Occurrence and estrogenicity of phenolics in paper-recycling process water: Pollutants originating from thermal paper in waste paper

Article abstract of DOI:10.1897/06-642R.1

Eight phenolics were detected in samples collected from areas where paper-recycling process water is discharged. The detected concentration levels were up to 270 μg/L and 230 μg/g in water samples and sediment samples, respectively, obtained from both the outfall of the paper-recycling process water and its downstream areas. In particular, totarol (compound 4), 2,4-bis(1-phenylethyl)phenol (compound 6), 4,4′-butylidenebis(6-t-butyl-m- cresol) (compound 7), 2,4-bis(1-phenylethyl)-6-chlorophenol (compound 8), and 4-hydroxy-4′-isopropoxydiphenyl sulfone (compound 9) were identified for the first time as environmental pollutants. The estrogenicities of the identified compounds were assessed by yeast two-hybrid assays incorporating either the human or medaka fish (Oryzias latipes) estrogen receptor α (hERα and medERα, respectively) and an estrogen receptor competitive enzyme-linked immunosorbent assay (ER-ELISA) both with and without metabolic activation by a rat liver S9 mix. Bisphenol A (compound 3) and 2-naphthol (compound 1) exhibited activity in the assays of both hERα and medERα without the S9 mix. The relative activity (%) to 17β-estradiol was 0.0015% for compound 3 and 0.0009% for compound 1 in the hERα assay and 0.027% for compound 3 and 0.0093% for compound 1 in the medERα assay. These compounds were attenuated by the S9 mix. The binding affinity was evaluated using an ER-ELISA. Compounds 3, 4, 6, and 7 exhibited affinity without the S9 mix. After exposure to the S9 mix, however, the binding affinity of compound 7 was eliminated by the S9 mix; those of compounds 3, 4, and 6 were attenuated; and that of compound 8 exhibited affinity. A comprehensive assessment of the estrogenicities of the phenolics originating from thermal paper and their implications for an aquatic environment may require an examination of the components of the phenolics, as in the present study.

Full text of DOI:10.1897/06-642R.1

Environmental Toxicology and Chemistry, Vol. 26, No. 11, pp. 2356–2366, 2007
2007 SETAC
Printed in the USA
730-7268/07 $12.00 .00
᭧
0
ϩ
OCCURRENCE AND ESTROGENICITY OF PHENOLICS IN PAPER-RECYCLING
PROCESS WATER: POLLUTANTS ORIGINATING FROM THERMAL PAPER
IN WASTE PAPER
M
ASANORI
Institute for Environmental Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Shizuoka Institute of Environment and Hygiene, Kita-Ando 4-27-2, Aoi-ku, Shizuoka 420-8637, Japan
T
ERASAKI,*† FUJIO
S
HIRAISHI,‡ HITOSHI
F
UKAZAWA,§ and MASAKAZU
M
AKINO
†
†
‡
§
(
Received 22 December 2006; Accepted 29 May 2007)
Abstract—Eight phenolics were detected in samples collected from areas where paper-recycling process water is discharged. The
detected concentration levels were up to 270 g/L and 230 g/g in water samples and sediment samples, respectively, obtained
from both the outfall of the paper-recycling process water and its downstream areas. In particular, totarol (compound 4), 2,4-bis(1-
phenylethyl)phenol (compound 6), 4,4 -butylidenebis(6- -butyl- -cresol) (compound 7), 2,4-bis(1-phenylethyl)-6-chlorophenol
compound 8), and 4-hydroxy-4 -isopropoxydiphenyl sulfone (compound 9) were identified for the first time as environmental
pollutants. The estrogenicities of the identified compounds were assessed by yeast two-hybrid assays incorporating either the human
or medaka fish (Oryzias latipes) estrogen receptor (hER and medER , respectively) and an estrogen receptor competitive
enzyme-linked immunosorbent assay (ER-ELISA) both with and without metabolic activation by a rat liver S9 mix. Bisphenol A
compound 3) and 2-naphthol (compound 1) exhibited activity in the assays of both hER and medER without the S9 mix. The
relative activity (%) to 17 -estradiol was 0.0015% for compound 3 and 0.0009% for compound 1 in the hER assay and 0.027%
for compound 3 and 0.0093% for compound 1 in the medER assay. These compounds were attenuated by the S9 mix. The binding
␮
␮
Ј
t
m
(
Ј
␣
␣
␣
(
␣
␣
␤
␣
␣
affinity was evaluated using an ER-ELISA. Compounds 3, 4, 6, and 7 exhibited affinity without the S9 mix. After exposure to the
S9 mix, however, the binding affinity of compound 7 was eliminated by the S9 mix; those of compounds 3, 4, and 6 were attenuated;
and that of compound 8 exhibited affinity. A comprehensive assessment of the estrogenicities of the phenolics originating from
thermal paper and their implications for an aquatic environment may require an examination of the components of the phenolics,
as in the present study.
