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Environmental risk assessment of using

organic wastes as soil amendments: effects on
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Conference Paper September 2015

DOI: 10.13140/RG.2.1.1790.9849

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Environmental risk assessment of using organic wastes as soil amendments:
effects on aquatic organisms exposed via the soil-water pathways to aquatic
systems

Ana Simes
CFE - Centre of Functional Ecology, Department of Life Sciences
University of Coimbra, Coimbra, Portugal
email: acarolinafsimoes@gmail.com

Rui Ribeiro
CFE - Centre of Functional Ecology, Department of Life Sciences
University of Coimbra, Coimbra, Portugal
email: rui.ribeiro@zoo.uc.pt

Paula Alvarenga
Departamento de Tecnologias e Cincias Aplicadas
Escola Superior Agrria, Instituto Politcnico de Beja, Portugal
LEAF Centro de Investigao em Agronomia, Alimentos, Ambiente e Paisagem
Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal
email: paula.alvarenga@ipbeja.pt

Patrcia Palma
Departamento de Tecnologias e Cincias Aplicadas
Escola Superior Agrria, Instituto Politcnico de Beja, Portugal
CIMA - Centro de Investigao Marinha e Ambiental, Faculdade de Cincias e Tecnologia,
Universidade do Algarve, Faro, Portugal
email: ppalma@ipbeja.pt

Matilde Moreira-Santos
CFE - Centre of Functional Ecology, Department of Life Sciences
University of Coimbra, Coimbra, Portugal
email: matilde.santos@zoo.uc.pt

ABSTRACT
The interest in the recycling of organic wastes (OW) in agricultural soils is growing. The
present study aimed to contribute to the environmental risk assessment of using organic
wastes as soil amendments, by assessing effects on aquatic organisms. An experimental field
was amended with sewage sludge-SS, mixed municipal solid waste compost-MMSWC and
agricultural waste compost-AWC; doses were 6, 12 and 24 ton SS dry matter/ha, while
MMSWC and AWC doses were adjusted for the same amount of organic matter/ha. Soil
eluates were prepared to perform a battery of seven toxicity tests with key taxonomic and
functional species. Unexpectedly, based on chemical/microbiological legislated limits, no
toxicity was observed for all OW amendment doses, although up to a 35% D. magna feeding
inhibition was observed for all three wastes. In conclusion, most likely due to complex

Corresponding author

1
interactions occurring under natural circumstances, it is important to integrate chemical,
microbiological and ecotoxicological approaches.

KEYWORDS
Organic wastes recycling; soil amendment; legal recommended doses; soil eluates; battery of
ecotoxicity tests; short-term sublethal responses

INTRODUCTION
In the past years there has been a growing concern regarding the disposal of organic wastes
(e.g. sewage sludge, agro-industrial sludge, municipal solid waste, compost) in landfills due
to the resultant contamination of air, soil and water [1, 2, 3], with the European Union
legislation on this matter being increasingly tighter. Council Directive 99/31/EC was issued in
1999 [4] aiming to prevent and reduce the adverse environmental impacts of landfills while
defining goals to be achieved by the member states regarding the deposition of organic
residues in landfills; it targeted a reduction to 35% of the total amount of biodegradable
organic waste going to landfills in 1995 by 2020.

One of the most welcome solutions for the environmental problems caused by the deposition
of organic wastes in landfills is their recycling in agriculture as soil amendments [5, 6, 7].
Organic wastes increase the humus content of the soil and, by consequence, increase the water
retention capacity and improve the soil structure, which are fundamental soil attributes for
root penetration and appropriate drainage (porosity) and aeration [8]. Although limit values
for the most hazardous and common contaminants present in organic wastes have been
established at the European level [9] and by member states [10, 11], chemical analyses should
be integrated with ecotoxicity tests, allowing establishing cause-effect relationships between
contaminants and observed biological responses.

