ORIGINAL PAPER

Use of waste materials as nursery growing mediafor Pinus halepensis production

Pilar Manas • Elena Castro • Pau Vila •

Jorge de las Heras

Received: 5 May 2009 / Revised: 13 November 2009 / Accepted: 9 December 2009 / Published online: 29 December 2009

� Springer-Verlag 2009

Abstract Peat moss is gradually being replaced by other

materials as a growing medium in forest nurseries due to

economic and ecological constraints. In this study, six

different mixtures were tested, mixing peat moss (P) and

pine bark (B) with digested sewage sludge (S) activated

sewage sludge (A) and paper mill sludge (M), as growing

media for Pinus halepensis seedlings; three different waste

doses were applied. Seed germination percentage, seedling

growth and foliar nutrient content after 1 year in a green-

house and percentage survival after transplanting were

recorded. The influence of base substrate (P or B) on ger-

mination percentage changed in different ways according

the type of waste. The order of the different applied mix-

tures by suitability (germination rate and seedling growth)

from best to worst was as follows: activated sludge with

peat, activated sludge with pine bark, sewage sludge with

peat, sewage sludge with pine bark, paper mill sludge

with pine bark and finally paper mill sludge with peat.

Keywords Pinus halepensis � Sewage sludge �Paper mill sludge � Nursery � Germination

Introduction

Fertilization of nutrient-depleted and degraded forest soils

may be required to sustain human utilization of forest

resources. Thus, application of sewage sludge compost

directly on soils has been tested both in forests (Meghelli

2006; Meyer et al. 2004; Valdecantos 2005) and in pastures

(Mosquera-Losada et al. 2001). Biosolids from municipal

wastewater sludge have been widely used over recent years

(Sanchez-Monedero et al. 2004) as a soil base substrate.

Another material used recently has been paper mill sludge.

As is the case with sewage sludge, most studies consist of

paper mill sludge application directly onto soils in order to

determine the effects on plants and soils (Feldkirchner

et al. 2003; O’Brien et al. 2003). However, few studies

have reported on the use of these waste products for

growing forest seedlings (Chong and Lumis 2002; Manas

et al. 2009). In nurseries, peat and natural soils are most

commonly used as substrates for growing ornamental

plants and forest seedlings. Nevertheless, these materials

are being fully or partially replaced with various organic

waste products such as municipal solid waste compost,

sewage sludge, pine bark, sawmill residues and rice hull

(Ingelmo et al. 1998). The main reason for this change is

the high price of good quality horticultural peat, especially

in countries without natural peat moss and the uncertain

availability of peat in the near future due to environmental

constraints (Abad et al. 2001). In some European countries,

the application of compost is an alternative to the use of

inorganic fertilizers since commercial compost production

has increased and compost quality has improved (Borken

et al. 2004). It has been shown that applications of sewage

sludge (usually composted sewage sludge) and pine bark in

different proportions can improve the growth of pine

seedlings a few months after planting (Hernandez-

Communicated by A. Merino.

P. Manas (&) � E. Castro � J. de las Heras

Departamento de Produccion Vegetal y Tecnologıa Agraria-

CREA, Universidad de Castilla-La Mancha, Campus

Universitario s/n, 02071 Albacete, Espana

e-mail: mariap.manas@uclm.es

P. Vila

Facultad de Biologıa, Universidad de Murcia, Murcia, Espana

123

Eur J Forest Res (2010) 129:521–530

DOI 10.1007/s10342-009-0349-4

Apaolaza et al. 2005). However, the response depends on

the sewage sludge and mixtures used, and there is only

limited information on the use of these growing media in

forest container nurseries (Manas et al. 2009).

The main goal of this paper is to assess suitability of

some alternatives to traditional substrates used in forest

nurseries, including industrially and domestically derived

organic amendments, in order improve sustainability.

Pinus halepensis Mill., (Aleppo pine) has been selected for

this purpose, since it is one of the most important tree

species in Mediterranean forests. The aim is to evaluate the

effect of three factors in the growth of pine tree seedlings:

(1) base substrate: peat (P) or pine bark (B); (2) type of

sludge: sewage sludge (S), activated sewage sludge (A) and

paper mill sludge (M); and (3) different proportions of

sludge and standard (peat or bark) medium.

Materials and methods

Design of experiment and localization

The field trial was carried out in a 6.8 m 9 20 m green-

house located in Albacete (SE Spain). Five different

materials were used to make growing media mixtures: Two

principal base substrate: peat (P) and pine bark (B); two

waste products from wastewater treatment plants (WWTP):

sewage sludge from Albacete WWTP (S) and activated

sewage sludge from Alcazar de San Juan WWTP in Central

Spain (A); and the final residual of a waste paper (post-

consumer) manufacturing process: paper mill sludge (M).

The raw peat material was homogeneous light Sphagnum.

The peat substrate was not amended in any other way (e.g.

no liming or fertiliser added). The characteristics of the

peat substrate were as follows: 90 g l-1 dry density, 95%

pore volume, 80% water volume-10 cm and 15% air vol-

ume-10 cm. The pine bark was ground to a grain size of

around 0.5 mm. The origin of wastewater in Albacete and

Alcazar de San Juan is mixed: 70% of domestic origin and

30% industrial in both cities, approximately. Sewage

sludge from the Albacete WWTP was processed by a

trickling filter bed system with a fixed bed of plastic media

over which wastewater flows downwards and is mixed with

a layer or film of microbial slime covering the bed media.