Keywords—Phenolics
Thermal paper
Paper-recycling process water
Estrogenicity
Yeast two-hybrid assay
INTRODUCTION
tems [14,15]. In Japan, the annual production of special paper,
including thermal paper, is approximately 132,000 tons
Effluents and process waters from paper-recycling indus-
tries are complex matrices containing several compounds.
These compounds originate primarily from either the wood
extractives or the additives used in the paper production pro-
cess. In a previous study, the chemical characterization of the
process waters from a paper mill industry revealed the presence
of lignans [1], fatty acids [2], phytosterols [1,3,4], and resin
acids [1–5]. The use of chlorine as a bleaching agent results
in the formation of dioxin-like compounds [6] and chlorinated
phenolics [6–8], such as chlorophenols, chloroguaiacols, chlo-
rocathecols, and chlorinated bisphenol A (BPA) [9]. Several
studies also have examined the organic additives used in the
paper production process [6,10]. Those studies generally fo-
cused on agents such as surfactants and biocides, which must
be added in large amounts to eliminate the problems related
to microbial growth during the treatment of closed water cir-
cuits. These compounds also are highly toxic to several aquatic
species, as demonstrated in previous studies that considered
different parameters, such as their lethal toxic effects and the
resulting physiological and reproductive disorders [10–13].
Thermal paper is widely used in an increasing number of
applications such as point-of-sale receipts, admission ticket-
vending machines, medical records, baggage tags, fax paper,
and delivery and food receipts generated by point-of-sale sys-
(
http://www.jpa.gr.jp). Thermal paper consists of a smooth
base paper with a thin coating that contains a leuco dye and
a phenolic developer as well as other substances. The phenolic
developer and leuco dye react on the application of heat, re-
sulting in the formation of a visible color. Bisphenol A is
widely used in the paper industry as a phenolic developer.
Recently, several chemicals that are structurally similar to BPA
also have been used. Many of these chemicals are bisphenol
S derivatives, the two phenolic rings of which are joined to-
gether with sulfur [16]. Bisphenol S derivatives have excellent
stability against high temperature and are resistant to sunlight
[14]. In addition, hindered phenol compounds are employed
as image stabilizers in dyes used for thermal paper [17]. Phe-
nolic developers in particular may exhibit estrogenic properties
similar to that of estradiol; their phenolic hydroxyl groups and
limited water solubility, at least in part, impart estrogenicity
[
18]. If thermal paper is mixed as an impurity with waste paper
stock, it may remain in the aquatic compartment and, conse-
quently, be discharged along with the process water. The oc-
currence of phenolic developers in the environment, however,
which went unnoticed for a long time, is caused by an increase
in the production of thermal paper during the last decade.
Moreover, only limited spectroscopic analysis data are avail-
able for phenolic additives in thermal paper, and to our knowl-
edge, no study on their estrogenicity has hitherto been reported.
Therefore, the objective of the present study was to develop
*
To whom correspondence may be addressed
(
terasaki@u-shizuoka-ken.ac.jp).
Published on the Web 6/14/2007.
2
356

Phenolics in paper-recycling process water
Environ. Toxicol. Chem. 26, 2007
2357
a gas chromatography–mass spectrometry (GC-MS) method
and to authentic synthetic standards using fractionation by gel
permeation chromatography to establish, to our knowledge for
the first time, the occurrence of phenolic developers, which
generally are used in the manufacture of thermal paper, in
samples collected from areas where paper-recycling process
water is discharged. In addition, we assessed the estrogenicities
of the identified compounds using yeast two-hybrid assays
incorporating the human or the medaka fish (Oryzias latipes
)
estrogen receptor (hER and medER , respectively) and an
␣
␣
␣
estrogen receptor competitive enzyme-linked immunosorbent
assay (ER-ELISA).
MATERIALS AND METHODS
Chemicals
2
-Naphthol (compound 1), drometrizole (compound 2),
BPA (compound 3), abietic acid (compound 5), and 4,4 -bu-
tylidenebis(6- -butyl- -cresol; compound 7) were purchased
from Wako Pure Chemical Industries (Osaka, Japan). Totarol
(
(
Fig. 1. Location of sampling sites and outfall in Shizuoka Prefecture,
Japan. Arrows indicate the direction of water flow.
