Science of the Total Environment 444 (2013) 3242

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Science of the Total Environment

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Microbial degradation of the pharmaceutical ibuprofen and the herbicide 2,4-D in

water and soil Use and limits of data obtained from aqueous systems for predicting
their fate in soil
Cristobal Girardi a, 1, Karolina M. Nowak a, Otoniel Carranza-Diaz a, b, Benjamn Lewkow a, Anja Miltner a,,
Matthias Gehre c, Andreas Schffer d, Matthias Kstner a
a
UFZ Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstrae 15, 04318 Leipzig, Germany
b
UFZ Helmholtz Centre for Environmental Research, Department of Analytical Chemistry, Permoserstrae 15, 04318 Leipzig, Germany
c
UFZ Helmholtz Centre for Environmental Research, Department of Isotope Biogeochemistry, Permoserstrae 15, 04318 Leipzig, Germany
d
Department of Environmental Biology and Chemodynamics, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

H I G H L I G H T S

Contaminant degradation in soil and aqueous systems follows different kinetics.

Maximum mineralisable amount in soil and aqueous systems is similar.
Maximum mineralisation rates are 3.5 times faster in aqueous systems than in soil.

a r t i c l e i n f o a b s t r a c t

Article history: The persistence of chemicals is a key parameter for their environmental risk assessment. Extrapolating their
Received 30 May 2012 biodegradability potential in aqueous systems to soil systems would improve the environmental impact as-
Received in revised form 13 November 2012 sessment. This study compares the fate of 14/13C-labelled 2,4-D (2,4-dichlorophenoxyacetic acid) and ibupro-
Accepted 13 November 2012
fen in OECD tests 301 (ready biodegradability in aqueous systems) and 307 (soil). 85% of 2,4-D and 68% of
Available online 21 December 2012
ibuprofen were mineralised in aqueous systems, indicating ready biodegradability, but only 57% and 45% in
Keywords:
soil. Parent compounds and metabolites decreased to b2% of the spiked amounts in both systems. In soil,
Biodegradation rates 36% of 2,4-D and 30% of ibuprofen were bound in non-extractable residues (NER). NER formation in the abi-
Mineralisation otic controls was half as high as in the biotic treatments. However, mineralisation, biodegradation and abiotic
Risk assessment of chemicals residue formation are competing processes. Assuming the same extent of abiotic NER formation in abiotic
Non-extractable residues and biotic systems may therefore overestimate the abiotic contribution in the biotic systems. Mineralisation
Labelled compounds was described by a logistic model for the aquatic systems and by a two-pool rst order degradation model for
OECD tests 301, 307 the soil systems. This agrees with the different abundance of microorganisms in the two systems, but pre-
cludes direct comparison of the tted parameters. Nevertheless, the maximum mineralisable amounts deter-
mined by the models were similar in both systems, although the maximum mineralisation rate was about 3.5
times higher in the aqueous systems than in the soil system for both compounds; these parameters may thus
be extrapolated from aqueous to soil systems. However, the maximum mineralisable amount is calculated by
extrapolation to innite times and includes intermediately formed biomass derived from the labelled carbon.
The amount of labelled carbon within microbial biomass residues is higher in the soil system, resulting in lower
degradation rates. Further evaluation of these relationships requires comparison data on more chemicals and
from different soils.
2012 Elsevier B.V. All rights reserved.

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4-DCP, 2,4-dichlorophenol; ANOVA, Analysis of Variance; BSTFA, bis-trimethylsilyltriuoroacetamide; DegT50, degradation half
life; EA-IRMS, elemental analysis-isotope ratio monitoring mass spectrometry; EMEA, European Medicines Agency; ERA, Environmental risk assessment; GC-C-IRMS, gas chromatography
combustionisotope ratio monitoring mass spectrometry; GC/MS, gas chromatography/mass spectrometry; KOW, octanol-water partitioning coefcient; LSC, liquid scintillation counting;
MCPA, 2-methyl-4-chlorophenoxyacetic acid; MM, mineral medium; NER, non-extractable residues; PEC/PNEC, predicted environmental concentration/predicted no-effect concentration;
REACH, Registration, Evaluation, Authorisation and Restriction of Chemicals; SPE, solid phase extraction; SS, suspended solids; TOC, total organic carbon; WHC, water holding capacity.
Corresponding author. Tel.: +49 341 235 1763; fax: +49 341 235 1471.
E-mail addresses: cristobal.girardi-lavin@ufz.de (C. Girardi), anja.miltner@ufz.de (A. Miltner).
1
Present address: Fundacin Chile, Gerencia de Agua y Medio Ambiente.

