Removal of Chlorophenolics From Pulp and Paper

Mill Wastewater Through Constructed Wetland

Ashutosh Kumar Choudhary*1, Satish Kumar2, Chhaya Sharma3

ABSTRACT: This study evaluates the treatment efficiency of horizontal subsurface flow (HSSF) constructed wetland for the removal of
AOX (adsorbable organic halides) and chlorophenolics from pulp and
paper mill wastewater. The dimensions of HSSF constructed wetland
were 3.5 m in length, 1.5 m in width, and 0.28 m in depth, with surface
area of 5.25 m2. The HSSF constructed wetland unit was planted with an
ornamental plant species, Canna indica. Under hydraulic retention time
(HRT) of 5.9 days, the average AOX removal was 89.1%, and 67% to
100% removal of chlorophenolics from pulp and paper mill wastewater
was achieved. The complete removal of 2,3-dichlorophenol, 3,4dichlorophenol, 2,3,5-trichlorophenol, 2,4,6-trichlorophenol, 3,5-dichlorocatechol, 3,6-dichlorocatechol, and 4,5,6-trichloroguaiacol was
observed. Some of the chlorophenolics were found to accumulate in the
plant biomass and soil. The evapotranspiration rate varied from 6.7 to
12.7 mm day1 during the experimental period. The mass balance of
chlorophenolics was also studied in constructed wetland system. Water
Environ. Res., 85, 54 (2013).
KEYWORDS: AOX, constructed wetland, Canna indica,
chlorophenolics, paper mill wastewater.
doi:10.2175/106143012X13415215907419

Introduction
Indian pulp and paper mills discharge a large volume of high
pollution load wastewater to the environment, which may cause
deleterious environmental effects upon direct discharge. In the
pulp and paper industry, wood preparation, pulping, pulp
washing, bleaching, and coating operations are the major source
of pollution (Catalkaya and Kargi, 2007). Effluents of paper
industry contain suspended solids (1050 kg/ton of air dried
pulp [ADP]), biochemical oxygen demand (BOD) (1040 kg/ton
of ADP), chemical oxygen demand (COD) (20200 kg/ton of
ADP), adsorbable organic halides (AOX) (04 kg/ton of ADP),
toxicity, color, and high concentration of nutrients that cause
eutrophication in receiving water (Catalkaya and Kargi, 2008;
Pokhrel and Viraraghavan, 2004). Pulping, bleaching (chlorination and extraction stage), and chemical recovery sections are
the major sources of color, mainly because of lignin and
derivatives of lignin. Effluent originating from the first extraction
stage is highly colored and typically accounts for 80% of the
*1

Department of Paper Technology, Indian Institute of Technology

Roorkee, Saharanpur-247001, U.P., India; e-mail: akchoudhary.env@
gmail.com.

2
Department of Paper Technology, Indian Institute of Technology
Roorkee, Saharanpur, India.
3
Department of Paper Technology, Indian Institute of Technology
Roorkee, Saharanpur, India.

54

color, 30% of BOD, and 60% of COD of the mills total pollution
load. This high load of color is not only aesthetically
unacceptable but also inhibits the natural process of photosynthesis in the wastewater receiving streams because of absorbance
of sunlight. This leads to the adverse effects on the aquatic biota
(Bajpai and Bajpai, 1994).
Among the various sections, bleaching section wastewater
accounts for the largest fraction of toxicity (Catalkaya and Kargi,
2008). Chlorobleaching (using chlorine, ClO2, or other chlorine
compounds such as calcium or sodium hypochlorite) of wood
pulp causes formation of chlorinated organic compounds
(Sharma et al., 1997). More than 500 different chlorinated
organic compounds have been identified including chlorate,
chloroform, chlorophenolics (chlorophenols, chlorocatechols,
chloroguaiacols, chlorosyringols, chlorosyringaldehydes, and
chlorovanillins), chlorinated resin and fatty acids, chlorinated
hydrocarbons, and dioxins and furans (Freire et al., 2003). In
wastewater, these compounds are estimated collectively as AOX.
Chlorophenolics are formed primarily as a result of chlorination
of the lignin remaining in the pulp after pulping (Savant et al.,
2005). The nature and concentration of chlorophenolics formed
depends on the nature of the residual lignin and pulp bleaching
conditions (Voss et al., 1980). These are hydrophobic in nature
and have been shown to bioaccumulate in aquatic organisms.
Toxicity of chlorophenolics depends on the position of chlorine
atoms relative to the hydroxyl group on benzene ring. Many
authors reported the presence of toxic pollutants in fish and
their toxic effect on fish such as mutagenicity, respiratory stress,
liver damage, genotoxicity, and death when exposed to pulp and
paper mill wastewater (Erisction and Larsson, 2000). The
conventional treatment for Indian pulp and paper mills include
primary and secondary (aerobic/anaerobic biological systems)
treatment. Some of the pollutants in pulp and paper industry
wastewater are recalcitrant, thus, conventional biological treatment processes are not efficient for their remediation. It has
been observed that the secondary treated wastewater still
contains high organic load, color, and chlorinated organic
compounds that impart toxicity to the wastewater. To meet
discharge limits and to protect wastewater-receiving bodies,
pulp and paper mills are forced to adopt advanced treatment
systems.
Constructed wetlands are simple and low-cost wastewater
treatment systems that use natural processes using shallow
(usually less than 1 m deep) beds, plants, substrate (soil, sand,
and gravels), and a variety of microorganisms to improve
wastewater quality. Constructed wetlands are capable of
reducing contaminants including inorganic and organic matter,
toxic compounds, metals, and pathogens from different
Water Environment Research, Volume 85, Number 1

