Journal of Hazardous Materials 296 (2015) 120–127

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Evaluation of PCB dechlorination pathways in anaerobic sediment

microcosms using an anaerobic dechlorination model
Hale Demirtepe a , Birthe Kjellerup b , Kevin R. Sowers c , Ipek Imamoglu a,∗
a
Department of Environmental Engineering, Middle East Technical University, Ankara, Turkey
b
A. James Clark School of Engineering, Department of Civil and Environmental Engineering, University of Maryland at College Park, College Park,
MD 20742, USA
c
Institute of Marine and Environmental Technology, Department of Marine Biotechnology, University of Maryland Baltimore County, Baltimore,
MD 21202, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Anaerobic dechlorination of PCBs in

sediment microcosms modeled.
• Major pathways and congeners
active in dechlorination quantified.
• Dechlorination rates, toxicity change
and terminal products evaluated.
• Anaerobic dechlorination modeling
can support bioremediation efforts.

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

Article history: A detailed quantitative analysis of anaerobic dechlorination (AD) pathways of polychlorinated biphenyls
Received 24 December 2014 (PCBs) in sediment microcosms was performed by applying an anaerobic dechlorination model (ADM).
Received in revised form 10 April 2015 The purpose of ADM is to systematically analyze changes in a contaminant profile that result from micro-
Accepted 11 April 2015
bial reductive dechlorination according to empirically determined dechlorination pathways. In contrast
Available online 14 April 2015
to prior studies that utilized modeling tools to predict dechlorination pathways, ADM also provides
quantification of individual pathways. As only microbial reductive dechlorination of PCBs occurred in the
Keywords:
modeled laboratory microcosms, extensive analysis of AD pathways was possible without the complicat-
Polychlorinated biphenyls (PCBs)
Anaerobic dechlorination model (ADM)
ing effect of concurrent physico-chemical or other weathering mechanisms. The results from this study
Dechlorination pathway showed: (1) ninety three AD pathways are active; (2) tetra- to hepta-chlorobiphenyl (CB) congeners were
Sediment microcosm common intermediates in several AD pathways, penta-CBs being the most frequently observed; (3) the
highest rates of dechlorination were for penta-CB homologs during the initial 185 days; (4) the dominant
terminal products of AD were PCB 32(26–4), 49(24–25), 51(24–26), 52(25–25), 72(25–35), 73(26–35) and
100(246–24), (5) potential toxicity of the sediment was reduced. ADM serves as a powerful tool not only
for a thorough analysis of AD pathways, but also for providing necessary input for numerical fate models
(as a degradation term) that investigate dechlorination products or outcome of natural attenuation, or
bioremediation/bioaugmentation of PCB-impacted sediments.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Polychlorinated biphenyls (PCBs) are among the persistent

∗ Corresponding author at: Department of Environmental Engineering, Middle
organic pollutants (POPs) regulated by the Stockholm Convention
East Technical University, 06531 Ankara, Turkey. Tel.: +90 312 210 5861;
[1]. They were produced between 1930 and 1993 at an estimated
fax: +90 312 210 2646.
E-mail address: ipeki@metu.edu.tr (I. Imamoglu). amount of 1.3 million tons worldwide [2]. Many industries have

