Growth, yield and solute content of barley in soils treated with sewage sludge under semiarid Mediterranean conditions

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Field Crops Research 94 (2005) 224–237

Growth, yield and solute content of barley in soils treated with

sewage sludge under semiarid Mediterranean conditions

M. Carmen Antolın a,*, Inmaculada Pascual a, Carlos Garcıa b,Alfredo Polo c, Manuel Sanchez-Dıaz a

aDepartamento de Fisiologıa Vegetal, Facultad de Ciencias, Universidad de Navarra, Irunlarrea s/n, 31008 Pamplona, SpainbDepartamento de Conservacion de Suelos y Agua y Manejo de Residuos Organicos, Centro de Edafologıa y Biologıa

Aplicada del Segura, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, SpaincDepartamento de Contaminacion y Quımica Ambiental, Centro de Ciencias Medioambientales, CSIC,

Serrano 115 dpdo. 28006 Madrid, Spain

Received 1 April 2004; received in revised form 18 January 2005; accepted 19 January 2005

Abstract

Agricultural soils from many parts of the Mediterranean region are subjected to progressive degradation. Application of

sewage sludge provides not only a means for its disposal but also improves soil fertility and physical properties, causing an

increase in crop yield. A field experiment was carried out from 1998 to 2001, to investigate the effects of sewage sludge

application to barley (Hordeum vulgare L.) var. Sunrise crops on the relationships between plant physiology and some soil

properties. Treatments were: (1) fertilization with a conventional inorganic fertiliser (M); (2) 15 t ha�1 of sludge in 1998 only

(RS); (3) cumulative sewage sludge application, i.e., repeated applications of 15 t ha�1 every year (CS); and (4) unamended soil

as control (C). Cumulative application of sewage sludge to barley crop increased grain yield significantly, which might be

associated with improved early establishment of seedlings. The plants had higher dry matter yields and leaf protein

concentrations from the beginning of development to ear emergence. These CS plots had lower pH, and increased total

organic C (TOC), cation exchange capacity (CEC) and DTPA-extractable heavy metals. This treatment also improved soil

microbiological properties, such as basal respiration, microbial biomass and some soil enzyme activities (urease, BAA-protease,

phosphatase and b-glucosidase), which promote the recycling of nutrients for crop. Sewage sludge had a positive but short

residual effect after only 1-year application. Results indicate that relatively low application rates of sewage sludge could be used

for several years to maintain crop production in Mediterranean-type climates. However, there was a significant increase of grain

heavy metal concentrations that must be taken into consideration under long-term applications of sludge.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Barley; Leaf solutes; Sewage sludge; Soil enzymes; Yield; Heavy metals

* Corresponding author. Tel.: +39 948 425600; fax: +39 948 425649.

E-mail address: [email protected] (M.C. Antolın).

0378-4290/$ – see front matter # 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.fcr.2005.01.009

M.C. Antolın et al. / Field Crops Research 94 (2005) 224–237 225

1. Introduction

The use of wastes in agriculture and for land

reclamation is increasingly being identified as an

important issue for both soil conservation and residual

disposal. Most sewage wastes contain valuable

nutrients that could be used to improve soil fertility.

Agricultural practices often leads to a gradual

decrease in soil organic matter content, with the

consequent decrease of soil fertility through an

impoverishment of the physical, chemical and

biological properties of soils (Perucci et al., 1997).

This problem is especially serious in Mediterranean

climate zones where high temperatures during the

summer promote high annual mineralization of

organic matter (Garcıa et al., 2000). In Spain, the

abundance of carbonate-rich soils, with their low

organic matter content, favours the application of

sewage sludge as an organic amendment and nutrient

supply to soil with a relatively small risk of pollution

(Navas et al., 1998; Garcia-Gil et al., 2004).

Many papers have been published on the beneficial

effects of sewage sludge amendment on crop yield and

some soil physical and chemical properties, such as

improved soil structure, increased soil moisture and

porosity, provision of plant nutrients, increased humus

content and cation exchange capacity (Logan et al.,

1997; Navas et al., 1998; Barzegar et al., 2002; Speir

et al., 2003). Incorporation of organic materials, such

as sewage sludge, into soil also promotes its biological

activity (Saviozzi et al., 1999). Microbial activity and

soil fertility are generally closely related because it is

through the biomass that the mineralization of the

important organic elements (C, N and P) occurs (Ros

et al., 2003). However, the main problems of an

excessive application of sewage sludge are plant

toxicity due to accumulation of heavy metals in soils

(Jarausch-Wehrheim et al., 1999; McGrath et al.,

2000) but also the increase in its salt content (Hao and

Chang, 2003).