0048-9697/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2012.11.051
 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
1. Introduction
According to the new European legislation, all chemicals have to pass
an environmental risk assessment (ERA) for registration and marketing
authorisation purposes in the framework of the REACH (Registration,
Evaluation, Authorisation and Restriction of Chemicals) and EMEA
(European Medicines Agency) programmes (EMEA, 2006; OECD, 2005).
Biodegradation data are essential for this evaluation since biodegrada-
tion is one of the most important processes for real removal of
chemicals from the environment. Standardised screening tests such as
the OECD 301 tests for ready biodegradability (OECD, 1992) are used
by industry, authorities and scientic institutions for characterising
degradation in the environment (Aronson et al., 2006; Boethling et al.,
1995). These tests are conducted in liquid systems for 28 days and are
supposed to allow general predictions of the biodegradation behaviour
of organic chemicals in both aquatic and terrestrial compartments
(Guhl and Steber, 2006). Ready biodegradability tests provide limited
opportunity for biodegradation and therefore a positive result in such
a test may indicate that the tested compound is biodegraded easily in
the environment (De Bruijn and Struijs, 1997). Recently, more and
more information about the interpretation of these data and their po-
tential use for prediction is available (Boethling et al., 2009, 1995;
Howard and Banerjee, 1984; Tunkel et al., 2000). However, the implica-
tions of these tests for terrestrial ecosystems still are not entirely clear
(Drer et al., 1996; Howard and Banerjee, 1984). They generally use
high chemical concentrations, standardised mineral salts media, and
the chemical as the sole source of carbon and energy. These arbitrary,
but not necessarily realistic, assumptions considerably compromise
the predictive power of such tests for real environmental compart-
ments (Ahtiainen et al., 2003).
For a realistic estimate of biodegradation and the fate of a chemical
in soils, simulation tests such as the OECD 307 (OECD, 2002) are much
more appropriate. Biodegradation in soil is a complex process which
cannot be described adequately by short-term experiments and simple
models (Drer et al., 1996). Environmental biodegradation data for
chemicals in the soil compartment have not yet been determined for
most of the commercially available chemicals (Aronson et al., 2006).
In particular, detailed mass balances as well as information about trans-
formation products and non-extractable residue (NER) formation are
missing (Boethling et al., 2009). The reason for this lack of data is the
complexity and diversity of soils, which results in higher efforts and
cost (e.g. labelled compounds) for soil tests. As abundant data are avail-
able for aqueous systems, transferring data from aqueous to soil sys-
tems would eliminate the need for numerous simulation tests. This,
however, requires a proper transfer function from the aqueous to the
soil system in order to obtain realistic estimates.
In order to start the comparison of biodegradation of pollutants in
aqueous and soil systems, mass balances of carbon turnover need to
be established in both systems, ideally by using standardised OECD
301 and 307 test protocols. For this study, two model compounds
with data on their environmental fate already available were selected:
ibuprofen (one of the most consumed pharmaceuticals) and 2,4-
dichlorphenoxyacetic acid (2,4-D; one of the most applied herbicides).
Even though 2,4-D is regarded as readily biodegradable in aqueous sys-
tems (EPA, 2010), residues are detected in surface water (IFEN, 2004).
In a rainbow trout vitellogenin assay, 2,4-D was the only pesticide out
of four which showed estrogenic activity if administered as a single sub-
stance (Xie et al., 2005). In soil biodegradation experiments, half of the
2,4-D initial concentration was mineralised after 8 days; the other half
remained as NER (Lerch et al., 2009), with unknown composition and
thus environmental risk. Results for ibuprofen biodegradation are con-
troversial. Richardson and Bowron (1985) classied ibuprofen as inher-
ently degradable in aqueous systems, whereas Quintana et al. (2005)
reported no degradation within 28 days. Moreover, ibuprofen appeared
to be readily degraded (>95%) in waste water treatment plants but
exhibited a half-life of 20 days in lake water (Buser et al., 1999). Recently,
33
it has been shown that chronic exposure of aquatic organisms to ibupro-
fen affects reproduction of these organisms (Han et al., 2010), and a PEC/
PNEC (predicted environmental concentration/predicted no-effect con-
centration) ratio>1 was reported for ibuprofen (Stuer-Lauridsen et al.,
2000); indicating that this compound may represent a risk for the envi-
ronment. In soil, half-lives between 1 and 6 days have been reported
(Xu et al., 2009). However, few studies applied labelled compounds,
and no data from studies using ring-labelled compounds are available,
which would provide information about the general carbon turnover
of the aromatic ring as the most stable part of the molecule.
Therefore, the aim of the present work was to directly compare the
microbial biodegradation of both stable-isotope and radio-(ring)-
labelled ibuprofen and 2,4-D in water and soil systems. The comparison
was performed using the OECD tests 301 (OECD, 1992) and 307 (OECD,
2002) in order to compare biodegradation data obtained from
water-based tests in the soil environment. The results provide a rst
conceptual approach for the assessment of the fate of the two model
compounds in the two different environmental systems. The approach
aims at increasing our understanding of biodegradation processes and
kinetics in soil and aqueous systems and in particular to open the option
to use ready biodegradability data obtained from aqueous systems for
estimating the biodegradation of a given compound in soil.
2. Material and methods
2.1. Chemicals and soil
Chemicals were obtained in p.a. quality from VWR (Darmstadt,
Germany) or Sigma-Aldrich (Munich, Germany) unless otherwise stated.
2-hydroxyibuprofen (chemical purity 99.8%) was purchased from LGC
GmbH (Luckenwalde, Germany), 13C6-2,4-dichlorophenoxyacetic acid
(2,4-D) and 13C6-ibuprofen (both with 99 at% 13C and 98% chemical puri-
ty) from Alsachim (Illkirch, France), 14C6-2,4-D (10 mCi/mmol, 98 at.%
of 14C) from Hartmann Analytik (Braunschweig, Germany), 13C2-sodium
13
acetate (99 atom% C) from Cambridge Isotope Laboratories Inc. (Andover,
USA), and [U- C]-sodium acetate (50 mCi/mmol, 98 atom % 14C) from
14
Biotrend GmbH (Cologne, Germany).
The reference soil used was the A horizon of a Haplic Chernozem from
the agricultural long-term experiment Statischer Dngungsversuch
(Bad Lauchstdt, Germany), which had been fertilised with 20 t per hect-
are of farmyard manure every second year for more than 100 years. The
soil contained 21% clay, 68% silt, 11% sand, 0.17% total N, 2.1 wt.% total
organic C (TOC) and had a pH of 6.6 and a water holding capacity
(WHC) of 37.5% (Nowak et al., 2011). The material was sieved to 2 mm
for the experiment.
2.2. Incubations in mineral medium
Biodegradation experiments in aqueous systems were performed
according to the OECD guideline 301B (OECD, 1992). Basically, four dif-
ferent incubations were performed for 2,4-D and six for ibuprofen:
a) standard mineral medium (MM) spiked with 14C6-2,4-D or 13C6-
ibuprofen, b) sterilised MM with the labelled test compound (sterile
control, to test for abiotic degradation), c) MM with labelled acetate
(positive control), d) MM with labelled acetate and the unlabelled test
compound (inhibition test), e) MM with unlabelled ibuprofen (control),
and f) non-amended MM (blank). Each treatment was inoculated with
diluted fresh activated sludge (adjusted to 10 mg L1 of suspended
solids) from a municipal wastewater treatment plant (Klrwerk
Rosental, Leipzig, Germany). The chemicals were spiked from stock so-
lutions (2,4-D dissolved in acetone, ibuprofen and acetate in MM). The
nal concentration of the chemicals in MM was 20 mg L1; the initial
radioactivity in the 2,4-D experiment was 5.5 kBq per system. 300 mL
