Olive pulp and its effluents suitability for soil amendment.

Abstract

Olive pulp (OP) and its effluents produced after digestion processes were characterised and their suitability as soil amendment materials were investigated. Results showed that OP and its effluent for hydrogen (EH2) and methane production (ECH4) contain high amount of organic matter, remarkable concentration of nutrients and negligible content of heavy metals. Decreasing concentrations of low molecular weight phenols (monomeric phenols) and increasing amount of humic-like materials were found passing from OP to EH2 and ECH4. The effects on both wheat seed germination and seedlings growth were also investigated. Addition of OP at the highest doses delayed both seed germination and seedling growth. These effects decreased when the OP and its effluents were incorporated into the soil. On the contrary an enhancement of seedlings growth was detected by addition of EH2 and ECH4. Enhancement effects also were found out by addition of lower OP concentrations. The phytotoxic effects decreased when the products were incorporated into the soil.

Full text

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Journal of Hazardous Materials A138 (2006) 211–217
Olive pulp and its effluents suitability for soil amendment
A. Nastri, N.A. Ramieri, R. Abdayem, R. Piccaglia, C. Marzadori, C. Ciavatta∗
Dipartimento di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum, Universit` a di Bologna, Viale G. Fanin 40, I-40127 Bologna, Italy
Received 18 October 2005; received in revised form 3 May 2006; accepted 4 May 2006
Available online 15 June 2006
Abstract
Olive pulp (OP) and its effluents produced after digestion processes were characterised and their suitability as soil amendment materials
were investigated. Results showed that OP and its effluent for hydrogen (EH2) and methane production (ECH4) contain high amount of organic
matter, remarkable concentration of nutrients and negligible content of heavy metals. Decreasing concentrations of low molecular weight phenols
(monomeric phenols) and increasing amount of humic-like materials were found passing from OP to EH2and ECH4. The effects on both wheat
seed germination and seedlings growth were also investigated. Addition of OP at the highest doses delayed both seed germination and seedling
growth. These effects decreased when the OP and its effluents were incorporated into the soil. On the contrary an enhancement of seedlings growth
was detected by addition of EH2and ECH4. Enhancement effects also were found out by addition of lower OP concentrations. The phytotoxic
effects decreased when the products were incorporated into the soil.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Olive pulp; Phenolic compounds; Physicochemical characterisation; Seed germination; Wheat seedling growth
1. Introduction
The olive oil industry is extremely important from an envi-
ronmental sustainability point of view because of the significant
amount of wastes produced that lead to economic, technical and
organisational constraints in the adoption of an environmentally
sustainable disposal [1–3].The technology for olive oil extrac-
tionhasprogressedsignificantlysincethebeginningofthe1970,
when the three-phase centrifugation system appeared. It sepa-
rates the oil from an aqueous phase (olive mill wastewater) and
a solid phase (olive husk) in a continuous process. The main
inconvenience of this system is a large production of wastew-
aters characterised by a heavy pollutant load [2–5]. Numerous
attempts have been made in order to reduce their environmental
impact,suchastreatingthemphysically,chemicallyandbiologi-
callyorapplyingthemdirectlytosoils;neverthelessthedisposal
of wastewaters continues to be a very difficult process [4].
In the 1990, the olive oil industry has adopted a new con-
tinuous centrifugation system with a two-phase decanter, which
separatestheoilfromasolidhumidby-productcalledolivepulp
(OP), constituted by variable quantities of olive pulp, stones,
∗Corresponding author. Tel.: +39 051 2096201; fax: +39 051 2096203.
E-mail address: claudio.ciavatta@unibo.it (C. Ciavatta).
residual oil and vegetative waters [6,9]. Even though the direct
application of OP onto land is an inexpensive way of disposing
andrecyclingnutrientsandorganicmatter,itmightbeasourceof
pollution and could have an unfavourable environment impact.
Many studies have reported the toxic effects of oil by-products
on plants and soil microbial activity, because of their content of
monomeric phenolics, fatty acids and mineral salts [5–12].
An interesting alternative for a sustainable disposal of OP is
its use in energy recovery. It is a good potential energy source
because of its high polysaccharide content and can produce
ethanol, hydrogen and methane through aerobic and anaerobic
digestion [13]. However, only when the by-products remaining
at the end of the digestion processes are eliminated might their
use in agriculture as amendments be feasible.
