Environmental Pollution 128 (2004) 429435

www.elsevier.com/locate/envpol

Priming eects on PAH degradation and ecotoxicity during a

phytoremediation experiment
Erik J. Jonera,*, Doris Hirmannb, Oliver H.J. Szolarb, Dragana Todorovicb,
Corinne Leyvala, Andreas P. Loibnerb
a
LIMOS (Laboratoire des Interactions Microorganismes-Mineraux-Matie`re Organique dans les Sols)CNRS UMR 7131,
Henri Poincare University, Faculty of Science, PO Box 239, F-54506 Vandoeuvre-les-Nancy Cedex, France
b
IFA-Tulln, Konrad Lorenz Strasse 20, A-3430 Tulln, Austria

Received 20 February 2003; accepted 12 September 2003

Capsule: Priming eects during set-up of bioremediation laboratory experiments may largely surpass treatment eects.

Abstract
An experiment was conducted to distinguish priming eects from the eects of phytoremediation of a creosote-polluted soil. The
concentration of 13 polycyclic aromatic hydrocarbons (PAHs), and their combined soil toxicity (using four bioassays), was deter-
mined on recently excavated, homogenized soil and on such soil subjected to a time-course phytoremediation experiment with
lucerne. The results showed a high priming eect, with minor positive and synergistic eects of planting and fertilization on PAH
degradation rates. At the end of the experiment, PAH degradation reached 86% of the initial 519 mg PAHs kg 1. Two of the four
toxicity tests (bioluminescence inhibition and ostracod growth inhibition) corroborated the chemical data for residual PAHs, and
indicated a signicant reduction in soil toxicity. We conclude that priming eects can easily surpass treatment eects, and that an
unintentional pre-incubation that ignores these eects can jeopardize the full quantitative assessment of in situ bioremediation of
contaminated soil.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Bioremediation; Creosote; Ecotoxicity; Ostracod test; Polycyclic aromatic hydrocarbons

1. Introduction and in view of the forthcoming enlargement of the

Large areas of soil are polluted with recalcitrant
organic substances that pose environmental problems
due to their toxicity and tendency to disperse through
wind and water erosion. Phytoremediation is a technol-
ogy that combines low costs with ecient erosion con-
trol and biodegradation of a wide range of organic
pollutants, thus reducing the risk that these substances
represent for human health (Cunningham et al., 1997).
A wide range of parameters that inuence the eciency
of phytoremediation still remains to be identied.
Important research eorts on this area are made both in
North America and in Europe to respond to increasingly
severe standards imposed by environmental legislators,
* Corresponding author at present address: Norwegian Forest
Research Institute, Hgskoleveien 12, N-1432 Aas, Norway. Tel.:
+47-6494-9191; fax: +47-6494-2980.
E-mail address: erik.joner@skogforsk.no (E.J. Joner).
0269-7491/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2003.09.005
European Union to include former USSR associated
states that have substantial environmental problems
(van der Lelie et al., 2001).
Assessment of bioremediation eciency may be based
on spiking experiments where experimentally intro-
duced pollutants are deliberately left in contact with soil
for weeks or months to age, and thus become less
bioavailable and comparable to the same compounds in
environmental samples (Hatzinger and Alexander, 1995;
Jin et al., 1999; Roper and Pfaender, 2001). On the
other hand, the degradation of targeted compounds in
environmental samples may be attempted in experi-
ments where industrially polluted soil is used directly in
the laboratory or pilot scale reactors. The latter usually
requires that the environmental samples are homo-
genized and mixed to reduce pollutant heterogeneity.
This is a necessity to avoid an excessively high number
of samples and analysis for initial characterization and
monitoring of treatment eects, which is the only other
430 E.J. Joner et al. / Environmental Pollution 128 (2004) 429435