http://dx.doi.org/10.1016/j.jhazmat.2015.04.033
0304-3894/© 2015 Elsevier B.V. All rights reserved.
 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127
used PCBs in products such as dielectric fluids, hydraulic fluids,
flame retardants and adhesives [3]. PCBs released into the envi-
ronment eventually end up adsorbed to sediment particles in the
aquatic environment [4] where they bioaccumulate in the food
chain and act as potential neurotoxins, endocrine disruptors, and
carcinogens [5]. Despite their inherent chemical stability PCBs
have been shown to undergo various transport and transformation
mechanisms such as physicochemical (transport, solubilization and
volatilization [6–8]) and biological (aerobic and anaerobic degra-
dation [9–11]). Although bioremediation of PCBs has received
significant attention, it is still not a commonly used technique.
Current treatments include monitored natural attenuation, where
natural attenuation capacity of indigenous microorganisms is uti-
lized (e.g., Lake Hartwell, SC, or Hudson River, NY). More traditional
techniques that rely on significant environmental disruption, such
as dredging and/or capping, are non-sustainable and less cost effec-
tive [12].
Anaerobic dechlorination (AD) is performed by several
halorespiring bacteria [10] via the replacement of chlorines with
hydrogen atoms [11]. The halorespiring bacteria mainly target
extensively chlorinated congeners at the meta and para posi-
tions. However, ortho chlorine removal is also possible, but not as
common as meta and para dechlorination [12]. Reductive dechlori-
nation of extensively chlorinated congeners reduces the potential
toxicity (based on the 12 dioxin-like congeners [13] and non-
dioxin-like ortho-substituted congeners [14]) and bioaccumulation
of PCBs [9,11]. In addition, aerobes such as Burkholderia xenovorans
will subsequently cleave the aromatic ring of congeners usually
having five or fewer chlorines with biphenyl dioxygenase after
anaerobic reductive dechlorination in the 2,3- or 3,4-positions
[15,16]. Dechlorination on its own would not result in complete
destruction of PCBs, but the products of anaerobic halorespiration
(those having less than five chlorines) are generally more suscep-
tible to complete mineralization by aerobic bacteria with genes
encoding the biphenyl degradation pathway. Therefore, AD of PCBs
holds great potential for pioneering bioremediation of contami-
nated sediments.
Since commercial PCB mixtures detected in the environment are
a complex group of compounds, composed of over 100 congeners
per sample, investigation of their environmental degradation is
facilitated by the use of mathematical tools. In recent years, studies
directed towards modeling degradation mechanisms to evaluate
the fate of pollutants in the environment have been performed.
With each of these studies, PCB dechlorination in sediments was
examined [17–20]. While some of these studies aimed to iden-
tify possible dechlorination pathways [19,20], others attempted
to quantify individual pathways [17,18], by using weathered PCB
data. Previous reports did not test ADM to comprehensively analyze
dynamic changes in dechlorination pathways over time in purely
anaerobic sediments prepared under laboratory conditions. Apply-
ing an anaerobic dechlorination model on controlled laboratory
PCB data would enable predictions based on the probability, tempo-
ral trend and quantification of anaerobic dechlorination pathways.
This study aims to present an in-depth quantitative analysis of
AD pathways of PCBs in sediment microcosms composed of Balti-
more Harbor (BH) sediments incubated for 500 days [21]. As only
microbial reductive dechlorination of PCBs was taking place in
these anaerobic laboratory microcosms, extensive analysis of AD
pathways was made possible without the complicating effect of
concurrent aerobic degradation, physico-chemical interactions or
other weathering mechanisms. This study aims to provide 1) a bet-
ter understanding of the AD pathways, 2) a quantitative assessment
of AD rate, 3) an evaluation of the change in overall PCB toxicity in
sediments, 4) an identification of the dechlorination end products.
In this study, the anaerobic dechlorination model (ADM) previ-
ously developed [17,18,22] was applied. ADM serves as a powerful
121
tool not only for a thorough analysis of AD pathways, but also for
providing necessary input for numerical fate models (as a degra-
dation term) that investigate dechlorination products or outcome
of natural attenuation, or bioremediation/bioaugmentation of PCB-
impacted sediments.
2. Materials & methods
2.1. Microcosm sediment data
Sediment data used in the study was from a microcosm exper-
iment, conducted with Baltimore Harbor (BH) sediments [21].
Briefly, 10 ml of anaerobic low saline mineral medium with a
mixture of formate, acetate and propionate as electron donors pre-
pared in 25 ml anaerobe tubes was inoculated with 2 g wet wt
sediment and sealed under N2 –CO2 (80:20) with Teflon septa. Aro-
clor 1260 (AccuStandard, CT) was added to a final concentration
of 100 ppm in acetone. Microcosms were incubated statically at
30 ◦ C in the dark. Triplicate sediment microcosms were sampled
after incubation for 0, 88, 185, 278 and 500 days by vortexing the
culture to suspend the sediment and removing 0.5 ml of culture
with a 1-ml glass pipette. Samples were extracted with hexane,
passed through a copper–Florisil column and analyzed using a gas
chromatograph equipped with a Ni63 electron capture detector as
described previously [23]. PCB congeners were quantified with a
10-point calibration curve using 2,2 ,3,4,4 ,5,6,6 -CB (10 ppm) as an
internal standard [23]. A total of 177 congeners were detected in
89 chromatographic peaks. To avoid quantifying irrelevant and bio-
logically improbable AD pathways in the model 49 congeners were
deleted based on empirical data [24] or because they are neither
present in Aroclor 1260 nor probable dechlorination products. The
average PCB profile for microcosms is shown in Fig. A1 of Supple-
mentary material.
2.2. Anaerobic dechlorination model (ADM)
ADM was originally developed by Imamoglu et al. [17,25] and
modified by Bzdusek et al. [18,26] Additionally, a second modi-
fication has been conducted within the scope of this study [22].
As an initial validation study, the model was tested on a limited
laboratory PCB data set [25], applied primarily to environmental
sediment samples from Ashtabula River [17], Fox River [25], Lake
Hartwell [18] and Sheboygan River [26]. The general principles of
the model explained in detail in Imamoglu et al. [17,25], Bzdusek
et al. [18,26] and Demirtepe [22] are summarized below.
The model aims to predict an “anaerobically dechlorinated PCB
profile” as similar to the sample source as possible. During AD, a
PCB congener is transformed from one congener to another without
destroying the biphenyl structure. Therefore, the mass balance of
the parent congener being dechlorinated and the product congener
being formed is maintained. The set of dechlorination pathways
were used by the model to alter the original PCB profile based on
the mass balance between congeners. These pathways were applied
to the PCB profile, after it had been normalized to 1000 mol permil
(mol ‰) [17]. PCB analysis results were in mol%. Hence, before being
used in the model, these are converted to mol‰ to bring them to
an equal base.
The model is modified from its original form, such that the
dechlorination pathways are now defined by the targeted chlorines
(i.e., chlorines on meta and para positions, either singly or doubly
flanked) rather than dechlorination processes (i.e., N, H, H , M, Q,
P, and LP) defined in the literature [27,28], as was previously the
case [17,18]. Both versions of the model yielded satisfactory results,
yet in the previously used version, extra dechlorination pathways
had to be included, which were not defined by the dechlorination
122 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127
Table 1
Goodness of fit criteria of measured PCB profiles vs. predicted profiles for quantified anaerobic dechlorination pathways in different time periods during microcosm operation.
Results were shown as comparison with initial profile and between sequential time periods.a
Time periods (Model runs)
Comparison with the t = 0–88 days
initial profile (day 0) t = 0–185 days
t = 0–278 days
t = 0–500 days
Sequential time periods t = 88–185 days
t = 185 –278 days
t = 278–500 days
a
Qr : percent improvement in similarity on reactive congeners, cos :coefficient of proportional similarity, R2 : coefficient of determination.
processes, but known to occur in BH sediments. Hence, the modifi-
cation to produce a wider range of possible pathways was deemed
necessary. Subsequently, the model alters the original profile with
respect to possible dechlorination pathways. Each altered profile
was then compared with the sample profile to obtain the most
similar profile.
Inputs of the model were the sediment microcosm PCB con-
gener profile at a given time point (sample profile), the original
PCB congener profile (i.e., PCB profile at t = 0 day), the list of con-
geners analyzed in all samples, and the possible reactions among
these congeners. The outputs of the model were an anaerobically
dechlorinated PCB congener profile (altered profile), dechlorination
reactions with quantification values associated with them (i.e., in
terms of mol‰), and the goodness of fit criteria indicating the sim-
ilarity between the sample and the altered profiles (i.e., input vs.
output).
The objective function is to minimize the sum of square of dif-
ferences (S) between the resulting altered profile and the sample
profile:

m
2
S= (yj − xj )
j=1
where yj is the concentration of altered congener j (mol‰), xj is the
concentration of sample congener j (mol‰), and m is the number
of congeners [25]. In the data set, there were 84 congener groups
with co-elution and a total of 129 individual congeners (Fig. A1 of
Supplementary material). Alterations on the sample profile were
made according to the biologically confirmed AD pathways input
to the model. That is, if a dechlorination pathway was not shown to
occur in any environmental study, meaning that the pathway was
unlikely to occur, it was eliminated from the pathway list and not
quantified. For example, there were no studies reporting dechlo-
rination of 2345 to 234, hence this pathway was not included in
the model. Additionally, unflanked-Cl or ortho-Cl removal was dis-
regarded, since their occurrence in the environment was rare [29].
The AD pathways were constituted by assuming all PCB congeners
were equally bioavailable at the concentration measured. After
alteration of the original PCB profile, its similarity with the sam-
ple profile was measured using the following indicators: percent
improvement in similarity, Q [25], cosine  coefficient of propor-
tional similarity [30], and the coefficient of determination, R2 [31]. A
satisfactory model fit is defined by cosine  and R2 close to 1 and Q as
high as possible. Alterations were performed on complete congener
profiles. Since profiles were normalized to 1000 mol‰, the result-
ing altered profile and quantification of pathways were also on the
same basis. The model also identified reactive congeners as the ones
that took part in the dechlorination pathways either as a parent or
as a product compound. A second separate normalization was car-
ried out on the PCB profiles with these reactive congeners to utilize
it during the calculation of goodness of fit criteria. Hence, the calcu-
lation of the goodness of fit criteria was based on both the complete
congener list and the reactive congeners; such as Q for 84 analyzed
Qr Cos  R2
95.05% 0.99 0.99
94.08% 0.97 0.91
89.5% 0.93 0.83
97.3% 0.99 0.96
93.7% 0.97 0.91
94.3% 0.99 0.99
76% 0.92 0.82
congeners and Qr for 82 reactive ones. Co-elution of congeners
was disregarded during the identification of dechlorination path-
ways. All 129 congeners identified in chromatographic peaks were
treated as individual congeners, and they were allowed to appear
in any possible pathways. During quantification of pathways, the
most likely co-eluting congener was included in the model. The
most likely congeners were based on the composition of Aroclor
1260 [32] and the possible dechlorination products. In cases where
most likely co-eluting congener could not be determined, the one
having lowest IUPAC number in the co-eluting group was used.
ADM provided averages and standard deviations (SD) of quantifi-
cation values, derived from 100 shuffles of reaction sequences, for
each pathway. Therefore, the average and relative standard devia-
tion (RSD) values of each pathway were the most important outputs
of ADM to evaluate the pathways.
3. Results and discussion
3.1. Initial assessment of dominant anaerobic dechlorination
pathways
(1)
An average PCB profile of three microcosms was used for mod-
eling, since initial assessment of the model with three individual
microcosms revealed that the quantified pathways for each were
not significantly different from each other (p > .05). In order to
identify the dominant dechlorination pathways in the microcosm
sediments, initial model runs were made between day 0 and 500.
The predominant AD pathways both in the environment and in the
laboratory were typically singly/doubly flanked meta/para chlorine
removal. Therefore, these were individually tested on the aver-
age PCB profile as a first step. None of the individual mechanisms
represented the changes observed in the microcosm sediments sat-
isfactorily as indicated by the poor model fit (data not shown).
Hence, combinations of these mechanisms were applied. Unlikely
pathways that were not shown to occur in biological studies were
eliminated, as noted above. As a result, the combination of all
pathways, dechlorination of congeners in positions of singly and
doubly flanked meta and para chlorines, provided the best fit for
the average congener profile at day 500. A minor improvement
in the model fit resulted from excluding one possible dechlorina-
tion pathway from pathway list. During modeling of BH sediment
microcosms, PCB 101(245–25) was identified as forming two prod-
ucts, PCB 49(24–25) and 52(25–25). From a previously conducted
study with BH sediments, it was known that PCB101 only dechlo-
rinated to PCB49 and not PCB52, while PCB52 was formed from
PCB 92(235–25) [24]. Hence, the pathway PCB 101(245–25) to PCB
52(25–25) was excluded to favor the dechlorination of PCB101 to
49. By doing so, a better estimation of the levels of both PCB49
and 52 in the predicted PCB profile at day 500 was possible, which
resulted in an improved model fit. A comparison of the PCB pro-
files at day 0 and 500 is shown in Fig. 1a, while the measured vs.
modeled day 500 (using PCB dechlorination from singly & doubly
 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127 123

Fig. 1. PCB congener profiles of (A) measured day 0 vs. 500, (B) day 500 measured vs. predicted by the model. Cos  and R2 values are provided to observe the similarity
between profiles, percent improvement in similarity (Q) values are provided to show success of fit of model.