of the spiked MM was incubated in 500 mL Schott bottles in the dark
at 20C for 28 days. Samples were periodically (every 3 days) ushed
with humid CO2-free air in order to provide the O2 necessary for
34 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
microbial respiration. The CO2 in the gas leaving the bottles was trapped
in 20 mL 1 M NaOH. The bottles were destructively sampled after 3, 6,
20 and 28 days (only after 20 and 28 days for the sterile controls) in
the 2,4-D experiment and after 6, 13, 20 and 28 days (only after
28 days for the sterile controls) in the ibuprofen experiment.
2.3. Soil incubation experiments
Biodegradation experiments in soil were based on the OECD guide-
line 307 (OECD, 2002). Four different incubations were performed for
each compound under study: a) soil with the 13C-labelled test compound,
b) soil with the unlabelled compound (control), c) non-amended soil
(blank), and d) sterilised soil with the 13C-labelled test compound (sterile
control). Blank and control systems (b and c) allowed a correction for the
natural abundance of 13C in the soil samples. In order to mimic the dom-
inant pathway into soil (Kmmerer, 2001), ibuprofen was added to the
soil with stabilised sludge (equivalent to the 5 t/ha allowed in Europe),
whereas 2,4-D was added directly to the soil from the stock solution
(2,4-D dissolved in acetone and ibuprofen in acetonitrile). In order to
minimise the toxic effect of the solvents on the soil biota, only 10% of
the total amount of the soil was spiked and care was taken to completely
evaporate the solvent. The spiked soil was then mixed with the remaining
soil. The nal concentration of each chemical in soil was 20 mg kg1 of
soil. Forty grammes of soil were incubated in 1000 mL Schott bottles in
the dark, at 20C and 60% of the WHC, for 64 (2,4-D) and 90 (ibuprofen)
days. The bottles were periodically ushed with humid CO2-free air in
order to provide the O2 necessary for soil respiration. The CO2 in the gas
leaving the bottles was trapped in 40 mL 1 M NaOH. The bottles were de-
structively sampled after 0, 2, 4, 8, 16, 32 and 64 days (only after 32 and
64 days for the abiotic systems) in the 2,4-D experiment and after 0, 2,
7, 14, 28, 59 and 90 days (only after 28 and 90 days for the abiotic sys-
tems) in the ibuprofen experiment.
14 13 13
2.4. Mineralisation of C6-2,4-D, C6-2,4-D and C6-ibuprofen
Mineralisation in the experiments with 13C-label was determined
periodically by measuring total inorganic carbon in the NaOH traps
using a Shimadzu TOC-5050 Total Organic Carbon Analyser. For the
13
C-labelled samples, the isotopic composition of CO2 was determined
by GCcombustionisotope ratio mass spectrometry (GCCIRMS).
2 mL of the NaOH from the traps were acidied with 400 L phosphoric
acid (85%) in 15 mL crimp cap vials. Head-space samples (100250 L)
were analysed by GCCIRMS (Herrmann et al., 2010). For 14C-2,4-D
experiments in MM, the amount of 14CO2 in the NaOH traps was deter-
mined by means of liquid scintillation counting (LSC) with UltimaGold
scintillation cocktail and a Wallac 1414 scintillation counter (Perkin
Elmer Wallac GmbH, Freiburg, Germany).
2.5. Label in MM and in suspended solids
Samples from the OECD 301 tests were ltered over 0.22 m cellulose
lter in order to determine the amount of label in suspended solids (SS).
Filtered (dissolved label only) and unltered (dissolved+suspended
label) samples were measured by means of either LSC for radiolabelled
samples or elemental analysisisotope ratio monitoring mass spectrom-
etry (EA-IRMS) in a Euro EA Elemental Analyzer (Eurovector, Germany)
coupled to a MAT 253 IRMS (Thermo Fisher Scientic, Germany) for
13
C-labelled samples.
2.6. Extractable and non-extractable residues in soil
Soil extractions were performed using an ASE 200 accelerated solvent
extraction system (Dionex, Sunnyvale, CA) equipped with 11 mL stainless
steel extraction vessels, according to (Radjenovi et al., 2009) with slight
modications. Five grammes of soil were mixed with Hydromatrix and
with 20 g of the internal standard 2-methyl-4-chlorophenoxyacetic acid
(MCPA). The samples were extracted with methanol-water (1:1, v/v) at
the following operating conditions: extraction temperature, 100C;
extraction pressure, 100 bar; preheating period, 5 min; static extraction
period, 10 min; number of extraction cycles, 3; solvent ush, 150% of
the cell volume; and nitrogen purge, 150 s. A subsample of the extract
(crude extract) was used for 13C analysis; the remaining sample was
diluted with MilliQ water until the solvent content was b5% for chemical
analysis (see Section 2.7). The extractable (evaporated crude and pure
extracts) and non-extractable residues were determined by EA-IRMS or
LSC as described above.
2.7. Chemical analyses
MM and diluted soil extracts were acidied to pH 2 and puried
by means of solid phase extraction (SPE). The SPE cartridges
(CHROMABOND EASY, 200 mg; Macherey-Nagel, Dren, Germany)
were conditioned with 5 mL of methanol and 5 mL of Milli-Q water.
After sample application, they were washed with 10 mL of Milli-Q
water. The columns were eluted twice with 5 mL of methanol-
acetonitrile (1:1, v/v) for 2,4-D and with 5 mL of methanol-
tetrahydrofuran (1:1, v/v) for ibuprofen. A subsample of the puried
extract was used for 13C analysis by EA-IRMS. The extracts were evapo-
rated under a gentle stream of nitrogen. Samples were silylated with
40 L acetonitrile and 80 L bis-trimethylsilyltriuoroacetamide
(BSTFA) for 10 min at 60C. Target chemicals were determined and quan-
tied by means of gas chromatographymass spectrometry (GCMS)
using an Agilent 7890A GC equipped with a HP-5MS column (30 m
250 m0.25 m; Agilent Technologies, Bblingen, Germany), following
the temperature programme of Zwiener et al. (2002). The isotopic com-
positions of the parent compound and its metabolites were determined
by means of GCCIRMS. The compounds were separated on a BPX-5 col-
umn (50 m0.32 m0.5 m; SGE; Darmstadt, Germany) using a Ther-
mo Scientic Trace GC ULTRA with the following temperature
programme: 60C (2 min), 160C (0 min) at 20C/min, 260C (0 min)
at 6C/min, and 300C (5 min) at 20C/min.
2.8. Data analyses, mass balance and biodegradation kinetics
All experiments were performed in triplicates; the results are
presented as means with standard deviation. Mineralisation, extract-
able and non-extractable residues were quantied at each sampling
date. The amount of 13C in each fraction was estimated according to
Lerch et al. (2009). Calculation of the recovery at each sampling
date allowed setting up a full mass balance and describing the kinet-
ics of compound degradation. The total recovery of the label in the
mass balances for all experiments ranged from 80 to 109%, data cor-
rection was therefore not necessary for setting up the mass balances.
The behaviour of the mineralisation kinetics of the organic com-
pounds in mineral medium followed a sigmoidal curve. Therefore, a
logistic model was used to t the mineralisation of the contaminants
and to calculate the corresponding rates. Mineralisation rates as well
as half-lives were calculated from the resulting regression. The logis-
tic model equation is as follows:
a
y 1
1 e
tm
k
where y is the cumulative mineralisation (% of initial) at time t (days),
a is the maximum mineralisation (%) at the nal plateau of the curve,
k is the kinetic rate constant (d), m is the time at which half of the
maximum mineralisation has occurred (d). The degradation half-life
(DegT50) is obtained by solving the appropriate rearrangement of
Eq. (1) for y = 50%.
The mineralisation of the organic compounds in soil proceeded with
fast rates in the beginning and continuously slowed down during the
experiment. The data were therefore tted to a two-pool exponential
 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
degradation model. In this model, the compound is (mathematically)
partitioned into two pools degrading according to rst order kinetics,
each with its own degradation rate constant. In addition, the model
implicitly includes a third, stable pool which is not mineralised at all
as the percentages of the two mineralised pools do not necessarily
sum up to 100%. The model equation is as follows:
   