Ј
t
m
ramine T (0.56 g) in ethanol (2 ml) for 4 h at 60ЊC. The mixture
compound 4) and N, O-bis(trimethylsilyl)trifluoroacetamide
BSTFA) were purchased from Sigma-Aldrich Japan (Tokyo,
was acidified with 1 M hydrochloric acid and then extracted
with ethyl acetate. The organic solution was washed with brine
and dried over sodium sulfate. After the removal of the solvent
under reduced pressure, the residue was chromatographed on
a silica gel column (length, 500 mm; inner diameter, 30 mm)
with toluene as the eluent. The fractions containing compound
Japan). Methanol-d4 was purchased from Cambridge Isotope
Laboratories (MD, USA), chloroform- from Acros Organics
Tokyo, Japan), pyrene- 10 from Kanto Chemicals (Tokyo, Ja-
pan), silica gel 60 (0.040–0.063 mm for column chromatog-
raphy) from Merck (Darmstadt, Germany), and Sephadex
LH-20 from Amersham Biosciences (Tokyo, Japan). The sol-
vents were of high-performance liquid chromatography grade
and were not further purified.
d
(
d
௢
8
were collected, and the solvent was evaporated to yield yel-
1
low oil (0.18 g). For H-NMR (methanol-
d
),
␦ values were
1
1
.49 to 1.50 (multiplet, 6H, 2
H, CH), 4.50 to 4.52 (multiplet, 1H, CH), 6.88 (double dou-
1.70, 6.30 Hz, Ar-H), 6.94 (double doublet, 1H,
2.30, 4.60 Hz, Ar-H), and 7.11 to 7.22 (multiplet, 10H,
ϫ CH ), 3.97 to 4.00 (multiplet,
3
blet, 1H,
J
J ϭ
Synthesis and characterization
ϭ
ϩ
2
,4-Bis(1-phenylethyl)phenol (compound 6), 2,4-bis(1-
phenylethyl)-6-chlorophenol (compound 8), and 4-hydroxy-
-isopropoxydiphenyl sulfone (compound 9) were synthe-
sized and characterized by proton nuclear magnetic resonance
Ar-H); high-resolution FAB
tensity, 100%; calculated for 336.1282, C H O Cl) and
ϩ MS m/z (M ): 336.1252 (in-
3
5
2
2
21
4
Ј
338.1216 (intensity, 35%; calculated for 338.1252,
3
7
C H O Cl).
2
2
21
1
(
H-NMR) spectroscopy operating at 500 MHz (ECA-500
4-Hydroxy-4Ј-isopropoxydiphenyl sulfone (compound 9)
spectrometer; JEOL, Tokyo, Japan), GC (HP6890A GC sys-
tem; Agilent Technologies, Tokyo, Japan)-MS (JMS-AM-
SUN200; JEOL), and high-resolution fast atom bombardment-
was obtained by the etherification of 4,4-dihydroxydiphenyl
sulfone [20]. A solution of potassium hydroxide (0.27 g) and
potassium carbonate (0.33 g) in water (1 ml) was added to a
solution of 4,4-dihydroxydiphenyl sulfone (1.2 g) in toluene
(4 ml); 2-bromopropane (0.88 g) was then added dropwise
MS (FAB
ϩ MS; JMS-700 instrument; JEOL).
2
,4-Bis(1-phenylethyl)phenol (compound 6) was obtained
by the condensation of phenol with styrene in strongly acidic
media [19]. Styrene (2.1 g) was added to a solution of phenol
with stirring at 90ЊC. Subsequently, the mixture was refluxed
for 15 h and poured into a 10% potassium hydroxide solution.
The resultant mixture was neutralized with sulfuric acid and
extracted with ether. The solvent was evaporated to yield a
(
0.94 g) in concentrated sulfuric acid (0.1 g) over a period of
6
1
0 min at 110
ЊC, and the mixture was then heated for 2 h at
50ЊC. After cooling, the mixture was acidified with 1 M hy-
white powder (compound 9; 1.0 g), which was crystallized
1
drochloric acid and then extracted with ethyl acetate. The or-
ganic layer was washed with brine and dried over sodium
sulfate. After the removal of the solvent under reduced pres-
sure, the residue was chromatographed on a silica gel column
from hexane/ether (melting point, 121–123
(chloroform- ), values were 1.33 (doublet, 6H,
CH ), 4.56 (septet, 1H, 5.70 Hz, CH), 6.89 (double
2.05, 9.15 Hz, Ar-H), and 7.80 (double doublet,
Њ
C). For H-NMR
d
␦
J ϭ 6.30 Hz,
2
ϫ
J ϭ
3
triplet, 4H,
J ϭ
ϩ
(
length, 500 mm; inner diameter, 30 mm) and eluted with
4H, 2.05, 9.15 Hz, Ar-H); mass (MS) m/z: 292 (M ).