means to reduce variability in the resulting data. Mixing
and sieving of soil do however introduce other qualita-
tive changes than homogeneity: aggregates are broken
up, air is introduced into the soil, organisms and nutri-
ents are brought into contact, volatiles are lost, etc.
These changes all contribute to a priming eect where
degradation is boosted (Joner et al., 2002; Kuzyakov et
al., 2000). In this situation one has two possibilities for
initiation of an experiment: either start up immediately
after homogenization to assess treatment eects (inocu-
lation, nutrient addition, planting or others) that may
coincide with the priming eect, or wait and initiate the
experiment at a later time when the priming eect has
ceased. The former has the disadvantage that a large
priming eect may mask smaller treatment eects, while
the latter has the disadvantage of losing the most reac-
tive or bioavailable parts of the pollutant in question for
which the treatment may have the largest eect. Under
eld conditions, where priming eects are largely
absent, the imposed treatments may be eective, but
this can perhaps not be demonstrated convincingly in
the laboratory due to confounding priming eects.
We have addressed the question of priming eects
during phytoremediation in a time-course experiment
using a sub-soil from an old, abandoned railroad sleeper
treatment plant contaminated with polycyclic aromatic
hydrocarbons (PAHs). Plants were introduced either
immediately after soil homogenization, or after a delay
of 5 weeks. Besides the impact of planting, the eects of
added mineral nutrients were addressed in a factorial
design. Priming and treatment eects were assessed as
changes in residual PAH concentrations in soil and as
changes in soil toxicity using a range of bioassays. These
were followed up to 15 weeks at four points in time and
compared with abiotic controls where biodegradation
was blocked with a respiration inhibitor.
2. Materials and methods
2.1. Experimental soil
A sub-soil (from the vadose zone and deeper than 2
m) was excavated from a former railroad sleeper plant
in Austria where PAH contaminants had been seques-
tered under largely anaerobic conditions since a large
accidental creosote spill during World War II. The
duration of shipment, storage (4  C) and preparation of
the soil was minimized, and the experimental treatments
imposed within 4 days. Soil preparation included air
drying to ca. 50% of the water-holding capacity, sieving
(< 2 mm) and mixing, after which samples were taken
for PAH analyses and initial ecotoxicity measurements.
Some physical and chemical data on the experimental
soil are presented in Table 1.
2.2. Experimental design

Table 1 The experiment comprised two overlapping parts,

Characteristics of the experimental soil at the start of the experiment
each with a full factorial design (Fig. 1). Factors inclu-
Texture ded in both parts were planting (plants or no plants),
Sand 34% fertilization (fertilization or no fertilization) and harvest
Silt 57%
time (5 and 10 weeks after sowing), all with ve replicates.
Clay 9%
Organic C 3.0% Abiotic controls (fertilized and unfertilized pots; each
Inorganic C 3.1% n=4) were included where the soil solution contained
Total N 0.38% 2% NaN3. The two overlapping parts were dis-
1
NH4N 0.4 mg kg tinguished by a 5-week delay in sowing, during which
1
NO3N 6.8 mg kg
1
the pots of the delayed part were watered and incubated
PO4P (in CAL extracts) 14.7 mg kg
pH (in 0.01 M CaCl2) 7.2
Water-holding capacity 75%

PAHs (no. of aromatic rings)