y 1 1e 1t 2 1e 2t
where y is the cumulative mineralisation (% of initial) at time t (days),
1 and 2 are the kinetic rate constants (d1) for the rapidly
mineralised and the slowly mineralised pool, respectively, and 1 and
2 are the percentages of initial substrate (% of initial) attributed to
the rapidly mineralised and the slowly mineralised pool, respectively.
As 1 and 2 do not necessarily sum up to 100%, this model implicitly
assumes a third refractory pool, which does not change over the time
of the experiment. Eq. (2) does not allow for a straightforward analyti-
cal solution for DegT50, which is therefore read from plots of the
parameterised version of Eq. (2).
One-way ANOVA tests were performed in order to identify differ-
ences in mineralisation and between pure and crude extracts. All cal-
culations and data analyses were performed with the MATLAB
software. The parameters were estimated from a set of three replicate
samples. Differences were regarded as statistically signicant for all
tests if p b 0.05. Condence intervals ( 95%) and standard deviations
were also computed.
3. Results
14
3.1. Biodegradation of C6-2,4-D in MM (OECD 301)
Mineralisation of 14C6-2,4-D in MM displayed a sigmoidal kinetics
with a lag phase (day 011), a phase of intensive degradation (days
1220) and a nal phase showing a plateau (from day 21) (Fig. 1a).
The initial lag phase indicated adaptation of the degrading microbes, be-
fore 2,4-D was degraded exponentially. Finally, the 2,4-D was exhausted
and the mineralisation ceased. The logistic model tted the data very
well, although the tted parameters carry some uncertainty due to the
large standard deviation of the mineralisation determined for day 20
(Table 1). The high uncertainty at this time may be a result of slight dif-
ferences in the lag times of the replicate microcosms, which result in a
large variation in mineralisation in this steep part of the curve. At the
end of the incubation, 85.0% of the 14C6-2,4-D was mineralised. The
mineralisation rate constant obtained for the logistic model (Eq. (1))
was 1.522 d and the corresponding DegT50 was 18.6 d (Table 1).
2,4-D at the applied concentration (20 mg kg1) only slightly inhibited
Fig. 1. Mineralisation of 14C6-2,4-D and 14C-acetate in mineral medium according to the OECD test 301 protocol. Panel (a) shows mineralisation under biotic () and abiotic ()
conditions together with the tted logistic equation and the corresponding 95% condence intervals; black symbols and lines indicate mineralisation, grey symbols and lines indi-
cate total recovery, and panel (b) shows the mineralisation of acetate alone (E) and acetate in the presence of 2,4-D ().
35
acetate mineralisation until the third day of incubation (Fig. 1b;
p b 0.05). From day 3 onwards no signicant inhibition was observed
(p >0.05), suggesting that the herbicide and its degradation products
are not, or only slightly, toxic for the microbial community of activated
sludge. In the abiotic systems, only 0.03% of the applied 14C6-2,4-D
was mineralised, indicating minimal abiotic mineralisation of 2,4-D. In
contrast, the incorporation of the label from 2,4-D into suspended solids
after 28 days was higher in the abiotic incubations (23.7%; Table S1)
2
than in the biotic ones (3.9%). This, however, accounts for a lower per-
centage of the label remaining in the medium on day 28 in the abiotic
(24.3%) than in the biotic incubations (73.6%).
13
3.2. Biodegradation of C6-2,4-D in soil (OECD 307)
The biotic mineralisation of 13C6-2,4-D in soil started immediately,
without any lag phase (Fig. 2a). The rates were fast at the beginning
and slowed down progressively. The mineralisation of 2,4-D proceeded
until the end of the experiment, when 57.6% of the initially added 2,4-D
was mineralised. The data could be tted by the two-pool exponential
degradation model (Eq. (2)). The rapidly mineralised pool of 2,4-D
amounted to 47% of the initially added 2,4-D, and the slowly
mineralised pool to 32.5% (Table 2). According to the model, the resid-
ual pool of 20.3% is not mineralised. The fast and the slow pool were de-
graded with degradation rate constants of 0.087 d1 and 0.004 d 1,
respectively. DegT50 of the combined process was 37.7 d.
In parallel to the mineralisation, NER were formed (Fig. 2b). The label
in the crude extract fraction decreased rapidly with time (Fig. 2c). At
time 0, 88% of the initial 13C amount could be extracted. The extractabil-
ity decreased to 40% after 8 days and to 8% after 64 days. The amount of
label in the puried extract, which mainly corresponds to parent com-
pound and metabolites, also decreased quickly, from 86% at the begin-
ning to 5.6% at day 8 and to 2% at the end of the incubation (Fig. 2d).
These results are consistent with the GC-MS analysis of 2,4-D and its
metabolites in the extracts (Table S2). Only the parent compound (all
sampling dates) and the metabolite 2,4-dichlorophenol (2,4-DCP; days
2 and 4) were detected.
A rapid increase (from 4.3% to 26%) in the amount of NER was ob-
served until day 8 (Fig. 2b), and a stable level was reached on day 16.
Finally, NER accounted for 36% of the initially added 13C label.
In the abiotic systems, mineralisation was low (2.5% of the initial 13C;
Fig. 2) but 2,4-D was also transformed: 11% of the initial 13C6-2,4-D was
found as 13C6-2,4-DCP after 32 and 64 days (Table S2). NER amounted to
18.6% of the applied 13C at the end of the incubation, much less than in
the biotic systems. On day 0, the crude extract fraction accounted for
95% of the applied 13C. This fraction decreased to 68% at the end.
Moreover, crude and puried extracts were not signicantly different
(p>0.05). It has to be considered, however, that in the biotic systems,
36 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242