Theaimofthisresearch,withintheUEBIOTROLL1Project,
was the chemical characterisation of olive pulp (OP) and the
effluents arising from its anaerobic digestions for the produc-
tion of hydrogen (EH2) and methane (ECH4) and to assess
their effect on the germination and the early stages of wheat
1Research funded by the European Commission (FP5, Quality of life and
management of living resources—key action 5), Contract no. QLK5-CT-2002-
02344—integrated biological treatment and agricultural reuse of olive mill
effluents with the concurrent recovery of energy sources (BIOTROLL).
0304-3894/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2006.05.108

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A. Nastri et al. / Journal of Hazardous Materials A138 (2006) 211–217
development, so as to examine whether they can be re-used in
agriculture.
2. Materials and methods
Theolivepulp(OP)wascollectedfromatwo-phasedecanter
mill and was submitted to two different fermentation processes
resulting in one effluent from hydrogen production (EH2) and
one from methane production (ECH4) [12]. Urea and disodium
phosphate were added during the biotransformation processes
to make up for N and P deficiencies. Detailed information on
the two digestion processes has been reported by Gavala et al.
[13].
Varying amounts of fresh samples of OP, EH2and ECH4
were: (i) freeze-dried to perform the chemical analyses; (ii)
stored at 4◦C for direct use as a fresh material; (iii) frozen at
−20◦C for further analysis.
2.1. Analytical methods
In order to chemically characterise OP, EH2and ECH4, the
following parameters were determined: dry matter (d.m.) con-
tent, ash content, pH, electrical conductivity (EC), total lipid
content, total content of phenolic compounds. The analyses of
the most important low molecular weight phenols (LMWP),
organic carbon (TOC), extracted organic carbon (TEC), humic-
like and fulvic-like acids (HLA+FLA), nitrogen (N), ammo-
nium and nitrates and the most representative macro and
micronutrients were also performed. The d.m. content, ash con-
tent,pHandECweredetermineddirectlyusingthefreshmateri-
als,whiletheotheranalyseswerecarriedoutonthefreeze-dried
products.
The three materials were dried in an oven at 105◦C for 24h,
toaconstantweight,forthed.m.determinationandat600◦Cfor
24hfortheashcontentanalysis.ThepHandECweremeasured
in a water extract, 3:50 (w/v) and 1:10 (w/v), respectively. The
total lipid content was determined using the Soxhlet method
with petroleum ether as extractant [14]. Total content of phe-
nolic substances, expressed as gallic acid, was determined by
means of the Folin Ciocalteau method using water+methanol
(1:1) in a ratio 1:20 (w/v) as extracting solution for the col-
orimetric determination [15,16]. Low molecular weight phe-
nols (LMWP) were characterised by HPLC after extraction at
pH 7 with a buffer solution of 0.03M EDTA–Na2+0.06M
KH2PO4+0.04MNa2HPO4·2H2Oat120◦Cfor1handacidifi-
cationwith10MHCl.Thephenoliccompoundsidentifiedwere:
oleuropein (the most important phenol in olive fruits and leaves,
but also found in a lower quantity in olive oil residues) and the
following LMWP (<350Da): hydroxytyrosol, tyrosol, vanillic
acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid,
gallicacidandvanillin;catecholwasnotseparatedduetoitshigh
photolability.Theanalyseswereperformedat280nmand25◦C,
with a variable UV–vis detector. Separations were achieved on
a Phenomenex C18 reversed-phase column (150mm×4.6mm,
5mm). A gradient of water:acetic acid (100:1, v/v) as solvent
A and methanol:acetonitrile:acetic acid (95:5:1, v/v/v) as sol-
vent B at a constant flow rate of 1.5mLmin−1as mobile phase
was used, according to the following elution program: isocratic
elution with 5%B for 2min, followed by a gradient to 25%B
over 5min, with a further stepwise gradient to 40%B at 8min,
50%Bat10minand100%Bat5min,heldfor5minandreturned
to initial conditions over 5min [17,27]. The quantification of
compounds was performed by external standard calibration.
