Application of the OECD 301F respirometric test for

the biodegradability assessment of various potential

endocrine disrupting chemicals
Athanasios S. Stasinakis
a,
*
, Anastasios V. Petalas
a,b
, Daniel Mamais
b
,
Nikolaos S. Thomaidis
c
a
Department of Environment, Water and Air Quality Laboratory, University of the Aegean, University Hill, Mytilene 81 100, Greece
b
Department of Water Resources, Faculty of Civil Engineering, National Technical University of Athens, 5 Iroon Polytechniou Street, Zografou,
Athens 15773, Greece
c
Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Athens 15771, Greece
Received 11 December 2006; received in revised form 2 August 2007; accepted 3 August 2007
Available online 18 September 2007
Abstract
The biodegradability of several potential endocrine disrupting compounds, namely 4-n-nonylphenol (4-n-NP), nonylphenol monoetho-
xylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), peru-
orooctanoate (PFOA) and peruorononanoate (PFNA) was evaluated in this study, using OECDmethod 301F(manometric respirometry
test) and activated sludge as inoculum. According to the results, 4-n-NP and BPA meet the strict denition of ready biodegradability and
they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7 5.7%,
n = 3), TCS (52.1 8.5%, n = 3) and NP1EO (25.9 8.1%, n = 3), indicating their possible biodegradation in wastewater treatment sys-
tems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable
compound showed the absence of co-metabolic phenomena during 4-n-NP, BPA and TCS biodegradation. Using rst order kinetics to
describe biodegradation of the target compounds, half-lives of 4.3 0.6, 1.3 0.2, 1.8 0.5, 6.9 2.6 days were calculated for 4-n-
NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio scheri showed that biodegradation of 4-n-NP,
NP1EO, BPA and TCS is a simultaneous detoxication process, while possible abiotic or biotic transformations of NP2EO, DEHP,
PFOA and PFNA during respirometric test resulted to signicant increase of their toxicities.
2007 Elsevier Ltd. All rights reserved.
Keywords: Biodegradation kinetics; Respirometry; Wastewater; Activated sludge; EDCs
1. Introduction
Several synthetic organic compounds used in everyday
life products, such as surfactants, personal-care products,
plasticizers and industrial additives, are commonly
detected in municipal wastewater and they present signi-
cant research interest due to their extensive use and to their
toxicological properties (Birkett and Lester, 2003).
Nonylphenol polyethoxylates (NPnEOs, where n indi-
cates the number of ethoxy units) are an important group
of non-ionic surfactants that are widely used in many com-
mercial and household functions, including detergents, cos-
metic products and textiles (Birkett and Lester, 2003).
Several studies have proved that biotransformations of
long chain NPnEOs occurring in sewer system and primary
clarier, result in accumulation of shorter chain metabolic
intermediates including nonylphenol (NP), nonylphenol
monoethoxylate (NP1EO) and nonylphenol diethoxylate
(NP2EO) (Ahel et al., 1994; Langford et al., 2005). These
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.08.002
*
Corresponding author. Tel.: +30 22510 36257; fax: +30 22510 36246.
E-mail address: astas@env.aegean.gr (A.S. Stasinakis).
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 34583467
compounds have been classied as endocrine disruptors by
several organizations (Birkett and Lester, 2003), while NP
has been listed as a priority substance in the Water Frame-
work Directive (EU, 2001). Regarding NP, this compound
is a mixture of dierent branched and linear chain isomers
(ortho-, meta- or para-), with the most common ring iso-
mers being the para isomers (4-NPs). Recent studies have
shown that 4-n-NP is a strong estrogenic isomer (Vetillard
and Bailhache, 2006), commonly detected in wastewater
treatment systems (WWTSs) (Gatidou et al., 2006) and it
is usually used as the representative compound of this cate-
gory (Ying et al., 2003; Ballesteros et al., 2006; Gatidou
et al., 2006).
Bisphenol A (BPA) is a monomer of polycarbonate plas-
tics widely used in the cover coating on food and drink
packages, dental sealants and baby-milk bottles and it pos-
sesses estrogenic and antiandrogenic activity (Birkett and
Lester, 2003). Triclosan (TCS) is a broad spectrum antimi-
crobial agent that is used in personal-care products such as
soaps, toothpastes and deodorants, as well as in textiles
(Singer et al., 2002). Phthalic acid esters are industrial
chemicals serving as additives in polyvinylchloride, poly-
vinylacetate, cellulosic and polyurethane resins (Birkett
and Lester, 2003). Among them, DEHP (di-(2-ethyl-
hexyl)-phthalate) is often detected in WWTSs (Fauser
et al., 2003) and it has xeno-estrogenic, carcinogenic and
mutagenic eects (Beliles et al., 1989). Finally, peruoro-
chemicals have been used in a variety of industrial and
household products, such as re-ghting foams, textile
and paper coatings, shampoos and conditioners (Houde
et al., 2006). Among them peruorooctanoate (PFOA)
and peruorononanoate (PFNA) have been recently
detected in WWTSs (Sinclair and Kannan, 2006), they
can be bioaccumulated and biomagnied through food
webs and they have shown potentials for reproductive
interference in animal experiments (Guruge et al., 2006;
Houde et al., 2006).
Despite the often detection of these compounds in
WWTSs, there are few studies investigating their biodegra-
dation in continuous-ow activated sludge experiments.
This is possibly due to the fact that these experiments are
time consuming, costly and require specialised technical
equipment. On the other hand, OECD ready biodegra-
dability tests (OECD, 1993) are commonly used to obtain
C OH
CH
3
CH
3
OH