The present study main aim was to contribute to the environmental risk assessment of using
organic wastes as soil amendments, by assessing potential effects on aquatic organisms,
which may be exposed to contaminants and pathogenic organisms by drift, runoff, drainage or
leaching. To conduct the study under more realistic amendment scenarios, an agricultural soil
was amended with three organic wastes, sewage sludge (SS), mixed municipal solid waste
compost (MMSWC) and agricultural waste compost (AWC), and subsequently cultivated
with the ryegrass Lolium perenne. To accomplish the main study aim, aqueous extracts, i.e.,
eluates, were produced from the various solid matrices, as eluates indirectly simulate the
mobilization of contaminants via the soil-water pathways to aquatic systems [12, 13], serving
as early indicators for the contamination of soil porewater and of surface and groundwater
[12]. Then, an extensive battery of seven single-species laboratory sublethal toxicity tests was
performed, as a means to fully identify the ecological receptors potentially at most risk in
aquatic systems adjacent to agricultural soils. Results of the present study revealed a general
lack of toxicity for all tested amendment doses (6, 12 and 24 ton dry matter/ha) of all three
organic wastes, except in the toxicity test based on the growth of the microalga and on the
feeding of the cladoceran.

MATERIALS AND METHODS

Organic wastes
The three organic wastes used in the present study (SS, MMSWC and AWC) were selected
because all have important practical advantages concerning the improvement of the quality of
the soil and guaranteed logistics of transport and application on a yearly basis. A detailed

2
physico-chemical characterization of the three organic wastes was previously performed and
is presented in Table 1 [14]; it should be stressed that AWC presented a Ni concentration
slightly above legislated limits.

Table 1. Physico-chemical characterization of the three organic wastes selected for the present
study according to Portuguese legislation [11, 14].
Legal
Parameter SS MMSWC AWC
Limit [11]
pH 7.4 7.8 8.3 -
Conductivity (mS/cm) 1.23 7.19 6.12 -
OM (%) 74.3 39.5 41.3 -
N (%) 6.2 2.1 1.8 -
Agronomic C/N 5.9 9.3 11.8 -
parameters P total (% P2O5) 6.9 3.6 3.5 -
-1
K total (g K2O kg ) 14.5 10.5 45.6 -
Na (g kg-1) 1.6 32.1 29.8 -
-1
Ca (g kg ) 27.6 83.4 30.2 -
-1
Mg (g kg ) 7.9 14.0 37.5 -
Cd (mg kg-1 DM) <0.3 3.0 1.1 20
Cr (mg kg-1 DM) <5.6 14.5 99.1 1000
Cu (mg kg-1 DM) 155.8 179.5 54.9 1000
-1
Metals Hg (mg kg DM) <1.2 0.63 <0.28 16
Ni (mg kg-1 DM) 22.5 29.2 360.5 300
Pb (mg kg-1 DM) <5.6 202.3 17.4 750
Zn (mg kg-1 DM) 581.1 473.5 412.8 2500
LAS (mg kg-1 DM) 12 16 6 2600
NPE (mg kg-1 DM) <40 <40 <40 50
Organic -1
PCB (mg kg DM) <0.021 <0.021 <0.021 0.8
Compounds -1
PAH (mg kg DM) 2.23 0.532 <0.160 6
PCDD/F (ng TE kg-1 DM) 4.7 16 5 100
Escherichia coli (CFU g-1 ) 4.3 x 104 1 x 10 1 x 10 1000
Pathogenic
Salmonella spp
microorganisms Present Absent Absent Absent
(Present/Absent 50 g-1)

Agricultural soil amendment

The amendment doses were 6, 12 and 24 ton SS dry matter/ha (D6, D12 and D24,
respectively), and the amendment doses of MMSWC and AWC were calculated in order to
deliver the same amount of organic matter (OM) per unit area of soil (11, 23 and 45 ton dry
matter/ha of MMSWC and 11, 22 and 43 ton dry matter/ha of AWC). A control treatment
without organic waste amendment was also performed (D0). The lowest dose (D6) was
chosen because of being the recommended agronomic dose in Portuguese legislation [10] and
the other two doses were chosen to fully explore the use of the wastes as soil amendments,
while keeping OM increments in the soil within optimal agronomic levels. SS was chosen as
the reference for the exact doses of 6, 12 and 24 ton dry matter/ha because it was the richest in
OM. Since OM in soils greatly influences contaminants bioavailability, particularly of metals
[15], guaranteeing equal increments in OM across organic wastes within the same dose was
critical to establish more precise cause-effect relationships between waste toxicity and
organism responses.