The possible combinations of soil types and plant

species are very large, thus the variety of both plants

and soils must be considered for the optimum use of

sewage sludge as a fertiliser (Schmidt, 1997). For

example, some studies indicated that sewage sludge

application could be useful for cereal crops grown in

different soils (Barbarick and Ippolito, 2000; Christie

et al., 2001; Barzegar et al., 2002). In addition, it has

also been reported that continued sludge application in

arid zones, with neutral to slightly alkaline soils, has

merits as a potential fertiliser due to relatively low

plant metal uptake under these conditions, especially

when the sludge came from an agricultural area

(Unger and Fuller, 1985).

Therefore, the general objective of this work was to

study sewage sludge application to barley crops

growing in an alkaline, degraded soil, characteristic of

most of the Mediterranean climate zones. The work

was carried out over a 3-year period with the aim to

understanding relationships between plant physiology

and some soil properties under field conditions.

Finally, the relationship between the content of

specific organic solutes in leaves and some aspects

of plant yield will be discussed.

2. Material and methods

2.1. Experimental design

Field experiments were carried out for 3 years

(1998–2001) in an experimental field in Larraga,

Navarra (northern of Spain) (latitude: 4283303100N;longitude: 185104100W) at a mean altitude of 450 m

above sea level. The climate is semiarid Mediterra-

nean (Papadakis, 1966) with an average annual

rainfall of 500 mm, occurring mostly in autumn and

spring and a mean annual temperature of 13 8C(Fig. 1). The soil was classified as Gypsic Haploxerept

(Soil Survey Staff, 1998) with a composition of 26.7%

sand, 51.1% silt and 22.3% clay. The sewage sludge

was collected at Larraga wastewater plant, which

processes wastewater corresponding to 2667 person

equivalents per year. The sludge had been anaerobi-

cally digested and dried to 30% of dry matter. The

main characteristics of the soil and sludge are shown

in Table 1.

A fully randomised design with three replications

within treatments was employed. Field plots

(3 m � 15 m each one) were sown with winter barley

(Hordeum vulgare L.) cv. Sunrise. Treatments were:

(1) fertilization normally applied to this crop,

consisting of a basic fertiliser (30 kg N ha�1,

70 kg P2O5 ha�1 and 90 kg K2O ha�1) applied at

sowing (November) and 80 kg N ha�1 in January

(M); (2) 15 t ha�1 (dry matter basis) sewage sludge in

M.C. Antolın et al. / Field Crops Research 94 (2005) 224–237226

Fig. 1. Monthly rainfall and mean temperature (8C) during the threeyears of the experiment in Larraga (Spain). Included are mean

temperature (8C) and total rainfall in the season (mm) for each

year during the growing period of barley.

October of 1998 (residual sludge, RS); (3) 45 t ha�1

sewage sludge as three repeat applications of 15 t ha�1

every year (cumulative sludge, CS); and (4) una-

mended soil as a control (C).

Table 1

Chemical characteristics of soil and sewage sludge

Parameters Soil Sewage sludge

pH (H2O) 8.3 7.9

Electrical conductivity (dS m�1) 0.4 1.2

Total organic carbon (g kg�1) 7.6 183

Available N (mg kg�1) 8.1

Available P (mg kg�1) 26.5

Available K (mg kg�1) 159

Total N (g kg�1) 22.2

Total P (g kg�1) 16.6

Total K (g kg�1) 4.7

Mn (mg kg�1) 345 226

Zn (mg kg�1) 54.4 731

Cu (mg kg�1) 14.3 205

Pb (mg kg�1) 16.0 80.5

Cd (mg kg�1) 0.2 <3.0

Cr (mg kg�1) 29.2 52.0

Ni (mg kg�1) 19.4 <25.0

The plots were ploughed each year to incorporate

the fertiliser or sewage sludge into the soil. Ploughing

was done in opposite directions in alternate years to

limit the mixing of the soil in adjacent plots to the

edges of the plots. Sewage sludge was applied with a

spreader and rotovated into the top 20 cm of soil 1-

month before planting. Seeds of barley were sowed

each year in the first week of November (seed density:

200 kg ha�1). The crops were grown using all

recommended inputs of herbicides for optimum yield.

During crop development, four sampling times

were used: at the four-leaf development stage (Zadoks

scale 14) (Zadoks et al., 1974); at ear emergence

(Zadoks scale 49); at anthesis (Zadoks scale 65); and

at grain maturity (Zadoks scale 94). The crop was

harvested in the third week of June. Soil samples (0–

20 cm) were taken randomly using a soil probe

(3.6 cm diameter, 18 cm deep). Each soil sample

consisted of a mixture of 10 soil cores selected from

each plot at harvest. All plant and soil samples were

collected from the central part of each plot to avoid

edge effects.

2.2. Soil measurements

Plant residues and stones were removed from soil

before use. Field moist samples of each plot were

bulked, mixed and split into two sub-samples. One

sub-sample was air dried, sieved (<2 mm) and stored

for subsequent chemical analysis. Another sub-sample

was sieved (<2 mm) and stored at 2 8C for biological

and biochemical determinations.