In activated sludge produced in Alcazar de San Juan

WWTP, atmospheric air or pure oxygen is bubbled through

the primarily treated sewage (or industrial wastewater),

combined with organisms to develop a biological floc

which reduces the organic content of the sewage. Both

types of wastewater sludge were anaerobically digested

and dried to remove 70% of the water content. Paper mill

sludge is the final waste product of the paper-making

process, and the major effluent from a paper mill is a

suspension of paper fibres in water. The processing of

second-hand paper makes paper for printing and rejections

(40% of dampness). The rejections are pressed and have

solid aspect composed of 45% cellulose, 23% kaolin, 27%

calcium carbonate, 4% bentonite, talcum, feldespate and

sands and 1% of ink and other compounds used in printing

and packing paper. Plastic containers (35 cavities, each

with a capacity of 220 cm3) disinfected in water and bleach

(1:1) were filled with the following mixtures of growing

media: (1) peat (P) ? sewage sludge (S) at 10% (P-10S),

25% (P-25S) and 50% (P-50S); (2) pine bark (B) ? sew-

age sludge at 10% (B-10S), 25% (B-25S) and 50%

(B-50S); 93) peat ? activated sewage sludge (A) at 10%

(P-10A), 25% (P-25A) and 50% (P-50A); (4) pine

bark ? activated sewage sludge at 10% (B-10A), 25%

(B-25A) and 50% (B-50A); (5) peat ? paper mill sludge

(M) at 10% (P-10M), 25% (P-25M) and 50% (P-50M); (6)

pine bark ? paper mill sludge at 10% (B-10M), 25%

(B-25M) and 50% (B-50M). Seedlings were not fertilized

in any case (neither organic nor inorganic fertiliser) to

avoid potential treatment interactions. Containers filled

with only either peat or pine bark were used as controls (P

and B, respectively). For all combinations, nine containers

(replicates) were used, in total 9 9 35 = 315 cavities per

treatment. Filled containers were randomly set on benches

inside the greenhouse and one P. halepensis seed was sown

in each cavity in May 2005. Seeds were obtained from the

Alcaraz-Segura mountain Range, located in the South-

western area in the province of Albacete which had a

germination capacity of 95% (based on official seed test

results). Germination conditions were the same for all

treatments (natural daylight), and the average solar radia-

tion during the emergence period was 26.65 MJ m-1. The

pots were irrigated up to field capacity three times weekly

using a micro-sprinkler head system. Irrigation water cor-

responded to a Ca-SO4 facie and C2-S1 classification,

meaning it is medium-salinity, low-sodium water (United

States Salinity Laboratory 1954).

Experimental design, sampling and measurement

The experimental design was a nine replicate (nine con-

tainers) of eighteen different growing mixtures (eighteen

treatments) and two controls (P and B). Germination, based

on cotyledon emergence, was monitored from day 24 to

day 61 after sowing. One year after sowing, three groups of

five seedling samples from each treatment were randomly

collected to report height and collar diameter (300 seed-

lings) and analyse chemical parameters in foliar tissue.

Fifteen seedlings representing each growing medium

treatments (total 300 plants) were randomly selected and

planted in the field (2 m apart) in early May 2006

(348 days after sowing). Survival percentage of the

522 Eur J Forest Res (2010) 129:521–530

123

seedlings was recorded 2 months after planting (414 days

after sowing).

Analytical methods

Three samples of growing media mixtures were analysed

for pH, organic matter, total N, extractable P, Na, P, Zn,

Ca, Mg, Fe, Cu and Mn. Different analytical methods were

used depending on the parameter. pH was measured with a

previously calibrated CRISON GLP22 pH meter and

1:2 w v-1 suspension of soil in water. The other variables

we determined (shown with their respective methods) were

organic matter (Walkley and Black method), total N

(Kjeldahl procedure), extractable P (Olsen method), Na

and K (atomic emission spectroscopy method after an acid

digestion), and Zn, Ca, Mg, Fe, Cu, and Mn (atomic

absorption spectroscopy method).

Chemical properties based on three samples of mixtures

are shown in Tables 1, 2 and 3. Plant height was measured

from the root collar to the base of the terminal bud. For

stem diameter, two measurements were taken from each

group of five plants. Stem diameter for each plant was the

mean of two measurements, each at 90� (approx) to each

other. Foliar tissue were oven-dried at 100�C, initially for

an hour, later to 75�C for 48 h until a constant weight was

obtained. The dried samples were used to determine the

chemical concentrations in the plants (nutrients: N, P, K,

Ca, Mg; and micronutrients: Fe, Mn, Zn, Cu), using the

same approach as that described earlier for the growing

media mixtures.

Statistical procedure

A factorial ANOVA was used to study the effects of

treatment variables: base substrate, type of sludge and

different proportions. The factorial ANOVA was multi-

level in order to examine the effects of multiple indepen-

dent variables. To ensure that data came from a normal

distribution, standardized skewness and standardized kur-

tosis values were verified. Percentage values were trans-

formed by arcsine. For germination percentage, seedling

height and diameter Fisher’s least significant difference

(LSD) procedure was used to compare mean (P \ 0.05).

Results

Germination percentage

Pine seed germination (based on cotyledon emergence)

showed significant differences depending on the base

substrate (peat or bark), sludge type, the interaction

between base substrate and type of sludge as well as the

interaction between proportion and type of sludge

(Table 4). The interaction between base substrate and type

of sludge as well as the interaction between proportions

and type of sludge over germination percentage were

stronger than each factor separately.

The base substrate effect on germination percentage

varied with the type of waste used. In the case of paper mill

sludge (Fig. 1a), the germination percentage was higher in

bark than in peat mixtures, but in contrast, the germination

percentage was higher in peat mixtures when activated

sewage sludge was used (Fig. 1b) with the P-10A treatment

resulting in the highest value (86%). There was no differ-

ence between peat and bark with sewage sludge additions

(Fig. 1c). In activated sludge mixtures, germination per-

centage decreased significantly with the highest activated

sludge proportions (Fig. 1b) and the proportions of paper

mill sludge in the pine bark (Fig. 1a). There were no sig-

nificant differences according to the doses of waste in

sewage sludge mixtures (Fig. 1c).