Choudhary et al.

wastewaters (Crites et al., 1997; Grismer et al., 2003; Song et al.,

2001). Reduction or removal of contaminants is accomplished by
diverse treatment mechanisms including sedimentation, filtration, chemical precipitation, adsorption, microbial interactions,
and uptake or transformation by plants (Watson et al., 1989;
Choudhary et al., 2011a). Microorganisms play an important role
in biochemical transformation and removal of toxic contaminants from wastewater added to wetlands (Field and Alvarez,
2008; Kivaisi, 2001). The efficiency of constructed wetlands to
remove the contaminants from the wastewater mainly depends
on the root zone interactions between soil, contaminants, plant
roots, and a variety of microorganisms. These systems have more
aesthetic appearances than traditional wastewater treatment
systems (Langergraber, 2008; Kadlec et al., 2000). The treatment
efficiency of these systems mainly depends on the wetland
design, hydraulic retention time (HRT), type of contaminant,
microbial interactions, and the climatic factors.
In our earlier communication we have studied the optimization of HRT for the treatment of pulp and paper mill wastewater
using horizontal subsurface flow (HSSF) constructed wetland
(Choudhary et al., 2011b, 2011c). The HRT of 5.9 days was found
to be optimum for the removal of COD (81% to 94%) and color
(95% to 99%). The main objective of the present study was to
remove the AOX and chlorophenolics from pulp and paper mill
wastewater through HSSF constructed wetland under optimized
HRT (5.9 days). The removal of BOD, COD, and color from
paper mill wastewater was also studied. The additional objectives
were to analyze the soil and plant material for the existence of
chlorophenolics and to study the mass balance of chlorophenolics in the treatment system. The evapotranspiration rate from
constructed wetland unit during the experimental period was
also studied.
Materials and Methods
The chlorophenols used were obtained from Aldrich (Milwaukee, Wisconsin) and Sigma (St. Louis, Missouri). The
chlorocatechols, chloroguaiacols, chlorovanillins, chlorosyringaldehydes, and chlorosyringols were supplied by Helix Biotech
Corporation (Richmand, B.C., Canada). Solvents (i.e., n-hexane,
acetone, methanol, and diethyl ether methyl) used were highperformance liquid chromatography grade. Analytical grade
acetic anhydride was used after double distillation. Other
reagents used for experimental studies were of analytical reagent
grade. Standard solutions of chlorophenols were prepared in
acetone: water (10:90) solution.
For this study, a horizontal subsurface flow constructed
wetland unit was constructed. The dimensions of HSSF
constructed wetland unit were 3.5 m in length, 1.5 m in width,
and 0.28 m in depth, with surface area of 5.25 m2. The detailed
configuration of HSSF constructed wetland unit was given
elsewhere (Choudhary et al., 2011c). The constructed wetland
unit was equipped with inlet and outlet hydraulic structures.
Wastewater inflow was through perforated poly(vinyl chloride)
pipe, placed across the entire width at the upstream side of the
constructed wetland unit so that wastewater flow had a uniform
distribution across unit. The outlet structure of the unit was an
orifice (5 cm diameter) at the bottom of the downstream end of
the unit. The plant species (i.e., Canna indica) was collected
from the nearby region and planted in both the units by hand at
an interval of 30 cm. At the time of plantation the average height
and density of the plants were 15 cm and 7 m2 respectively.
January 2013

The HSSF constructed wetland unit was loaded with water for
five weeks prior to the application of the wastewater to support
growth of the plant species. Wastewater (after primary
treatment) from the pulp and paper industry was loaded
subsequently to the unit for 9 months (April 2010 to December
2010). For the initial first month, HSSF constructed wetland unit
was left to acclimatize and stabilize before data collection to
allow the development of root zone microbial population. From
May 2010 to October 2010, the experimental data was collected
to obtain the optimum HRT for the treatment of pulp and paper
mill wastewater (Choudhary et al., 2011b, 2011c). From
November 2010 to December 2010, treatment of wastewater
was conducted under optimum HRT (5.9 days). The hydraulic
retention time (HRT) was calculated by eq 1 (U.S. Environmental Protection Association, 1993):
HRT

nLWd
Q

where n is the effective porosity of the media, L is the length of

the unit, W is the width of the unit, d is the average depth of the
bed, and Q is the average flow of wastewater through the unit.
The porosity of the substrate (n) was determined at the
beginning of the study and was estimated to be 0.27 (27%).
Inlet water flow measurements were made daily. Inlet
structure of the HSSF constructed wetland unit was fitted with
a gate valve that enabled manual adjustment of the flows. The
inlet water flows from constructed wetland was measured by
using a cylinder and a stopwatch. The flow was adjusted to the
desired value if it deviated from the set flow rate value. Outlet
flow was measured twice monthly. Readings were taken after
every two hours during the 24 hours of the chosen day and the
mean was used to represent the outflow for the whole day.
Evapotranspiration has an important role in controlling the
water budget of the constructed wetland (Kadlec and Knight,
1996). Evapotranspiration was calculated every month during
this experimental period as the difference between inlet and
outlet flow rates. The mass removal by the treatment system was
calculated by eq 2 as follows:
Mass removal Ci Qi  Ce Qe