Soil and sludge pH were analysed in H2O

suspensions (1:2.5, w/v, in soil and 1:6 in sludge).

Electrical conductivity (EC) was measured in 1:1 and

1:6 water extract for soil and sludge, respectively.

Total organic C (TOC) was determined by oxidation

with K2Cr2O7 in a concentrated H2SO4 medium

and measurement of dichromate excess using

(NH4)2Fe(SO4)2 (Yeomans and Bremmer, 1989).

Cation exchange capacity (CEC) was determined by

the sodium acetate method (Bower et al., 1952).

Available N (N–NO3� and N–NH4

+) was extracted

with 1 M KCl and determined spectrophotometrically

in the filtered extracts. The N–NO3� was measured

after mechanical shaking for 2 h at 50 8C, by the

difference in absorbance between 220 and 270 nm.

The N–NH4+ was quantified by the phenol


hypochlorite method measuring the absorbance at

640 nm (Solorzano, 1969).

Microbial biomass C was determined by the

fumigation-extraction method (Vance et al., 1987).

Respiration rates were measured in hermetically sealed

flasks, in which a 50-g soil sample moistened to 60% of

theirwaterholdingcapacity,waskept inthedarkat28 8Cfor 14 days. The CO2 emitted was measured with an

infrared gas analyser (Ros et al., 2003).

Urease activity was measured as the amount of

NH4+ released from 0.5 g soil after incubation for

90 min with the substrate urea (6.4%) at 30 8C, in2 ml of borate buffer (0.1 M, pH 10) (Kandeler and

Gerber, 1988). Protease activity was measured as the

amount of NH4+ released from 0.5 g soil after

incubation for 90 min with the substrate N-a-

benzoyl-L-argininamide (0.03 M) at 40 8C, in 2 ml

of phosphate buffer (0.1 M, pH 7) (Nannipieri et al.,

1980). Phosphatase and b-glucosidase activities

were measured by spectrophotometrical determina-

tion at 398 nm of the amounts of p-nitrophenol

released from 0.5 g soil after incubation at 37 8C for

90 min with the substrates p-nitrophenyl phosphate

disodium (0.115 M), and p-nitrophenyl-b-D-gluco-

pyranoside (0.05 M), respectively, in 2 ml of maleate

buffer (0.1 M, pH 6.5) (Masciandaro et al., 1994).

The reaction was stopped by cooling at 2 8C for

15 min. Following this, 0.5 ml of 0.5 M CaCl2 and

2 ml of 0.5 M NaOHwere added and the mixture was

centrifuged at 2000 � g for 5 min. For each enzyme

assay, a blank experiment was performed which

consisted of adding the substrate to the soil sample

after incubation and immediately prior to stopping

the reaction.

For total heavy metal concentrations, plant and soil

samples were digested first with 65% HNO3 and 72%

HClO4 (Walst, 1971) and then, with 40% HF. The

‘plant available’ metal concentrations in soil were

determined after extraction with 0.005 M DTPA

(Lindsay and Norvell, 1978). All plant and soil

material digests and soil extracts were analysed for

Cd, Cu, Cr, Mn, Ni, Pb and Zn using inductively

coupled plasma mass spectrometry (ICP-MS).

2.3. Plant measurements

At each sampling time, 10 plants per plot were

collected and rapidly frozen �80 8C until analysis.

Measurements of organic solutes were made on the

youngest fully mature leaves of the main shoot. All

analyses were carried out in duplicate in each

sample.

Samples of 0.1 g of fresh leaves were ground in

an ice-cold mortar and pestle containing

potassium phosphate buffer (50 mM, pH 7.5).

The homogenates were filtered through four layers

of cheesecloth and centrifuged at 3500 � g at 4 8Cfor 15 min. The supernatant was collected and

stored 4 8C for protein, total soluble sugars (TSS)

and proline determinations (Irigoyen et al., 1992).

Leaf solute protein was measured by the protein

dye-binding method of Bradford (1976) using

bovine serum albumin as a standard. Total soluble

sugars (TSSs) were analysed by reacting 0.25 ml of

the supernatant with 3 ml of freshly prepared

anthrone and placing in boiling water for 10 min.

After cooling, the absorbance at 625 nm was

determined in a spectrophotometer (Yemm and

Willis, 1954). Free proline determination of the

supernatant was measured by reacting 1 ml of

the supernatant with 5 ml of freshly prepared

ninhydrine and placed in boiling water for

45 min. Free proline was estimated spectrophoto-

metrically at 515 nm (Paquin and Lechasseur,

1979).

Yields were obtained by removing all plants from

three randomly selected samples of 0.5 m2 in each

plot at harvest. Plant dry matter was determined after

drying at 70 8C to constant weight. Plant samples for

nutrient analysis were collected at harvest and

plant N was measured using an elemental EA 1108

analyser.