3
The field experiment consisted in dividing an agricultural soil into four blocks (i.e., four
replicated strips of soil) separated by buffer zones, so that each block comprised one replicate
(with an area of 12.5 m2) of each of the 10 different treatments, i.e., D0, plus three doses (D6,
D12 and D24) times three organic wastes (SS, MMSWC and AWC). The total isolated area
was plowed and immediately after amended with the organic wastes. After one month of
stabilisation (T0), one composite sample of approximately 10 Kg soil wet weight was
collected from each of the 10 treatments and immediately after the soil was cultivated with
the ryegrass Lolium perenne L. After three months (T1) a second composite soil sample was
collected following exactly the same procedures.

Eluates preparation
Eluates preparation was carried out in accordance with a recommended protocol [16]. A
mixture of amended soil and distilled water (1:10 ratio, w/v, based on the solid matrice dry
weight) was shaken in a magnetic stirrer during 12 h, centrifuged at room temperature (twice
20 min at 3600g) and the supernatant collected as eluate and stored either at 4 C in the dark
to be used within three weeks of preparation or at -20 C until use (within 10 months).

To perform the ecotoxicological characterization of the amended soils, eluates were prepared
immediately upon the arrival of the composite soil samples to the laboratory, both at T0 and
T1. Only the 100% eluate concentration was tested, with all doses within each organic waste
and sampling tested simultaneously.

Ecotoxicological tests
To extensively evaluate the ecological receptors potentially at most risk in aquatic systems
adjacent to agricultural fields amended with the three organic wastes, a battery of laboratory
toxicity tests employing key taxonomic and functional species was used and is presented in
Table 2. The guidelines in which the toxicity testing procedures adopted were based are
presented in Table 3. All test organisms were obtained either from dormant stages (V. fischeri,
S. polyrhiza, B. calyciflorus, and H. incongruens) or standard laboratory cultures (P.
subcapitata, H. attenuata and D. magna).

Table 2. Detailed description of the battery of laboratory toxicity tests selected for the present
study.
Taxonomic Functional Exposure
Species Endpoint
group group period
Vibrio Luminescence
bacteria decomposer 5 min
fischeri (Lt)
Pseudokircheneriella primary Specific growth rate
microalgae 72 h
subcapitata producer (/d)
Spirodela primary Growth
macrophyte 72 h
polyrhiza producer (mm2/72 h)
Brachionus primary
rotifer 48 h Population growth rate (/d)
calyciflorus consumer
Hydra secondary Post exposure feeding
cnidarian 48 h
attenuata consumer (nauplii/hydranth/30 min)
Daphnia primary Feeding
cladoceran 24 h 5
magna consumer (x 10 cells/juvenile/24 h)
Heterocypris primary Specific growth rate
ostracod 6d
incongruens consumer (/day)

4
 Table 3. Standard guidelines or standard operational procedures followed to perform the
laboratory toxicity tests of the present study.

Ecotoxicological test Standard guidelines/Standard operational procedures

Microtox 81.9% basic test protocol for water samples

5-min V. fischeri
(http://www.sdix.com/uploadedFiles/Content/Products/Water_
luminescence
Quality_Tests/Microtox-SOLO-Manual.pdf)
72-h P. subcapitata
[17, 18]
growth
Spirodela Duckweed Toxkit standard operational procedure
72-h S. polyrhiza growth
(http://www.microbiotests.be/toxkits/Spirodelastp.pdf)
Rotoxkit F Chronic standard operational procedure
48-h B. calyciflorus
(http://www.microbiotests.be/toxkits/RotoxkitFshort-
population growth
chronicstp.pdf)
48-h H. attenuata
[19, 20]
postexposure feeding
24-h D. magna feeding
[21, 22]
test
6-d H. incongruens Ostracodtoxkit F standard operating procedure
growth (http://www.microbiotests.be/toxkits/OstracodtoxkitFstpl.pdf)