1

where Ci (mg L ) is the influent concentration of a pollutant, Qi

(L day1) is the influent flow rate, Ce (mg L1) is the effluent
concentration of a pollutant, and Qe (L day1) is the effluent
flow. The total loading of chlorophenolics to the HSSF
constructed wetland was calculated by multiplying the total
wastewater feed during experimental period with the average
concentration of total chlorophenolics. The total mass content
of chlorophenolics accumulated in soil was calculated by
multiplying the total weight of the HSSF constructed wetland
soil (oven dried) with the concentration of chlorophenolics in
unit weight of soil (oven dried). Similarly, the total mass content
of chlorophenolics accumulated in plant biomass was calculated
by multiplying the total weight of the plant biomass harvested
(oven dried) from constructed wetland with the concentration of
chlorophenolics in unit weight of plant biomass (oven dried).
Degradation of chlorophenolics in the wetland unit was
calculated by eq 3 as follows:
Degradation TLc  Sc  Pc  Oc

where TLc is the total loading of chlorophenolics to the wetland

55

Choudhary et al.

Table 1Gas chromatography and mass spectrometer conditions for the analysis of chlorophenolics.
Gas Chromatography
Parameters
Detector
Carrier gas (flow rate)
Sample injected (lL)
Injection mode
Column dimensions
Film thickness
Injector temperature (8C)
Column temperatures (8C)

Mass Spectrometer
Conditions
Mass spectrometer
Helium (1 mL min1)
1
Split less
30 m 3 0.25 mm
0.25 lm
210
45 for 1 min
45 to 280 at 68C min1
280 for 25 min

unit, Sc is the mass of chlorophenolics accumulated in the

wetland soil, Pc is the mass of chlorophenolics accumulated in
the plant biomass and, Oc is the total mass of chlorophenolics
came out through outflow wastewater.
Wastewater samples were collected at the inlet and the outlet
of the constructed wetland system. Wastewater samples were
analyzed immediately in the laboratory for pH, COD, biochemical oxygen demand, color, AOX, and chlorophenolics. Constructed wetland plant biomass and soil samples were collected
after two days on the completion of the experiment and analyzed
for chlorophenolics. pH was determined by Toshniwal pH meter
and color measurement was performed spectrometrically on a
Analytic Jena spectrophotometer (Spekol 2000; Wembley,
England). For COD and BOD determination standard methods
were employed (Clesceri et al., 1998). AOX was determined by
Dextar AOX analyzer (Thermo Electron Corporation, Waltham,
Massachusetts).
Extraction of chlorophenolics from the wastewaters was
performed by simple modification of the procedure suggested
by Lindstrom and Nordin (1976). The inlet and outlet
wastewaters (1000 mL) were adjusted to pH 2 with dilution
H2SO4 and extracted with 400 mL of 90:10 diethyl ether/acetone
mixture for 48 h, with intermittent shaking. For chlorophenolics
extraction from plant material and constructed wetland soil,
samples were oven dried at 1058C for 4 h. Samples of oven dried
plant biomass (7 g) and of oven dried soil (10 g) were weighed in
cellulose thimble and placed in a Soxhlet apparatus. The samples
were then extracted for 24 h with methanol (250 mL) (Alonso et
al., 1998). Following extraction, the solvent phase was then
reduced to 5 mL by using vacuum rotatory evaporator. Then 4.5
mL distilled water was added to the solvent and again reduced to
4.5 mL by evaporation of solvent phase by using a vacuum
rotatory evaporator.
After extraction, all samples of chlorophenolics were derivatized by using 0.5 mL acetic anhydride (Abrahamsson and Xie,
1983) injected into the TR-5 fused silica capillary column (as
acetyl derivatives) using an auto sampler (AI 3000, Thermo
Electron Corporation, Waltham, Massachusetts) and were
analyzed by using gas chromatography coupled with mass
spectrometer (Trace GC Ultra-DSQ, Thermo Electron Corporation, Waltham, Massachusetts). The various chlorophenolics
were first identified by matching their mass spectrum with that
obtained from the National Institute of Standards and Technology library. Once main peaks were identified, pure standard
solutions of target compounds (as acetyl derivatives) were
56

Parameters
Ionisation mode
Ionising energy (eV)
Scan range (m/z)
Scan speed (amu/sec)
Fore pressure (mTorr)

Conditions
Electron impact
70
42 to 336
216.7
38 to 45

injected into the gas chromatographymass spectrometer

system for determining retention times. The gas chromatography and mass spectrometer conditions are given in Table 1. The
corresponding retention time and base peak values for standard
chlorophenolics are given in Table 2.
Soil adsorption and absorption experiments were carried out
in 2 L beakers by adding 1 kg of oven dried soil in 1 L
chlorophenolics solution of known concentration. To know the
effect of microbes, experiments were carried out in 2 L beakers
containing known concentration of chlorophenolics solution.
Plants were collected from the constructed wetland, and root
zone of the plants (as a source of microbes) were kept in
chlorophenolics solution, for 48 h and 96 h, respectively. Air
supply was also provided to the solution by a small aerator. After
Table 2Retention time of various chlorophenolic compounds
(as acetyl derivatives) in TR-5 glass capillary column.
Serial
number
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.