The TOC was determined with the Springer-Klee method using
1/3M K2Cr2O7as oxidant [18]. The TEC and the HLA+FLA
were extracted in NaOH+Na4P2O70.1M; a polyvinylpyroli-
done column was used to separate the humic-like substances
from the non-humic substances, 1/3M K2Cr2O7was used as
oxidant[19,20].Thetotalnitrogencontentwasdeterminedusing
an elemental analyser. Ammonium and nitrate were measured
after extraction with hydrochloric acid (1:1)+water and steam
distillation with MgO and Devarda alloy [21]. Total phosphorus
was measured colorimetrically according to the Murphy–Riley
method [22]. The other macro and micronutrients were deter-
mined by inductively coupled plasma optical emission spec-
troscopy (ICP-OES) after digestion with nitric acid 65%.
Allresultsaremeansoftestsperformedintriplicateandhave
been reported with their standard deviation.
2.2. Germination and seedling growth
TostudytheeffectsofOP,EH2andECH4onwheat(Triticum
durum L., cultivar Duilio), two different experiments were car-
ried out: a paper study and a soil study. The three by-products
were applied at different doses, usually utilized for wheat fertil-
isation, in order to assess if there were any positive or negative
or no effects on seed germination and seedling growth of wheat,
with or without soil as growth medium. Both studies were held
in a 100mm×10mm Petri dishes, in a growth chamber settled
at 21◦C from 6 to 20h and at 15◦C from 20 to 6h, with a pho-
toperiod of 14h/10h light/dark cycle. The procedure based on
the rules for seed testing [23] was used.
2.2.1. Paper study
The experiment was carried out using a germination
paper disk as growth medium. The three fresh by-products
(OP–EH2–ECH4), adjusted to the same water content, were
spread on the germination paper at doses of 60, 120 and
180kgNha−1(standard N doses for wheat fertilisation) and at
70tonnesha−1(corresponding to: 420, 273 and 175kgNha−1
for OP–EH2–ECH4, respectively) and 80m3ha−1(correspond-
ing to: 559, 329 and 210kgNha−1for OP–EH2–ECH4, respec-
tively) of materials, and were compared to a no-nitrogen control
(C=0kgNha−1). The latter two doses, expressed like tonnes
or m3ha−1of material, correspond to Italian legal limits for
the application of sludges from agro-industrial origin (Law
no. 99/1992) to which OP was compared and for olive mill
wastewaters (Law no. 574/1996) to which EH2 and ECH4
were compared, respectively. The agronomic doses of 60, 120
and 180kgNha−1correspond to the addition of 9.4, 18.8 and
28.2tonnesha−1of fresh OP, of 15.4, 30.8 and 46.2tonnesha−1
of fresh EH2and of 24, 48 and 72tonnesha−1of fresh ECH4,
respectively. Before use, all the materials were shaken to avoid
sedimentationandensurehomogeneity.Distilledwaterwasused

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A. Nastri et al. / Journal of Hazardous Materials A138 (2006) 211–217
213
as a control (C). Soon after spreading the three by-products on
germination paper, 20kernels of durum wheat were placed in
each Petri dish. The dishes were initially kept in the dark for 4
days and subsequently the photoperiod was adjusted as previ-
ously reported. They were watered with deionised water when
necessary. The number of germinated seeds was counted daily
until the end of the germination process and then calculated as
a percentage of seeds sown. The criterion to establish germi-
nation was the emergence of a primary root and a coleoptile
the same length as the seed [23]. The seedlings were grown for
10 days after which the following parameters were recorded:
shoot height, root and shoot dry biomass (at 105◦C for 24h)
and number of abnormal seeds, i.e. those seeds characterised by
seedlings with weak development or deformed structure [23].