2.4. Statistical analysis

For each property data were submitted to a two-

factor analysis of variance (ANOVA). The variance

was related to the main treatments (year and

amendment type) and to the interaction between

them. Means � standard errors were calculated, and

when the F ratio was significant, the least significant

difference (LSD) test was applied using the SPSS

statistical package version 9.0 for Windows 98.

Using the same data, a correlation analysis was also

calculated, to evaluate the extent of association and

its significance.


3. Results

3.1. Weather characteristics of the growing seasons

The temperature regime during the experimental

period was typical of a Mediterranean climate zone

and was quite similar in the 3 years studied (Fig. 1).

Rainfall was high during the autumn and winter

months, which is important for recharging soil water.

However, the rainfall regime was quite different over

each entire season. For instance, the 1999/2000

growing season had higher rainfall during spring,

whereas the 2000/2001 growing season had little

rainfall at this time, but had high precipitation between

October and February.

3.2. Soil properties

Sewage sludge applications affected significantly

some soil chemical properties, e.g., pH, total organic C

(TOC) and cation exchange capacity (CEC) (Table 2).

Thus, cumulative sludge (CS) plots have decreased pH

and increased TOC and CEC. A residual effect was

observed for CEC.

Table 2

Soil chemical properties after harvest in unamended (C), mineral fertilised (

treated plotsa

Year Treatment pH EC (dS

1998/1999 C 8.6 a 0.45 a

M 8.6 a 0.55 a

RS 8.4 a 0.57 a

CS 8.4 a 0.57 a

1999/2000 C 8.5 a 0.37 a

M 8.5 a 0.35 a

RS 8.5 a 0.52 a

CS 8.3 b 0.55 a

2000/2001 C 8.6 a 0.35 a

M 8.6 a 0.51 a

RS 8.4 a 0.59 a

CS 8.1 b 0.54 a

Year *** ns

Amendment type ns ns

Interaction ns ns

In 1999, RS and CS had the same values because there was a unique trea Values are mean of three data. Within each parameter and year, means

LSD).** Significant at 0.01 probability level.*** Significant at 0.001 probability level.

The cumulative effect of multiple applications of

sludge over a 3-year period was evident in the

biochemical and microbiological soil properties at the

time of the harvest (Fig. 2). Results showed that basal

respiration, microbial biomass C and activities of

some hydrolases were significantly stimulated by

repeated sludge additions (CS).

Soil available N (N–NH4+ and N–NO3

�) always

remained significantly higher in CS plots than in other

treatments. This effect was more evident after 3 years

of sludge addition. A residual effect was observed only

in 2000 (Table 3). On the other hand, cumulative

sludge application increased soil DTPA-extractable

heavy metals especially, Cu and Zn (Table 4). This

increase became significant from the second year of

sludge addition. There were no differences in Cr and

Ni (data not shown).

3.3. Plant solutes, and yield and its components

There were no differences in the shoot dry matter of

barley between amendments treatment applied during

the first year of study (Fig. 3). However, the following

years cumulative sludge (CS) and mineral fertiliser

M), residual sewage sludge (RS) and cumulative sewage sludge (CS)

m�1) TOC (mg 100 g�1) CEC (meq 100 g�1)

0.67 a 6.67 a

0.66 a 6.45 a

0.78 a 6.66 a

0.78 a 6.66 a

0.74 ab 7.32 ab

0.71 b 5.77 b

0.89 ab 5.95 b

1.03 a 8.39 a

0.84 b 6.36 c

0.78 b 6.18 c

0.91 b 8.16 b

1.23 a 8.69 a

** **

** ns

ns ns

atment; ns: not significant.

followed by the same letter are not significantly different (p < 0.05,


Fig. 2. Basal respiration, microbial biomass C and some enzyme activities at the end of the three years of experiment in unamended (C) mineral

fertilised (M), residual sewage sludge (RS) and cumulative sewage sludge (CS) treated plots. Different letters indicate significant differences

between treatments at p � 0.05 (LSD).

Table 3

Soil and plant nitrogen concentrations in unamended (C), mineral fertilised (M); residual sewage sludge (RS) and cumulative sewage sludge (CS)

treated plots during the seasons studieda (otherwise as in Table 2)

Year Treatment Straw N (g kg�1) Grain N (g kg�1) Soil N–NH4+ (mg kg�1) Soil N–NO3

� (mg kg�1)