Name of
compound

Retention
time (min)

Base peak
(m/z)

3-Chlorophenol
4-Chlorophenol
2,6-Dichlorophenol
2,5-Dichlorophenol
2,4-Dichlorophenol
2,3-Dichlorophenol
3,4-Dichlorophenol
4-Chloroguaiacol
2,4,5-Trichlorophenol
2,3,6-Trichlorophenol
2,3,5-Trichlorophenol
2,4,6-Trichlorophenol
4,5-Dichloroguaiacol
2,3,4-Trichlorophenol
4,6-Dichloroguaiacol
3,6-Dichlorocatechol
3,5-Dichlorocatechol
3,4,6-Trichloroguaiacol
3,4,5-Trichloroguaiacol
4,5,6-Trichloroguaiacol
5,6-Dichlorovanillin
Pentachlorophenol
Tetrachloroguaiacol
Trichlorosyringol
Tetrachlorocatechol
2,6-Dichlorosyringaldehyde

14.20
14.36
16.52
16.96
16.98
17.69
18.27
18.70
19.07
20.01
20.17
20.31
21.19
21.23
22.27
22.50
22.77
23.16
24.40
25.07
25.85
26.22
26.66
26.96
28.31
28.59

127.9
127.9
161.9
161.9
161.8
161.8
161.9
157.9
195.8
195.8
195.9
195.8
191.9
195.8
191.9
177.9
177.9
225.9
225.8
225.9
219.9
265.7
261.8
255.8
247.8
249.9

Water Environment Research, Volume 85, Number 1

Choudhary et al.

Table 3Average values of the wastewater parameters at the

inlet and outlet of the horizontal subsurface flow constructed
wetland for the HRT of 5.9 days. Minimum and maximum values
are indicated in the brackets.
Parameters
pH
Chemical oxygen demand (mg L1)
Color (platinum-cobalt unit)
Biological oxygen demand a (mg L1)
Adsorbable organic halides (mg L1)
Chlorophenolics (lg L1)
Chloride (mg L1)
a

Inleta6SD
7.7
1011
2553
248
16.5
40.00
516

6
6
6
6
6
6
6

0.17
82
238
15
3.50
5.98
54

Outleta6SD
7.5 6
122.7 6
100.7 6
17.0 6
1.8 6
4.02 6

0.1
24
25
2
0.23
0.29

n 4.

experiments solutions were analyzed for remaining chlorophenolics.

Results and Discussion
The characteristics of the wastewater collected from the
inflow and outflow of HSSF constructed wetland at HRT of 5.9
days are shown in Table 3. Table 4 shows the average inflow and
outflow concentration and average removal efficiencies of
individual chlorophenolics. Before adding wastewater to the
constructed wetland unit, blank experiments were carried out
for AOX and chlorophenolic compounds. These compounds
were not detected from the constructed wetland unit.
At HRT of 5.9 days, the average wastewater pH values
obtained for the inlet and outlet of unit were 7.760.17 and
7.560.1, respectively, with an observed decrease of 0.2 units in
pH from inflow to outflow. This decrease in pH value from inlet
to outlet may be because of the formation of some acidic
components. Bojcevska and Tonderski (2007) also reported the
similar trend of pH during the sugar industry wastewater
treatment by constructed wetland. The average value of inflow
color was 25536238 platinum-cobalt units and the average color
removal was 96.1%. The color of the pulp and paper mill
wastewater is mainly because of the lignin and its derivatives.
The probable reason for the color removal from the wastewater
through constructed wetland is filtration and adsorption of
organic matter on the substrate and decomposition of lignin and
its derivatives by microbial processes. Calheiros et al. (2009)
reported up to 90% color reduction for the treatment of tannery
wastewater through horizontal subsurface flow wetland at the
HRT of 7 days, which is comparable with our results.
The average values of inflow COD and BOD5 were 1011682
mg L1 and 248615 mg L1, respectively. The average COD and
BOD5 removal efficiencies of the unit were 87.9% and 95.6%,
respectively. The organic load removal is thought to occur
rapidly through physical processes such as settling and
entrapment of organic particulate matter in the void spaces of
the substrate. The substrate is the main supporting material for
plant and microbial growth. In our constructed wetland system
we used sand and fine gravels that promoted greater growth of
plants; thus, organic matter removal was achieved with high
efficiency. The microorganisms attached to the root zone of the
plants also play an important role in the degradation of organic
matter (i.e., conversion of organic carbon to simple compounds),
and for this purpose, oxygen is probably supplied by the root
zone of Canna indica. As our results show (Table 5), soil
January 2013