Foliar nutrients

N content in pine needles varied significantly depending on

the base substrate type (peat or bark) and their proportions

(10, 25 and 50%), type of sludge (S, A and M) added and

the interactions between them (Table 5). In general, a

higher foliar N content in growing media mixtures with

peat was observed (Table 6). In the mixtures that contained

peat and sludge from WWTP (A and S), the highest N

content was obtained with P-25A treatment (20.1 mg g-1)

and P-50S (21.4 mg g-1); these values were two times that

of the control (P). With paper mill sludge (M) combina-

tions, N foliar content was similar or less than the control.

In pine bark mixtures, the highest nitrogen content values

were obtained at highest S and A proportions (10.5 and

18.5 mg g-1, respectively).

Base substrate and type of waste affected foliar P con-

tent (Table 5), but the impact of sludge proportions was

less clear. No differences were recorded for P-M mixtures

but additions of activated sewage sludge and sewage

sludge increased the phosphorus concentration in seedlings

grown on peat-based mixtures (Table 6). In the same way,

all additions to bark increased the phosphorus concentra-

tion compared to control, especially in A mixtures. Base

substrate and type of sludge had more influence on foliar K

content than did different proportions (Table 5), but base

substrate-proportions and base substrate-type interactions

had a significant effect also. Foliar K content in seed-

lings grown in the peat control (2.75 mg g-1) was always

higher than those in P-M, P-A and P-S (Table 6), although

this value is quite similar to others, such as P-50M

(2.67 mg g-1) or P-50A (2.72 mg g-1). This was not the

case in substrate mixtures with pine bark, where K content

Eur J Forest Res (2010) 129:521–530 523

123

Ta

ble

1C

hem

ical

char

acte

rist

ics

of

the

gro

win

gm

edia

mix

ture

sw

ith

pap

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slu

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PP

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MP

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MP

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MB

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B-2

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pH

5.1

7±

0.0

55

.91

±0

.06

6.3

6±

0.0

17

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4.9

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56

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67

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.(m

mh

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30

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20

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Org

anic

mat

ter

(%)

83

±1

2.1

27

3.5

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5.2

67

2±

15

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70

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21

.47

72

±1

.56

85

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1.5

68

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81

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5

N(%

)0

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1.2

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0.0

51

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0.0

61

.27

±0

.30

1.5

4±

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70

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0.4

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60

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P2O

5(m

gk

g-

1)

27

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75

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32

2±

87

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30

6±

23

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31

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65

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28

9±

45

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18

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25

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13

9±

36

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17

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K2O

(mg

kg

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10

79

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22

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89

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8.1

57

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56

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79

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45

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89

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91

01

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25

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69

38

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57

97

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8.8

8

CaO

(%)

0.1

7±

0.0

11

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±0

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1.7

5±

0.1

02

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.60

1.0

9±

0.2

30

.31

±0

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0.4

9±

0.0

50

.95

±0

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Mg

O(%

)0

.09

±0

.01

0.1

2±

0.0

10

.12

±0

.04

0.1

3±

0.0

10

.12

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0.0

8±

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10

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2

Na

(mg

kg

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61

6±

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73

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9.9

81

03

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65

0.5

81

18

9±

23

5.4

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78

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96

18

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5.7

87

15

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51

05

1±

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3

Fe

(mg

kg

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71

1±

66

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72

9±

75

.45

93

5±

32

8.4

71

10

5±

26

5.7

85

02

±4

7.5

64

71

±6

9.5

67

82

±8

9.2

21

20

1±

98

9.4

0

Mn

(mg

kg

-1)

6±

0.0

52

3±

0.1

22

2±

0.2

3.2

24

5±

1.6

24

4±

1.6

94

0±

5.9

83

5±

6.3

94

3±

5.6

6

Zn

(mg

kg

-1)

9±

0.0

22

7±

0.8

94

8±

5.6

87

5±

4.5

91

0±

5.1

42

4±

6.6

92

7±

4.5

83

9±

6.8

8

Cu

(mg

kg

-1)

1±

0.0

15

2±

0.0

66

7±

7.2

11

24

±1

2.5

72

±0

.66

39

±6

.54

60

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.68

11

8±

66

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C:N

13

7±

25

.87

34

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35

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.01

32

±3

.87

27

±8

.90

13

0±

25

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10

6±

57

.11

81

±5

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K:M

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10

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0.0

3±

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10

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0.5

5±

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20

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±0

.04

0.3

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0.0

5

Ca:

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29

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10

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12

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6±

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23

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78

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AB

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pH

5.1

7±

0.0

55

.65

±0

.02

7.3

6±

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36

.93

±0

.33

4.9

4±

0.4

56

.22

±5

.12

6.9

5±

6.3

27

.36

±5

.66

E.C

.(m

mh

os

cm-

1)

0.2

7±

0.0

10

.44

±0

.12

1.3

2±

0.2

10

.63

±0

.03

0.2

6±

0.0

10

.37

±0

.08

0.6

6±

0.4

40

.91

±0

.05

Org

anic

mat

ter

(%)

83

±1

2.1

26

8±

1.5

66

6.2

±6

.52

74

.7±

5.4

77

2±

1.5

67

7.9

±6

.66

73

±6

.57

67

.2±

8.6

5

N(%

)0

.35

±0

.06

1.3

7±

0.2

33

.19

±0

.33

1.9

1±

0.2

31

.54

±0

.57

0.5

1±

0.2

41

.11

±0

.12

1.7

9±

0.0

5

P2O

5(m

gk

g-

1)

27

9±

75

.15

83

1±

56

.87

14

40

4±

44

7.1

22

16

1±

55

6.3

52

89

±4

5.8

79

75

±6

5.8

74

56

6±

68

7.5

18

14

7±

54

7.5

2

K2O

(mg

kg

-1)