BPA
O
Cl
Cl OH
Cl
TCS
O
O
O
O
CH
3
CH
3
CH
3
CH
3
DEHP
C
9
H
19
OH
4-n-NP
C
9
H
19
OCH
2
CH
2
OH
NP1EO
C
9
H
19
OCH
2
OCH
2
CH
2
OH
NP2EO
C
O
OH CF
3
(CF
2
)
5
CF
2
PFOA
C
O
OH CF
3
(CF
2
)
6
CF
2
PFNA
Fig. 1. Chemical structures of the studied compounds.
A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467 3459
a rst characterization of organic compounds in terms of
their accessibility to microbial degradation (Alexy et al.,
2004; Guhl and Steber, 2006) and play an important role
in the EU environmental classication of chemicals (EC,
1993), as well as in environmental risk assessment (EC,
2003). These tests are performed in a batch mode, con-
taining a mineral medium, the test substance as the sole
carbon source and a low concentration of a microbial inoc-
ulum. Due to the aforementioned stringent test conditions,
it is generally assumed that chemicals meeting the pertinent
biodegradation criteria will be rapidly biodegraded in the
environment.
Among the OECD tests for ready biodegradability, the
manometric respirometry test (OECD 301F) has been
widely used, providing good reproducibly, precision and
accuracy (Guhl and Steber, 2006; Lapertot and Pulgarin,
2006). In this test, biodegradation is calculated for each tar-
get compound from the measured BOD. A BOD value
equal or higher than 60% of theoretical oxygen demand
(ThOD), obtained within 28 days, is regarded as evidence
of ready biodegradability. This threshold level has to be
reached in a 10-day window within the 28-day period of
the test. The 10-day window begins when the degree of bio-
degradation has reached 10% of the ThOD and must end
before or at the day 28 of the test (OECD, 1993). The
determination of BOD is advantageous because it is a
direct biological parameter of aerobic degradation in con-
trast to other ready biodegradability tests using dissolved
organic carbon (DOC) removal (such as OECD 301A,
301E), where the elimination of an organic substance from
water allows only indirect conclusions about biodegrada-
tion, including mechanisms such as adsorption onto sur-
faces or/and suspended solids (Guhl and Steber, 2006).
Moreover, due to the high data density providing by the
continuously BOD recording (360 values in a period of
28 days), these tests provide sucient data for calculating
accurate rst order kinetics in the degradation phase (Rich-
terich and Steber, 2001). According to a recently published
paper (Boethling and Lynch, 2006), kinetic terms such as
rate constant, half-life and lag phase should be reported
in OECD biodegradation studies, additionally to biodegra-
dation percentage at a xed time.
The objective of this study was to evaluate the ready
biodegradability of 4-n-NP, NP1EO, NP2EO, BPA, TCS,
DEHP, PFOA and PFNA (Fig. 1) by activated sludge,
using OECD method 301F. Biodegradation percentages
and other kinetic terms (lag phase, rate constant and
half-life) of the target compounds were calculated in
28-days manometric respirometry tests. In addition, bio-
degradation tests in the presence of a readily biodegradable
compound were performed for 4-n-NP, BPA and TCS to
investigate their possible eects on heterotrophic bacteria
as well as the existence of co-metabolic phenomena. More-
over, experiments were performed using Vibrio scheri bio-
assay to investigate possible toxicity change of the target
compounds prior and after termination of manometric res-
pirometry tests.
2. Methods
2.1. Chemicals
Stock and working solutions of the target compounds
were prepared in methanol HPLC grade (Merck, Ger-
many) by weighting appropriate amounts of 4-n-NP
(99.5%, Labor Dr. Ehrenstorfer-Schafers, Germany, CAS
No: 104-40-5), NP1EO (99%, Labor Dr. Ehrenstorfer-
Schafers, Germany, CAS No: 27986-36-3), NP2EO (99%,
Labor Dr. Ehrenstorfer-Schafers, Germany, CAS
No: 20427-84-3), BPA (>97%, Buchs, Switzerland, CAS
No: 80-05-7), TCS (>97%, Fluka, Germany, CAS No:
3380-34-5), DEHP (99%, Aldrich, WI, USA, CAS
No: 117-81-7), PFOA (97%, Alfa Aesar, Germany, CAS
No: 335-67-1), PFNA (95%, Alfa Aesar, Germany,
CAS No. 375-95-1) and stored at 20 C. Sodium acetate
trihydrate, allylthiourea, trace elements and mineral salts
used in biodegradation and toxicity tests were of analytical
grade and purchased from Merck (Germany).
2.2. Activated sludge inoculum
Activated sludge was collected from a municipal WWTS
(Mytilene, Lesvos). The inoculum was preconditioned to
reach the endogenous respiration rate, by washing once
with tap water, diluting to a concentration of 3 g l
1
dry
matter and nally aerating for 2 days (Reuschenbach
et al., 2003). This starved activated sludge suspension was
further diluted to the inoculum concentration given in
OECD protocol (OECD, 1993).
2.3. Biodegradation experiments
Manometric respirometry tests were carried out in the
Sensomat system (AQUALYTIC