adsorption and absorption plays an important role in the

removal of organic compounds for the wastewater, which further
increases the removal efficiency of the constructed wetland
system. Daniels (2001) reported an 80% to 85% reduction in the
COD of tannery wastewater (inlet COD ranging from 1 000 to 2
000 mg L1) after a 5-day retention time through subsurface flow
constructed wetland. Mantovi et al. (2003) reported removal
efficiencies greater than 90% for COD (859-2312 mg L1) by
treatment through constructed wetland for dairy parlor effluent
and domestic wastewater. Prabhu and Udayasoorian (2007)
reported BOD (64% to 77%) and COD (49% to 62%) removal
from pulp and paper mill wastewater by using constructed
wetland at HRT of 48 h (2 days). At HRT of 6 days,
Sirianuntapiboon and Jitvimolnimit (2007) reported the BOD
removal up to 92.3% from wastewater by using subsurface flow
constructed wetland planted with Canna species. Konnerup et al.
(2009) also used Canna species for the treatment of domestic
wastewater through subsurface flow constructed wetland and
reported 83%66% COD removal at HRT of 4 days. Our results
are in line with the literature.
The average AOX and chlorophenolics concentration values
of inflow wastewater were 16.563.50 mg L1 and 40.065.98 lg
L1, respectively, and the average removal was 89% and 90%,
respectively. Six categories of chlorophenolic compounds
(chlorophenols, chlorocatechols, chloroguaiacols, chlorosyringaldehyde, chlorosyringol, and chlorovanillin) were detected in
the inflow and outflow wastewater of the HSSF constructed
wetland as shown in Table 4. Among the various chlorophenolics
in the inflow wastewater, 2,4,5-trichlorophenol contributed to
the highest concentration (11.2864.29 lg L1) followed by 2,4,6trichlorophenol (5.0161.23 lg L1), 3-chlorophenol (3.9061.87
lg L1), 2,4-dichlorophenol (3.1760.90 lg L1), 2,6-dichlorophenol (2.7961.10 lg L1), 4,5-dichloroguaiacol (2.5860.86 lg
L1), 2,6-dichlorosyringaldehyde (2.1460.85 lg L1), and 4chlorophenol (1.6060.77 lg L1). Other chlorophenolics were
present in minor quantities.
Among all categories, chlorophenols contributed to highest
share with quantity of 30.52 lg L1 in the inflow wastewater. The
results indicate that approximately 79% of the identified
compounds in the inflow wastewater were di- and trichlorophenolic compounds. In the outflow wastewater of HSSF
constructed wetland, 21 chlorophenolic compounds were
detected. 2,4,5-trichlorophenol contributed to the highest
concentration (1.8260.21 lg L1). The quantities of other
chlorophenolic compounds detected in the outflow wastewater
were in the range of 0.01 to 0.31 lg L1. After wetland treatment,
67% to 100% removal of chlorophenolics was achieved (Table 4).
Lowest removal was observed in case of 2,3,6-trichlorophenol
(67%) followed by 2,3,4-trichlorophenol (77%), tetrachloroguaiacol (77%), 2,4,5-trichlorophenol (84%), and tetrachlorocatechol
(84%). The removal of pentachlorophenol (which is most toxic in
the group of chlorophenolics) was achieved at level of 89%. The
complete removal of 2,3-dichlorophenol, 3,4-dichlorophenol,
2,3,5-trichlorophenol, 2,4,6-trichlorophenol, 3,5-dichlorocatechol, 3,6-dichlorocatechol, and 4,5,6-trichloroguaiacol was
achieved.
In constructed wetland treatment system, the AOX and
chlorophenolic compounds from wastewater may be removed by
a number of processes including adsorption and absorption,
oxidationreduction, microbial interactions, and uptake by
plants when passed through the substrate of constructed
57

Choudhary et al.

Table 4Average values of the chlorophenolics at the inlet and outlet of the horizontal subsurface flow constructed wetland for the
hydraulic retention time of 5.9 days.
Concentration (lg L1)
Serial number
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
a

Inleta 6 SD

Name of compound
3-Chlorophenol
4-Chlorophenol
2,6-Dichlorophenol
2,5-Dichlorophenol
2,4-Dichlorophenol
2,3-Dichlorophenol
3,4-Dichlorophenol
4-Chloroguaiacol
2,4,5-Trichlorophenol
2,3,6-Trichlorophenol
2,3,5-Trichlorophenol
2,4,6-Trichlorophenol
4,5-Dichloroguaiacol
2,3,4-Trichlorophenol
4,6-Dichloroguaiacol
3,6-Dichlorocatechol
3,5-Dichlorocatechol
3,4,6-Trichloroguaiacol
3,4,5-Trichloroguaiacol
4,5,6-Trichloroguaiacol
5,6-Dichlorovanillin
Pentachlorophenol
Tetrachloroguaiacol
Trichlorosyringol
Tetrachlorocatechol
2,6-Dichlorosyringaldehyde

3.90
1.60
2.79
0.75
3.17
0.02
0.15
0.79
11.28
0.06
0.43
5.01
2.58
0.26
0.21
0.33
0.08
0.27
0.04
0.01
1.61
1.10
0.22
0.27
0.93
2.14

Table 5Removal of chlorophenolics from aqueous solution by

the soil adsorption and absorption process.
Serial number

Name of compound
2,5-Dichlorophenol
2,6- Dichlorophenol
2,4,5-Trichlorophenol
2,3,5-Trichlorophenol
Pentachlorophenol
3,5-Dichlorocatechol
Tetrachlorocatechol
4,5,6-Trichloroguaiacol
4,5-Dichloroguaiacol
Tetrachloroguaiacol

Ci* (lg/L)
10.90
13.15
9.85
10.62
9.96
10.20
8.49
11.65
10.14
10.08

6
6
6
6
6
6
6
6
6
6

*initial concentration, 4 h, 1 kg oven dried soil/L

58

1.87
0.77
1.10
0.36
0.90
0.01
0.04
0.24
4.29
0.03
0.19
1.23
0.86
0.17
0.13
0.17
0.02
0.07
0.01
0.006
0.66
0.44
0.04
0.05
0.17
0.85

