble and

500
Mn a a
400 ab
b
300
c c
200
d d d
100

0
0.5 Fe

0.4
a
0.3 a ab
0.1 M CaCl2-extractable metal concentration in soil (µg g dry soil-1)

0.2 abc
abc
0.1
c bc bc c
0.0
350 Zn a a
300 b b
250
200
c
150 c
2D Graph 2
100
50 d d d
0
0.5 Pb

0.4

0.3 a
ab
ab ab
0.2 ab
ab ab
0.1
b ab
0.0
10 Cu day 1
day 31
8 day 83
day 119
6 a a
4
2 b b bc c
c c c
0
Non-amended Non-amended +C +M
(El Vicario) (Reading)

Fig. 2. Concentrations of metals in 0.1 M CaCl2 -extracts of soil used to grow C. album. Samples were taken on day 1, from the initial
soil before mixing (with compost or manure), and on days 31, 83 and 119. Seeds from the Reading and El Vicario populations of C.
album were both sown (on day 31) in control soil. The El Vicario population was also sown in soil amended with compost (+C) or
manure (+M). All values are means of 6 replicates.
 D.J. Walker et al. / Chemosphere 57 (2004) 215–224
exchangeable metal levels (Shuman, 1999; Yoo and
James, 2002), but in the current study there was no
significant effect of compost or manure on CEC (data
not shown). The effects of organic amendments on heavy
metal solubility also depend greatly upon the degree of
humification of their OM and their effect upon soil pH
(Narwal and Singh, 1998; Alm as et al., 1999; Shuman,
1999; Walker et al., 2003). However, in the current
study, the effects of compost and manure on soil pH
seemed to be the main determinants of Zn and Mn
availability and uptake. So, the pH effect was greater
than that of metal complex formation by soluble OM.
The effect of the compost with respect to metal avail-
ability was limited in this soil due to its inability to
prevent acidification.
The availability and plant uptake of Pb were reduced
by the organic amendments (Figs. 1 and 2). This may
have been a consequence of the immobilisation of Pb by
humified OM, in the compost-treated soil, and, in the
case of manure, due to increased soil pH and the for-
mation of insoluble carbonates and/or phosphates with
CO2 and soluble phosphates, respectively, released
during mineralisation of the OM (Ross, 1994; Walker
et al., 2003).
Considered together, the tissue samples collected
from the field and those of the pot experiment show that
C. album did not accumulate Cu and Pb in shoot dry
matter above 50 and 25 mg kg
1 , respectively. While
plants collected in the field could tolerate a Zn concen-
tration in shoots of 1168 mg kg
1 , concentrations in
excess of 2000 mg kg
1 appeared to be phytotoxic, under
the conditions of the pot experiment.
4. Conclusions
The cow manure was capable, in the short-term, of
preventing soil acidification and decreasing heavy metal
bioavailability, and could help to facilitate the initial re-
vegetation of soils contaminated by pyritic mine waste
and liable to acidification, such as the one at El Vicario.
The increase of soil pH caused by manure addition was
the main factor reducing metal availability. It is vital to
monitor the availability of soil heavy metals following
OM application, as changes occur with time, due to
microbiologically-mediated transformations of organic
compounds (Alm as et al., 1999; Tordoff et al., 2000).
The present results indicate that C. album could be
employed as an indicator species for this purpose,
mainly for Zn and Mn.
Acknowledgements
The authors are grateful to Dr. R.J. Froud-Williams
(School of Plant Sciences, University of Reading, UK),
223
for supplying seeds of a population of C. album from a
non-contaminated site (‘‘Reading’’), and to Angeles
Escolar and Antonia Garc
ıa, for technical assistance.
This work was financed by the Spanish Ministry of
Science and Technology (project no. REN2001-1113-
C02-02), and by the Junta de Andaluc
ıa.
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