Table 1 Therefore it can be assumed that ibuprofen is adsorbed reversibly to the

Parameter estimates from the logistic model for the biodegradation of 2,4-D and ibu- activated sludge particles or can be degraded in sorbed state (Kimura
profen in aqueous systems model.
et al., 2007). Consistent with the presence of a lag phase, no metabolites
a
equation : y
a were found in the biotic incubations until day 20 (Table S3).
1 e
tm
k
Abiotic mineralisation (1.3% after 28 days) was very low (Fig. 3a),
and 23.5% of the applied 13C6-ibuprofen was found adsorbed to
Incubation k(d1) m(d) a(%) R2 SSE DegT50
(d) suspended solids (Table S3). The acetate degradation curve and the in-
hibition test curve were not signicantly different (p > 0.05; Fig. 3b).
2-4 D 1.522 0.083 18.03 1.025 85.06 2.045 1.000 0.140 18.6
(biotic) Ibuprofen thus did not inhibit the microbial activity at the applied
Ibuprofen 3.541 1.236 13.44 2.077 63.55 4.192 0.958 177.8 18.1 concentration.
(biotic)

Estimates standard deviation (n = 3); R2: coefcient of determination, and SSE: sum
of squared errors of prediction.
a
For further explanation, see Section 2.
a substantial portion of the added 2,4-D was mineralised. Metabolisation
and abiotic sequestration are therefore competing processes during NER
formation, and the abiotic treatments can never quantitatively reect
the abiotic processes in the biotic systems.
3.3. Ibuprofen degradation in MM (OECD 301)
Mineralisation of 13C6-ibuprofen in MM followed logistic kinetics
(Fig. 3a). Until day 6, the mineralisation remained less than 10% of the
initially applied label, indicating a lag phase due to the adaptation of
the microorganisms. This is also reected by a 7-day lag phase
(mineralisationb 10% of initial) predicted by the logistic model. The bio-
degradation rate constant calculated by Eq. (1) was 3.542 d and the cor-
responding DegT50 was 18.1 d (Table 1). Incorporation of the 13C label
into suspended solids was high on day 6, due to sorption of ibuprofen
to suspended sludge particles and incorporation into biomass (Table
S3). The proportion of 13C in suspended solids decreased with time.
3.4. Ibuprofen degradation in soil (OECD 307)
The biotic mineralisation of 13C6-ibuprofen in soil started immediate-
ly without any lag phase (Fig. 4a). Similar to what we found for 2,4-D, the
degradation rate was high at the beginning of the incubation and slowed
down continuously. At the end of the experiment, 45.2% of the initially
added 13C6-ibuprofen had been mineralised (Table S4). The data could
be described by the two-pool exponential degradation model (Fig. 4a).
For this compound, the rapidly mineralised pool amounted to 52.3% of
the label added initially and the slowly mineralised pool was almost neg-
ligible (Table 2). As a result, a relatively large percentage (47.7%) of the
added compound was predicted to remain in the refractory pool which
is not mineralised at all. For both actively degraded pools of the model,
the predicted degradation rate constants were lower than for 2,4-D
(0.024 d 1 for the fast pool and 0.003 d1 for the slow pool). Both the
lower percentage of mineralisable label (1 +2) and the lower degra-
dation rate constants resulted in a slower degradation of ibuprofen in
comparison to 2,4-D and thus in a longer DegT50. In agreement with
the measured data, the model predicts a DegT50 longer than the exper-
imental period; it was estimated at 129.6 d.
The amount of label in the crude extracts decreased from 95.6% of
the applied 13C label on day 0 to 13.4% on day 90 (Table S4, Fig. 4c).

Fig. 2. Fate of 13C6-2,4-D in soil under biotic () and abiotic () conditions according to the OECD test 307 protocol. a) mineralisation (data points and tted two-pool exponential
degradation model with 95% condence intervals), b) non-extractable residues (black symbols and lines) and total recovery (grey symbols and lines), c) extractable amount before
purication and d) extractable amount after purication.
 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242 37

Table 2
Parameter estimates from the two-compartment model for the biodegradation of 2,4-D and ibuprofen in soil systems model.
   
.equationa : y 1 1e1 t 2 1e2 t

Incubation R2 SSE 1 2 1 2 DegT50

(d1) (d1) (% mineralised of initially applied substrate) (% mineralised of initially applied substrate) (d)

2-4 D (biotic) 0.982 73.1 0.087 0.022 0.004 0.006 47.22 1.88 32.47 16.31 37.7
Ibuprofen (biotic) 0.991 37.7 0.024 0.004 0.003 0.012 52.33 1.50 0.006 0.205 129.6

Estimates standard deviation (n = 3), R2: coefcient of determination, and SSE: sum of squared errors of prediction.
a
For further explanation, see Section 2.

On day 0, the puried extract (mainly parent compound and metab-
olites) accounted for 81.0% of the initially added amount, on day 28
for 9.7% and on day 90 for 0.8% (Fig. 4d). The GC-MS data show that
the amount of ibuprofen decreased rapidly until day 28 and remained
very low until the end of the experiment (0.5%, Table S4). The metab-
olite 2-hydroxyibuprofen was detected from day 2 until day 28 with a
maximum of 7% of the initial 13C6-ibuprofen equivalents.
At the beginning of the incubation, only 2.7% of the applied 13C
label was detected as NER (Fig. 4b). The amount of 13C-NER increased
steadily until day 28, and then remained constant at 30% of the initial-
ly added label until the end.
In the abiotic systems, mineralisation accounted for only 3.9% at the
end of the incubation, and only 15.4% of the applied 13C was found in
NER (Fig. 4a, b). No metabolites were detected, and crude and puried
extracts were not signicantly different from each other (p>0.05).
Again, the abiotic controls do not exactly reect the abiotic processes
in the biotic systems because metabolisation and abiotic sequestration
are competing processes during NER formation.
3.5. Modelling results
The mineralisation of the two compounds was well described by
the logistic model for the aqueous systems and by the two-pool expo-
nential degradation model for the soil systems. R 2 was higher than
0.95 in all cases. The logistic model was needed in order to describe
the mineralisation in the aqueous systems, because the ready biode-
gradability tests in aqueous systems (OECD 301) showed a pro-
nounced lag phase for both compounds, which is consistent with
the low microbial biomass in the inoculum for degradation. According
to the model parameters (see Table 1), 2,4-D was degraded faster
(lower k in Eq. (1)) and to a higher extent (higher a) than ibuprofen.
In the soil system, no lag phase was observed for either compound,
and the two-pool exponential degradation model tted the data well.
Again, 2,4-D was degraded faster (higher values for both pools in
Eq. (2)) and to a higher extent (higher percentage of initial label in the
two modelled pools, 1 +2).
The fact that different models tted compound degradation in aque-
ous systems and in soil indicated that different factors control biodegra-
dation in the two systems. This implies that comparison of compound
degradation in the two systems is not straightforward. However, as
both models are ultimately derived from the Monod kinetics (using dif-
ferent simplications; Simkins and Alexander, 1984), they are still com-
parable from a theoretical point of view. In practice, however, the tted
parameters lump together different variables of the Monod equation.
We therefore decided to compare the maximum mineralisable amounts
and the maximum rates for the two models, which can be easily derived
from the parameterised model equations. In the case of the logistic
model, the maximum mineralisable amount is given by the parameter
a in Eq. (1), and the maximum mineralisation rate is the rst derivative
of Eq. (1) at t = m. In case of the two-pool exponential degradation
model, the maximum mineralisable amount is given by 1 + 2 in
Eq. (2), and the maximum degradation rate is the rst derivative of
Eq. (2) at t = 0. This comparison shows that the maximum degradation
rate as predicted by the models is substantially reduced in the soil ex-
periments compared to the aqueous system incubations (Table 3). The
maximum degradation rate in the aqueous systems was 3.3-fold and
3.6-fold higher than in the soil systems for 2,4-D and ibuprofen, respec-
tively. This suggests that maximum degradation rates in soil can be es-
timated from those in water by assuming a reduction factor of about 3.5.
In contrast, the maximum mineralisable amount as predicted by the
models is only slightly lower in soils than in aqueous systems. For this
comparison, it has to be considered that the maximum mineralisable
amount is a theoretical value reached after innite time. As the degrada-
tion rates are lower in soil than in aqueous systems, and microbial bio-
mass derived from the labelled carbon is intermediately formed, the
actually detected mineralisation in the soil systems at the end of the