0.31
0.12
0.29
0.09
0.18

0.09
1.82
0.02
0.20
0.11
0.06
0.02

0.04
0.01
0.10
0.12
0.05
0.02
0.15
0.12

6 0.09
6 0.03
6 0.05
6 0.02
6 0.07
ND
ND
6 0.03
6 0.21
6 0.01
ND
6 0.02
6 0.009
6 0.01
6 0.003
ND
ND
6 0.008
6 0.007
ND
6 0.01
6 0.02
6 0.005
6 0.007
6 0.01
6 0.01

Removal (%)
92.1
92.5
89.6
88.0
94.3
ND
ND
88.6
83.9
66.7
ND
96.0
95.7
76.9
90.5
ND
ND
85.2
75.0
ND
93.8
89.1
77.3
92.6
83.9
94.4

n 4; ND not detected.

wetland. To determine the extent these processes (i.e., soil

adsorption/absorption and microbial degradation) are effective
for the removal of chlorophenolics, we carried some studies in
the laboratory. The studies indicate that soil adsorption/
absorption effectively removed the chlorophenolics from the
solution. The removal ranged from 36.5% to 77.9%, as shown in
Table 5. It was observed that this process is more effective for
highly chlorinated chlorophenolics as compared to lower
chlorinated chlorophenolics. The degree of sorption and its rate
depends on the characteristics of both the organic compounds
(e.g., organic carbon partition coefficient (KOC)) and the solid
surface (e.g., plants, substrate, and litter) (Imfeld et al., 2009;

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6

Outleta 6 SD

0.32
0.79
0.58
0.87
0.70
1.19
0.34
0.86
0.66
1.22

Removal (%)
38.4
36.5
52.9
55.7
77.9
37.2
68.4
44.9
38.9
70.9

Karickhoff, 1981). The results of microbial degradation studies

are shown in Table 6. Examination of data shows that 19.9% to
28.8% and 30.6% to 43.3% removal of chlorophenolics were
achieved after the time interval of 48 h and 96 h, respectively. As
organic in nature, biological degradation and transformation are
the two main processes for removal of AOX and chlorophenolics in constructed wetlands. The extent of microbial
degradation or transformation of organic compounds within a
constructed wetland is also expected to strongly depend on the
physicochemical properties (e.g., water solubility and concentration) of the contaminant (Imfeld et al., 2009). The typical
values of water solubility and log KOW for different categories of
chlorophenolics are shown in Table 7. It is well documented in
literature that a variety of microorganisms (e.g., Pseudomonas
sp., Sphingomonas sp., Flavobacterium sp., Novosphingobium
lentum, Pseudomonas sp., Rhodococcus chlorophenolicum,
Sphingomonas chlorophenolica, Desulfitobacterium hafniense,
and Mycobacterium sp.) have the ability to interact, both
chemically and physically, with the substances leading to
structural changes or complete degradation of the chlorophenolics compound (Czaplicka, 2004; Field and Alvarez, 2008;
Wiren et al., 2002). These compounds become soluble after
some initial reductive dechlorination steps, and thus can be
easily degraded by microbial interactions (aerobic/anaerobic).
Both aerobic and anaerobic microbial degradation processes
simultaneously take place in the constructed wetlands. Under
anaerobic conditions, chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates
Water Environment Research, Volume 85, Number 1

Choudhary et al.

Table 6Removal of chlorophenolics from aqueous solution by

biosorption and microbial decomposition process.

1999). Prabu and Udayasoorian (2007) reported the removal of

chlorophenols, from pulp and paper mill wastewater through
constructed wetland planted with Phragmitis australis, Cyperus
pangorei, and Typha latifolia, was 87%, 81%, and 72%,
respectively (at HRT of 48 h).
During this study, 26 chlorophenolic compounds were fed to
the wetland unit through pulp and paper mill wastewater. Of
these, 14 chlorophenolic compounds of four different categories
(chlorophenols, chloroguaiacols, chlorosyringaldehyde, and chlorovanillin) were detected in the plant biomass (Canna indica)
harvested from wetland unit (Table 8). Among the various
chlorophenolics, 5,6-dichlorovanillin contributed to the highest
accumulation in the plant biomass (623.00614.62 lg OD Kg1)
followed by 2,6-dichlorosyringaldehyde (222.00610.07 lg OD
Kg1) and 4-chloroguaiacol (154.0066.90 lg OD Kg1). Other
chlorophenolics were accumulated in minor quantities. Among
all categories, chlorovanillin contributed to the highest share with
53.4%, followed by chlorosyringaldehyde (19.0%), chloroguaiacols
(14.3%), and chlorophenols (13.4%). Among chlorophenols,
monochlorophenols accumulated to the highest extent in the
plant biomass (i.e., 38.4%).
Uptake of any organic compound into plant tissue is
predominantly affected by the lipophilic nature, which can be
characterized by the octanolwater partition coefficient (Kow)
(Ryan et al., 1988). Generally, organic compounds with a log
KOW between 0.5 and 3 are absorbed best by plants (Trapp and
Karlson, 2001). Organic compounds with a log Kow . 4 are not
thought to be significantly absorbed by the plant unit membrane
because of retention within the root epidermis (Trapp, 1995). In
this study, only mono, di, and trichlorophenolics were detected
in the plant biomass. It is because of this reason that for mono,
di, and trichlorophenolics the log KOW , 4 and for tetra and
pentachlorophenolics the log KOW . 4 (Shiu et al., 1995; Xie et
al., 1984).
In soil samples collected from wetland unit, all six categories
of chlorophenolic compounds (chlorophenols, chlorocatechols,
chloroguaiacols, chlorosyringaldehyde, chlorosyringol, and
chlorovanillin) were detected (Table 8). 2,6-dichlorosyringaldehyde (45.263.08 lg OD Kg1) contributed to the highest
concentration in soil samples followed by trichlorosyringol
(23.561.64 lg OD Kg1), 4-chloroguaiacol (20.661.32 lg OD
Kg1), pentachlorophenol (11.660.54 lg OD Kg1), 3,6-dichlorocatechol (11.261.03 lg OD Kg1), 3,5-dichlorocatechol
(9.260.93 lg OD Kg1), and 2,4,5-trichlorophenol (5.4060.30
lg OD kg1).
The accumulation of chloroorganics in soil shows its low
biodegradation by microbes and low or no water solubility,
resulting from binding to the soil matrix (Leigh et al. 2006;
Campanella et al., 2002). Adsorption of organic compounds to
soil may result from the physical or chemical adhesion of
molecules to the surfaces of solid bodies, or from partitioning of
dissolved molecules between the aqueous phase and soil organic