1998/1999 C 6.7 a 14.5 a 1.7 a 5.1 a

M 6.5 a 14.4 a 1.2 a 5.2 a

RS 6.4 a 17.0 a 1.5 a 6.4 a

CS 6.4 a 17.0 a 1.5 a 6.4 a

1999/2000 C 3.4 ab 9.1 b 1.0 b 7.2 b

M 3.5 a 12.5 a 2.6 a 8.1 b

RS 3.4 ab 12.6 a 2.3 ab 10.0 b

CS 3.1 b 12.8 a 2.3 ab 17.7 a

2000/2001 C 2.1 c 11.3 bc 2.8 b 2.0 c

M 2.9 b 11.9 ab 2.3 b 2.3 bc

RS 3.3 a 9.7 c 2.8 b 4.5 b

CS 3.9 a 13.8 a 3.7 a 10.1 a

Year *** *** *** ***

Amendment type ns ns ns ***

Interaction ** ns ns **

ns: not significant.a Values are mean of three data. Within each parameter and year, means followed by the same letter are not significantly different (p < 0.05,

LSD).** Significant at 0.01 probability level.*** Significant at 0.001 probability level.


Table 4

Soil DTPA-extractable heavy metals in unamended (C), mineral fertilised (M), residual sewage sludge (RS) and cumulative sewage sludge (CS)

treated soils during the seasons studieda (otherwise as in Table 2)

Year Treatment Cd (mg kg�1) Cu (mg kg�1) Mn (mg kg�1) Pb (mg kg�1) Zn (mg kg�1)

1998/1999 C 0.012 a 1.67 a 4.24 a 1.43 a 1.69 b

M 0.012 a 1.33 a 4.19 a 1.40 a 1.15 b

RS 0.013 a 1.51 a 5.04 a 1.29 a 6.11 a

CS 0.013 a 1.51 a 5.04 a 1.29 a 6.11 a

1999/2000

C 0.013 b 1.19 b 4.66 a 1.34 b 0.77 b

M 0.013 b 1.19 b 4.17 a 1.39 ab 0.39 b

RS 0.016 ab 1.34 b 4.27 a 1.21 b 1.52 b

CS 0.018 a 2.39 a 4.76 a 1.71 a 4.47 a

2000/2001

C 0.013 b 1.43 b 6.57 b 1.75 ab 2.41 b

M 0.016 ab 1.30 b 7.14 ab 1.60 ab 2.46 b

RS 0.016 ab 1.48 b 6.96 ab 1.36 b 3.41 b

CS 0.021 a 3.47 a 7.44 a 2.13 a 8.72 a

Year ** *** *** *** ***

Amendment type ** *** *** ** **

Interaction * *** ns ** ***

ns: not significant.a Values are mean of three data. Within each parameter and year, means followed by the same letter are not significantly different (p < 0.05,

LSD).* Significant at 0.05 probability level.** Significant at 0.01 probability level.*** Significant at 0.001 probability level.

(M) significantly increased shoot dry matter produced

with respect to control plants (C). Residual sludge

(RS) treatment led to higher shoot dry matter during

the second year but not during the third year.

In general, leaf solute content showed marked

seasonal patterns (Figs. 4–6). Leaf protein concentra-

tion strongly decreased during growth season (Fig. 4).

With the exception of first year of experiment, sludge-

treated plants had higher leaf proteins than did the C

and M plants in the first developmental stages, e.g., in

young plants (Zadoks scale 14) in 1999/2000 and at

ear emergence (Zadoks scale 49) in 2000/2001. The

greatest free proline content was found in young

plants, especially in 2000, and then, it tended to

decrease through to maturity (Fig. 5). Except in 1998/

1999, CS and M plot plants had higher proline

concentration than C plants during some of the

developmental phases. The pattern of total soluble

sugars (TSS) in leaves differed for each year assayed

(Fig. 6). Thus, significant differences were found only

in 2000/2001, where leaf TSSs were always lower in

CS plot plants than in other treatments. The TSS

concentration increased at anthesis in all treatments,

especially in C plot plants.

Yield of barley tended to increase in sludge and

mineral fertiliser amended plots in all years, but these

positive effects were more evident in 2000 (Table 5).

In consequence, the interaction between year and

amendment type was significant. Thus, yields in CS

and RS plots increased by 47 and 40%, respectively, in

comparison to unamended plots, and CS plots had

higher yield than did M plots (23%). In 2001, CS and

M plots achieved similar yields, but there was no

residual effect of the sludge in the RS plots. Most of

the yield components increased in crops of CS and M

treated plots in 2000 and 2001 seasons although each

component responded in a different way (Table 6).

Thus, there were significant interactions between

amendment type and year. The number of ears per unit

area was the component most enhanced in the CS

treated plots.

Plant N concentrations were generally higher in all

amended treatments than in C (Table 4). Grain heavy

metal concentrations varied little in CS treated plants


Fig. 3. Shoot dry matter of barley in unamended (C) mineral

fertilised (M), residual sewage sludge (RS) and cumulative sewage

sludge (CS) treated plants during growth for each year. The bars

indicate standard error (S.E.) of the mean S.E. values lower than

10% were not represented. Asterisks indicate significant differences

with respect to control plants within each year at p � 0.05 (LSD).