ZN, Tintometer
GmbH, Germany), which is based on a manometric princi-
ple and uses innovative piezo-resistive pressure sensor tech-
nology. Owing to microbial activity, oxygen is taken from
the gas phase of the hermetically sealed reaction vessels,
while carbon dioxide released from respiration is absorbed
by KOH in a small tube and the resulting reduction in air
pressure inside the closed system is measured. From the
resulting data, the BOD in the system can be directly calcu-
lated in mg l
1
according to the following equation (Reus-
chenbach et al., 2003; Vahaoja et al., 2005)
BOD
MO
2

RT
m
V
t
V
I
V
I
a
T
m
T
0

DpO
2
1
where M(O
2
) is the molecular weight of oxygen (32000
mg mol
1
), R is the gas constant (83.144 l mbar
mol
1
K
1
), T
0
is the reference temperature (273.15 K),
T
m
is the incubation temperature (293.15 K), V
t
is the total
volume of the test vessel (ml), V
I
is the liquid volume in the
test vessel (ml), a is the Bunsen absorption coecient
(0.03103) and Dp(O
2
) is the dierence of the oxygen partial
pressure (mbar).
3460 A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467
Biodegradation experiments were performed in eight
experimental cycles according to the experimental protocol
presented in Table 1. Biodegradation of each target com-
pound was studied in triplicate in AQUALYTIC

asks
(Table 1, Test Suspension). Initially, solution of each com-
pound in methanol (12 ml) was poured into AQUALY-
TIC