flanked meta/ para positions) data is shown in Fig. 1b together with
the results of their respective goodness of fit indicators.The mod-
eled pathways were also applied to the sterile (abiotic) microcosm
data between day 0 and 500. There was not a significant differ-
ence between day 0 and 500 for abiotic microcosms (t(83) = −3.52,
p > .05). Also, the chlorine per biphenyl (Cl/b) change between these
days were lower than 2% (Cl/b0 = 6.28, Cl/b500 = 6.16). Hence, the
observed shift in the controls could not be explained with the mod-
eled AD pathways.
3.2. Detailed pathway analysis of anaerobic dechlorination
The microcosms showed different dechlorination patterns with
time, hence, ADM was applied progressively for each time inter-
val, i.e., between 0 and 88 days, 0 and 185 days, etc. and between
time intervals 88 and 185 days, 185 and 278 days, 278 and 500
days on the average microcosm PCB data using singly and dou-
bly flanked meta and para dechlorination pathways. The goodness
of fit criteria of observed vs. modeled PCB congener profiles are
presented in Table 1. Here, the cosine  and R2 values for the ini-
tial time period are the best, then a poorer fit is observed for the
intermediate time periods followed by a better final day prediction.
The apparent good fit between 0 and 88 days is because no major
change occurred in PCB pattern during this time, due to a lag period
before AD started [11]. ADM predicted the 0–500 day time period
most successfully, as expected, since the dechlorination pathways
identified with biological studies rely on comparison of initial PCB
profiles (e.g., Aroclors) and a final dechlorinated profile [33,34].
There were a total of 250 possible AD reactions of which a min-
imum of 226 were quantified as non-zero. Some of these non-zero
quantified pathways emerged as minor with low average and high
standard deviation values. Therefore, a classification was neces-
sary. Quantification values were sorted for each time interval and
median values of each were used as a reference point. Furthermore,
RSD values associated with the pathways were used as an indica-
tion of prevalence; lower RSD values indicating greater prevalence.
As a result, any pathway whose quantification exceeded the median
value and had an RSD value lower than 100 was defined as a major
pathway. Broadly, a total of 93 major pathways were assessed for
the time interval between day 0 and 500. The complete list of
major dechlorination pathways quantified in BH sediment micro-
cosms is given in Table A1 and depicted in Fig. 2. Among these
major pathways, the ones adopting the cut-off criteria for all time
periods, i.e., days 0–88, 88–185, 185–278 & 278–500 were found
to be consistently predominant during the dechlorination process
(Fig. 2a). Additionally, some of the major pathways were found
to be predominant initially, during the time periods 0–88 and
88–185 d, not conforming to the criteria for final time period(s)
124 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127

A 23456-236 2346-2356 Octa

2345-245 2356-245 2356-234 2356-235 23456-25 2345-234 2346-236 2346-235 Hepta

245-245 2345-24 235-245 2356-24 236-245 234-236 2356-23 2356-25 2345-23 2346-25 234-245 236-246 235-246 Hexa

245-24 245-34 236-24 345-25 236-23 236-25 245-23 Penta

245-4 24-26 25-35 23-26 Tetra

34-2 Tri

2-3 Di

B 2356-236 2346-245 2345-236 2345-234 Hepta

2345-24 235-245 236-236 2356-24 2356-26 2356-23 245-246 2345-26 2346-25 2356-35 2345-23 2346-34 Hexa

235-24 236-26 245-26 236-24 245-34 2356-2 246-25 236-35 246-34 Penta

24-26 245-3 24-34 Tetra

24-3 Tri

C 23456-236 2345-2345 Octa

2345-245 2345-236 2345-235 Hepta

2345-25 235-236 236-246 234-236 245-245 2356-34 Hexa

235-35 246-26 234-26 236-24 234-34 245-24 236-34 Penta

25-35 245-2 234-3 23-34 234-4 Tetra

24-2 23-4 Tri

2-4 Di

Fig. 2. (A) Consistently predominant dechlorination pathways during entire incubation period, (B) Initially predominant pathways, (C) Predominant pathways after 185 days
of microcosm incubation. Congeners appearing in three or more pathways are shown in boldface, coplanar congeners identified by WHO as toxic dioxin-like properties [13]
are shown in underlined italic.