10

79

±1

22

.14

17

8±

68

.41

27

16

±2

22

.32

32

8±

63

.25

89

.1±

5.6

99

97

±6

6.9

81

48

5±

63

2.4

71

48

5±

25

8.4

1

CaO

(%)

0.1

7±

0.0

11

.48

±0

.23

0.8

9±

0.1

51

.95

±0

.23

1.0

9±

0.2

30

.22

±0

.13

0.3

6±

0.0

10

.55

±0

.15

Mg

O(%

)0

.09

±0

.01

0.1

6±

0.0

20

.27

±0

.05

0.2

1±

0.0

20

.12

±0

.05

0.1

1±

0.0

10

.23

±0

.06

0.2

7±

0.0

4

Na

(mg

kg

-1)

61

6±

15

.58

53

6±

85

.65

16

91

±5

6.7

46

62

±2

3.2

25

78

±2

5.6

96

23

±6

5.6

49

89

±5

6.7

41

32

7±

22

1.3

3

Fe

(mg

kg

-1)

71

1±

66

.26

20

96

±4

56

.12

85

96

±6

65

.41

46

04

±4

78

.55

50

2±

47

.56

18

83

±8

73

.32

40

07

±6

54

.78

92

88

±4

47

.54

Mn

(mg

kg

-1)

6±

0.0

53

3±

2.3

24

7±

5.8

77

1±

1.1

44

4±

1.6

94

3±

6.3

44

5±

4.5

65

1±

6.5

3

Zn

(mg

kg

-1)

9±

0.0

22

99

±6

8.1

42

68

±4

1.2

38

25

±8

7.6

51

0±

5.1

44

8±

7.6

81

08

±5

0.2

22

57

±5

4.7

8

Cu

(mg

kg

-1)

1±

0.0

15

8±

5.6

59

±1

.56

15

9±

14

.77

2±

0.6

61

3±

6.1

23

±6

.34

58

±4

.68

C:N

13

7±

25

.87

29

±6

.21

2±

2.2

23

±5

.47

27

±8

.90

88

±5

.87

38

±4

.55

22

±5

.64

K:M

g0

.63

±0

.01

0.0

5±

0.0

20

.43

±0

.22

0.0

7±

0.0

30

.03

±0

.01

0.4

6±

0.0

50

.31

±0

.07

0.2

8±

0.1

1

Ca:

Mg

1±

0.0

27

±0

.04

2±

0.1

27

±1

.33

6±

0.0

21

±0

.06

1±

0.2

21

±0

.58

Av

erag

eco

nce

ntr

atio

nv

alu

esar

eg

iven

on

ad

ryw

eig

ht

bas

is.

Pp

eat,

Aac

tiv

ated

sew

age

slu

dg

e,B

pin

eb

ark

.(n

=3

)

524 Eur J Forest Res (2010) 129:521–530

123

depended on the different components (A) and proportion

applied (B-M) so, in this case, B-M and B-S mixtures

resulted in higher values for K.

On the other hand, Ca foliar content varied with base

substrate, proportions and type of sludge, but the interac-

tion effects were not significant (Table 5). Besides, the

sludge amendment increased Ca levels in leaves with the

highest value in P-25A (10.09 ± 0.960 mg g-1) (Table 6).

In mixtures with peat, higher values were obtained in P-M

(9.16 mg g-1 in P-25M), P-A (10.0 mg g-1 in P-25A) andTa

ble

3C

hem

ical

char

acte

rist

ics

of

the

gro

win

gm

edia

mix

ture

sw

ith

sew

age

slu

dg

e

PP

-10

SP

-25

SP

-50

SB

B-1

0S

B-2

5S

B-5

0S

pH

5.1

7±

0.0

56

.35

±0

.23

6.9

1±

0.1

57

.45

±1

.56

4.9

4±

0.4

55

.86

±3

.23

7.1

2±

3.2

27

.69

±0

.23

E.C

.(m

mh

os

cm-

1)

0.2

7±

0.0

10

.62

±0

.05

0.4

5±

0.0

61

.06

±0

.02

0.2

6±

0.0

10

.33

±0

.12

0.4

3±

0.1

50

.39

±0

.06

Org

anic

mat

ter

(%)

83

±1

2.1

27

0.4

±4

.78

71

.7±

6.3

25

3.5

±5

.66

72

±1

.56

79

.1±

8.9

56

5±

3.2

17

3.8

±5

.62

N(%

)0

.35

±0

.06

1.3

7±

0.0

51

.4±

0.2

33

.2±

0.2

31

.54

±0

.57

0.3

1±

0.1

50

.63

±0

.22

1.0

8±

0.3

2

P2O

5(m

gk

g-

1)

27

9±

75

.15

73

0±

47

.89

85

4±

41

.53

92

69

±5

54

.62

28

9±

45

.87

30

0±

56

.58

59

3±

65

.23

89

1±

56

.47

K2O

(mg

kg

-1)

10

79

±1

22

.14

16

8±

23

.65

22

5±

65

.55

10

60

±7

53

.41

89

.1±

5.6

98

68

±6

9.3

21

01

3±

65

8.6

56

72

±5

6.4

7

CaO

(%)

0.1

7±

0.0

11

.82

±0

.11

1.7

7±

0.2

31

.27

±0

.26

1.0

9±

0.2

30

.24

±0

.14

1.1

9±

0.2

51

.07

±0

.01

Mg

O(%

)0

.09

±0

.01

0.1

7±

0.0

70

.16

±0

.04

0.3

1±

0.0

60

.12

±0

.05

0.0

8±

0.0

20

.14

±0

.09

0.1

2±

0.0

6

Na

(mg

kg

-1)