Fig. 4. Leaf soluble protein concentration of barley in unamended

(C), mineral fertilised (M), residual sewage sludge (RS) and cumu-

lative sewage sludge (CS) treated plants during growth for each year.

Otherwise as in Fig. 2.

for Cd, Cr, Mn, Ni and Pb (data not shown) but grain

concentrations of Cu and Zn increased significantly

(Fig. 7). These increases were evident from two

applications of sludge (2000). The residual effect was

observed only for Zn in 2001.

4. Discussion

4.1. Soil properties

Long-term sludge application improves physical

and chemical properties of soils due to the addition of

organic matter (Logan et al., 1997; Barzegar et al.,

2002; Veeresh et al., 2003). With respect to chemical

properties, our study showed that cumulative sludge

(CS) plots have decreased pH, probably due to

nitrification of N–NH4+ from the sludge (Stamatiadis

et al., 1999). In addition, CS application increased

TOC and CEC and a residual effect was observed for

CEC. This effect generally is due to the high CEC of

the sewage sludge (Saviozzi et al., 1999). It has also

been demonstrated that the annual addition of organic

amendments, such as sewage sludge, improved

fertility of degraded soils of Mediterranean climate

zones, due to positive effects on soil biological and

biochemical properties (Garcıa et al., 2000). Similarly,

our data showed that the cumulative applications of

sludge over a 3-year period had a significant impact on

biochemical and microbiological soil properties

measured at the end of experimental period. Thus,

basal respiration and microbial biomass C were

significantly stimulated in CS plots. The increase in

basal respiration can be attributed to the incorporation

of easily biodegradable organic matter and nutrients,


Fig. 5. Leaf proline concentration of barley in unamended (C),

mineral fertilised (M), residual sewage sludge (RS) and cumulative

sewage sludge (CS) treated plants during growth for each year.

Otherwise as in Fig. 2.

ig. 6. Leaf total soluble sugars (TSS) concentration of barley in

namended (C), mineral fertilised (M), residual sewage sludge (RS)

nd cumulative sewage sludge (CS) treated plants during growth for

ach year. Otherwise as in Fig. 2.

able 5

ield of barley cv. Sunrise in unamended (C), mineral fertilised (M);

esidual sewage sludge (RS) and cumulative sewage sludge (CS)

eated plots during the seasons studieda (otherwise as in Table 2)

reatments 1999 (t ha�1) 2000 (t ha�1) 2001 (t ha�1)

2.43 a 2.24 c 1.54 b

2.60 a 3.24 b 2.85 a

S 3.33 a 3.79 ab 1.84 b

S 3.33 a 4.19 a 3.41 a

ear **

mendment type ***

nteraction *

s: not significant.a Values are mean of three data. Within each column, means

ollowed by the same letter are not significantly different (p < 0.05,

SD).* Significant at 0.05 probability level.* Significant at 0.01 probability level.* Significant at 0.001 probability level.

which stimulated the indigenous soil microbiota, and

to addition of exogenous microorganisms that provide

nutrients and organic substrates that will stimulate the

soil microflora (Banerjee et al., 1997; Garcia-Gil et al.,

2004).

In general, enzyme activity of a soil depends on the

level of extracellular enzymes present, the amount of

active enzymes within dead cells and associated cell

fragments, and the level of activity associated with

living cells. Urease and protease activities are

involved in the hydrolysis of N compounds to NH4+

using urea-type and low-molecular-weight protein

substrates, respectively. Even though plants contain

urease activity, the activity in soils is considered

mainly of microbial origin (Klose and Tabatabai,

2000). The increases in urease and protease activities

in CS plots may be related to the N organic compounds

incorporated with the sludge and root exudates after

cropping, because root exudates can contain amino

acids and small peptides, which would stimulate these

F

u

a

e

T

Y

r

tr

T

C

M

R

C

Y

A

I

n

f

L

*

**


Table 6

Yield components for three consecutive annual winter barley crops

grown from 1999 to 2001a (otherwise as in Table 2)

Year Treatment Ears

(m�2)

Grains

per ear

Grain

weight (mg)

1998/1999 C 390 a 17.0 a 39.6 a

M 418 a 16.0 a 38.4 a

RS 403 a 20.0 a 42.8 a

CS 403 a 20.0 a 42.8 a

1999/2000 C 463 b 13.3 b 36.6 b

M 623 ab 13.2 b 38.3 ab

RS 562 ab 17.7 a 38.2 ab

CS 682 a 15.6 ab 39.3 a

2000/2001 C 430 c 9.8 a 37.0 b

M 664 b 10.4 a 41.2 a

RS 463 c 10.5 a 38.3 ab

CS 772 a 10.5 a 42.2 a

Year *** *** **

Amendment type *** *** **

Interaction ** * ns

ns: not significant.a Values are mean of three data. Within each parameter and year,

means followed by the same letter are not significantly different

(p < 0.05, LSD).* Significant at 0.05 probability level.** Significant at 0.01 probability level.*** Significant at 0.001 probability level.