(Fig. 2b), while some became predominant after the initial time Table 2
Dechlorination mechanisms from chlorobiphenyl groups in major anaerobic dechlo-
period(s) had passed (Fig. 2c). Moreover, seven AD pathways had
rination pathways observed by the model. Previous observations in BH sediment
RSD values higher than 100, which means the parent congener microcosms were also noted.
was dechlorinated into possibly more than one daughter con-
Chlorobiphenyl group targeteda Chlorine removal mechanism
gener. These are shown with dashed arrows in Fig. 2c. All pathways
labeled as “initially predominant” in this study constitute thermo- 2345- double-flanked meta or para removalb
dynamically favorable reactions as discussed by Chen et al. [35], 236-, 2356- ortho-flanked meta removalb
245- para-flanked meta removalb
either as a congener or as chlorobiphenyl groups. Not all path-
2346- double-flanked meta removalb
ways labeled “consistently predominant” are among these. On the 245- meta-flanked para removal
other hand, Fagervold et al. [24] could not find a significant rela- 23456- double-flanked meta removal
tionship (p > .05) between dechlorination rates and differences in 2346- meta-flanked para removal
34- meta-flanked para removal
the estimated Gibbs free energy of formation (as given by Holmes
a
et al. [36]) between parent and daughter PCB congeners. Ini- Targeted chlorine is shown as underlined boldface.
b
tial dechlorination reactions and their rates are likely mediated Mechanisms also observed in a previously conducted BH sediment microcosm
study by Fagervold et al. [24].
by kinetics due either to bioavailability (i.e., aqueous concentra-
tion) or the saturation constant of the microorganisms for specific
congeners. results of the ADM. Among the major AD pathways (93 path-
When the chlorine positions of dechlorinated congeners were ways), the appearance frequency of congeners (either as parent
evaluated comparatively between Fig. 2a–c, a number of impor- or product) was evaluated. Congeners 49(24–25), 92(235–25),
tant observations were made. For example, while preference for 101(245–25), 137(2345–24), 146(235–245), and 180(2345–245)
removal of chlorines from singly-flanked para positions does were observed in four different AD pathways. In addition, other
not change over the course of AD, it increases for chlorines sit- tetra- to hepta-CB congeners, which appeared in three dif-
uated in singly-flanked meta positions. Also, while chlorines in ferent AD pathways were 42(23–24), 71(26–34), 84(236–23),
doubly-flanked meta positions were targeted at the initial stages 90(235–24), 91(236–24), 99(245–24), 110(236–34), 118(245–34),
of AD, there were still a significant number of pathways quantified 134(2356–23), 139(2346–24), 141(2345–25), 150(236–246),
showing removal of chlorines in doubly-flanked para positions 170(2345–234) and 174(2345–236). These congeners are labeled
at later stages of the AD. When the dechlorination mechanisms as “major substrates of anaerobic dechlorination”, and depicted in
of the major pathways were evaluated, chlorines in positions Fig. 2.
listed in Table 2 were found to be targeted. While part of these The identification of these major dechlorination pathways and
were also observed in a previous BH sediment microcosm study major substrates have a number of important implications: (1)
[24], a more comprehensive list can now be presented using close monitoring of these pathways and congeners will enable
 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127
Table 3
Average anaerobic dechlorination rates of homolog groups for all time periods.
Homolog Anaerobic dechlorination rates (mol‰ Cl/day)
group
Initial Intermediate Intermediate
t = 0–88 d t = 0–185 d t = 0–278 d
Tri-CB 0.0028 0.0183 0.0158
Tetra-CB 0.0018 0.0250 0.0206
Penta-CB 0.0069 0.0433 0.0307
Hexa-CB 0.0079 0.0320 0.0236
Hepta-CB 0.0063 0.0226 0.0179
Octa-CB 0.0020 0.0077 0.0069
Nona-CB 0.0007 0.0027 0.0029
Deca-CB 0.000 0.0036 0.0062
improved monitoring of PCB attenuation during bioremediation of
contaminated sediments; (2) the possibility of stimulation of these
pathways could be investigated so that AD can proceed at a faster
rate or until the substrates for aerobic degradation are obtained;
and (3) examination of the consistently observed pathways will
help to understand whether further dechlorination can occur or AD
has come to a plateau. It should also be noted that enhancement of
the analytical methods to avoid co-elution of these congeners dur-
ing PCB analysis would help improve the monitoring and modeling
process.
3.3. Evaluation of anaerobic dechlorination rates
The AD rate was calculated with the PCB profiles as the
change in chlorine per biphenyl per day (Cl/bp/d) between dif-
ferent time periods. The average rate for all microcosms was
0.0043 ± 0.0005Cl/bp/d. When compared to the rates reported in
literature, spiked BH microcosm sediments were found to have a
rate close to that reported for Hudson River sediment microcosms
spiked with a mixture of Aroclors 1242/1254/1260 [4]. Spiked Hud-
son River sediments had a rate of 0.0348 Cl/bp/wk, which was
approximately 2.6 times faster than the rate of unspiked, weathered
Hudson River sediments at 0.0133 Cl/bp/wk [4]. Another laboratory
study [37,38] reported St. Lawrence sediments freshly spiked with
Aroclor 1248 had rates between 0.013 and 0.027 Cl/bp/d, which was
one order of magnitude faster than the BH microcosm sediments.
In contrast, the in situ AD rate of weathered PCBs in Lake Hartwell
was much lower with an average of 0.000258 Cl/bp/d [39].
The rate of AD was also calculated using the average quantifica-
tion values of dechlorination pathways for different time periods
in terms of mol‰ chlorines per day. Dechlorination rates for each
homolog, at each time period for the average PCB profile were cal-
culated. The time period from day 0 to day 185 showed the highest
dechlorination rate. For this time period, the average dechlorina-
tion rate for the removal of one chlorine atom from any homolog
was 0.019 mol‰ Cl/day. Typically, AD pathways with penta-CBs
as the parent congener showed the highest dechlorination rates
(>0.007 mol‰ Cl/day) for different time periods. Moreover, dechlo-
rination pathways having hexa-, hepta- and tetra-CBs as parent
congeners also emerged among the fastest observed.
Lastly, the dechlorination rates in microcosm sediments