Removal (%)
Serial
number
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Name of compound
2,5-Dichlorophenol
2,6- Dichlorophenol
2,4,5-Trichlorophenol
2,3,5-Trichlorophenol
Pentachlorophenol
3,5-Dichlorocatechol
Tetrachlorocatechol
4,5,6-Trichloroguaiacol
4,5-Dichloroguaiacol
Tetrachloroguaiacol

Ci* (lg/L)
10.90
13.15
9.85
10.62
9.96
10.20
8.49
11.65
10.14
10.08

6
6
6
6
6
6
6
6
6
6

After 48 h

After 96 h

22.1
23.0
24.3
25.0
27.3
21.8
28.2
25.9
19.9
22.8

43.3
41.4
36.2
34.5
34.6
43.3
33.6
35.3
34.7
30.6

0.32
0.79
0.58
0.87
0.70
1.19
0.34
0.86
0.66
1.22

*initial concentration.

are available. Under aerobic conditions, both lower and higher

chlorinated phenols can serve as sole electron and carbon
sources supporting growth (Field and Alvarez, 2008). The
aerobic degradation rate is relatively slower for high-chlorinated
compounds in comparison to low-chlorinated compounds
(Amon et al., 2007). The biodegradation of pentachlorophenol
is faster in anaerobic than in aerobic conditions. In anaerobic
conditions, dechlorination of pentachlorophenol led to the
formation of a mixture of di, tri, and tetrachlorophenols
(DAngelo and Reddy, 2000). In aerobic conditions, highchlorinated phenolics are resistant to biodegradation because
chlorine atoms interfere with the action of many oxygenase
enzymes, which generally initiate the degradation of aromatic
rings (Copley, 1997). Several aerobic chlorophenolics degrading
bacteria have been isolated from contaminated soils such as
Arthrobacter sp., Flavobacterium sp., and Sphingomonas chlorophenolica (Yang et al., 2006; Miethling and Karlson, 1996).
Higgblom et al. (1988) reported the o-methylation of chlorinated
guaiacols after p-dechlorination/hydroxylation by Rhodococcus
chlorophenolicus.
Our study shows that plants uptake play a direct role in the
removal of chlorinated organic compounds from pulp and paper
mill wastewater but indirectly they may provide aerobic
conditions to the microorganisms in the root zone for the
degradation of chlorophenolics. In this study, comparatively low
removal was observed in case of 2,3,6-trichlorophenol, 4,5,6trichloroguaiacol, 3,4,5-trichloroguaiacol, 2,3,4-trichlorophenol,
and tetrachloroguaiacol, as highly halogenated organic compounds are extremely resistant to decomposition, because of
their low solubility in water and the lack of a structural site for
enzyme attachment for biotransformation or degradation.
However, there is substantial evidence that pentachlorophenol
degrades readily under the alternating aerobic and anaerobic
conditions that exist in constructed wetland soils (DeBusk,

Table 7Typical values of water solubility and log KOW for different categories of chlorophenolics.
Parameter*
Water solubility 258C (mg/L)
Log KOW

MCP

DCP

TCP

TeCP*

PCP

3 222 to 7 391
2.15 to 2.16

1 829 to 4 500
2.63 to 2.80

438 to 1200
3.27 to 3.72

103 to 1192
3.61 to 4.59

14
5.12

*Calculated by EPI suite (U.S. EPA); MCP Monochlorophenolics; DCP Dichlorophenolics; TCP Trichlorophenolics; TeCP Tetrachlorophenolics; and
PCP Pentachlorophenolics
January 2013

59

Choudhary et al.

Table 8Chlorophenolics detected in plant biomass and soil

samples.
Concentration
(lg oven dried kg1)
Serial
number
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
12.
13.
14.
15.
16.
17.
18.
19.