2.2.2. Soil study
The second experiment was carried out in addition to the
paper study, to evaluate whether the effects exerted by the three
by-products on germination and seedling growth change as a
consequence of their interactions with soil. The test was per-
formed in the same condition of the paper study (Section 2.2.1),
but in this case 50g of soil was used in each Petri dish as growth
medium. The soil was a top layer of Typic Ustipsamment soil
[24], sampled at a depth of 0–40cm from an area of Southern
Italy characterised by large-scale olive oil production. Before
use, the soil was air-dried and crushed to pass through a 2mm
sieve[25].Theanalyseswerecarriedoutaccordingtothechem-
ical methods of soil analysis [25] and the main physical and
chemical characteristics of the soil are reported in Table 1. At
thebeginningoftheexperimenttheOP–EH2–ECH4,adjustedto
the same water content, were mixed with the soil at agronomic
dosesof60,120and180kgNha−1,thatwerecomparedtoano-
nitrogen control (C=0kgNha−1). Distilled water was used as
a control. Some days before the experiment started, the soil was
moistened with deionised water up to its water holding capac-
ity (20%, p/p) to stabilise microbial activity. Twenty kernels of
durum wheat per Petri dish were placed on the soil at 0 (T0), 15
(T15), 30 (T30) and 60 (T60) days after soil treatment with the
threeoliveoilby-products,inordertoassesswhethertherewere
Table 1
Physical and chemical properties of the Typic Ustipsamment soil [24]
Parameter
Texture
Sand (%)
Silt (%)
Clay (%)
pH (pH-unit)
Electrical conductivity, EC (dSm−1)
Total nitrogen, TNK (mgkg−1)
Available P (mgkg−1)
Exchangeable K (mgkg−1)
Total organic carbon, TOC (%)
Organic matter, OM (%)
Total extracted organic carbon, TEC (%)
Humic-like substances, HLA+FLA (%)
Humification rate, HRa(%)
80
7
13
7.60
0.17
600
12.3
112
0.48
0.79
0.31
0.24
50
aHR=(HLA+FLA)/TOC×100.
any differences in phytotoxicity over time as a consequence of
their degradation. The experiment was carried out in the same
way as the paper study. The germination percentage was mea-
sured daily until the end of the germination process. After 10
days, the seedlings were removed from their substrate and were
rinsedwithwatertoremovesoilparticles.Theshootheight,root
and shoot dry biomass and number of abnormal seedlings were
then determined.
Both experiments were performed in triplicate using a com-
pletelyrandomiseddesign.Resultsweresubmittedtoaone-way
ANOVA, means were reported with their standard deviations
and compared by using the Student’s–Newman–Keuls test for
multiple comparisons.
3. Results and discussion
3.1. Chemical characterisation
The main chemical characteristics of the three by-products
are shown in Tables 2 and 3. The analyses performed on the
freeze-dried products have been expressed both for the dry mat-
ter and the fresh material.
OP has the highest dry matter content, whereas ECH4and
EH2show a significant reduction, respectively 9.8% and 4.6%,
both because OP was diluted to promote fermentation processes
andprobablybecausepartoftheorganicmatterwasusedbyfer-
mentationagentsasasubstrate(Table2).Theashcontentshows
a similar trend; however, in this case, there are no differences
betweenthetwoeffluents(Table2).OPandEH2haveacidicpH
while ECH4is moderately alkaline, probably, as a consequence
oftheadditionofureaduringtheanaerobicdigestions(Table2).
The EC is higher in OP and ECH4, whereas EH2shows a lower
value (Table 2).
Fortheremainingchemicalparametersdetermined,thethree
by-products were compared on a dry matter basis. Lipid con-
tent is higher in OP and EH2than in ECH4(Table 2). Total
phenolic compounds, TEC, HLA+FLA show an increase in
digested materials, whereas TOC quantities are similar in the
three by-products (Table 2). The trend observed suggests that
microorganisms, during the biological processes for hydrogen
and methane production, might have used a more readily avail-
able source of C (e.g. lipids, carbohydrate and polysaccharides)
as a preferential substrate probably transforming part of it into
a more recalcitrant form of C, such as phenolic and humic-like
substances. This hypothesis is also supported by the apparent
humification rate (AHR) that increases from 23% in OP to
37% in EH2and 53% in ECH4, and by the total phenolic com-
pounds (13.7% in OP, 25.7% in EH2and 32.6% in ECH4): both
parametersshowtheprogressivepolymerizationofthematerials
(Table 2).
To obtain more information on the phenolic content of the
three by-products, the most important LMWP (monomeric phe-
nols), which correspond to the most toxic phenolic fraction for
plants and soil microorganisms [8,11,26–28] were identified.
TheLMWPcontentvariesinthethreematerials(Fig.1).OPhas
the highest content of total LMWP: 2.37gkg−1d.m., whereas
EH2and ECH4contain respectively 1.22 and 0.96gkg−1d.m.