between sequential time periods, i.e. from day 0–88, 88–185,
185–278 and 278–500, were determined (Table 3). By evaluating
the dechlorination rates, the greatest change in profiles occurred
between days 88 and 185, with an average dechlorination rate of
0.037 mol‰ Cl/day for all homologs and a maximum of 0.080 mol‰
Cl/day observed for penta-CB. The values observed from Table 3
indicate that the dechlorination rates were not homogeneous
throughout the microcosm incubation period. After a relatively
slow period (a lag time), microorganisms were able to dechlorinate
125
Final Sequential time periods
t = 0–500 d
t = 88–185 d t = 185–278 d t = 278–500 d
0.0102 0.0372 0.0155 0.0058
0.0126 0.0468 0.0146 0.0247
0.0202 0.0804 0.0138 0.0133
0.0147 0.0559 0.0132 0.0097
0.0116 0.0390 0.0167 0.0068
0.0049 0.0148 0.0120 0.0047
0.0018 0.0068 0.0099 0.0020
0.0054 0.0181 0.0215 0.0095
PCBs. This also had bearing on the success of field studies aimed at
obtaining as complete a dechlorination as possible.
Factors affecting rate include actual aqueous phase concen-
tration (dependent on absolute concentration, aqueous solubility
and partition coefficient of sediment) and kinetics of uptake
and catabolism. Furthermore, bioavailability has an impact on
bioremediation rates of hydrophobic organic contaminants [40].
Bioavailability of organic compounds in environmental matrices
are affected by several factors, such as: physico-chemical proper-
ties of the compound, extent of sorption/desorption, composition
and structure of the matrix and type of microorganisms present in
that matrix [40,41]. Incorporation of bioavailability, through mea-
surement of aqueous phase concentrations [40], or other relevant
parameters was not within the scope of this study. Therefore, any
effect of bioavailability on dechlorination rates of homologs is not
considered.
Three predominant phylotypes of PCB halorespiring bacteria
and their selective activities were identified in BH sediment micro-
cosm enriched with Aroclor 1260: o-17 (flanked chlorines in ortho
and para positions); DEH-10 (double flanked chlorines in meta and
para positions, para flanked chlorines in meta position); SF-1 (dou-
ble flanked chlorines in the meta position, ortho flanked chlorines
in the meta position).
3.4. Evaluation of change in PCB toxicity
Change of PCB toxicity in microcosm sediments is handled
by monitoring of change in concentration of toxic dioxin-like
coplanar congeners from day 0 through day 500. This change is
then explained through dechlorination pathways quantified by
the ADM. The available data and results of ADM provided valu-
able information regarding AD pathways involving toxic dioxin-like
coplanar congeners (toxicity-related pathways). Although, ortho
dechlorination has the potential to increase toxicity due to for-
mation of co-planar congeners, the only biologically confirmed
[24,42,43] ortho-Cl removal pathways in BH do not result in an
increase in toxicity (e.g., PCB151 to 92, PCB92 to 72, PCB90 to
68). Reactions involving ortho dechlorination were not included in
this study because they are very rare and change in ortho-Cl per
biphenyl in microcosms is not statistically significant when com-
pared to total-Cl per biphenyl change (p > .05). Accordingly, toxicity
increase due to ortho-Cl is not expected. There were a total of 17
toxicity-related non-zero quantified pathways (Table A2), of which
two were among the major pathways (shown in italics in Fig. 2).
The pathways resulting in the conversion of a toxic congener to a
non-toxic or less toxic congener accounted for the decrease in tox-
icity. The pathways in which a toxic congener was converted into
another one, both having equal toxic equivalency factors (TEFs)
[13], lead to no net change in toxicity. Complete separation and
singular quantification of toxic congeners is imperative to make
accurate assessments on the change of the total toxicity. Overall,
126 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127
in the case for BH microcosm sediments, the final net toxicity was
observed to be lower than the initial toxicity in sediments, which
demonstrated the potential effectiveness of AD to reduce toxicity
if it is adopted as a bioremediation strategy.
Investigation of pathways affecting toxicity using ADM have
three essential implications: (1) pathways that achieve toxicity
reduction in anaerobic sediments (especially, those concerning
toxic congeners 105(234–34), 118(245–34), 156(2345–34), 167
(245–345) & 189 (2345–345) should be stimulated in the sedi-
ments, (2) risk reduction potential of AD in the sediments can
be predicted with ADM by examining the quantification of the
toxicity-related pathways, and (3) analytical methods which avoid
co-elution of especially toxic congeners with less toxic congeners
would provide a more accurate assessment of the net change in
toxicity of PCBs as a result of AD.
3.5. Terminal products of anaerobic dechlorination
Experimental microcosm data was evaluated to identify accu-
mulating congeners in sediments. For this purpose, a cut off
value was arbitrarily defined, as the congeners having higher
concentrations than mean plus one standard deviation of the
experimental PCB profile at day 500 (>36.0 mol‰). The accumula-
tion of these congeners can then be explained using dechlorination
pathways identified by the ADM. The aim was to study the chlorine
placement of these accumulating congeners (e.g., presence of
an unflanked meta or ortho chlorine) to devise ways to facilitate
their dechlorination. Typically, congeners having less than five
chlorines, 16/32(23–2/26–4), 22/51(23–4/24–26), 49(24–25),
64/68/71/72(236–4/24–35/26–34/25–35), 52/73(25–25/26–35)
and 67/100(245–3/246–24) were accumulated in microcosm
sediments at day 500. Since unflanked chlorine removal was not
considered in the model, congeners with only unflanked chlorines
were the expected terminal products of AD. Every co-eluted
congener group had at least one congener with only unflanked
chlorines in its structure. ADM points out the pathways by which
terminal products are transformed or have the potential for trans-
formation within the sediments. Congeners 32(26–24), 49(24–25),
51(24–26), 52(25–25), 72(25–35), 73(26–35) and 100(246–24)
were true terminal products of AD since they did not appear as the
parent congener in any quantifiable dechlorination pathway. These
congeners were also detected among the major dechlorination