Name of
compound
3-Chlorophenol
4-Chlorophenol
2,5-Dichlorophenol
2,4-Dichlorophenol
2,3-Dichlorophenol
3,4-Dichlorophenol
4-Chloroguaiacol
2,4,5-Trichlorophenol
2,3,5-Trichlorophenol
2,4,6-Trichlorophenol
4,6-Dichloroguaiacol
3,6-Dichlorocatechol
3,5-Dichlorocatechol
3,4,5-Trichloroguaiacol
5,6-Dichlorovanillin
Pentachlorophenol
Trichlorosyringol
2,6-Dichlorosyringaldehyde

Plant 6 SD
34.71 6
25.29 6
19.14 6
0.14 6
13.71 6
12.86 6
154.00 6
7.57 6
30.86 6
11.86 6
8.57 6

4.00 6
623.00 6

222.00 6

2.93
2.72
1.15
0.02
1.15
1.53
6.90
1.27
2.45
1.23
1.41

0.34
14.62

10.07

Soil 6 SD

0.50 6
2.50 6
0.50 6

20.60 6
5.40 6

2.10 6

11.20 6
9.20 6

2.22 6
11.60 6
23.50 6
45.20 6

0.04
0.11
0.03

1.32
0.30
0.23
1.03
0.93
0.12
0.54
1.64
3.08

matter. The extent of adsorption depends on the compounds

hydrophobic nature and the chemical structure, the organic
carbon content, composition of soil organic matter (Imfeld et al.,
2009), and the organic carbon partition coefficient (Koc)
(Karickhoff, 1981). The adsorption properties of chlorophenolics
are controlled mainly by their degree of substitution and the
resultant hydrophobicity. Cheng et al. (2008) reported effect of
pH on the adsorption behavior of p-chlorophenol on soil. The
pH of substrate is a major factor affecting the fate and transport
of chlorophenols in constructed wetlands; as the pH value
increases, the degree of ionization of a compound also increases
(Cheng et al., 2008).
The approximate total mass content of chlorophenolics
loaded to HSSF constructed wetland was 1 017 mg. Out of
which, 0.2% got accumulated in plant biomass, 27.4% got
accumulated in the soil, 4.2% came through outlet of the HSSF
constructed wetland, and approximately 68.2% was degraded as
shown in Figure 1. Results show that large fraction of the total
loading got degraded in HSSF constructed wetland at HRT of 5.9
days. The mass balance of a particular organic compound
depends on the distribution of that compound in different
components of the constructed wetland systems. The partition
of an organic pollutant between the water and organic phases is
generally correlated with various properties, such as the water
solubility and the octanol or water partition coefficient. The
kinetics of different physical (adsorption/absorption), chemical
(oxidation/reduction), and biological processes (aerobic/anaerobic) decides the actual degradation or accumulation in the
constructed wetland treatment system. In early stages of
constructed wetland operation, sorption onto soil substrate is
higher because of the high adsorption capacity of previously
unexposed substrate (Omari et al., 2003). When no sorption
desorption equilibrium is reached, the system acts as a sink for
the contaminants. After attaining steady-state conditions,
60

Figure 1Mass balance of chlorophenolics: (A) accumulated in

plant biomass, (B) accumulated in soil, (C) in outlet wastewater,
and (D) degraded in horizontal subsurface flow constructed
wetland.

pollutants will still be retained by reversible sorption processes,

but further net loss of pollutants will not occur. This retention
may increase the pollutants residence time within the constructed wetland and support biodegradation by increasing
exposure to microorganisms (Imfeld et al., 2009).
Evapotranspiration was measured from May 2010 to December 2010 as shown in Figure 2. Evapotranspiration was not
measured in the month of September because of heavy rainfall.
The highest evapotranspiration was observed in the month of
June (12.7 mm day1) and lowest in the month of December (6.7
mm day1). The evapotranspiration rate increased from May to
June, decreased from June to November, and became nearly
constant in December. During the experimental period (May
2010 to December 2010), minimum and maximum temperature
of the day was also observed (Figure 3). The highest temperature
(44.28C) was observed in the month of June. From July to
September, the difference between maximum and minimum day
temperature was comparatively low (58C to 168C).

Figure 2Monthly evapotranspiration of the horizontal

subsurface flow constructed wetland during the experimental
period.
Water Environment Research, Volume 85, Number 1

Choudhary et al.

References

Figure 3Variation of minimum and maximum temperature of

the day during the experimental period.
The growth and biomass of the vegetation affects the water
balance of the wetland unit (Konnerupa et al., 2009). It means
that the greater the biomass, the greater will be the evapotranspiration. The rate of evapotranspiration depends on the physical
evaporation from the constructed wetlan unit surface and the
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loss because of evapotranspiration during the experimental
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Conclusion
HSSF constructed wetland was found an effective treatment
technology for the remediation of pulp and paper mill
wastewater. At HRT of 5.9 days, 67% to 100% removal of
chlorophenolics from pulp and paper mill wastewater was
achieved. Some of the chlorophenolics were found to accumulate in the plant biomass and soil. The main mechanisms for the
AOX and chlorophenolics are adsorption and absorption,
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the root zone of the plants. Mass balance studies show that
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constructed wetland unit and a small proportion accumulated
in the plant species Canna indica. Evapotranspiration played a
significant role in controlling the water budget of the system and
showed a dependence on climatic conditions.
Acknowledgment
The authors gratefully acknowledge the research grant for this
study, which was provided by the Ministry of Environment and
Forest, Government of India.
Submitted for publication May 9, 2012; accepted for
publication June 9, 2012.
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Water Environment Research, Volume 85, Number 1