61

6±

15

.58

64

9±

54

.63

64

9±

56

.78

98

9±

98

.56

57

8±

25

.69

59

8±

33

.62

69

4±

63

.54

65

1±

56

.78

Fe

(mg

kg

-1)

71

1±

66

.26

42

05

±5

64

.87

43

20

±4

47

.62

47

53

±6

63

.54

50

2±

47

.56

34

86

±3

37

.15

42

04

±5

32

.66

47

53

±6

25

.47

Mn

(mg

kg

-1)

6±

0.0

58

7±

8.9

48

9±

5.6

31

02

±2

5.6

64

4±

1.6

91

00

±2

2.1

41

02

±2

1.4

71

02

±2

3.1

4

Zn

(mg

kg

-1)

9±

0.0

26

91

±4

7.5

47

25

±7

8.5

29

11

±3

2.2

21

0±

5.1

46

95

±3

6.5

97

82

±6

5.5

79

11

±9

8.5

5

Cu

(mg

kg

-1)

1±

0.0

11

60

±2

3.5

51

70

±4

7.8

72

01

±4

1.2

12

±0

.66

16

2±

56

.87

18

2±

23

.66

20

1±

63

.14

C:N

13

7±

25

.87

30

±4

.62

30

±6

.32

10

±2

.54

27

±8

.90

15

0±

14

.65

60

±1

.23

40

±4

.56

K:M

g0

.63

±0

.01

0.0

5±

0.0

20

.06

±0

.02

0.1

5±

0.0

50

.03

±0

.01

0.5

6±

0.0

60

.30

±0

.03

0.2

9±

0.0

1

Ca:

Mg

1±

0.0

28

±1

.11

8±

4.6

53

±1

.23

6±

0.0

22

±1

.16

±2

.36

6±

4.3

3

Av

erag

eco

nce

ntr

atio

nv

alu

esar

eg

iven

on

ad

ryw

eig

ht

bas

is.

Pp

eat,

Sse

wag

esl

ud

ge,

Bp

ine

bar

k.

(n=

3)

Table 4 P values for base substrate (1), doses (2), type of sludge (3)

and interactions (1)–(2), (1)–(3), (2)–(3) and (1)–(2)–(3) in emergence

percentage, height and diameter

Emergence

percentage

Height Diameter

Base substrate:

peat or bark (1)

\0.001a \0.001a 0.006a

Proportions (2) 0.128 \0.001a \0.001a

Type of sludge (3) \0.001a \0.001a \0.001a

(1)–(2) 0.199 0.339 0.105

(1)–(3) \0.001a 0.001a 0.664

(2)–(3) \0.001a \0.001a \0.001a

(1)–(2)–(3) 0.771 \0.001a \0.001a

a Denotes significant differences

(B)c d

e de d

bcb

a

0

20

40

60

80

100

%

(C)

abcababcbcabcabca

0

20

40

60

80

100

%

(A)ddd

bcaba

cda

0

20

40

60

80

100

P B P-10A P-25A P-50A B-10A B-25A B-50A

P B P-10S P-25S P-50S B-10S B-25S B-50S

P B P-10M P-25M P-50M B-10M B-25M B-50M

%

Fig. 1 Emergence of seedlings 61 days after sowing on a paper mill

sludge, b activated sewage sludge and c sewage sludge media. Means

within each medium type with the same letter are not significantly

different (P \ 0.05) according to the LSD test

Eur J Forest Res (2010) 129:521–530 525

123

P-S (8.29 mg g-1 in P-25S) compared to the control

(5.64 mg g-1). Combinations with pine bark showed the

same tendency: higher values were recorded in B-M

(8.35 mg g-1 in B-50M), B-A (7.87 mg g-1 in B-25A) and

B-S (8.05 mg g-1 in B-25S) mixtures compared with B.

The Mg foliar content values depended only on the pro-

portions and the interaction between base substrate and

type of sludge (Table 5). Pine trees grown in peat moss

mixtures (Table 6) yielded Mg content values similar to

those recorded for plants grown in bark-based mixtures. As

observed for Ca, any type of sludge favoured a greater

level of Mg in leaves and, in general, highest foliar Mg

content was recorded in 25% waste proportions except for

B-M mixtures. Mg content in P-M (Table 6) was higher

than in the control. In P-A and P-S, the highest values were

obtained in P-25A (4.71 ± 0.28 mg g-1) and P-25S

(4.55 ± 0.20 mg g-1). In B-A and B-S mixtures Mg val-

ues were higher than in the control or B-M mixtures except

B-50S. Foliar sodium content depended on the proportion

of sludge and the interaction between base substrate and

proportions. It was lower in pine bark mixtures, where the

type of sludge did not show differences among treatments

and, as seen for Ca and Mg, the highest foliar Na content

was observed in 25% waste proportions except for B-M.

Table 5 P values for base substrate (1), doses (2), type of sludge (3) and interactions (1)–(2), (1)–(3), (2)–(3) and (1)–(2)–(3) in chemical foliar

content (N, P, K, Ca, Mg, Fe, Cu, Mn and Zn)

N P K Ca Mg Na Fe Cu Mn Zn

Base substrate: peat or bark (1) 0.001a \0.001a \0.001a 0.006a 0.159 0.203 0.594 0.044a \0.001a \0.001a

Proportions (2) \0.001a 0.467 0.174 \0.001a 0.001a 0.003a 0.912 0.013a 0.001a 0.008a

Type of sludge (3) 0.002a \0.001a 0.003a 0.002a 0.467 0.159 0.007a 0.022a 0.001a \0.001a