Fig. 3. Mineralisation of 13C6-ibuprofen and 13C2-acetate in mineral medium according to the OECD test 301 protocol. Panel (a) shows mineralisation under biotic () and abiotic
() conditions together with the tted logistic equation and the corresponding 95% condence intervals; black symbols and lines indicate mineralisation, grey symbols and lines
indicate total recovery, and panel (b) shows the mineralisation of acetate alone ( ) and acetate in the presence of ibuprofen ().
38 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
Fig. 4. Degradation of 13C6-ibuprofen in soil under biotic () and abiotic () conditions according to the OECD test 307 protocol. a) mineralisation (data points and tted two-pool
exponential degradation model with 95% condence intervals), b) non-extractable residues (black symbols and lines) and total recovery (grey symbols and lines), c) extractable
amount before purication and d) extractable amount after purication.
experiment was lower than the maximum mineralisable amount calcu-
lated from the model.
4. Discussion
In order to evaluate the environmental fate of the model com-
pounds 2,4-D and ibuprofen, we compared results from OECD ready
biodegradability tests (OECD 301) with those from simulation tests
in soil (OECD 307). Not only mineralisation rates were determined,
but also metabolite proles and the carbon distribution during bio-
degradation. For both compounds, the mineralisation in the aqueous
systems could be modelled using a logistic equation, whereas they
were mineralised according to a two-pool exponential degradation
model in the soil systems indicating different degradation behaviour
in the two systems. The tted parameters can therefore not be direct-
ly compared. However, these parameters were used to calculate the
maximum mineralisable amount and the maximum mineralisation
rate for each compound in each system. The parameters allowed a
comparison between the compounds and the systems; we will dis-
cuss here some options identied for the prediction of biodegradation
in soil on the basis of data obtained from tests in aqueous systems.
Table 3
Comparison of maximum degradation rates and amounts for aqueous and soil systems.
Maximum mineralised percentage
__________________________
% of initial
2,4-D
Aqueous system 85.06 2.05
Soil system 79.69 16.41
4.1. Biodegradation of 2,4-D and ibuprofen in aqueous systems
According to the OECD 301 test, 2,4-D can be classied as readily bio-
degradable because it met the criterion of more than 60% mineralisation
(71.2%) within a 10-day window. Mineralisation was extremely rapid
after a pronounced lag phase and reached 85% at the end of the experi-
ment. In contrast, ibuprofen mineralisation started earlier, proceeded
with a slower rate and reached only 68% at the end of the experiment.
Thus, although 68% of the added ibuprofen was mineralised after
28 days under the OECD 301 test, this compound does not full the cri-
terion of rapid degradation in a 10 day-window. It is therefore classied
as easily degradable. However, according to the latest revision of the
OECD guidelines (OECD, 2006), it is justied to waive the requirement
of 60% mineralisation within a 10-day window in the case of ibuprofen,
because two isomers of it occur in the environment. Of these two iso-
mers, the pharmacologically inactive (R)-(-)-isomer is the more persis-
tent one (Buser et al., 1999). The adaptation period in the aqueous
system was shorter than in case of 2,4-D. This may be due to previous
exposure of the sludge microbial community to the pharmaceutical
but not to the herbicide in the municipal wastewater treatment plant.
The environmental risk of ibuprofen has been classied very differ-
ently in the past. Recently, Mige et al. (2009) published a database
Maximum mineralisation rate
________________________ ____________________________
d1 ___________________________
Ibuprofen 2,4-D Ibuprofen
63.55 4.19 13.97 0.83 4.48 1.59
52.34 1.51 4.24 1.07 1.26 0.21
 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
about the fate of pharmaceuticals in wastewater treatment plants. Re-
moval percentages are reported for 50 molecules, including ibuprofen.
70% removal was reported for ibuprofen, albeit with high variation be-
tween the studies. Moreover, as previously mentioned, a PEC/PNEC
ratio > 1 was determined for ibuprofen (Stuer-Lauridsen et al., 2000);
indicating that this compound may represent a risk for the environ-
ment. Our results, however, clearly show that ibuprofen is easily or
readily biodegradable in aqueous systems, and, based on these data, ap-
parently does not pose a risk for the environment. These results illus-
trate the strong variability between different studies. Our data will
also help to overcome the lack of studies in soil which hampers under-
standing the actual fate of contaminants in the environment.
Although the mineralisation kinetics of the two compounds dif-
fered considerably, the logistic model (Eq. (1)) allows a good estima-
tion of the mineralisation of both compounds. The different kinetics
are reected in the tted parameters, showing a longer time shift
(m) and a higher maximum mineralisation (a), but a lower rate con-
stant (k) for 2,4-D, indicating faster mineralisation of 2,4-D than ibu-
profen, but a more pronounced lag phase in the case of 2,4-D.
4.2. Biodegradation of 2,4-D and ibuprofen in soil systems
In contrast to the mineralisation in the aqueous systems,
mineralisation of both compounds started immediately in the soil sys-
tems. It can therefore be assumed that the soil microbial community
was able to use both 2,4-D and ibuprofen as carbon and energy sources
or to degrade the compounds cometabolically (Robertson and
Alexander, 1994). The presence of 2,4-D degrading organisms can be
easily explained because the soil used had been treated with structural-
ly related herbicides (e.g. MCPA and Dichlorprop; Merbach, UFZ, per-
sonal communication, 2010) which may have supported the growth
of adapted microbiota in the soil. Degrading organisms have also been
found in soils never exposed to the herbicide (Vieubl Gonod et al.,
2003). In addition, 2,4-D and ibuprofen differ in their solubility in H2O
(2,4-D: 600 mg/L; Villaverde et al., 2008; ibuprofen: 21 mg/L;
Yalkowski and Dannenfelser, 1992). Therefore, 2,4-D is present at
higher concentration than ibuprofen in the soil solution, which may re-
sult in a higher availability for degrading microbes and thus faster bio-
degradation (Semple et al., 2004). The rate of mineralisation was
highest in the beginning of the experiment, when 2,4-D was available
and the growth of its degraders was not limited by substrate availabili-
ty. Consistent with previous studies (Lerch et al., 2009; Vieubl Gonod
et al., 2003), the biodegradation decreased continuously throughout
the incubation concomitantly with decreasing availability of 2,4-D and
its metabolites, which were rapidly formed by both biotic and abiotic
reactions (Boivin et al., 2005).
Similarly, no lag phase was observed for ibuprofen. Although it is not
very likely that the soil had been exposed to ibuprofen prior to our incu-
bation study, mineralisation started immediately without any lag phase
and at relatively high rates. Reasons for the lack of an adaptation phase
to ibuprofen may be a previous exposure of soil microorganisms to the
pharmaceutical after the application of biosolids (Topp et al., 2008) and/
or to unspecic (cometabolic) microbial degradation activities against
aromatic compounds (Robertson and Alexander, 1994). Again, the
mineralisation rate was highest in the beginning and then decreased
over time. Similar to the situation in the aqueous systems, ibuprofen
in soil was mineralised at lower rates and to a lower extent than
2,4-D. The period of high microbial activity was longer in the case of ibu-
profen than of 2,4-D, coinciding with the periods of highest availability
of the parent compound and its metabolite 2-hydroxyibuprofen (Table
S4). As a consequence, DegT50 of ibuprofen in soil (130 days) was
much longer than for 2,4-D (38 days).
In a study by Richter et al. (2007), NER formation and mineralisation
of ibuprofen were faster during the rst 20 days of incubation, but the
nal mineralisation was lower (38%) than in the present study. This
may be explained by the use of methyl-labelled ibuprofen in the study
39
by Richter et al. (2007), whereas we used ring-labelled ibuprofen.
Murdoch and Hay (2005) described a degradation pathway of ibuprofen
by bacteria isolated from a waste water treatment plant, involving the
initial loss of the C3-methyl group. Such a pathway would explain the
faster loss of the 13C-label from the methyl group than from the aromatic
ring. The fast elimination of unlabelled ibuprofen from soil reported by
Xu et al. (2009) can be attributed to the fact that experiments with
unlabelled compounds do not allow distinguishing between sorption
and degradation.
In parallel with the degradation of the compounds, the extracted
amount decreased. In addition to the compounds added and their me-
tabolites, the extracts contained label bound to particulate organic mat-
ter, including biomass residues which were removed by the applied
purication method. Extractable 2,4-D in the puried extract decreased
to very low amounts after 8 days of incubation. The metabolite 2,4-DCP
could be detected after 2 and 4 days in substantial amounts, but not
thereafter, which is consistent with the results of Lerch et al. (2009)
who extracted only 6% of the applied 2,4-D-derived C after 8 days and
0.1% at the end of the experiment.
In line with the slower mineralisation, extractable ibuprofen de-
creased more slowly: substantial amounts were extracted until day
14, but from day 28, only low amounts of ibuprofen were extractable.
The metabolite 2-hydroxyibuprofen accumulated more slowly and to
a lesser extent than 2,4-DCP in the case of 2,4-D. This is related not
only to the slower metabolisation of ibuprofen than 2,4-D, but also to
the higher persistence of 2,4-DCP compared to 2-hydroxyibuprofen.
The differences between the biotic and the abiotic system show that
microbial activity was the main cause for the elimination of the com-
pounds. In addition to mineralisation, this activity resulted in signicant
NER formation during the experiments. This is reected by the fact that
NER formation was much higher in biotic soils than in abiotic ones dur-
ing the initial degradation phases and showed a rapid increase through-
out the experiment. At the end of the incubation, almost 40% of the
2,4-D-derived 13C in the biotic systems was found in NER, which is com-
parable to earlier observations (Boivin et al., 2005). NER formation in
the ibuprofen experiment was somewhat lower (30% at the end of the
experiment), but in a similar range.
In contrast to biotic soils, NER amounts in abiotic soils were low at
the beginning and showed a very slow increase towards the end, indi-
cating that 2,4-D and ibuprofen were not rapidly physico-chemically
stabilised in soil, which indicates competition between sequestration
and biodegradation. A moderate sorption tendency has been reported
for ibuprofen in a soil with similar texture (Kreuzig et al., 2003). Our
data and previous results (Xu et al., 2009), however, illustrate low sorp-
tion or entrapment of this compound to soil. Therefore, adsorption of
the two compounds to soil does not seem to play a major role in their
dissipation and sequestration and, apparently, does not control their
bioavailability. This is also reected by the signicantly lower amounts
of NER formed in the abiotic controls (19% in the case of 2,4-D and 15%
for ibuprofen) than in the biotic incubations. Abiotic degradation was