products of another BH sediment microcosm study by Fagervold
et al. [24], together with some other tetra-CBs. Accumulation of
tetra-CBs were the indication of purely anaerobic conditions in
microcosm sediments, since if there were any aerobic activity,
many tetra-CBs would degrade due to the available carbon posi-
tions to dioxygenase attack [15]. To enable complete degradation
of PCBs in sediments, identification of terminal AD products and
relevant dechlorination pathways are necessary.
4. Conclusion
• With the use of ADM, a detailed analysis of dechlorination
pathways for Aroclor 1260 in BH sediment microcosms was per-
formed. Since BH is a reduced environment and the laboratory
microcosms were prepared under purely anaerobic conditions,
modeling such PCB data provided a chance to evaluate complete
dechlorination pathways occurring in these sediments.
• Pathways that initially need to get started for dechlorination to
proceed were identified by the model together with the pathways
that were not actively occurring. Among 250 possible pathways,
93 were shown to occur as the major reactions in BH microcosm
sediments. From those, pathways that yield net accumulated con-
geners were identified, which indicated the need for stimulation
of dechlorination for certain congeners. By these results in the
ADM, aerobic degradation can be incorporated if the environmen-
tal sediments were to be bioremediated with the aim of complete
degradation of PCBs.
• The major congeners that were common intermediates of dechlo-
rination pathways were identified as PCB 49(24–25), 92(235–25),
101(245–25), 137(2345–24), 146(235–245), and 180(2345–245),
so that monitoring of these congeners would be useful for pro-
gression of degradation in contaminated sediments.
• Dechlorination rate was found to be highest, between days
88–185 after the initial lag between 0–88 days. Penta-CBs showed
the highest rate for almost all time periods.
• Toxicity reduction pathways were identified by the model and
sediment toxicity was shown to be reduced. Stimulation of these
pathways would be necessary for risk reduction in sediments.
• Terminal products of dechlorination in BH microcosm sediments
were revealed to be 32(26–4), 49(24–25), 51(24–26), 52(25–25),
72(25–35), 73(26–35) and 100(246–24).
• Until now, ADM was used to explain and evaluate the existing
situation in the sediments, but as this study demonstrates there
is potential for it to be used as a predictive tool to forecast the
results of AD prior to bioremediation. In this way, the model
would be implemented as a part of risk reduction evaluation
for the contaminated sediments. Ultimately, predictive modeling
tools combined with biological studies have the potential to opti-
mize bioremediation such that halorespiring microorganisms can
be selected to target specific congeners, or to assess the site-
specific environmental conditions and microbial consortia that
would be required for complete dechlorination, by bioaugmen-
tation and/or biostimulation.
Acknowledgements
This study was supported in part by U.S. Department of
Defense, Strategic Environmental Research and Development Pro-
gram Project Number ER-2135 to BVK. and Environmental Security
Technology Certification Program Number ER-201215 to KRS. We
thank Dr. Dilek Demirtepe Saygılı for her help during statistical
analyses.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jhazmat.
2015.04.033.
References
[1] Stockholm Convention, Stockholm Convention Website,
<http://chm.pops.int/TheConvention/ThePOPs/>, (2014).
[2] K. Breivik, A. Sweetman, J.M. Pacyna, K.C. Jones, Towards a global historical
emission inventory for selected PCB congeners–a mass balance approach 3.
An update, Sci. Total Environ. 377 (2007) 296–307,
http://dx.doi.org/10.1016/j.scitotenv.2007.02.026.
[3] M.D. Erickson, Introduction: PCB properties, uses, occurrence, and regulatory
history, in: L.W. Robertson, L.G. Hansen (Eds.), PCBs Recent Advances in
Environmental Toxicology in Health Effects, The University Press of Kentucky,
Kentucky, 2001, pp. xi–xxx.
[4] D.A. Abramowicz, M.J. Brennan, H.M. Van Dort, E.L. Gallagher, Factors
influencing the rate of polychlorinated biphenyl dechlorination in Hudson
River sediments, Environ. Sci. Technol. 27 (1993) 1125–1131.
[5] S.H. Safe, Polychlorinated biphenyls (PCBs): environmental impact,
biochemical and toxic responses, and implications for risk assessment, Crit.
Rev. Toxicol. 24 (1994) 87–149.
[6] J.C. Colombo, N. Cappelletti, A. Barreda, M.C. Migoya, C.N. Skorupka, Vertical
fluxes and accumulation of PCBs in coastal sediments of Rio De la Plata
estuary, Argentina, Chemosphere 61 (2005) 1345–1357,
http://dx.doi.org/10.1016/j.chemosphere.2005.03.090.
[7] A. Li, K.J. Rockne, N. Sturchio, W. Song, J.C. Ford, H. Wei, PCBs in sediments of
the Great Lakes –distribution and trends, homolog and chlorine patterns, and
 H. Demirtepe et al. / Journal of Hazardous Materials 296 (2015) 120–127
in situ degradation, Environ. Pollut. 157 (2009) 141–147,
http://dx.doi.org/10.1016/j.envpol.2008.07.014.
[8] G. Sanders, J. Hamilton-taylor, K.C. Jones, PCB and PAH dynamics in a small
rural lake, Environ. Sci. Technol. 30 (1996) 2958–2966.
[9] D.A. Abramowicz, Aerobic and anaerobic PCB biodegradation in the
environment, Environ. Health Perspect. 103 (1995) 97–99.
[10] J. Borja, D.M. Taleon, J. Auresenia, S. Gallardo, Polychlorinated biphenyls and
their biodegradation, Process Biochem. 40 (2005) 1999–2013,
http://dx.doi.org/10.1016/j.procbio.2004.08.006.
[11] J. Wiegel, Q. Wu, Microbial reductive dehalogenation of polychlorinated
biphenyls, FEMS Microbiol. Ecol. 32 (2000) 1–15
http://www.ncbi.nlm.nih.gov/pubmed/10779614
[12] K.R. Sowers, H.D. May, In situ treatment of PCBs by anaerobic microbial
dechlorination in aquatic sediment: are we there yet? Curr. Opin. Biotechnol.
24 (2013) 482–488, http://dx.doi.org/10.1016/j.copbio.2012.10.004.
[13] M. Van den Berg, L.S. Birnbaum, M. Denison, M. De Vito, W. Farland, M. Feeley,
et al., The 2005 World Health Organization reevaluation of human and
Mammalian toxic equivalency factors for dioxins and dioxin-like compounds,
Toxicol. Sci. 93 (2006) 223–241, http://dx.doi.org/10.1093/toxsci/kfl055.
[14] J.P. Giesy, K. Kannan, Dioxin-like and non-dioxin-like toxic effects of
polychlorinated biphenyls (PCBs): implications for risk assessment, Crit. Rev.
Toxicol. 28 (1998) 511–569,