Fluorene (3) 31.6 mg kg 1
Phenanthrene (3) 94.9 mg kg 1
Anthracene (3) 101.3mg kg 1
Fluoranthene (4) 140.3mg kg 1
Pyrene (4) 78.8 mg kg 1
Benz[a]anthracene (4) 18.3 mg kg 1
Chrysene (4) 19.2 mg kg 1
Benzo[b]uoranthene (5) 11.3 mg kg 1
Benzo[k]uoranthene (5) 4.2 mg kg 1
Benzo[a]pyrene (5) 10.0 mg kg 1
Benz[a,h]anthracene (5) 0.0 mg kg 1
Benzo[g,h,i]perylene (6) 5.7 mg kg 1 Fig. 1. Graphic presentation of the experimental design with symbols
Indeno[1,2,3-c,d]pyrene (6) 3.8 mg kg 1 representing harvest times for each treatment. Vertical dotted lines indi-
Sum of 13 PAHs 519.4 mg kg 1 cate samples that are common for two treatments. NF=not fertilized,
F=fertilized.
 E.J. Joner et al. / Environmental Pollution 128 (2004) 429435
under the same temperature and humidity conditions as
their counterparts sown at the start of the experiment.
2.3. Preparation and maintenance
Pots lined with polyethylene bags were lled with
moist soil equivalent to 700 g dry weight, sown with 10
pre-germinated seeds of lucerne (Medicago sativa L.)
and brought to 75% of water-holding capacity, using
deionized water or a nutrient solution (Hewitt, 1953).
Pots were maintained in a growth chamber at this water
content by adding water or nutrient solution by weight
three times per week. Growth chamber conditions
included 350 mmol m 2 s 1 photosynthetically active
radiation, a day/night cycle of 16/8 h at 21/18  C and
70% relative humidity (RH).
2.4. PAH analysis
PAHs were extracted from soil using Soxhlet extrac-
tion with CHCl3 (10 g soil, 4 h, cumulative percolation
volume > 2 l), and analysed on an HPLC (Hewlett
Packard 1050) tted with a 250 mm C-18 Vydac col-
umn, using 3D uorescence detection (HP 1100) as
described by Szolar et al. (2002).
2.5. Toxicity tests
Soil elutriates were prepared from moist soil (10 g dry
weight) shaken with 25 ml double distilled water for 24
h on an orbital shaker (8 rev min 1) at room temper-
ature. The soil suspension was transferred to glass cen-
trifuge test tubes (Corex1, USA) and centrifuged
(2000g, 30 min, 20  C). The clear supernatant was used
immediately for bioassays. The bioluminescence assay
(LUMIStox luminescent bacteria test, Dr. Lange, Dus-
seldorf, Germany) was performed according to the
manufacturer (DIN, 1993). Briey, it employed dupli-
cate measurements on 0.8 ml elutriate (pH 78) from
each sample (n=4) amended with 2% NaCl and 0.2 ml
of a bacterial (Vibrio scheri NRRL-B-11177) suspen-
sion incubated at 15  C (cooled using LUMIStherm
LTG 053) for 30 min. Bioluminescence was measured
with a luminometer (LUMIStox LPG 259) and lumi-
nescence inhibition recorded relative to a 2% (w/v)
NaCl control solution.
An algae test was carried out according to Environ-
ment Canada (1992), using Pseudokirchneriella sub-
capitata (previously Selenastrum capricornutum) and a
modied Gorhams medium (dela Cruz, 2001) contain-
ing mineral nutrients. An algal stock solution was culti-
vated in asks containing modied Gorhams stock
medium diluted 1:10, placed on an orbital shaker (90
rev min 1) in an incubation chamber (75 mmol m 2 s 1,
14/10 h light/dark cycle, 23  C, 50% RH). Inoculum
was taken from a pre-culture set up 4 days in advance.
431
The inner 60 wells of transparent 96-well microtiter
plates (LUMITRAC 600, Greiner Labortechnik, Aus-
tria) were lled with 205 ml double distilled water (con-
trol) or soil elutriate, 5 ml stock medium, and 10 ml algal
inoculum, yielding an initial concentration of 1.0104
cells ml 1. Six wells were lled for each sample. Periph-
eral wells were lled with 220 ml water. The microtiter
plates with lids and packed in transparent plastic bags
were placed on a glass plate continuously illuminated
from below (40 mmol m 2 s 1) and incubated for 72 h
(25  C). Algal growth was measured with a cell counter
(SYSMEX EUROPE GmbH, Norderstedt, Germany),
and toxicity reported as percentage growth inhibition
relative to the controls. Tests were considered as valid if
the number of algal cells in the control had increased by
a factor of more than 16, and pH did not change by
more than 1.5 units.
The ostracod test (OSTRACODTOXKIT FTM) was
purchased from MicroBioTests Inc., Nazareth, Bel-
gium. This test was originally developed for sediment
testing, but in this study used with PAH-contaminated
soil. Ten recently hatched ostracods (Heterocypris
incongruens) were transferred into each well of a 12-well
plate containing an algal suspension (provided) as food
supply. Instead of the prescribed 300 ml sediment (man-
ufacturers procedure), 400 mg soil were placed in each
well. Uncontaminated soil from Lower Austria served
as a control. Four measurements were made per repli-
cate sample. After 6 days at 25  C in the dark, surviving
organisms were counted and length increment mea-
sured. Toxic eects are reported as percent mortality
and percentage growth inhibition.
2.6. Statistical tests
Plant data and PAH concentrations were subjected to
ANOVA and dierences between treatments tested with
Fischers PLSD test. Toxicity data were compared and
tested with Students t-test or with MannWhitney Rank
Sum test.
3. Results
Initial toxicity of the soil was high, but within the
range where changes could be detected, according to all
the applied toxicity tests (Lumistox test: 54% inhibition,
algae test: 91% inhibition, ostracod tests: 88% mortal-
ity and 67% growth inhibition, Fig. 3). During the rst
5 weeks, the total concentration of PAHs fell drasti-
cally, and only between 24 and 38% (124197 mg kg 1)
of the initial amounts remained, depending on treat-
ment (Fig. 2). The proportion remaining was lowest for
PAHs with four aromatic rings (1215%), followed by
the three-ring PAHs (2855%). Far less change in the
concentration of higher molecular weight PAHs was
432 E.J. Joner et al. / Environmental Pollution 128 (2004) 429435