asks and it was allowed to stand at room

temperature for complete methanol evaporation (Hayashi
et al., 2005). Afterwards, appropriate volumes of the min-
eral medium consisted of KH
2
PO
4
, K
2
HPO
4
, Na
2
H-
PO
4
12H
2
O, NH
4
Cl, MgSO
4
7H
2
O, CaCl
2
and
FeCl
3
6H
2
O (OECD, 1993) were added to each ask
and the mixture was subjected to ultrasonication (Hayashi
et al., 2005). The concentrations of the target compounds
ranged between 10 and 50 mg l
1
(Table 1), while the inoc-
ulum concentration was 30 mg l
1
dry matter (OECD,
1993). To prevent nitrication, allylthiourea was added in
all asks at concentration of 10 mg l
1
. Addition of ally-
lthiourea is not considered in the original OECD 301F,
however it has been proved to be an eective inhibitor of
nitrication processes (Reuschenbach et al., 2003) and it
has been often used in respirometric biodegradation tests
(Reuschenbach et al., 2003; Vahaoja et al., 2005).
In each experimental cycle, a biotic control was used to
check BOD due to endogenous respiration, while a positive
control was used to check inoculum viability (Table 1). For
4-n-NP, BPA and TCS, a toxicity control was also used to
investigate possible eects of the target substances on
heterotrophic bacteria that degrade readily biodegradable
compounds and to study whether co-metabolic phenomena
enhance biodegradation of the target substances (Alexy
et al., 2004). All the experiments were carried out in ther-
mostatically controlled conditions (20 C), while the culti-
vation medium was stirred to maintain biomass in
suspension.
To calculate biodegradation percentage at the end of
each test (28th day), the amount of oxygen taken up by
the microbial population in test suspension (corrected for
uptake by biotic control, run in parallel) was expressed as
percent ThOD
Degradation % 100
BODBOD
blank
ThOD
2
where BOD is biochemical oxygen demand of the test sus-
pension (mg l
1
), BOD
blank
is biochemical oxygen demand
of the biotic control (mg l
1
) and ThOD is theoretical oxy-
gen demand required when the target compound is com-
pletely oxidized (mg l
1
).
First order kinetics was applied in the degradation phase
and half-lives of the target compounds were estimated
using the following equations:
BOD
r
BOD
ult
e
k
1
t
3
t
1=2

ln 2
k
1
4
where BOD
ult
is the ultimate BOD (mg ThOD l
1
), BOD
r
is the BOD remaining at time t (BOD
ult
BOD, mg l
1
),
k
1
is the degradation rate constant (day
1
) and t
1/2
is the
half-life (days).
The degradation rate constant (k
1
) was evaluated by
plotting ln
BODr
BOD
ult

vs. t (R
2
> 0.95). This model has been
used several times in the past, describing other organic
compounds biodegradation (Staples et al., 1999; Stasinakis
et al., 2005).
2.4. Vibrio scheri toxicity experiments
To investigate possible toxicity changes of the target
compounds after OECD biodegradability tests, bacterial
bioluminescence bioassay, V. scheri (strain NRRL-B
11177) was used (DIN 38412-34, 1991) and percentage
inhibition was calculated using samples prior starting and
after terminating manometric respirometry tests.
In particular, samples of the target substances were
taken prior to the biodegradation tests and were diluted
Table 1
Experimental protocol used in manometric respirometry tests
Target
compounds
Initial concentration
(mg l
1
)
Concentration as ThOD
(mg l
1
)
Test suspension
a
(n = 3)
Biotic control
b
(n = 1)
Positive control
c
(n = 1)
Toxicity control
d
(n = 1)
4-n-NP 30.0 89.5
NP1EO 30.0 83.5
NP2EO 25.6 67.7
TCS 10.0 13.5
BPA 35.0 88.4
DEHP 35.0 90.5
PFOA 50.0 27.8
PFNA 50.0 28.4
The number of replicates is given in parenthesis.
a
Test suspension contained mineral medium, inoculum, nitrication inhibitor and the target compound.
b
Biotic control contained mineral medium, inoculum and nitrication inhibitor.
c
Positive control contained mineral medium, inoculum, nitrication inhibitor and sodium acetate trihydrate (217 mg l
1
or 102 mg l
1
as ThOD).
d
Toxicity control contained mineral medium, inoculum, nitrication inhibitor and sodium acetate trihydrate (217 mg l
1
or 102 mg l
1
as ThOD) and
the target compound.
A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467 3461
(2% NaCl aqueous solution, pH 7.0 0.2) at concentration
levels that were sucient for monitoring their inhibitory
eects on V. scheri. The dilution rates of the initial con-
centrations tested in the V. scheri bioassay equalled to
1:20, 1:5, 1:4.3, 1:3.5, 1:12.5, 1:2, 1:2 and 1:2 for 4-n-NP,
NP1EO, NP2EO, BPA, TCS, DEHP, PFOA and PFNA,
respectively, resulting in nominal concentrations of 1.5,
6.0, 6.0, 10, 0.8, 17.5, 25 and 25 mg l
1
. The bacterial sus-
pension and the prepared dilution series were maintained
at 15 C in a thermoblock prior to testing. Bioluminescence
was measured using a luminometer (Lumistox 300, Dr.
Lange GmbH) after 30 min incubation in the standard bio-
luminescence assay and inhibition percentages were calcu-
lated. A similar procedure was followed soon after
terminating manometric respirometry tests. Samples were
initially ltrated and afterwards diluted at the same ratio
with that reported above. All the experiments were per-
formed in triplicate.
3. Results and discussion
3.1. Validation and reproducibility of manometric
respirometry test
A ask containing sodium acetate trihydrate was used as
positive control in each experimental cycle (Table 1) for
checking the activity of the inoculum (OECD, 1993). In
all experimental cycles, sodium acetate was rapidly biode-
graded and, within 28 days, the calculated BOD reached
80.1 4.3% (n = 8) of the ThOD (Fig. 2). Similar biodeg-
radation of sodium acetate has been reported in the litera-
ture (Richterich and Steber, 2001). The above results
indicate that microbial biomass used in respirometric tests
was viable, all tests were valid according to the OECD pro-
tocol and satisfactory reproducibility of the method was
achieved.
3.2. Nonylphenols biodegradation
To investigate 4-n-NP biodegradation, three AQUA-
LYTIC