Fig. 7. Grain Cu and Zn concentrations in barley in unamended (C)

mineral fertilised (M), residual sewage sludge (RS) and cumulative

sewage sludge (CS) treated plants. Different letters indicate sig-

nificant differences between treatments within each year at p � 0.05

(LSD).

activities (Bonmanti et al., 1985; Lynch and Whipps,

1990). Phosphatase is an enzyme responsible for

hydrolysing inorganic and organic P compounds so

that they become available to plants. Phosphatases are

substrate-inducible and the intensity of excretion by

plant root and microorganisms is determined by their

requirement for orthophosphate. In our study, the

highest activity for this enzyme was measured in CS-

treated plots and was possibly due, at least in part, to

higher enzyme activity of barley roots, as indicated by

Johansson et al. (1999). b-Glucosidase is an enzyme

that catalyses the hydrolysis of b-glucosides in soils.

This enzyme plays an important role in the decom-

position of plant remains. The increase in b-

glucosidase activity observed in CS plots was possibly

related with the increased mineralization of organic

matter added with the sludge, which may provide to

substrates for b-glucosidase. Their hydrolysis pro-

ducts (sugars) are important energy sources for

microorganisms in soils (Ros et al., 2003). In our

work all enzymatic activities studied increased

significantly in CS treated plots. This agrees with

results reported by others showing improved enzyme

activity of soil after addition of several organic

amendments including sewage sludge (Perucci et al.,

1997; Johansson et al., 1999). There were good

correlations between the activities of hydrolases and

soil biochemical properties. Thus, basal respiration

was significantly correlated with urease (r = 0.82,

p < 0.001), protease (r = 0.69, p < 0.001), phospha-

tase (r = 0.84, p < 0.01) and b-glucosidase (r = 0.68,

p < 0.001). In addition, microbial biomass C was

correlated with urease (r = 0.56, p < 0.01), protease

(r = 0.65, p < 0.01) and phosphatase (r = 0.64,

p < 0.01).

Cumulative sewage sludge application increased

soil DTPA-extractable heavy metals (Cd, Cu, Zn),

with the increases of Cu and Zn being especially

significant. This would have been responsible for

increased uptake of these elements by barley plants,

and accumulation in the grain, indicated the existence

of a notable translocation of these metals from the

vegetative to reproductive organs (Jarausch-Wehr-

heim et al., 1999; Benıtez et al., 2001). However,

although Zn and Cu seemed to be the most mobile

elements (Moreno et al., 1996; Brofas et al., 2000),

in our work their values were always lower than

the upper critical amounts established for barley


(150–520 mg g�1 Zn and 14–25 mg g�1 Cu) (Beckett

and Davis, 1977). It is important to note that there was

no proportional relationship between increased

DTPA-extractable concentrations of Zn and Cu and

increases in their amounts in grain. The alkaline pH

and high CEC of the soil could have led to strong

adsorption of these heavy metals, decreasing their

solubilization, leaching and availability to plants and

favouring their accumulation in cultivated sites as

reported by others (Saviozzi et al., 1999; Benıtez et al.,

2001).

4.2. Plant development and yield

Different (organic and inorganic) amendments

resulted in more vigorous winter barley establishment

and greater plant weights compared with plants grown

in unamended plots. The positive effect of repeated

sewage sludge applications (CS) during three con-

secutive years was particularly striking. In the case of

the residual sludge (RS) treatment, mineralization of

the added organic matter released enough nutrients to

enhance growth of plants in the following year. This

residual effect had disappeared by the last season.

Similarly, other authors have observed that the long-

term effects of a single application of sludge are

generally only observed when the addition of organic

N is large, because the organically bound N had a half-

life of 1 year (Hall, 1984).

Proteins are important for N storage in plants.

Soluble total leaf protein concentrations were high

until about anthesis; then, protein levels decreased

probably because the assimilated N is translocated

rapidly to the developing grain (Carreck and Christian,

1991). As a general trend, sludge-treated plants had

higher leaf proteins than those from C and M

treatments during the first developmental stages,

e.g., from young plants to ear emergence. This

coincided with high straw and grain N at harvest and

greater available N in soil, possibly because post-

anthesis N uptake and its contribution to grain N was

greater with increasing amounts of N applied at the

seedling stage (Bulman and Smith, 1993).