observed after 5 weeks (ca. 87% remained). The lowest
PAH concentrations after 5 weeks were observed in the
treatment that had been planted and fertilized, while the
highest concentrations were found in the treatment
without plants or added mineral nutrients. Dierences
between treatments were largest for three-ring PAHs,
and least for six-ring PAHs. Two out of four bioassays
indicated no change in toxicity during the rst 5 weeks
(the algae growth test and ostracod mortality test),
while the Lumistox test showed half the initial biolumi-
nescence inhibition, and the ostracod growth test
showed a reduction from 67 to 48% inhibition (data for
fertilized and non fertilized treatments were mostly not
signicantly dierent, and only the former are pre-
sented; Fig. 3). During the rest of the experiment, PAH
concentrations continued to decrease, though at a
slower rate. The lowest total concentration attained
after 10 weeks was 94 mg kg 1 (planted/fertilized-treat-
ment), and the lowest concentration after 15 weeks was
74 mg kg 1 (observed in two treatments; non-planted/
fertilized, and planted/fertilized). The concentration of
four-ring PAHs in the most ecient treatments repre-
sented only 5% of the abiotic control after 15 weeks,
while three-, ve- and six-ring PAHs represented 17, 62
and 87% of their control values, respectively. The abio-
tic control treatment contained 90% of the initial PAHs
after 15 weeks, and the main part of the loss (8%) was
due to a reduced concentration of three-ring PAHs.
The general trend of bioluminescence inhibition
resembled the time-dependent decrease in total PAH
concentration, with a steep and signicant fall during
the rst 5 weeks, and less changes during the rest of the
experiment. A single signicant treatment eect was
observed with this test: at 15 weeks non-planted/ferti-
lized soil had higher bioluminescence inhibition than
fertilized soil supporting plants from 5 to 15 weeks. The
ostracod growth inhibition test showed a similar sig-
nicant decrease in toxicity with time, though the rela-
tive changes were smaller. The algae growth test showed
a fairly constant, high (8594%) inhibition throughout
the whole experiment, while the ostracod mortality test
showed a high (7199%) toxicity at the two rst har-
vests and a steep drop at 10 weeks, increasing again
towards the end of the experiment. No treatment eects
were observed on toxicity according to the ostracod
mortality test, whereas the algae growth test indicated a
lower toxicity in the planted versus unplanted treat-
ments at 5 weeks (P=0.021, data not shown), and in the