Data analysis
A two-way ANOVA or two-way nested ANOVA was conducted for each waste with time
(T0, T1) and dose (D0, D6, D12, D24) as main factors followed by planned comparisons to
test for the effects of one main factor within the other; when necessary the one-tailed
Dunnetts multiple comparison test was performed to verify the existence of significant
differences between the control dose and each amended dose within each time, whereas
differences between both time periods within each dose were assessed by the two-tailed
planned comparisons. Prior to all analysis of variance, the assumptions of normality (Shapiro
Wilks test) and homoscedasticity (Barletts test) were checked.

RESULTS
All laboratory toxicity tests fulfilled the validity criteria for control performance required in
the respective procedures. In general, no toxicity was observed for all amendment doses of the
organic wastes towards the aquatic organisms used, although waste toxicity up to around a
35% feeding inhibition was observed for D. magna for all three wastes, mainly at D12 and
D24.

Surprisingly, the growth of the microalga was significantly inhibited in D12 of MMSWC at
T1 (by 40 to 60%) and D6 of AWC, but never at D24. It is likely that such effect was
associated to the presence of pathogenic organisms (e.g. fungi) which at the high dose were
affected by the presence of contaminants. Studies focusing on decay rates of pathogenic
organisms in organic wastes treatment revealed it is dependent on factors, such as
temperature, pH, volatile fatty acids, species, available nutrients, and time, which make it
difficult to predict the presence or not of pathogens [23, 24].

5
DISCUSSION
Results from the battery of toxicity tests performed with the eluates from the soils amended
with the organic wastes SS, MMSWC and AWC revealed a general lack of toxicity for all
tested doses (D6, D12 and D24), the only exception being the feeding of the cladoceran. The
feeding of D. magna was significantly inhibited at all doses of SS (by 17-34% at T0 and 11-
17% at T1) and AWC (by 28-32% at T0, except at D6, and 13-34% at T1. For eluates from
soils amended with MMSWC significant effects on feeding were only observed at T1 (by
28% at D12 and 26% at D24). The fact that D. magna is a non-selective filter feeder able to
feed on particles in suspension, which may had adsorbed to the microalgae and may induce
inhibitions of feeding [25], can possibly explain the sensitivity of the cladoceran.

The general lack of toxicity observed at the field scale was somehow unexpected, based on
the toxicity results of the eluates prepared from the pure wastes (unpublished results), on the
high Ni concentration of AWC and on the pathogenic microorganisms content of SS (see
Table 1); in effect in every AWC amended dose the Portuguese legal limit of application of
Ni per year (3 kg/ha/year) was exceeded (around 4, 8 and 16 kg/ha/year, for D6, D12 and D24
respectively). The observed results were likely associated to the complex interactions
occurring under natural circumstances, mainly related with the overall increase of the soil OM
content and pH and with changes in physical properties, which influence contaminants
bioavailability and consequent mobility [26]. Such interactions make it hard to predict how
contaminants may evolve in the soil matrix, emphasizing the importance of the integrated use
of chemical and ecotoxicological tests.

CONCLUSIONS
In general, no toxicity was observed for either amendment doses (D6, D12 and D24),
although waste toxicity up to around a 35% feeding inhibition was observed for D. magna for
all three wastes. The general lack of toxicity observed at the field scale was unexpected, based
on the toxicity results when eluates were prepared from the pure wastes, but were likely
associated to the complex interactions occurring under natural circumstances, which make it
hard to predict how metals bioavailability and mobility in soil may evolve, and thus in
interpreting toxicity tests results. Overall it is possible to conclude that all organic wastes used
in the present study, except SS, can be used as soil amendments without posing a risk to
aquatic communities via exposure to runoff/leachate, provided recommendations and
specifications (mainly in terms of waste composition and amendment dose) defined in
legislation are followed.

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