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A. Nastri et al. / Journal of Hazardous Materials A138 (2006) 211–217
Table 2
Chemical characteristics of olive pulp (OP) and effluents derived from its anaerobic digestions for the production of hydrogen (EH2) and methane (ECH4)
ParametersOP EH2
ECH4
f.m.a
d.m.a
f.m. d.m.f.m. d.m.
Dry matter (%)
Density (kgdm−1)
Ash (%)
pH
Electrical conductivity (dSm−1)
Total lipids (gkg−1)
Phenolic compounds (gkg−1)
TOC (C %)
TEC (C %)
HLA+FLA (C %)
cAHR (%)
Total nitrogen (gkg−1)
NH4-N (mgkg−1)
NO3-N (mgkg−1)
Carbon/nitrogen (C/N) ratio
Total P2O5(gkg−1)
K2O (gkg−1)
SO3(gkg−1)
28.4±0.67b
1.092±0.10
1.50±0.10
5.39±0.01
1.27±0.06
55
3.9
16.6
5.9
3.8
23
6.4
15
6.8
9.8±0.09
1.054±0.07
0.55±0.07
5.12±0.02
0.58±0.00
24
2.5
6.0
3.7
2.2
37
3.9
81
8.0
4.6±0.77
1.048±0.06
0.60±0.00
8.05±0.06
1.13±0.03
4.0
1.5
2.6
1.8
1.4
53
2.5
479
1.2
194±7.1
13.7±0.39
58.4±0.53
20.6±1.10
13.4±0.29
23
22.4±0.8
51±4
24±3
24.4
3.4±0.07
20±0.5
3.64±0.18
249±6.9
25.7±0.92
61.8±0.57
37.5±0.23
22.3±0.64
37
40±1.8
823±58
82±4
13.5
10.7±0.15
41±0.1
5.57±0.20
81±6.6
32.6±2.48
56.3±3.66
39.9±0.45
29.8±0.53
53
54±1.2
10442±1555
27±1
8.7
25.8±0.45
60±0.3
9.03±0.95
0.97
6.0
1.03
1.05
40
0.55
1.18
3.0
0.41
af.m.=fresh material; d.m.=dry matter.
b±S.D.
cApparent humification rate, AHR (%)=(HLA+FLA)/TOC×100.
(Fig. 1, sum of LMWP). Hydroxytyrosol was the most detected
compound and its content decreases from OP through EH2to
ECH4. Ferulic acid shows a similar trend, even though the
value detected is lower than hydroxytyrosol (−53%). Tyrosol
was found only in OP and ECH4(−72% than hydroxytyrosol)
while it was practically negligible in EH2, probably as a conse-
quence of the biological fermentations involved. The quantity
of the remaining LMWP is low, less than 0.10gkg−1of d.m.;
p-cumaric acid was the only LMWP that remained undetected.
It is important to note that the OP is the material poorest in total
phenolic compounds (Table 2) and richest in LMWP (Fig. 1).
The consequence is that OP could have phytotoxic effects on
higher plants mainly during germination and seedling develop-
ment, due to the enhancing action of phenolic compounds on
seed dormancy [11]. These findings supported the hypothesis
thatbothbiologicalprocesses(EH2andECH4)contributetothe
microbial polymerization of LMPW towards phenolic macro-
molecules (humic-like materials) (Table 2).
TotalNcontent(Table2)thatispredominantlyintheorganic
form increases with OP digestion. A very large quantity of
ammoniumwasmeasuredinthedigestedeffluents(Table2)due
to the addition of urea at the beginning of the fermentation pro-
cess as a source of N for the microorganisms. Nitrate content is
negligibleinallthreeproducts(Table2).TheC/Nratioishigher
in OP (24.4) and decreases in the digested samples, with values
of 13.5 in EH2and 8.5 in ECH4(Table 2); this suggests that the
Table 3
Total macro and micronutrients and heavy metal content in the three olive oil by-products (mgkg−1)
Parameters OPEH2
ECH4
f.m.a
d.m.a
f.m.d.m. f.m.d.m.