asks containing 4-n-NP and other constituents

previously reported were used. After an initial lag phase
of 7.9 0.6 days, reecting the time required for either
the induction of specic metabolic enzymes or/and for
growth of species capable of metabolizing the target com-
pound (Richterich and Steber, 2001; Foulk and Bunn,
2007), 4-n-NP was aerobically biodegraded at a percentage
of 61.5 6.0% (n = 3) by day 28 (Fig. 2, Table 2). Degra-
dation reached 10% by day 8 and exceeded 60% by day 14,
in approximately 6 days (Fig. 2). These results marginally
meet the strict denition of readily biodegradation (OECD,
1993), thus 4-n-NP is not expected to be persistent in the
activated sludge process.
So far, in some published papers, NP is considered as a
stable molecule which is not rapidly degraded in activated
sludge systems (Birkett and Lester, 2003). However, it
should be mentioned that this observation has been
excluded by monitoring nonylphenols concentrations in
real WWTSs (Ahel et al., 1994) or using technical mixtures
of NPnEOs as parent compounds in laboratory experi-
ments (Esperanza et al., 2004). In both aforementioned
cases, NP appears in euents of activated sludge units as
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (days)
%

D
e
g
r
a
d
a
t
i
o
n
Sodium acetate 4-n-NP NP1EO NP2EO Sodium Acetate + 4-n-NP
Fig. 2. Aerobic biodegradation of nonylphenols using OECD method 301F. (Three replicates containing 4-n-NP, eight replicates containing sodium
acetate trihydrate as positive control, one replicate containing 4-n-NP and sodium acetate trihydrate (0.88:1 as ThOD) as toxicity control, three replicates
containing NP1EO and three replicates containing NP2EO.)
3462 A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467
an intermediate starting from NPnEOs and therefore a
mass balance becomes dicult since the produced mass
of NP can exceed its inuent mass. In this study, 4-n-NP
was used as the parent compound and the obtained results
are consistent with others investigators following a similar
protocol. Staples et al. (1999), using manometric respiro-
metry and technical NP as target compound, reported an
average NP biodegradation equal to 62%. Moreover, Tan-
ghe et al. (1998) observed signicant NP biodegradation in
a lab-scale activated sludge system, feding on 8.33 mg l
1
of technical NP and operating at various temperatures.
The half-life of 4-n-NP obtained in this series of experi-
ments was equal to 4.3 0.6 days (Table 2). This value was
signicantly lower than values reported in previous aerobic
experiments for other substrates such as seawater (Ekelund
et al., 1993), aquifer material (Ying et al., 2003) and sedi-
ment (Yuan et al., 2004), indicating the higher anity of
activated sludge for NP biodegradation.
Additionally to the experiments reported above, a toxi-
city control containing a mixture (0.88:1 as ThOD) of 4-n-
NP and sodium acetate trihydrate was used to investigate
4-n-NP biodegradation in the presence of a readily biode-
gradable compound. Up to the end of this experiment,
almost 63% of the mixture was biodegraded (Fig. 2), a
value which was lower than the biodegradation percentage
that was expected according to the previously reported
experiments (71%). Moreover, degradation reached 10%
by day 1 and exceeded 60% by day 25, indicating that a
prolonged time was required for the adequate biodegrada-
tion of the mixture (Fig. 2). According to the aforemen-
tioned results, it seems that the presence of a readily
biodegradable substance did not enhance 4-n-NP biodegra-
dation via a co-metabolic process. On the contrary, a
decreased biodegradation rate was observed possibly due