(1)–(2) 0.050a 0.737 0.005a 0.144 0.938 0.014a 0.982 0.101 0.083 0.678

(1)–(3) 0.012a \0.001a 0.003a 0.054 0.049a 0.319 0.190 0.008a 0.976 \0.001a

(2)–(3) 0.002a 0.036a 0.127 0.175 0.108 0.479 0.053 0.009a 0.065 0.083

(1)–(2)–(3) \0.001a 0.050a 0.815 0.106 0.228 0.187 0.956 0.013a 0.034a 0.001a

a Denotes significant differences

Table 6 Average macronutrient content in seedling needles (mg g-1 dry weight) for each growing media mixture following one season’s

growth

N P K Na Ca Mg

P 11.3 ± 0.5 0.29 ± 0.05 2.75 ± 0.18 296 ± 45.1 5.64 ± 0.60 3.93 ± 0.14

P-10M 11.1 ± 0.8 0.28 ± 0.02 2.25 ± 0.03 228 ± 60.9 8.34 ± 0.49 4.12 ± 0.15

P-25M 10.8 ± 0.3 0.28 ± 0.01 2.45 ± 0.24 417 ± 128 9.16 ± 0.77 4.50 ± 0.30

P-50M 9.6 ± 0.2 0.32 ± 0.02 2.67 ± 0.06 273 ± 3.9 8.92 ± 0.46 4.44 ± 0.12

P-10A 10.5 ± 4.1 1.63 ± 0.43 2.04 ± 0.17 187 ± 72.3 7.14 ± 0.23 4.17 ± 0.12

P-25A 20.1 ± 2.9 2.42 ± 0.34 2.28 ± 0.17 393 ± 27.2 10.0 ± 0.96 4.71 ± 0.28

P-50A 11.4 ± 1.9 1.44 ± 0.13 2.72 ± 0.32 198 ± 17.0 7.97 ± 0.79 3.68 ± 0.07

P-10S 9.6 ± 1.0 1.59 ± 0.13 1.78 ± 0.18 218 ± 73.3 5.98 ± 0.33 4.54 ± 0.22

P-25S 12.4 ± 0.7 1.01 ± 0.05 2.56 ± 0.22 269 ± 44.4 8.29 ± 0.36 4.55 ± 0.20

P-50S 21.4 ± 4.1 2.70 ± 0.41 2.26 ± 0.10 225 ± 42.7 7.97 ± 0.79 3.94 ± 0.51

B 9.6 ± 0.3 0.29 ± 0.02 2.94 ± 0.03 261 ± 62.1 5.80 ± 1.24 3.56 ± 0.10

B-10M 9.7 ± 1.0 0.39 ± 0.05 3.36 ± 0.32 307 ± 35.7 7.57 ± 0.87 4.24 ± 0.18

B-25M 10.5 ± 1.0 0.34 ± 0.02 3.06 ± 0.09 229 ± 21.6 8.24 ± 0.10 3.89 ± 0.23

B-50M 9.3 ± 0.1 0.33 ± 0.02 2.67 ± 0.14 186 ± 15.6 8.35 ± 0.34 3.69 ± 0.19

B-10A 11.2 ± 1. 3.79 ± 0.71 2.15 ± 0.25 231 ± 37.8 6.21 ± 0.55 4.10 ± 0.50

B-25A 8.3 ± 0.9 3.90 ± 0.95 2.38 ± 0.26 287 ± 100 7.87 ± 0.80 4.92 ± 0.92

B-50A 18.5 ± 3.7 4.05 ± 1.00 2.30 ± 0.40 281 ± 62.6 7.63 ± 0.43 4.29 ± 0.39

B-10S 6.7 ± 0.1 1.39 ± 0.31 3.02 ± 0.70 216 ± 51.3 5.24 ± 3.20 4.14 ± 0.03

B-25S 9.8 ± 2.0 1.18 ± 0.19 3.24 ± 0.55 240 ± 78.8 8.05 ± 0.61 4.43 ± 0.40

B-50S 10.5 ± 1.8 1.35 ± 0.20 2.68 ± 0.10 200 ± 22.5 7.05 ± 1.11 3.42 ± 0.35

526 Eur J Forest Res (2010) 129:521–530

123

The levels of Cu, Mn and Zn in leaves varied with base

substrate (peat or bark), proportions (10, 25 and 50%) and

interactions between some of these factors but Fe only

showed significant differences according the type of sludge

(Table 5). Plants grown in pine bark growing medium

mixtures had higher micronutrients (Fe, Cu, Mn and Zn)

than those from the peat moss growing medium. In this

case, any type of sludge in growing media caused a lower

level of these elements than plants from control treatments

(Table 7). The proportion factor did not show a clear trend

in foliar micronutrient content, but interactions between

this factor and the others considered were stronger.