Calcium (CaO)
Magnesium (MgO)
Sodium (Na2O)
Aluminum (Al)
Cadmium (Cd)
Cobalt (Co)
Chromium (Cr)
Copper (Cu)
Iron (Fe)
Manganese (Mn)
Nickel (Ni)
Lead (Pb)
Zinc (Zn)
1588
347
60
27.5
–c
–c
–c
4.3
123
8.2
<2
<2
6
5590±125b
1220±36
214±15
97±8
–c
<1
<0.5
15±2
433±32
29±4
5±1
4±1
21±3
727
262
910
10.5
–c
–c
0.15
2.75
66.3
2.9
0.7
0.5
6.6
7425±58
2675±14
9300±110
108±0.5
–c
<1
1.5±0.0
28.1±0.2
677±2.6
30.0±0.1
7.0±0.0
5.4±0.1
67±0.1
593
152
950
64
0.023
0.09
0.14
2.1
60.5
2.3
0.5
0.2
4.5
12911 ± 95
3317 ± 19
20710 ± 360
1392 ± 10
0.5 ± 0.0
2.0 ± 0.0
3.0 ± 0.0
45.7 ± 0.4
1317 ± 12
49.2 ± 0.4
10.5 ± 0.1
4.7 ± 0.4
97 ± 0.5
af.m.=fresh material; d.m.=dry matter.
b±S.D.
cDetection limit.

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215
Fig. 1. Concentration of the various free phenolic compounds.
two digested materials, and in particular ECH4, might release
in soil inorganic N more easily than OP. The concentration of
phosphorus (Table 2) increases markedly in the digested sam-
ples, as a consequence of the addition of phosphates as nutrients
formicroorganismsduringthemicrobiologicaltransformations.
The content of potassium and sulphur follows a similar trend to
that observed for phosphorus, even though the values are lower
(Table 2).
The content nutrient (Ca and Mg) can be considered negli-
gible for crop fertilisation, while the concentration of sodium
increases in the effluents (Table 3) as a consequence of the
addition of Na2HPO4 salt before the digestions. Among the
micronutrients, only the amount of iron, present in bioavailable
forms (organic matter Fe-complexes) could be of agronomic
interest. The amount of heavy metals, including the micronutri-
ents copper and zinc, is negligible (Table 3).
3.2. Germination and seedling growth
3.2.1. Paper study
The effects of the three by-products at the various doses both
on seedling germination and number of abnormal is shown in
Fig. 2. At the beginning of the germination process, 2 days
after sowing, the percentage of germinated seedlings decreased
markedly compared to the control with OP application at doses
Fig.2. Paperstudy:effectsofolivepulp(OP)andeffluentsfromhydrogen(EH2)
andmethaneproduction(ECH4)appliedatdifferentN-dosesonthegermination
(%) and on the number of abnormal seeds (%). Histograms of the same shape
with the same letter or without letter do not differ per p<0.05 (test SNK).
Table 4
Total amount of low molecular weight phenols (LMWP) (monomeric phenols)
added with the three olive oil by-products
DoseLMWP added (kgha−1)
OPEH2
ECH4
60kgNha−1
120kgNha−1
180kgNha−1
70tonnesha−1
80m3ha−1
5.9
11.9
17.8
47.1
58.8
1.8
3.7
5.5
8.4
10.1
1.1
2.1
3.2
3.1
3.7
higherthan60kgNha−1andwithEH2at80m3ha−1(p=0.01).
At the end of germination, 4 days after sowing, no phytotoxic
effects were found with any of the treatments. These results
demonstrate that OP is able to enhances the seed dormancy [11]
when the total amount of LMWP added was around 8kgha−1
(Table 4). OP had a negative effect not only on the germina-
tion energy, but also on the number of seeds characterised by
anabnormalgrowthofseedlings.Inparticular,withtheapplica-
tionof70tonnesha−1and80m3ha−1ofproduct,corresponding
to 47.1 and 58.8kgha−1of total LMWP added, the number of
abnormal seeds increased remarkably, compared to the control
(p=0.01).
The effects of the treatments on seedling growth are reported
in Fig. 3. OP at 70tonnesha−1and 80m3ha−1doses had a neg-
ative effect on plant development: the shoot height and shoot
weight decreased compared to the control by 55% and 53%,
respectively, and the root weight decreased by 37%. No dif-
ferences from the control were observed with the other doses
(p=0.01). On the contrary, with ECH4, for all doses and EH2,
at120and180kgNha−1,evenasignificantincreaseinseedling
growth was observed compared to the control (p=0.01).