to incomplete predominance of specialised 4-n-NP degrad-
ing bacteria or/and inhibitory eects of 4-n-NP on hetero-
trophic microorganisms that degrade sodium acetate
(Alexy et al., 2004).
The experiments for nonylphenol ethoxylates biodegra-
dation were conducted following the same experimental
protocol described above for 4-n-NP. Regarding NP1EO,
a signicant lag phase was initially observed and degrada-
tion reached 25.9 8.1% (n = 3) by the end of the test
(Fig. 2, Table 2). NP2EO was not biodegraded during
the test (Fig. 2, Table 2). To the best of our knowledge, res-
pirometric tests or other biodegradation experiments using
activated sludge as inoculum and NP1EO or NP2EO as
parent compounds have not been conducted previously.
However, Yuan et al. (2004), investigating biodegradation
of NP and NP1EO in river sediment, reported that NP bio-
degradation was higher than that of NP1EO. Moreover,
Esperanza et al. (2004) investigating nonylphenols removal
in pilot scale WWTSs reported that NP and NP1EO exhi-
bited elimination rates (sum of adsorption and biodegrada-
tion) around 90% and 70% in the aeration tank,
respectively, while NP2EO concentrations were increased,
indicating that its removal rate was lower than the rate
of its formation from the higher oligomers. According to
those ndings, it seems that the presence of ethoxylate
groups and the relative increase of molecular weight of
these compounds, decreases biodegradability of these
nonylphenols.
3.3. BPA biodegradation
In experiments using BPA as a sole carbon source, after
an initial lag phase, BPA was biodegraded at a percentage
equal to 87.8 6.9% (n = 3, Table 2). Degradation reached
10% by day 4.5 and exceeded 60% by day 6.2 (Fig. 3).
These results meet the strict denition of ready biodegra-
dability and BPA is not expected to be persistent in an acti-
vated sludge system. Similar results have been reported in a
previous study investigating ready biodegradability of BPA
in OECD 301F respirometric tests (West et al., 2001). The
half-life of BPA obtained in this series of experiments was
equal to 1.3 0.2 days (Table 2). Slightly higher half-lives
of BPA have also been reported for other substrates such
as river water (Kang and Kondo, 2002) and soil (Fent
et al., 2003), indicating the ready biodegradability of this
compound in the environment.
To investigate BPA biodegradation in the presence of a
readily biodegradable compound, a toxicity control con-
taining a mixture (0.87:1 as ThOD) of BPA and sodium
acetate trihydrate was used. Up to the end of this experi-
ment, 154.5 mg l
1
of BOD were consumed (81% of
ThOD), a value which was similar to the sum of BOD con-
sumed when BPA or sodium acetate trihydrate were stud-
ied solely (84% of ThOD). Biodegradation reached 10%
by day 1 and exceeded 60% by day 9, indicating that a pro-
longed time was required for the adequate biodegradation
of the mixture (Fig. 3). These observations indicate that
beside the fact that a prolonged time was required for mix-
ture biodegradation, possibly due to incomplete predomi-
nance of specialised degrading bacteria; no inhibitory
eect of BPA on heterotrophic microorganisms was
observed. The dierence in the observed and the expected
degradation percentage of the mixture could be attributed
Table 2
Degradation of dierent chemical compounds according to OECD 301F
manometric respirometry test and respective kinetic values (lag period,
kinetic constant and half-life)
Test
compound
a
Degradation
b
(%)
Lag period
b
(days)
k
1
-constant
b
(day
1
)
Half-life
b
(days)
4-n-NP 61.5 (6.0) 7.9 (0.6) 0.16 (0.02) 4.3 (0.6)
NP1EO 25.9 (8.1) 17.3 (0.7) >28
NP2EO n.b.
c