Seedling height, diameter and survival percentage

Seedling height was significantly affected by base sub-

strate, proportions of sludge, type of sludge and their

interactions but not the interaction between base substrate

and proportions (Table 4). In all these substrates, the

lowest seedling height values were recorded for paper mill

sludge (4.7 cm in the case of P-50M) (Fig. 2a), and no

treatment differences in seedling height were obtained for

those grown in B, P, P-M and B-M. Pines trees grown in A

mixtures showed greater height than those grown in other

wastes (25 cm in the case of P-25A) (Fig. 2b). In the case

Table 7 Average

micronutrients content in

seedling needles (mg kg-1 dry

weight) for each growing media

mixture one season’s growth

Fe Cu Mn Zn

P 90.26 ± 25.14 5.57 ± 2.49 92.56 ± 37.16 69.08 ± 39.73

P-10M 31.77 ± 3.92 2.73 ± 0.21 23.00 ± 11.41 27.73 ± 6.53

P-25M 24.53 ± 1.83 2.50 ± 0.08 8.73 ± 2.81 21.97 ± 0.33

P-50M 26.30 ± 1.87 2.37 ± 0.17 6.40 ± 1.26 25.70 ± 3.34

P-10A 29.87 ± 3.79 2.60 ± 0.22 2.20 ± 1.50 34.27 ± 9.53

P-25A 45.60 ± 1.07 2.37 ± 0.62 2.13 ± 0.62 18.10 ± 2.27

P-50A 29.60 ± 0.65 2.30 ± 0.16 0.01 ± 0.01 34.47 ± 1.75

P-10S 31.10 ± 3.47 2.67 ± 0.17 2.77 ± 1.86 39.50 ± 0.93

P-25S 29.23 ± 1.70 2.47 ± 0.05 2.77 ± 0.82 35.97 ± 1.88

P-50S 40.70 ± 2.27 2.13 ± 0.09 3.00 ± 0.79 23.90 ± 1.28

B 51.40 ± 13.73 3.10 ± 0.22 57.17 ± 19.68 39.50 ± 4.52

B-10M 26.77 ± 2.64 2.37 ± 0.24 39.86 ± 5.70 25.13 ± 2.71

B-25M 26.33 ± 2.09 2.40 ± 0.01 30.43 ± 4.60 25.97 ± 3.84

B-50M 25.47 ± 2.19 2.13 ± 0.17 23.20 ± 6.38 23.90 ± 2.62

B-10A 41.27 ± 4.88 2.33 ± 0.21 17.53 ± 7.01 30.23 ± 3.74

B-25A 55.13 ± 34.60 2.27 ± 0.31 29.33 ± 15.55 36.13 ± 7.96

B-50A 38.97 ± 4.62 3.20 ± 1.18 11.87 ± 4.92 29.43 ± 4.10

B-10S 32.43 ± 6.87 5.33 ± 1.59 42.63 ± 20.60 61.47 ± 8.82

B-25S 22.33 ± 0.45 2.13 ± 0.33 12.30 ± 3.99 40.23 ± 7.69

B-50S 35.53 ± 17.45 3.23 ± 0.29 5.03 ± 2.00 50.07 ± 7.17

(A)

abc a bcd ab ab

05

1015202530

cm

(B)

bc

ede

bcb

cd

05

1015202530

cm

(C)

a a

dc

e

b b

e

05

1015202530

cd d d

P B P-10M P-25M P-50M B-10M B-25M B-50M

a a

P B P-10A P-25A P-50A B-10A B-25A B-50A

P B P-10S P-25S P-50S B-10S B-25S B-50S

cm

Fig. 2 Height (cm) on a paper mill sludge, b activated sewage sludge

and c sewage sludge media. Means within each medium type with the

same letter are not significantly different (P \ 0.05) according to the

LSD test

Eur J Forest Res (2010) 129:521–530 527

123

of sewage sludge mixtures (S), the highest values

were recorded for plants grown with high proportions

(P-50S: 16.7 cm and B-50S: 17.1 cm) of sewage sludge

(Fig. 2c).

The results for diameter growth were similar to that

described for height. In general, the lowest values were

recorded in P, B, P-M and B-M (Fig. 3a), and the highest

values were obtained in seedlings growing in P-A and B-A

mixtures (Fig. 3b). In the case of sewage sludge mixtures,

P-10S and B-50S recorded highest diameters (2.7 and

2.9 mm, respectively) (Fig. 3c).

Figure 4 shows that survival percentage of pine seed-

lings after 2 months in the field differed depending on the

growing media used to grow them in the nursery. For

seedlings grown in the unaltered substrates (controls),

those grown on peat survived better (87%) than those

grown in bark. Mixtures containing paper mill sludge

(Fig. 4a) resulted in the lowest survival, lower than for the

controls (P and B). In contrast, plants grown in a mixture

containing waste paper water sludge (Fig. 4b, c) had sur-

vival levels up to 80%, such in those grown in P-A, B-A

and B-S mixtures (75–100%).

Discussion

Germination percentage

Germination may be more closely related to water avail-

ability and physical properties of the growing media, such

as porosity and particle size, than any other properties

(Bunt 1988). Although the factors that affect P. halepensis

seed germination are still not well known, several authors

have noted that the availability of nutrients and the litter

type have no significant effects (Broncano et al. 1998). The

additions of sewage sludge and paper mill sludge improved

the hydrophysical properties of peat moss and pine bark

(Manas et al. 2009), but they did not significantly improve

germination. On average, P-A growing media resulted in

the highest germination. In the case of B-A mixtures,

germination percentage decreased when the proportion of

activated sludge increased (B-10A [ B-25A [ B-50A).

This could be because this waste has a high nutritional

content and perhaps some chemical element or other factor

not detected in the analyses inhibited germination. In

contrast, a high proportion of paper mill sludge (P-M)

(A)

ab bc d cd ab b ab a

0

1

2

3

4

5

mm

(B)

a a

b

cde

bcb

de

0

1

2

3

4

5

mm

(C)

a a

db

cb b

d

0

1

2

3

4

5

P B P-10M P-25M P-50M B-10M B-25M

P B P-10A P-25A P-50A B-10A B-25A B-50A

P B P-10S P-25S P-50S B-10S B-25S B-50S

mm

B-50M

Fig. 3 Diameter (mm) on a paper mill sludge, b activated sewage

sludge and c sewage sludge media. Means within each medium type

with the same letter are not significantly different (P \ 0.05)

according to the LSD test

(A)

0

20

40

60

80

100

%

(B)

0

20

40

60

80

100

%

(C)

0

20

40

60

80

100

P B P-10M P-25M P-50M B-10M B-25M B-50M

P B P-10A P-25A P-50A B-10A B-25A B-50A

P B P-10S P-25S P-50S B-10S B-25S B-50S

%

Fig. 4 Survival percentage of Pinus halepensis seedlings grown on apaper mill sludge, b activated sewage sludge and c sewage sludge

media 2 months after planting in the field

528 Eur J Forest Res (2010) 129:521–530

123

resulted in high germination rates, maybe because this

mixture contains the highest proportion of inert material

with a large store of readily available water.