The toxicity of OP can be related to its greater content of
LMWP (Fig. 1, Table 4) that are characterised by the highest
toxic effects on plants and soil microorganisms [5,11]. Phe-
nols delay, but not completely inhibit the germination [11,29],
whereas they seem to affect seedling growth more than germi-
nation. The shoot and root parameters were markedly reduced
at both doses 70tonnesha−1and 80m3ha−1and this reduction
can only be attributed to the higher LMWP added (47.1 and
58.8kgha−1, respectively). The maximum quantity of LMWP
Fig.3. Paperstudy:effectsofolivepulp(OP)andeffluentsfromhydrogen(EH2)
and methane production (ECH4) applied at different N-doses on the shoot and
root dry biomass of the single seedling (mg) and on the shoot height (cm).

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A. Nastri et al. / Journal of Hazardous Materials A138 (2006) 211–217
Fig. 4. Soil study: effects of olive pulp (OP) and effluents from hydrogen (EH2)
and methane production (ECH4) applied at different N-doses and at different
times of wheat sowing on germination at 3 days (%).
added with ECH4and EH2(3.7 and 10.1kgha−1, respectively)
is less than the amount added with OP at 120kgNha−1.
3.2.2. Soil study
The effects of the three products applied at different doses
varied in function of wheat sowing time. At the beginning of the
germination process (Fig. 4), with sowing at T0, a significant
increase in germination energy (+35% compared to the con-
trol) was found with all treatments with the exception of EH2
at 180kgNha−1that does not differ from the control (p=0.01).
With sowing at T15 and T30 no differences among treatments
were witnessed, whereas with sowing at T60 a decrease in ger-
mination energy (−19% compared to the control) was observed
with the application of ECH4(p=0.01), probably as a conse-
quence of phytotoxic compounds released by the degradation of
the product. At the end of the germination process no signifi-
cantdifferencesamongtreatmentswereobservedatthedifferent
wheat sowing times, hence the products did not exert any nega-
tive effects on germination.
Theshootdryweightresponsetotheapplicationofthemate-
rials is shown in Fig. 5. When the wheat was sown at T0 and
at T30, a significant increase in shoot weight, compared to the
control,wasobservedwithECH4atthehighestdoses(p=0.05).
With sowing at T15 no negative effects were observed, whereas
when the wheat was sown at T60, a significant increase, com-
pared to the control, was found with ECH4at all doses (+18%)
and with EH2at 180kgNha−1(+19%) (p=0.01). Shoot height
does not vary in function of the different treatments applied.
Additionally, when the wheat was sown at T0 a significant
Fig. 5. Soil study: effects of olive pulp (OP) and effluents from hydrogen (EH2)
and methane production (ECH4) applied at different N-doses and at different
times of wheat sowing on shoot dry weight (mg).
increase of the root dry weight, compared to the control, was
observed with EH2(+37%) and ECH4(+35%) at doses of 120
and 180kgNha−1(p=0.01); no negative effects were found at
the other sowing times.
Germination and seedling development were not negatively
affected by the application of the three by-products, on the con-
trary, a stimulatory effect (biostimulant effect) on plant growth
was detected with the two effluents.
4. Conclusions
The possibility of using OP, EH2and ECH4as soil amend-
ment seems to be very promising. They have shown chemical
characteristics compatible with a potential use in agriculture,
due to the amount of organic carbon, nutrients and a negligible
concentration of heavy metals. Comparing OP to its effluents
EH2and ECH4an increase in the AHR was observed. From the
agronomic point of view the increase of AHR can be consid-
ered positive, because the addition of stable organic biomasses
improve the soil’s chemical, physical and biological properties.
Consideringtheeffectsonwheat,onlyOPathighestdosesof
70tonnesha−1and 80m3ha−1(never used for wheat) delayed,
but not inhibited, the germination and negatively affected
seedling growth. With EH2and ECH4, on the contrary, a bios-
timulant effect, with a significant increase in plant growth,
was detected. Therefore, at N doses normally used for wheat,
the three products did not exert any relevant phytotoxicity on
seed germination and seedling growth; moreover, the phyto-
toxiceffectsdecreasedwhentheproductswereincorporatedinto
the soil.
Acknowledgements
The authors would like to thank Drs. A. Simoni, L. Cavani
and D. Montecchio for their technical support and useful sug-
gestions.
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