TCS 52.1 (8.5) 16.5 (3.5) 0.38 (0.09) 1.8 (0.5)
BPA 87.8 (6.9) 4.3 (0.3) 0.53 (0.08) 1.3 (0.2)
DEHP 58.7 (5.7) 4.1 (0.7) 0.10 (0.03) 6.9 (2.6)
PFOA n.b.
c

PFNA n.b.
c

a
The number of replicate tests performed for each compound was three.
b
The standard deviation of the results is given in brackets.
c
No biodegradation was observed during the experiment.
A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467 3463
to slight variations in the quantity and quality of the ino-
culum used in each ask (Alexy et al., 2004).
3.4. TCS biodegradation
After an initial lag phase of 16.5 3.5 days, TCS was
aerobically biodegraded at a percentage equal to
52.1 8.5% (n = 3) and a half-life equal to 1.8 0.5 days
was calculated (Table 2). In the toxicity control containing
a mixture of TCS and sodium acetate trihydrate (0.13:1 as
ThOD), 85.5 mg l
1
of BOD were consumed, a value which
was similar to the sum of BOD consumed when TCS
(7.0 mg l
1
of BOD) or sodium acetate trihydrate
(81.7 mg l
1
of BOD) were studied solely. These results
indicated the absence of TCS inhibitory eects on hetero-
trophic microorganisms, as well as the absence of co-meta-
bolic phenomena in the presence of a readily biodegradable
compound.
According to OECD (1993), biodegradation results
reported above for TCS do not meet the strict denition
of ready biodegradability (BOD P60% of ThOD); how-
ever, they indicate a possible TCS biodegradation in a
WWTS. Moreover, in several recent studies it has been
argued that the pass criterion for OECD BOD tests should
be decreased to 50% (Bealing, 2002; Boethling and Lynch,
2006), a criterion which is fullled according to the present
results. This statement is supported by previous studies
investigating TCS fate in activated sludge systems. In a
one-week survey of a WWTS in Switzerland, Singer et al.
(2002) reported that TCS was bio-eliminated at a
percentage equal to 79%. Moreover, Federle et al. (2002)
investigating the fate of TCS in laboratory activated sludge
systems, supported that its greatest part was biodegraded.
The fact that OECD criteria were not fullled in the pre-
sent study could be possibly due to the experimental condi-
tions used (low biomass concentration, no biomass
acclimatization), which dier signicantly from the operat-
ing conditions occurring in an activated sludge system.
3.5. DEHPPFOAPFNA biodegradation
Respirometric tests containing DEHP showed that this
compound was biodegraded at a percentage equal to
58.7 5.7% (n = 3, Table 2). Although this partial
DEHP elimination does not meet the strict denition
of ready biodegradability according to OECD (1993), it
is possible that a faster process and ecient biodegrada-
tion could be achieved in the presence of high concentra-
tions of acclimatized microorganisms in a WWTS. This
hypothesis is proved by a previous study where signi-
cant DEHP bio-elimination (70%) was calculated
performing mass balances in a WWTS (Fauser et al.,
2003).
The half-life of DEHP obtained in this series of experi-
ments was equal to 6.9 2.6 days (Table 2). In previous
experiments investigating DEHP biodegradation during
anaerobic sludge digestion, signicantly higher half-lives
of DEHP were calculated (Gavala et al., 2003), indicating
the higher anity of activated sludge for its biodegradation
under aerobic conditions.
On the other hand, PFOA and PFNA were not biode-
graded at all during respirometric experiments (Table 2).
To the best of our knowledge, there are no data in the lite-
rature for the biodegradation potential of these compounds
in activated sludge process. However, Sinclair and Kannan
(2006) monitoring the mass ows of these compounds in
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (days)
%