Fig. 2. Concentrations of PAHs with 3, 4, 5 and 6 aromatic rings (see Table 1) in soil subjected to incubation with (P) or without (NP) plants and
with (F) or without (NF) additional mineral nutrients for 15 weeks. Plants were either sown immediately (010) or after a 5 week delay (515) and
harvested after 10 weeks growth. Abiotic controls were incubated with 2% sodium azide (NaN3). Bars are S.D., n=5.
 E.J. Joner et al. / Environmental Pollution 128 (2004) 429435 433

two planted/fertilized treatments relative to non-planted hand, the factors that were radically changed following
soil at 10 weeks (P=0.036, data not shown). excavation and sieving, like an increase in O2 avail-
Initial plant growth was slow, but higher for fertilized ability and a decrease in the content of volatiles with
than for non-fertilized treatments during the rst 5 potentially negative impact on biological activity, may
weeks (Table 2). At 10 weeks, plants grown without have impeded pollutant degradation in situ. The bioas-
fertilization were small and stunted, while plants receiv- says and normal appearance of plant seedlings at the
ing mineral nutrients were 38 times bigger. At the last start of the experiment indicated that such inhibitory
harvest (15 weeks), plants aged 10 weeks were twice as eects were not prohibitive of biological activity after
big as plants aged 10 weeks harvested at the second excavation, sieving and homogenization. Apparently, a
harvest. Root densities in soil were proportional to shoot compatible microora existed or established rapidly in
mass, but only fertilized plants grown from 5 to 15 the soil during preparation. This is commonly observed
weeks were close to exploiting the entire soil volume (Allard et al., 2000; Kastner et al., 1998), even though
(results not shown). Roots were examined for mycor- we expected that a prevailing anaerobiosis in situ would
rhizal colonization, but no mycorrhizal structures were
detected. Table 2
Plant growth on a creosote-polluted soil as a function of growth
period and mineral nutrient addition (S.D. in parentheses, n=5)
4. Discussion
Growth period Plant dry weight
The soil used in the present experiment was char- (weeks)
acterized by a very high initial PAH dissipation due No. nutrients Nutrients
added (mg) added (mg)
to priming eects, and low additional eects in response
to the imposed treatments. This was obviously because 05 68 (10) 125 (43)
neither readily available C (from root exudates) nor 010 203 (128) 1635 (108)
510 121 (16) 372 (88)
mineral nutrients were limiting for PAH degradation
515 428 (339) 3683 (204)
following excavation and homogenization. On the other