J ϭ
toluene/ethyl acetate (9/1, v/v). The fractions containing com-
Sampling locations and sample collection
pound 6 were collected, and the solvent was evaporated to
1
yield a colorless liquid (0.45 g). For H-NMR (methanol-
d
),
Fuji City (Shizuoka Prefecture) has 85 pulp and paper mills
and recycling paper-mill enterprises, which represents the larg-
est paper-mill industrial area in Japan. Process water (average
␦
3
values were 1.46 to 1.48 (multiplet, 6H, 2
.96 (m, 1H, CH), 4.47 to 4.48 (multiplet, 1H, CH), 6.65
2.85, 8.00 Hz, Ar-H), 6.80 to 6.83
multiplet, 1H, Ar-H), 6.91 (double doublet, 1H, 2.30,
.45 Hz, Ar-H), 7.04 to 7.11 (multiplet, 5H, Ar-H), and 7.14
ϫ CH ), 3.93 to
3
3
(
(
double doublet, 1H,
J
ϭ
flow, 570,000 m /d) is discharged via an outfall into the port.
J
ϭ
Samples were collected on two different dates (November
2005 and August 2006) from the outfall (site 1), upstream of
the outfall (site 2), the mouth of the port (site 3), and two
tributaries (sites 4 and 5) from which the rivers discharge into
the Pacific Ocean (Fig. 1). Sediment samples (500 g) were
7
ϩ
to 7.20 (multiplet, 5H, Ar-H); mass (MS) m/z: 302 (M ).
,4-Bis(1-phenylethyl)-6-chlorophenol (compound 8) was
obtained by the reaction of compound 6 (0.36 g) with chlo-
2

2
358
Environ. Toxicol. Chem. 26, 2007
M. Terasaki et al.
Table 1. Retention times, monitored ions, and quality-control data for trimethylsilyl derivatives of target compounds and partition coefficients
of the target compounds
Mass ions (
m
/
z
)b
Partition coefficientf
Log KOW Log KOC
Recovery
LOQ
d
c
r2e
No.
Compound name
Rt (min)a Quant Conf
(%
Ϯ
SD)
(␮g/L)
1
2
3
4
5
6
7
8
9
2-Naphthol
Drometrizole
Bisphenol A
Totarol
Abietic acid
2,4-Bis(1-phenylethyl)-phenol
12.4
18.0
19.0
20.1
20.7
20.8
22.0
22.1
22.8
201
282
357
343
256
359
483
393
307
216
297
372
358
374
374
526
408
364
92
94
96
95
99
102
94
108
94
Ϯ
12
4
2
1
6
3
1
5
2
0.09
0.07
0.10
0.06
1.60
0.08
0.19
0.06
0.12
0.9900
0.9912
0.9921
0.9905
0.9887
0.9810
0.9958
0.9909
0.9934
2.69
3.00
3.64
7.28
6.46
5.83
9.09
6.47
3.12
2.98
ϫ
ϫ
ϫ
ϫ
ϫ
ϫ
ϫ
ϫ
ϫ
103
5
4
5
4
6
7
6
3
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
1.01
7.52
6.59
2.20
3.63
2.59
6.00
9.54
10
10
10
10
10
10
10
10
4,4
2,4-Bis(1-phenylethyl)-6-chlorophenol
4-Hydroxy-4 -isopro-poxydiphenyl sulfone
Ј
-Butylidenebis(6-
t
-butyl-
m
-cresol)
Ј
a
Rt
Quant
ϭ
retention time.
for quantification; Conf
ϭ standard deviation.
b
ϭ
ϭ
for confirmatory purposes.
c
d
e
f
SD
LOQ
ϭ
limits of quantification.
2
r
ϭ
coefficient of determination.