apparently higher in soil systems than in aqueous systems, presumably
due to redox reactions with soil minerals (Guo et al., 2004; Park et al.,
1990). Nevertheless, the sterile systems do not reect the abiotic pro-
cesses in the biotic systems, as the fast degradation of 2,4-D and ibupro-
fen efciently competes with sorption. Kstner et al. (1999) have
already reported a higher abiotic NER formation in anthracene experi-
ments when fungal and bacterial metabolism was inhibited in compar-
ison to the active biotic turnover. Abiotic NER formation is thus
overestimated by the general approach of incubating separate abiotic
systems prescribed in the OECD protocols.
Microbial biomass residues have been shown to be responsible
for the majority of NER from 2,4-D (Nowak et al., 2011) and ibupro-
fen (Nowak et al., unpublished results). In these cases, virtually all
NER could be explained by the formation of biogenic residues from
the biomass of microorganisms which utilised the compounds as C
sources. As biogenic NER are in principle degradable/mineralisable,
40 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
this is consistent with the maximum mineralisation amounts pre-
dicted by the parameterised models. Such biogenic residues were
not found in the abiotic controls when analysing this fraction
(Nowak et al., 2011), additionally indicating that abiotic process
may not occur in a similar way if biotic reactions are present, and
that even sorbed compounds may be degraded. These ndings con-
tradict the suggestions of Boivin et al. (2005) that NER ageing and
the related reduction in bioavailability over time is due to physical
entrapment rather than irreversible chemical bonding.
4.3. Comparison of the degradation in aqueous and soil systems
For both compounds, mineralisation was faster and had a higher
extent in the aqueous systems than in the soil systems, resulting in
shorter DegT50 in the aqueous systems than in the soil systems. In
addition, the mineralisation kinetics differed considerably between
the systems: the time course of the degradation of 2,4-D and ibupro-
fen in aqueous systems suggested a sigmoid kinetics, whereas in soil
systems the process could be described best by a two-pool exponen-
tial model. This reects the different conditions in the test systems: in
the aqueous systems, the estimated biomass concentration (based on
10 mg/L suspended solids) corresponds to about half the added con-
centration of the chemicals (20 mg/L), whereas in the soil systems,
the biomass concentrations (estimated as ca. 1% of the soil TOC, i.e.
ca. 200 mg/kg soil) were about 10 times as high as the concentration
of the chemicals (20 mg/kg). These major differences resulted in a
substantial limitation of 2,4-D or ibuprofen degradation in the aque-
ous systems by the low abundance of degrading microorganisms in
the starting phase of the experiment. This situation, and thus the
time course of mineralisation in the aqueous systems, is best de-
scribed by the logistic model (Simkins and Alexander, 1984). In con-
trast, in the soil systems biomass was relatively high from the
beginning, and the growth on the substrate, which was much less
abundant than the initial biomass, did not result in a signicant in-
crease of the biomass. Degradation under these conditions can be de-
scribed by the rst-order exponential decay model (Simkins and
Alexander, 1984). Our data, however suggest that mineralisation of
the two compounds in soil cannot be well described if we assume a
single pool of the chemicals with a xed degradation rate constant
. For an improved t we had to assume that i) the chemicals parti-
tion into several pools, and that ii) each pool is mineralised following
rst-order kinetics with its own degradation rate constant i. The
model tted best if we assumed two pools which are degraded with
different rate constants, plus a third inert pool which is not degraded
at all. This third pool does not explicitly show up in the equation, but
is implied by the fact that 1 + 2 b 100%. Ageing of parent com-
pounds enhances NER formation via chemical and/or physical pro-
cesses (Sharer et al., 2003; Walker et al., 2005). In addition, biogenic
residues are formed during microbial degradation (Nowak et al.,
2011). This may partly explain the presence of several pools of the
compounds with different degradation rate constants. Another possi-
ble explanation lies in the inhomogeneous distributions of both the
chemicals and the microorganisms. All these factors result in limited
bioavailability of the compounds and thus in slower degradation or,
in extreme cases, in no degradation. As the inuence of these factors
may vary for different microenvironments, several pools of the
chemicals are formed.
4.4. Prediction of biodegradation in soil from data obtained in aqueous
systems
Frequently, ready biodegradability tests in aqueous systems are the
only source of information available for the prediction of the environ-
mental fate of chemicals in the environment. However, Aronson et al.
(2006) concluded that estimated half-lives should not be used for risk
assessment, but only for banning and prioritising chemicals. This is
supported by our results, where DegT50 of ibuprofen and 2,4-D were
similar in the aqueous systems, but clearly different in the soil systems.
A central assumption implemented for ERA within the REACH and
EMEA initiatives is that ready biodegradability in water can always be
transferred to soil. Screening tests appear to give fairly reproducible
qualitative results for chemicals that are either highly recalcitrant
(e.g. ciprooxacin; Girardi et al., 2011) or highly biodegradable
(2,4-D, this study); however, for less biodegradable chemicals the results
are extremely variable and difcult to interpret, as demonstrated by the
inconsistent data for ibuprofen.
The presented data set offers the unique possibility to compare the
degradation of 2,4-D and ibuprofen in aqueous systems and in soil
based on the modelling data. As stated above, both the kinetics and the
cumulative mineralisation differed between the systems: mineralisation
in the aqueous systems could be modelled by the logistic degradation
model (Eq. (1)), whereas a two-pool exponential degradation model
(Eq. (2)) described the soil data best. Both the logistic and the exponen-
tial decay model are derived from the Monod kinetics by simplications
of the original equation based on the conditions for degradation
(Simkins and Alexander, 1984). Nevertheless, it is not straightforward
to compare the models directly, because the tted parameters lump to-
gether several parameters from the Monod equation, and these are dif-
ferent for the two cases. In addition, the distribution of the chemicals
into different pools in the soil systems hampers the direct comparison
of the two models. We therefore did not compare the tted parameters
directly, but used the parameterised Eqs. (1) and (2) to calculate the
maximum mineralisable amount of each compound and the maximum
degradation rate in aqueous and soil systems (see Table 3). Both param-
eters can be easily derived from Eqs. (1) and (2): In Eq. (1), the maxi-
mum mineralised amount is a, and in Eq. (2) it is 1 +2. The
maximum degradation rates are the derivatives of the equations at
time m for the logistic model (i.e. at the inection point) and at time
0 for the two-compartment exponential decay model. These data allow
a direct comparison of the mineralisation in the aqueous systems and
in soil, although the conditions and thus the appropriate models differed
between the two systems. The comparison shows that in aqueous sys-
tems, the maximum mineralisable amount is slightly higher than in
soil systems, but the difference is only signicant for ibuprofen
(Table 3). In both cases, the differences are small (b 20% of the values),
thus it is reasonable to conclude that the maximum mineralisable
amount is only slightly lower, if at all, in soil than in water. This was
not expected, because in soil systems, usually NER are formed in addi-
tion to metabolites and biomass. However, this applies to innite reac-
tion times. In addition, a proportion of the pollutant-derived C is
incorporated intermediately into microbial biomass and subsequently
into biogenic residues. This explains the fact that the extents of biodeg-
radation in water (sum of mineralisation +suspended solids) and soil
(sum of mineralisation +biogenic NER) are similar at the end of the ex-
periments. More pronounced differences between the aqueous and the
soil systems were observed for the maximum mineralisation rates,
which were signicantly higher in the aqueous systems than in the soil
systems (Table 3). As for the maximum mineralisable amount, the max-
imum degradation rates are lower for ibuprofen than for 2,4-D, which is
reected by the overall slower mineralisation of ibuprofen than 2,4-D. A
closer inspection of the maximum degradation rates, however, shows
that for both compounds, the rates in the aqueous systems are about
3.5 times as high as in the soil systems. These data indicate that there
may be a relation between mineralisation kinetics in aqueous and soil
systems.
However, these results can only be regarded as a rst step towards a
transfer function for extrapolating water-based data in order to obtain
sound estimates of soil degradation. In general, it has to be taken into ac-
count that the designation of a chemical as persistent or non-persistent,
or the environmental fate of a chemical, cannot be considered an inher-
ent property of the compound which can be as easily determined as,
for example, Kow values (Boethling et al., 2009). This approach is not
 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
adequate, since the fate and degradation of a chemical is determined by a
combination of compound-specic properties and environmental condi-
tions (Boethling et al., 1995), including abiotic interactions, bioavailabil-
ity, microbial community, levels of microbial activity, concentrations of
the chemicals, soil type etc. All these factors result in considerable data
variation in simulation systems. For proper risk assessment, the effects
of these factors need to be studied using isotopically labelled compounds
with the labelled atom in the most stable position of the molecule, which
allows differentiating between dissipation and degradation. In addition
to degradation kinetics, DegT50 values should be reported rather than
dissipation half-lives (Boethling et al., 2009).
Therefore, the presented approach clearly needs to be complemented
by studying more soils and more chemicals in both systems. A possible
effect of the soil/chemical combination is indicated by the fact that the
DegT50 (i.e. the time when 50% of the added label was mineralised)
was similar for both compounds in the aqueous systems (ca. 18 days),
but in the soil systems it was more than three times longer for ibuprofen
(130 days) than for 2,4-D (38 days). Therefore, key soil parameters
affecting biodegradation (e.g. soil structure, water content, pH, redox po-
tential, microbial community) need to be dened and taken into account
in future studies.
Combining the effects of the chemical structure of a pollutant and
the soil properties affecting its biodegradation would be an important
step towards explaining why small differences in the chemical struc-
ture of the pollutants and in the experimental conditions may result
in pronounced differences of degradation rates. As a consequence,
statistically established correlations have been considered helpful
for roughly classifying chemicals, but were not used for prediction
purposes. In any case, detailed insight into the environmental fate
of pollutants denitely needs experimental studies. Therefore, our