http://dx.doi.org/10.1046/j. 1440-1770.2002.00185.x.
[15] D.L. Bedard, R. Unterman, L.H. Bopp, M.J. Brennan, M.L. Haberl, C. Johnson,
Rapid assay for screening and characterizing microorganisms for the ability to
degrade polychlorinated biphenyls, Appl. Environ. Microbiol. 51 (1986)
761–768.
[16] L. Bopp, Degradation of highly chlorinated PCBs by pseudomonas strain
LB400, J. Ind. Microbiol. 1 (1986) 23–29.
[17] I. Imamoglu, K. Li, E.R. Christensen, Modeling polychlorinated biphenyl
congener patterns and dechlorination in dated sediments from the Ashtabula
River, Ohio USA, Environ. Toxicol. Chem. 21 (2002) 2283–2291.
[18] P.A. Bzdusek, E.R. Christensen, C.M. Lee, U. Pakdeesusuk, D.L. Freedman, PCB
congeners and dechlorination in sediments of Lake Hartwell South Carolina
determined from cores collected in 1987 and 1998, Environ. Sci. Technol. 40
(2006) 109–119.
[19] S.C. Karcher, J.M. Vanbriesen, M.J. Small, Numerical method to elucidate likely
target positions polychlorinated biphenyl dechlorination, J. Environ. Eng.
(2007) 278–286.
[20] A.S. Hughes, J.M. Vanbriesen, M.J. Small, Identification of structural properties
associated with polychlorinated biphenyl dechlorination processes, Environ.
Sci. Technol. 44 (2010) 2842–2848.
[21] B.V. Kjellerup, C. Naff, S.J. Edwards, U. Ghosh, J.E. Baker, K.R. Sowers, Effects of
activated carbon on reductive dechlorination of PCBs by organohalide
respiring bacteria indigenous to sediments, Water Res. 52 (2014) 1–10,
http://dx.doi.org/10.1016/j.watres.2013.12.030.
[22] H. Demirtepe, Modeling Anaerobic Dechlorination of Polychlorinated
Biphenyls, MS Thesis, Middle East Technical University, Ankara, Turkey, 2012.
[23] S.K. Fagervold, J.E.M. Watts, H.D. May, K.R. Sowers, S. Carolina, Sequential
reductive dechlorination of meta-chlorinated polychlorinated biphenyl
congeners in sediment microcosms by two different chloroflexi phylotypes,
Society 71 (2005) 8085–8090, http://dx.doi.org/10.1128/AEM.71.12.8085.
[24] S.K. Fagervold, H.D. May, K.R. Sowers, Microbial reductive dechlorination of
aroclor 1260 in Baltimore harbor sediment microcosms is catalyzed by three
phylotypes within the phylum chloroflexi, Appl. Environ. Microbiol. 2 (2007)
3009–3018, http://dx.doi.org/10.1128/AEM.2958-06.
[25] I. Imamoglu, K. Li, E.R. Christensen, J.K. McMullin, Sources and dechlorination
of polychlorinated biphenyl congeners in the sediments of Fox River,
Wisconsin, Environ. Sci. Technol. 38 (2004) 2574–2583.
[26] P.A. Bzdusek, J. Lu, E.R. Christensen, PCB congeners and dechlorination in
sediments of Sheboygan River Wisconsin determined by matrix factorization,
Environ. Sci. Technol. 40 (2006) 120–129.
127
[27] D.L. Bedard, J.F. Quensen, Microbial reductive dechlorination of
polychlorinated biphenyls, in: L.Y. Young, C.E. Cerniglia (Eds.), Microbial
Transformation and Degradation Toxic Organic Chemicals, Wiley-Liss Inc.,
New York, 1995, pp. 127–216.
[28] D.L. Bedard, Polychlorinated biphenyls in aquatic sediments: environmental
fate and outlook for biological treatment, in: M.M. Haggblom, I.D. Bossert
(Eds.), Dehalogenation Microbial Processes Environmental Applications,
Kluwer Academic Publishers, Boston, 2003, pp. 443–465.
[29] D.L. Bedard, E.A. Pohl, J.J. Bailey, A.J.A. Murphy, Characterization of the PCB
substrate range of microbial dechlorination process LP, Environ. Sci. Technol.
39 (2005) 6831–6838.
[30] J.C. Davis, Statistics and Data Analysis in Geology, John Wiley & Sons, New
York, 2002.
[31] B.F. Manly, Statistics for Environmental Science and Management, Chapman &
Hall, Boca Raton, 2009.
[32] G.M. Frame, J.W. Cochran, S.S. Bøwadt, Complete PCB congener distributions
for 17 aroclor mixtures determined by 3HRGC systems optimized for
comprehensive, quantitative, congener-specific analysis, J. High Resolut.
Chromatogr. 19 (1996) 657–668, http://dx.doi.org/10.1002/jhrc.1240191202.
[33] D.L. Bedard, R.J. May, Characterization of the polychlorinated biphenyls in the
sediments of Woods Pond: evidence for microbial dechlorination of aroclor
1260 in situ, Environ. Sci. Technol. 30 (1996) 237–245,
http://dx.doi.org/10.1021/es950262e.
[34] J.F. Quensen, S.A. Boyd, J.M. Tiedje, Dechlorination of four commercial
polychlorinated biphenyl mixtures (aroclors) by anaerobic microorganisms
from sediments dechlorination of four commercial polychlorinated biphenyl
mixtures (aroclors) by anaerobic microorganisms from sedimentst, Appl.
Environ. Microbiol. 56 (1990) 2360–2369.
[35] I.M. Chen, F.C. Chang, M.F. Hsu, Y.S. Wang, Comparisons of PCBs
dechlorination occurrences in various contaminated sediments,
Chemosphere 43 (2001), http://dx.doi.org/10.1016/S0045-6535(00)417-3.
[36] D.A. Holmes, B.K. Harrison, J. Dolfing, Estimation of Gibbs free energies of
formation for polychlorinated biphenyls, Environ. Sci. Technol. 27 (1993)
725–731, http://dx.doi.org/10.1021/es00041a017.
[37] G.Y. Rhee, R.C. Sokol, C.M. Bethoney, Y.C. Cho, R.C. Frohnhoefer, T. Erkkila,
Kinetics of polychlorinated biphenyl dechlorination and growth of
dechlorinating microorganisms, Environ. Toxicol. Chem. 20 (2001)
721–726.
[38] R.C. Sokol, C.M. Bethoney, G.-Y. Rhee, Effect of aroclor 1248 concentration on
the rate and extent of polychlorinated biphenyl dechlorination, Environ.
Toxicol. Chem. 17 (1998) 1922–1926.
[39] V.S. Magar, R.C. Brenner, G.W. Johnson, J.F. Quensen, Long-term recovery of
PCB-contaminated sediments at the Lake Hartwell superfund site: PCB
dechlorination. 2. Rates and extent, Environ. Sci. Technol. 39 (2005)
3548–3554.
[40] W. Zhang, E.J. Bouwer, W.P. Ball, Bioavailability of hydrophobic organic
contaminants: effects and implications of sorption-related mass transfer on
bioremediation, Gr. Water Monit. Remediat. 18 (1998) 126–138.
[41] K. Nam, J.J. Kukor, Bioavailability of organohalides, in: M.M. Haggblom, I.D.
Bossert (Eds.), Dehalogenation Microbial Processes and Environmental
Applications, Kluwer Academic Publishers, Boston, 2003, pp. 291–302.
[42] M. Berkaw, K.R. Sowers, H.D. May, Anaerobic ortho Dechlorination of
Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor,
Appl. Environ. Microbiol. 62 (1996) 2534–2539
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1388898&tool
=pmcentrez&rendertype=abstract
[43] S.K. Fagervold, J.E.M. Watts, H.D. May, K.R. Sowers, Effects of bioaugmentation
on indigenous PCB dechlorinating activity in sediment microcosms, Water
Res. 45 (2011) 3899–3907, http://dx.doi.org/10.1016/j.watres.2011.04.048.