KOW
ϭ
octanol-water partitioning coefficient; KOC
ϭ
organic carbon adsorption coefficient. Estimated log KOW and KOC values were predicted
Version 3.20 software [21].
with the KOWWIN
௢
and PCKOCWin modules included with the U.S. Environmental Protection EPI Suite௢
௢
collected from the surface of the sediment layer by using an
Ekmann-Birge bottom sampler, and water samples (1 L) were
collected from the surface water. All samples were collected
in glass bottles with aluminum-lined stoppers, transported to
Folsom, CA, USA). The column temperature was initially
maintained at 60 C for 1 min. It was programmed to then attain
280 C at a rate of 10 C/min, with a final hold time of 7 min.
Helium was used as the carrier gas at a flow rate of 1.0 ml/
min, and the column head pressure was maintained at 8.0 psi.
Њ
Њ
Њ
the laboratory within 1 d, and stored at 4
within 10 d).
ЊC until analysis
(
The injector temperature was maintained at 250
injection volume was 1.0 l in the splitless mode. The electron
multiplier voltage for the MS was 1,988 V, and the interface
temperature was maintained at 280 C. Mass spectra were ob-
tained by electron-impact ionization at a voltage of 70 eV and
scanned over a range of 50 to 550 atomic mass units at
a rate of 1.5 scans/s, and the ion source temperature was main-
tained at 250 C. The phenolics in the samples were identified
ЊC, and the
␮
Extraction and fractionation
Freeze-dried sediment samples (10 g) were extracted by
ultrasonic extraction with methanol (four extractions, 10 ml/
extraction). After the removal of the solvent under reduced
pressure, the residue was dissolved in methanol (5 ml), and
the samples (1 ml) were subjected to gel permeation chro-
matography on a Sephadex LH-20/methanol column (length,
Њ
m/z
Њ
by comparing the GC retention times and mass spectra of the
samples with those of the authentic standards. These com-
pounds were quantified by correlating the ratio of the peak
area of the compound of interest to that of the internal standard
(pyrene-d10) with the calibration curve of the standard solution.
The standard calibration solution, containing compounds 1 to
9, was prepared in dichloromethane.
3
00 mm; inner diameter, 30 mm). Eighteen-milliliter fractions
were collected using a fraction collector (FR50N; Yamazen,
Osaka, Japan). Methanol was removed separately from each
fraction using a nitrogen stream. The fractions were pipetted
with methanol into a GC-MS vial and dried in a nitrogen
stream. The extracted samples were trimethylsilylated before
the GC-MS analysis. A total of 50
␮l of BSTFA was added
Quality assurance and quality control
to the vials containing the extracts. Following a 1-h reaction
at room temperature, the solvent in the vial was evaporated to
dryness under a nitrogen stream. The extracted samples were
All analyses were performed in two duplicates. The char-
acteristics of this method are shown in Table 1. The relative
standard deviation of the results obtained was within 12% for
recoveries and within 21% for the samples. The recoveries
reconstituted into dichloromethane (0.1 ml) containing 2
ml of internal standard (pyrene- 10) and then examined by GC-
MS.
␮g/
d
were determined using distillated water spiked to 5
recoveries varied between 92 and 108%, and all the standards
showed a linear range up to 320 g/L for abietic acid (com-
pound 5) and 100 g/L for the other compounds. The coef-
ficient of determination (
␮g/L. The
Water samples (100 ml) were adjusted to pH 3 with diluted
hydrochloric acid, filtered (GF/A glass microfiber filters;
Whatman Japan K.K, Tokyo, Japan), and extracted in dichlo-
romethane (two extractions, 25 ml/extraction) using a shaker
␮
␮
2
r
) ranged from 0.98 to 0.99 over five
concentration levels. The concentration of the lowest calibra-
tion standard was operationally defined as the limit of quan-
tification (LOQ). Each value of the octanol–water partition
coefficient and organic carbon adsorption coefficient was cal-
(
1
Recipro Shaker SR-2s; Taitec, Saitama, Japan) at 265 rpm for
0 min. After the removal of the solvent under reduced pres-
sure, the residue was treated in the same manner as the sed-
iment samples for the GC-MS analysis.
culated using the EPI Suite
samples (surface water of the Kusanagi River; 34
38 27 24 E) were routinely analyzed, and no contamination
௢
Version 3.20 software [21]. Blank
Њ
59 28 N,
Ј
Љ
Analytical methods
1
Њ
Ј
Љ
The standard and sample extracts were analyzed by GC
of the solvents or equipment was detected.