Foliar nutrients

Many nutrient ions remain in the growing medium, most

existing as cations. These nutrient ions remain in the

medium solution until plant roots take them up and utilize

them for growth and tissue maintenance or, because of

their positive electrical charge, they can become adsorbed

into negatively charged areas situated in certain types of

growing medium particles (Landis 1990; Neuschutz et al.

2006).

In this study, the mixtures with sewage and paper sludge

increased pH (to neutral values) and a higher nutrient

content depending on the composition of the mixtures and

doses. Previous tests showed than activated sludge pro-

duced in the WWTP of Alcazar de San Juan contributes to

an increase in pine needle N and P content (Manas et al.

2009). The high content of N, P and K in the P-25A

medium was also associated with high contents of these

ions in the needle tissue of seedlings grown on this med-

ium, suggesting that nutrient availability is high.

According to the classification of the macronutrient

concentration for the Pinus genera suggested by the FFCC

in the European continent (Stefan et al. 1997), the results

obtained for N fall into class 1 (B12 mg g-1) in most

cases. Only P-25S (12.4 mg g-1) can be classified as class

2 (12–17 mg g-1); and P-25A (20.1 mg g-1) and P-50S

(21.4 mg g-1) as class 3 ([17 mg g-1). The suitable range

for N concentration in P. halepensis needles suggested by

Bergmann (1993) and Furst (1997) is 10–20 mg g-1, and

the results obtained for P-S indicate an increase in the N

content as the sludge proportion is increased.

The results for P content were similar to those obtained

for N. According to Stefan et al. (1997), the suitable range

of P content in P. halepensis needles is 1–2 mg g-1, and

the results obtained for P-A, P-S and B-S were generally

within this range (it is important to note that the ranges

mentioned are specifically given for mature trees). How-

ever, the values for seedlings grown in P-M and B-M PB

were below the minimum of range. On the other hand, the

phosphorus of B-A pine needles was clearly higher than

the maximum recommended by Stefan et al. (1997). K

content was low in most cases, lower than the normal range

(3.57–6.88 mg g-1) is, and the highest values recorded for

B-10M (3.36 mg g-1) were just inside this range. How-

ever, calcium values were always within normal ranges

(3.28–7.22 mg g-1) or even higher. This could be a con-

sequence of the high presence of this cations in irrigation

water and in all the applied sludge.

One of the main risks of sewage sludge application in

plant production is the possibility of increasing micronu-

trient content in leaf tissues (de las Heras et al. 2005).

According to Stefan et al. (1997), most of the values

recorded were within the normal micronutrient range for

pine species. In the case of Fe, Cu, Mn and Zn, it is

important to note that the highest micronutrient levels in

the needles were recorded in the control mixtures (P and

B), except in the case of Zn (B-10S: 61.47 mg kg-1). In the

case of paper mill sludge, the micronutrient content in the

growing medium and in the needles were significantly

lower in relation to the control.

Seedling height, diameter and survival percentage

Differences in plant growth have been previously noted by

several authors who tested the different behaviour of soils

enhanced with sewage sludge (Siddique and Robinson

2003). In this study, peat-activated sludge mixtures showed

the highest values both for seedling height and shoot

diameter before planting, as in previous study (Manas et al.

2009). However, paper sludge media did not result in better

growth than that obtained using the standard (control)

media. This concurs with the results obtained by Chong

and Lumis (2002) in the nursery, although the growing

system was different (pot-in-pot). O’Brien et al. (2003)

showed that different applications of paper sludge on

agricultural soil could increase corn production, although it

did not increase the content of soil organic matter. In

general, use of pine bark in growing medium did not

improve seedling growth in this study. When pine bark is

fertilized with sewage sludge (or even inorganic NPK

fertilizer, as has been stated by Guerrero et al. 2002),

seedling height can be expected to increase significantly.

According to Manas et al. (2009), mixtures of activated

sewage sludge and peat mixtures are appropriate for pro-

ducing high quality pine seedlings with potential to grow

well after planting in the field. In this study, a higher

proportion of the plants grown in activated sludge (A) and

sewage sludge (S) mixtures survived after planting than

those grown in paper mill sludge (M) media. This suggests

that the reserve of nutrients supplied by wastewater treat-

ment plant wastes in the first stages in the greenhouse leads

to more vigorous plants than those grown in paper mill

sludge media that have low nutritional content.

Conclusion

In general, peat was a better substrate in the growing

medium mixtures than pine bark, and in most cases, 25%

waste plus peat yielded optimal results. For seedling

growth, foliar nutrient content and germination rate,

Eur J Forest Res (2010) 129:521–530 529

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activated sludge showed the best results compared to

sludge or paper mill sludge. The sludge produced by

wastewater treatment plants can be a source of fertilizer

since it has a significant content of N, P and organic matter

which appeared to be available to the plants, promoting

height and diameter growth in P. halepensis seedling in

this study. The activated sludge plus peat delivered the best

results, with the other media (in descending order from best

to worst) providing less favourable outcomes were acti-

vated sludge with peat, activated sludge with pine bark,

sewage sludge with peat, sewage sludge with pine bark,

paper mill sludge with peat and finally paper mill sludge

with pine bark. In this study, paper mill sludge did not

improve seedling emergence, height and diameter growth,

but waste products, such as some of those examined in this

study, show promise as an alternative for peat in container

nursery culture. Pine bark, used as a base medium, pro-

vided similar results to peat, suggesting that this medium

could be readily substituted for peat (which is a scarce,

non-renewable resource).

Acknowledgments The authors would like to thank to the labora-

tory of the Environment Science Centre (CSIC) and specifically Dr.

Alfredo Polo for help with the chemical analysis. Thanks to Stefanie

Kroll for reviewing the English text.

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