understanding of biodegradation processes and pathways in environ-
mental systems must be improved signicantly (Tunkel et al., 2000),
a statement which is still valid ten years after.
Existing guidance for the testing of chemicals places emphasis on
the screening phase. Although it is recognised that simulation tests
have greater environmental relevance, they are triggered only by a
negative result in the screening test. The presented results indicate
that it may be of advantage to extract more information from the
data obtained in the ready biodegradability tests than has been
done in the past. This may result in prediction tools which allow esti-
mating the fate of a chemical in soil from ready biodegradability data
and information on soil properties, and thus in a new approach for
the environmental risk assessment of chemicals. In critical cases,
however, the results of this extrapolation should always be conrmed
by simulation tests.
5. Conclusions
The models showed different biodegradation kinetics of 2,4-D and
ibuprofen in the two systems, soil and water, which are mainly related
to the different levels of microbial biomass present at the beginning of
the incubation in the tests. The models also demonstrated that for the
compounds studied, the maximum mineralisable amount and the max-
imum rate of mineralisation in soil and in aqueous systems were closely
related. However, the mineralisation actually detected in soil is much
lower, since the degradation resulting in intermediate carbon storage
within microbial biomass is not separately described by the model. In
addition, the degradation rates in soil are slower than in aqueous sys-
tems by a factor of about 3.5.
These ndings have to be veried with other compounds under
different environmental conditions, but they show that such predic-
tions are possible. However, in order to overcome the inconsistencies
of the existing data, wherever possible, we suggest that biodegrada-
tion tests in soil should be performed in order to provide a validated
assessment of the environmental risk of a chemical. In these tests, it is
essential that 13/14C-labelled compounds with the label in the most
41
stable position(s) of the molecules be used in order to obtain conser-
vative but realistic data. Only this approach allows distinguishing
between dissipation and degradation of a contaminant in soil, which
is necessary for risk assessment since only degradation ultimately
removes the compound from the system.
Another important issue is the need for a revision of the abiotic control
in soil guidelines for assessing biodegradation, since biotic degradation
competes with abiotic NER formation in the case of ready biodegradable
compounds, and such controls do not reect the actual abiotic contribu-
tion in biotic systems. The present concept of abiotic controls thus leads
to an overestimation of the abiotic contribution to NER formation.
Acknowledgements
The study was supported by the European Commission funding of
the RAISEBIO project (contract: MEST-CT- 2005-020984) under
Human Resources and Mobility Activity (6th Framework programme),
in particular the fellowships of C. Girardi and K. M. Nowak. The authors
thank U. Gnther, K. Ethner, F. Bratsch, A. Lopez and B. Wrz for the as-
sistance in the sample preparation and analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.scitotenv.2012.11.051.
References
Ahtiainen J, Aalto M, Pessala P. Biodegradation of chemicals in a standardized test and
in environmental conditions. Chemosphere 2003;51:52937.
Aronson D, Boethling R, Howard P, Stiteler W. Estimating biodegradation half-lives for
use in chemical screening. Chemosphere 2006;63:195360.
Boethling RS, Howard PH, Beauman JA, Larosch ME. Factors for intermedia extrapola-
tion in biodegradability assessment. Chemosphere 1995;30:74152.
Boethling R, Fenner K, Howard P, Klecka G, Madsen T, Snape JR, et al. Environmental
persistence of organic pollutants: guidance for development and review of POP
risk proles. Integr Environ Assess Manag 2009;5:53956.
Boivin A, Amellal S, Schiavon M, van Genuchten MT. 2,4-dichlorophenoxyacetic acid
(2,4-D) sorption and degradation dynamics in three agricultural soils. Environ
Pollut 2005;138:929.
Buser HR, Poiger T, Muller MD. Occurrence and environmental behavior of the chiral
pharmaceutical drug ibuprofen in surface waters and in wastewater. Environ Sci
Technol 1999;33:252935.
De Bruijn J, Struijs J. Biodegradation in chemical substances policy. In: Hales SG, Feijtel
T, King H, Fox K, Verstraete W, editors. Biodegradation kinetics: generation and use
of data for regulatory decision making. Brussels, Belgium: SETAC-Europe; 1997.
p. 3345.
Drer U, Haala R, Matthies M, Scheunert I. Mineralization kinetics of chemicals in soils
in relation to environmental conditions. Ecotoxicol Environ Saf 1996;34:21622.
EMEA. Guideline on the environmental risk assessment of medicinal products for
human use; 2006.
EPA. Technical factsheet on: 2,4 -D; 2010.
Girardi C, Greve J, Lamshft M, Fetzer I, Miltner A, Schffer A, et al. Biodegradation of
ciprooxacin in water and soil and its effects on the microbial communities. J Haz-
ard Mater 2011;198:2230.
Guhl W, Steber J. The value of biodegradation screening test results for predicting the elim-
ination of chemicals' organic carbon in waste water treatment plants. Chemosphere
2006;63:9-16.
Guo M, Papiernik SK, Zheng W, Yates SR. Effects of environmental factors on 1,3-
dichloropropene hydrolysis in water and soil. J Environ Qual 2004;33:6128.
Han S, Choi K, Kim J, Ji K, Kim S, Ahn B, et al. Endocrine disruption and consequences of
chronic exposure to ibuprofen in Japanese medaka (Oryzias latipes) and freshwater
cladocerans Daphnia magna and Moina macrocopa. Aquat Toxicol 2010;98:25664.
Herrmann S, Kleinsteuber S, Chatzinotas A, Kuppardt S, Lueders T, Richnow H-H, et al.
Functional characterization of an anaerobic benzene-degrading enrichment cul-
ture by DNA stable isotope probing. Environ Microbiol 2010;12:40111.
Howard PH, Banerjee S. Interpreting results from biodegradability tests of chemicals in
water and soil. Environ Toxicol Chem 1984;3:55162.
IFEN. Pesticides in water. Sixth Annual Report 2002 data, 42. Institut Franais de
l'Environnement; 2004.
Kstner M, Streibich S, Beyrer M, Richnow H-H, Fritsche W. Formation of bound residues
during microbial degradation of [14C]anthracene in soil. Appl Environ Microbiol
1999;65:183442.
Kimura K, Hara H, Watanabe Y. Elimination of selected acidic pharmaceuticals from
municipal wastewater by an activated sludge system and membrane bioreactors.
Environ Sci Technol 2007;41:370814.
Kreuzig R, Kullmer C, Matthies B, Hltge S, Dieckmann H. Fate and behaviour of phar-
maceutical residues in soil. Fresenius Environ Bull 2003;12:5508.
42 C. Girardi et al. / Science of the Total Environment 444 (2013) 3242
Kmmerer K. Drugs in the environment: emission of drugs, diagnostic aids and disin-
fectants into wastewater by hospitals in relation to other sources a review.
Chemosphere 2001;45:95769.
Lerch TZ, Dignac M-F, Nunan N, Bardoux G, Barriuso E, Mariotti A. Dynamics of soil mi-
crobial populations involved in 2,4-D biodegradation revealed by FAME-based sta-
ble isotope probing. Soil Biol Biochem 2009;41:7785.
Mige C, Choubert JM, Ribeiro L, Eusbe M, Coquery M. Fate of pharmaceuticals and
personal care products in wastewater treatment plants conception of a database
and rst results. Environ Pollut 2009;157:17216.
Murdoch RW, Hay AG. Formation of catechols via removal of acid side chains from ibu-
profen and related aromatic acids. Appl Environ Microbiol 2005;71:61215.
Nowak KM, Miltner A, Gehre M, Schffer A, Kstner M. Formation and fate of bound
residues from microbial biomass during 2,4-D degradation in soil. Environ Sci
Technol 2011;45:999-1006.
OECD. OECD guideline for testing of chemicals, Guideline 301: ready biodegradability;
1992.
OECD. OECD guidelines for testing of chemicals, guideline 307: aerobic and anaerobic
transformation in soil; 2002.
OECD. OECD guidance for industry data submissions on plant protection products and
their active substances; 2005.
OECD. Revised introduction to the OECD guidelines for testing of chemicals. section 3:
biodegradation and bioaccumulation. Part 1-Principles and Strategies Related to
the Testing of Degradation of Organic Chemicals; 2006.
Park KS, Sims RC, Dupont RR, Doucette WJ, Matthews JE. Fate of PAH compounds in two
soil types: inuence of volatilization, abiotic loss and biological activity. Environ
Toxicol Chem 1990;9:18795.
Quintana JB, Weiss S, Reemtsma T. Pathways and metabolites of microbial degradation
of selected acidic pharmaceutical and their occurrence in municipal wastewater
treated by a membrane bioreactor. Water Res 2005;39:265464.
Radjenovi J, Jeli A, Petrovi M, Barcel D. Determination of pharmaceuticals in sewage
sludge by pressurized liquid extraction (PLE) coupled to liquid chromatography-
tandem mass spectrometry (LC-MS/MS). Anal Bioanal Chem 2009;393:168595.
Richardson ML, Bowron JM. The fate of pharmaceutical chemicals in the aquatic envi-
ronment. J Pharm Pharmacol 1985;37:1-12.
Richter O, Kullmer C, Kreuzig R. Metabolic fate modeling of selected human pharma-
ceuticals in soils. CLEAN Soil, Air, Water 2007;35:495503.
Robertson BK, Alexander M. Growth-linked and cometabolic biodegradation: possible
reason for occurrence or absence of accelerated pesticide biodegradation. Pestic
Sci 1994;41:3118.
Semple KT, Doick KJ, Jones KC, Burauel P, Craven A, Harms H. Dening bioavailability
and bioaccessibility of contaminated soil and sediment is complicated. Environ
Sci Technol 2004;38:228A31A.
Sharer M, Park J-H, Voice TC, Boyd SA. Aging effects on the sorption-desorption characteris-
tics of anthropogenic organic compounds in soil. J Environ Qual 2003;32:138592.
Simkins S, Alexander M. Models for mineralization kinetics with the variables of substrate
concentration and population density. Appl Environ Microbiol 1984;47:1299306.
Stuer-Lauridsen F, Birkved M, Hansen LP, Holten Ltzhft HC, Halling-Srensen B. En-
vironmental risk assessment of human pharmaceuticals in Denmark after normal
therapeutic use. Chemosphere 2000;40:78393.
Topp E, Monteiro SC, Beck A, Coelho BB, Boxall ABA, Duenk PW, et al. Runoff of pharma-
ceuticals and personal care products following application of biosolids to an agri-
cultural eld. Sci Total Environ 2008;396:529.
Tunkel J, Howard PH, Boethling RS, Stiteler W, Loonen H. Predicting ready biodegrad-
ability in the Japanese ministry of international trade and industry test. Environ
Toxicol Chem 2000;19:247885.
Vieubl Gonod L, Chenu C, Soulas G. Spatial variability of 2,4-dichlorophenoxyacetic
acid (2,4-D) mineralisation potential at a millimetre scale in soil. Soil Biol Biochem
2003;35:37382.
Villaverde J, Kah M, Brouwn CD. Adsorption and degradation of four acidic herbicides
in soils from Southern Spain. Pest Manag Sci 2008;64:1299305.
Walker A, Rodriguez-Cruz MS, Mitchell MJ. Inuence of aging of residues on the avail-
ability of herbicides for leaching. Environ Pollut 2005;133:4351.
Xie L, Thrippleton K, Irwin MA, Siemering GS, Mekebri A, David C, et al. Evaluation of
estrogenic activities of aquatic herbicides and surfactants using an rainbow trout
vitellogenin assay. Toxicol Sci 2005;87:3918.
Xu J, Wu I, Chang AC. Degradation and adsorption of selected pharmaceuticals and per-
sonal care products (PPCPs) in agricultural soils. Chemosphere 2009;77:1299305.
Yalkowski SH, Dannenfelser RM. Aquasol database of aqueous solubility. Tucson, AZ:
College of Pharmacy, University of Arizona; 1992.
Zwiener C, Seeger S, Glauner T, Frimmel F. Metabolites from the biodegradation of
pharmaceutical residues of ibuprofen in biolm reactors and batch experiments.
Anal Bioanal Chem 2002;372:56975.