(
(
HP 6890 Series; Hewlett-Packard, Palo Alto, CA, USA)-MS
HP 6890 Series mass-selective detector; Hewlett-Packard) on
Yeast two-hybrid assays
a HP-5 fused silica capillary column (length, 30 m, inner di-
ameter, 0.32 mm; film thickness, 0.25 m; J&W Scientific,
The agonist activities of compounds 1 to 9 were examined
using the yeast two-hybrid assay system as developed by Ni-
␮

Phenolics in paper-recycling process water
Environ. Toxicol. Chem. 26, 2007
2359
Fig. 2. Total ion chromatograms of fractionated extracts: (a) Fraction 13, and (b) fraction 15. Gel permeation chromatography (Sephadex
௢ LH-
2
0; Amersham Biosciences, Tokyo, Japan) was used to obtain these extracts from the water sample at the outfall (site 1). IS internal standard.
ϭ
shikawa et al. [22] and modified using a yeast two-hybrid
centration (EC) ϫ 10, which is defined as the concentration
estrogenicity assay using yeast cells (Saccharomyces cerevi-
siae Y190) into which hER␣ or medER␣ and coactivator TIF2
had been introduced. This yeast two-hybrid assay was sepa-
rately conducted with and without possible activation/deacti-
vation by the S9 mix, as described previously [23]. In our
laboratory, research conducted subsequent to the amplification
of test solution required to produce a chemiluminescent signal
10-fold that of the blank control. Several compounds in an
assay exhibited a slight increase in the treatment to baseline
ratio; however, they did not reach a treatment to baseline ratio
of 10 even at higher concentrations and were defined as sus-
pected active. Relative affinity (as a % relative to 17
␤-estradiol
of the gene for medER
combinant plasmid for use in the yeast two-hybrid assay has
revealed that in general, the estrogenic activities of medER
relative to those of xenoestrogens) are higher than those of
hER
␣
and its introduction into a yeast re-
[E ]) was calculated by dividing the EC 10 of E in the
absence of the S9 mix by that of a test chemical. The working
range of an assay both with and without metabolic activation/
ϫ
2
2
␣
(
deactivation is 63 to 400 pM for E , 78 to 5,000 nM for trans-
2
␣
.
stilbene (tSB), and 313 to 20,000 nM for the target compounds.
The expression plasmids for the ligand-binding domain of
each hormone receptor and pGAAD424-TIF-2 were introduced
All the test chemicals were evaluated in three independent
experiments with two replicates each. The intra- and interassay
coefficients of variation were in the range of 0.6 to 20% and
2.9 to 28%, respectively. In the absence of the S9 mix, the
into the yeast lines that carried the
␤-galactosidase reporter
gene. To measure agonist activity in both assays, aliquots from
test chemical solutions (2 mM in dimethyl sulfoxide) were
positive control was E ; its 400 pM solution resulted in a
2
incubated for 4 h at 30
Њ
C, with yeast cells preincubated at 30
Њ
C
treatment to baseline ratio of 128 for hER
medER . In the presence of the S9 mix, the positive control
was SB (activated to 4-hydroxytrans-stilbene); its 2.5
solution resulted in a treatment to baseline ratio of 11 for hER
and 120 for medER . The potential metabolic transformation
␣ and 313 for
overnight in a modified synthetic dextrose medium (lacking
tryptophan and leucine). For metabolic activation/deactivation,
the test solutions were incubated with a S9 mix (4.8% S9;
Kikkoman, Noda, Japan) including 2.3 mM glucose-6-phos-
␣
t
␮M
␣
␣
phate (disodium salt), 2 mM
␤
-nicotinamide adenine dinucle-
of E by exposure to rat liver S9 mix resulted in a treatment
2
otide phosphate oxidized form, 60 mM Na PO , 40 mM
to baseline ratio of one in the yeast two-hybrid assay incor-
2
4
NaHPO , 10 mM KCl, 1 mM MgSO , and 4 mM 4-(2-hy-
porating the hER␣ to eliminate the estrogenicity. This revealed
4
4
droxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.2)
for 1 h at 37 C before incubation with the yeast cells. After
that the possible activation/deactivation by the S9 mix pro-
gressed.
Њ
the incubation period, a mixed solution of 2 mg/ml of zym-
olyase (Zymolyase 20T; Seikagaku, Tokyo, Japan) and a com-
mercial chemiluminescent reporter gene assay kit (Aurora Gal-
XE; ICN Pharmaceuticals, Costa Mesa, CA, USA) in the ratio
of 5:3, inducing chemiluminescence and enzymatic digestion,
was added followed by a light-emission accelerator reagent.