D
e
g
r
a
d
a
t
i
o
n
Sodium acetate BPA Sodium Acetate + BPA
Fig. 3. Aerobic biodegradation of BPA using OECD method 301F. (Three replicates containing BPA, eight replicates containing sodium acetate
trihydrate as positive control and one replicate containing BPA and sodium acetate trihydrate (0.87:1 as ThOD) as toxicity control.)
3464 A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467
WWTSs reported that they increased after secondary treat-
ment probably due to decomposition of a precursor com-
pound. Further simulation tests should be performed in
the future, investigating biodegradation of these com-
pounds under dierent operating conditions (acclimatized
biomass, existence of anoxic/anaerobic conditions).
3.6. V. scheri toxicity assessment
As it can be seen in Fig. 4, toxicity of 4-n-NP, NP1EO,
BPA and TCS on marine bacterium V. scheri was sig-
nicantly lower after termination of OECD manometric
respirometry tests, indicating that biodegradation of these
compounds is a simultaneous detoxication process. As a
result, euent wastewater disposal seems to possess lower
risk for deterioration of the nal receiver due to the pres-
ence of these compounds. Contrary to the above, toxicity
tests performed for the other compounds (NP2EO, DEHP,
PFOA and PFNA) showed that their toxicity was signi-
cantly increased after respirometric tests. Among these
compounds, as it was previously shown (Table 2), DEHP
was partially biodegraded, while NP2EO, PFOA and
PFNA were not biodegraded at all.
Regarding DEHP, it is likely that toxic metabolites are
produced during its biodegradation. In the literature, the
formation of persistent and toxic intermediates such as adi-
pic acid and 2-ethylhexanol has been reported (Nalli et al.,
2006). For the other three compounds, it is possible that
abiotic or biotic mechanisms were taken place in respiro-
metric tests, resulting on the production of more toxic
metabolites. Regarding NP2EO, its transformation to its
carboxylic acid analogue NP2EC has been reported (Potter
et al., 1999); however there are no available data in the
literature for the acute toxicity of this compound. Finally,
while it is well known that uorinated telomere alcohols
are probable precursor compounds that may undergo
transformation reactions leading to PFOA and PFNA
(Houde et al., 2006), to the best of our knowledge there
are no data regarding possible metabolites of these com-
pounds in the environment.
Further research should be performed in the future in
order to ll the gaps in the literature regarding transforma-
tion products of these compounds, as well as their toxico-
logical and chemical properties.
4. Conclusions
The objective of this study was to investigate biodegra-
dation of several synthetic organic compounds commonly
detected in WWTSs, using manometric respirometry test
(OECD method 301F). According to the results, 4-n-NP
and BPA are considered as readily biodegradable com-
pounds, NP1EO, BPA, TCS and DEHP were partially
biodegraded, while no biodegradation was observed for
NP2EO, PFOA and PFNA. Therefore for the non-readily
biodegradable substances, further simulation tests are
needed in order to investigate the fate of these compounds
in activated sludge systems. The use of V. scheri bioassay
revealed that biodegradation of 4-n-NP, NP1EO, BPA
and TCS is a simultaneous detoxication process. On
the contrary, toxicity of NP2EO, DEHP, PFOA and
PFNA increased after respirometric tests, indicating their
possible biotic or abiotic transformation to more toxic
metabolites.
0
20
40
60
80
4-n-NP NP1EO NP2EO BPA TCS DEHP PFOA PFNA
Target Compound
I
n
h
i
b
i
t
i
o
n

(
%
)
T= 0 days
T=28 days
Fig. 4. Percentage inhibition of identical dilution rates of the target compounds on marine bacterium Vibrio scheri at the start (day 0) and after
termination (day 28) of OECD 301F respirometric tests.
A.S. Stasinakis et al. / Bioresource Technology 99 (2008) 34583467 3465
Acknowledgements
This study was co-funded by the European Social Fund
(75%) and National Resources (EPEAEK-II) PYTHA-
GORAS I (25%). The authors would like to thank Mrs.
Sevasti Kotsifa and Mrs. Niki Papadopoulou for their
valuable help during the experiments.
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