Fig. 3. Soil toxicity assessed in samples taken during phytoremediation of creosote-polluted soil (only data for treatments receiving mineral nutri-
ents are presented) using four toxicity tests. Bars are S.D. (or 95% condence limits for Ostracod mortality), n=4.
434 E.J. Joner et al. / Environmental Pollution 128 (2004) 429435
limit the priming eects during the build-up of aerobic
PAH degrading microorganisms. Such a lag phase was
either very short, or unnecessary due to anaerobiosis
being facultative.
Another surprising observation was that PAHs were
highly bioavailable, in spite of the old age of the con-
tamination (> 50 years). This contradicts the general
consensus that aging of creosote/PAHs in soil reduces
the proportion that is prone to biodegradation (Allard
et al., 2000; Breedveld and Karlsen, 2000; Hatzinger and
Alexander, 1995). Organic matter was present as a
matrix for irreversible sorption, but the high capillarity
and near-saturated hydrologic conditions may have
moderated aging eects severely.
Faster and more exhaustive dissipation of PAHs,
irrespective of molecular size, was observed with this
soil compared with other creosote-polluted soils descri-
bed in the literature (e.g. Allard et al., 2000; Phillips et
al., 2000; Sayles et al., 1999) or other soils we have
assessed for remediation using plants (Joner et al., 2001,
2002, Joner and Leyval, 2003). This was particularly
evident for four-ring PAHs, which are normally less
reactive and biodegradable than lighter PAHs. Even the
concentration of ve-ring PAHs was reduced sig-
nicantly over a relatively short time (38% in 15 weeks),
with little or no eect of co-substrates provided by root
exudates. This is unusual (Joner et al., 2002, Sayles et
al., 1999), and the lack of dierences between planted
and unplanted treatments even more so, as ve-ring
PAHs are almost exclusively degraded by co-metabo-
lism (Cutright and Lee, 1994; Wilson and Jones, 1993)
for which root-derived C in exudates is an excellent
primary substrate (Banks et al., 1999; Cunningham et
al., 1997). Here, no such eect of roots was observed. In
fact, the only eect of plants that was observed was a
transitory increase in the dissipation rate of three-ring
PAHs.
While planting has frequently been reported as an
ecient means of enhancing degradation of PAHs in
soil (Anderson et al., 1993; Reilley et al., 1996; Schwab
and Banks, 1994), the addition of mineral nutrients has
given more variable results. Both negative (Kastner et
al., 1998; Johnson and Scow, 1999; Carmicheal and
Pfender, 1997) and positive (Liebeg and Cutright, 1999;
Phillips et al., 2000; Walworth et al., 1997) eects of N
and P on degradation of single PAHs have been repor-
ted. In the case of PAHs originating from creosote pol-
lution, Phillips et al. (2000) observed a positive eect
only of P amendments, with no or negative eects of N
or N+P. Breedveld and Sparrevik (2000), on the other
hand, noted a positive eect of N+P on degradation of
four-ring PAHs in a creosote polluted soil, but none on
three-ring PAHs. In our study, the results indicated the
opposite tendency for three- and four-ring PAHs, but
no dierences were statistically signicant for non-
planted treatments. Even the coupling of planting and
fertilization only had transitory positive eects, which
were limited to three-ring PAHs. Whether the pre-exist-
ing level of inorganic nutrients may explain these dif-
ferences, or the outcome depends on secondary eects
of osmotic stress and altered soil water potential (Wal-
worth et al., 1997) still remains to be resolved.
The two bioassays that reected PAH dissipation in
the present experiment (Lumistox and ostracod growth
inhibition) are both rapid, sensitive and relatively inex-
pensive, and they demand small sample volumes com-
pared to traditional soil tests with, e.g. earthworms.
While the Lumistox test has previously been used with
good results on polluted soils (e.g. Frische, 2003), the
ostracod test has to our knowledge not previously been
applied to soils. Experiments with other PAH-polluted
soils (Hirmann, 2003) do however indicate that it pro-
vides valuable information if included in toxicity
assessments. Within this study, the endpoint growth
inhibition allowed a more reliable estimation of the
toxicity compared to mortality, and data suggest ostra-
cod growth being an appropriate parameter for mon-
itoring biological remediation processes. A broader
screening of soils using this method would still be
necessary before it could be recommended for general
use in testing of contaminated soils.
The present results show that the magnitude of prim-
ing eects may largely surpass proper bio-treatment
eects, and that it is important to describe the changes
that take place during the initial phases of a soil reme-
diation eort. An unusually high dissipation was
observed during an initial priming phase, whether soil
was left undisturbed or subject to light perturbations
mediated by root penetration and exudation. This
extreme reactivity was most probably due to the nature
of the polluted soil that had been subject to conditions
that had been highly unfavorable for biological activity,
and stresses the necessity to assess the feasibility of
phytoremediation versus other bioremediation treat-
ments in pilot scale experiments prior to large-scale
eorts under eld conditions.
Acknowledgements
The authors beneted from a bilateral exchange grant
(Amadee 20/2002), and gratefully acknowledge this.
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