The intensity of the chemiluminescence produced by released
Estrogen receptor competitive enzyme-linked
immunosorbent assay
For the estrogen receptor, a competitive binding assay was
conducted using a ligand screening system (ER
kui, Japan) in accordance with the manufacturer’s protocol. In
brief, 30 l of stock solutions were serially diluted in a phos-
phate buffer (pH 7.2) and incubated with a receptor (24 nM,
20 l) and E solution (4.2 nM, 30 l) at 4 C for 1 h in 96-
well plates. The reaction solutions with estradiol–horseradish
peroxidase (50 l) were transferred to antibody plates coated
␣; Toyobo, Fu-
␮
␤
-galactosidase was measured using a 96-well plate luminom-
eter (ATTO JNR AB-2000; ATTO, Tokyo, Japan). The details
of the assay have been previously published [24].
␮
␮
Њ
2
Agonist activities were recorded relative to effective con-
␮

2
360
Environ. Toxicol. Chem. 26, 2007
M. Terasaki et al.
Fig. 3. Mass spectra of the trimethylsilyl (TMS) derivatives of compounds 1–9 in the water samples (site 1). Numbers represent the peak numbers
in Figure 2.
with an estradiol antibody (125 ng/well) and incubated at 4
Њ
C
sulfuric acid (1 M, 100 ␮l) was added to each well to stop the
for 1 h. After washing the wells with a washing solution (50
mM disodium phosphate, 150 mM NaCl, and 0.05% Tween-
reaction, and the absorbance of each solution was measured
at two wavelengths (450 and 655 nm). Diethylstilbestrol (DES;
300 nM) was used as a positive control.
For assays with metabolic activation/deactivation, the test
chemical solutions were treated in the same manner as in the
2
3
0 dilution buffer; 150
,3 ,5,5 -tetramethylbenzidine was added to each well, and the
C for an additional 20 min. Finally,
␮l), a substrate solution containing
Ј
Ј
plates were incubated at 37
Њ

Phenolics in paper-recycling process water
Environ. Toxicol. Chem. 26, 2007
2361
Table 2. Concentrations of compounds detected in samples collected from paper-recycling process water discharge areas
Outfall watersheda
Site 3
Site 1
Site 4
Water
Site 5
Water
g/L)
Site 2
Sediment Water
Water
Sediment
g/g)
No.
Compound name
Water
(
␮
g/L)
(
␮
(
␮
g/L)
(
␮
1
2
3
4
5
6
7
8
9
2-Naphthol
Drometrizole
Bisphenol A
Totarol
Abietic acid
2,4-Bis(1-phenylethyl)-phenol
0.63
13
4.1
0.70
270
4.4
2.7
82
27
1.1
8.4
2.2
2.4
230
4.6
1.5
NDb
ND
ND
ND
ND
ND
ND
ND
ND
Ͻ
LOQc
0.22
0.78
ND
0.16
0.31
0.20
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
39
Ͻ
11
LOQ
ND
0.10
4.4
0.28
ND
1.9
4,4
2,4-Bis(1-phenylethyl)-6-chlorophenol
4-Hydroxy-4 -isopro-poxydiphenyl sulfone
Ј
-Butylidenebis(6-
t
-butyl-
m
-cresol)
55
19
Ј
0.41
a
Site 1
ϭ
adjacent area; Site 2
not detected.
limit of quantification.
ϭ
upstream; Site 3
ϭ
downstream.
b
c
ND
ϭ
ϭ
LOQ
case of the yeast two-hybrid assays. The inhibition ratio was
calculated using the following equation: Inhibition ratio (%)
were not detected upstream (site 2) of the outfall and in the
other two tributaries (sites 4 and 5). Therefore, the occurrence
of phenolics at the mouth of the port originated from the paper-
recycling process water. The concentration levels at site 1
ϭ
(
B
Ϫ
S
)/(
negative control (1% dimethyl sulfoxide),
in the presence of a test chemical, and is the absorbance for
B
Ϫ
P
)·100, where
B
is the absorbance for the
S
is the absorbance
P
ranged from 0.63 to 270
1.1 to 230 g/g for the sediment sample. The concentration
levels at site 3 ranged from the LOQ to 39 g/L for the water
sample and from the LOQ to 11 g/g for the sediment sample.
␮g/L for the water sample and from
the positive control (DES). The concentration of a test chem-
ical yielding 50% inhibition (IC50) was calculated from a
sigmoidal curve fitted to the calculated inhibition rates. The
relative binding affinity (RBA) was calculated as follows: RBA
␮
␮
␮
A comparison of the concentrations suggested a reduction of
more than 80% for all the compounds in the downstream sam-
ples. In particular, the phenolics (compounds 4 and 6–9) were
detected for the first time as environmental contaminants in
paper-recycling process water. In Japan, compound 9 is widely
used as an alternative to BPA (compound 3) for the production
of thermal paper [27], because compound 3 is suspected to
have estrogenic properties similar to that of the natural sex
hormone [28]. Compound 9 was the major phenolic investi-
gated in the present study, and the concentration of compound
9 was much higher than that of compound 3 in all the samples.