The level of free proline in plant tissues can

drastically increase as a consequence of stresses such

as drought, salinity or low temperatures (Kuznetsov

and Shevyakova, 1999; Hare et al., 1999). The low

temperatures suffered by barley during winter could

have caused the accumulation of proline in leaves

observed each year. In our study, the increase in

proline was especially notable in the 1999/2000

growing season, and could be related with more severe

conditions of low rainfall and temperature presented

during this winter. In general, leaves of CS and M

amended plants showed higher proline concentrations

than those of C during their development, which might

also be related to the greater availability of N in these

soils. It has been reported that proline can also be a

reserve of N and a source of C in plants (Chiang and

Dandekar, 1995). Thus, the higher proline content in

CS and M might represent a strategy developed to

ensure the N and C supply to the grain.

Fructose, sucrose and, in particular, fructans are the

major water-soluble carbohydrates that are accumu-

lated in winter cereals (Suzuki, 2000). In our

experiment, differences were more appreciable in

the 2000/2001 growing season, where CS plants

exhibited lower TSS amounts until anthesis than did

the other treatments. After this, TSS decreased,

probably due to their mobilization to the grain. This

suggests that CS treated plants had a more accelerated

development with a high capacity for pre- and post-

anthesis translocation of TSS to grain. On the other

hand, it should be noted that the seasonal pattern of

TSSwas quite different in each year of the experiment.

Bearing in mind that leaf TSS amounts depend

primarily on photosynthesis, we consider that seasonal

TSS concentrations can be greatly influenced by

climatic variations each year. Indeed, we have

previously showed that a combination of multiple

factors such as high temperature, soil drought and

atmospheric humidity during the grain filling period

considerably changed the contribution of leaf and ear

photosynthesis to grain filling in barley (Sanchez-Dıaz

et al., 2002).

Yield of barley increased in sludge (CS) and

mineral (M) amended plots in comparison to

unamended plots. This increased grain yield was

primarily due to increased ear number per unit of area.

Yield from CS plots was even higher than from M

plots in the 1999/2000 growing season. The higher

yields in sludge-treated crops are usually attributed to

an improvement in the soil conditions, by the supply of

additional C from the sludge (Navas et al., 1998;

Christie et al., 2001). Furthermore, regular annual

applications of sludge have a cumulative effect on


residual N and increase the pool of soil mineralizable

N (Hall, 1984). The present work shows that there

were other factors that might have contributed to the

increased yield of CS plots. First, leaf protein

concentrations measured in young plants (Zadoks

scale 14) correlated with yield (r = 0.43, p < 0.05)

and ear number (r = 0.58, p < 0.05). Second, shoot

dry matter measured at ear emergence (Zadoks scale

49) strongly correlated with final grain weight

(r = 0.74, p < 0.001) and ear number (r = 0.79,

p < 0.001). These data suggest that sewage sludge

application might induce a superior performance of

barley crop by improving early seedling establishment

(Badaruddin et al., 1999).

Many authors studying the effects of management

on soil microbial activity have avoided associating the

results with plant yield (Fraser et al., 1988). However,

in our study grain yield was positively correlated with

almost microbiological and biochemical properties of

soil, e.g., basal respiration (r = 0.54, p < 0.01),

microbial biomass C (r = 0.51, p < 0.01), urease

(r = 0.55, p < 0.01), protease (r = 0.73, p < 0.001),

phosphatase (r = 0.55, p < 0.01) and b-glucosidase

(r = 0.81, p < 0.001). These good correlations show

that a measurable association could exist between crop

yield and soil microbial activity.

5. Conclusion

Repeated application of sewage sludge to an annual

barley crop produced an increase of grain yield, which

might be associated with improved early establish-

ment of seedlings. Plants have higher dry matter and

leaf protein concentrations from the beginning of

development to ear emergence. Sewage sludge

application also improved soil chemical, microbiolo-

gical and biochemical properties, which were reflected

in an increase of barley yield. Sewage sludge had a

positive but short residual effect 1 year after.

The results of this study indicate that relatively low

application rates of sewage sludge could be used for

several years to maintain crop production in Medi-

terranean-type climates. Continued sewage sludge use

on semi-arid zones may be an attractive option,

without risks, due to lower plant uptake of heavy

metals in these soils. However, there was a significant

increase of grain heavy metal concentrations that

should be taken into consideration if long-term

applications of sludge are proposed.

Acknowledgements

Authors thank Alberto Lafarga and J. Iraneta

(ITGA of Government of Navarra, Spain) for his

valuable comments and suggestions, A. Urdiain for his

excellent technical assistance in field measurements,

and NILSA (Navarra de Infraestructuras Locales, SA)

for providing sewage sludge and sludge analysis. I.

Pascual was the recipient of grants from Ministerio de

Educacion, Cultura y Deporte of Spain (Plan de

Formacion del Profesorado Universitario) (1999–

2002) and Asociacion de Amigos de la Universidad

de Navarra (2003). The Catedra Zurich of Medio

Ambiente supported this project.

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Documents

Growth, yield and solute content of barley in soils treated with sewage sludge under semiarid Mediterranean conditions