This suggests an increased use of compound 9 as an alternative
to BPA. The chlorinated compound 8 and its parent compound
(
%DES) ϭ (IC50 for DES)/(IC50 for test chemical)·100. The
working range of the assay is 15 to 60 nM for DES and 3.75
to 37,500 nM for the other chemicals.
All the test chemicals were evaluated in two independent
experiments, each with two replicates. The intra- and inter-
assay coefficients of variation were in the range of 1.3 to 8.5%
and 0.0 to 19%, respectively.
RESULTS AND DISCUSSION
Synthesis
Three compounds identified as contaminants in the samples
1
were synthesized and characterized ( H NMR spectroscopy and
(
compound 6) were detected at the outfall (site 1) and down-
stream (site 3) of the downfall. In samples obtained from site
, the concentration of compound 8 was 12- to 19-fold that
MS). Their purities were measured by GC-MS and confirmed
to be 98% or greater.
1
Occurrence of phenolic compounds in samples
of compound 6. This suggests that compound 6 reacts easily
with agents containing chlorine during the paper-recycling pro-
cess to provide the chlorination by-product. Compound 2 also
was detected in the samples obtained from sites 1 and 3. The
In the extracts from a water sample (site 1), the total ion
chromatograms for phenolic additives that were fractionated
by gel permeation chromatography on a Sephadex LH-20 are
shown in Figure 2. The MS data of the compounds identified
in the sample are shown in Figure 3. Fractions 13 and 14
contained phenolic developers [17,19,20] used in the manu-
facture of thermal paper, such as compounds 3, 6, 7, and 9,
and diterpenes (compounds 4 and 5) from wood (Fig. 2a).
Fraction 15 contained naphthol (compound 1) and drometri-
zole (compound 2), which are used in the manufacture of
thermal paper as an intermediate [25] for a sensitizer and as
a ultraviolet stabilizer [26], respectively (Fig. 2b).
The concentrations of the compounds present in the samples
collected from the study areas are summarized in Table 2.
Higher concentrations were observed in site 1, because it is
close to the outfall of the effluent that includes paper-recycling
process water. Site 3, which is at the mouth of a port, receives
water from the outfall, site 4, and site 5. All but two compounds
concentration of compound 2 in the water at site 1 was 13
L, which is comparable to the concentration in the water down-
stream of a chemical processing plant (30 g/L) [29]. Com-
pound 1 was predominant in the sediment samples, with con-
centrations of 1.1 g/g at site 1 and 0.16 g/g for the down-
␮g/
␮
␮
␮
stream samples. In the manufacture of thermal paper,
compound 1 is used as an intermediate for the sensitizer [25].
In addition, this compound is considered to be an intermediate
in the photodegradation of naphthalene and in the cometa-
bolization of naphthalene by microorganisms [30]. Naphtha-
lene is a ubiquitous environmental contaminant. Thus, the
source of compound 1 may be not only a raw material orig-
inating in the manufacture of paper but also a secondary trans-
formation product of naphthalene. Compound 5 is not a phe-
nolic compound; however, it had the highest concentration of
all the compounds present in the samples that were investi-
(
compounds 4 and 7) also were found at site 3, whereas they

2
362
Environ. Toxicol. Chem. 26, 2007
M. Terasaki et al.

Phenolics in paper-recycling process water
Environ. Toxicol. Chem. 26, 2007
2363
Fig. 4. Agonist activity of identified compounds, 17␤-estradiol (E ), and trans-stilbene in yeast two-hybrid estrogenicity assay incorporating the
2
human estrogen receptor
␣ (a and b) without rat liver S9 treatment and (c and d) with rat liver S9 treatment. Each point represents the mean of
three independent experiments, each with two replicates.
gated. The concentration of compound 5 in the water at site
for each concentration. In the yeast two-hybrid estrogenicity
assay incorporating medER without the rat liver S9 mix, two
1
was comparable both with and within the range of the con-
␣
centrations previously reported for mill process water in the
United States [12].
compounds exhibited agonist activity, with a treatment to base-
line ratio of 374 for compound 3 and 271 for compound 1 at
a concentration of 20,000 nM, the highest concentration tested.
In addition, compound 6 was suspected to have agonist activity
because of a treatment to baseline ratio of six at the highest
concentration tested. In the presence of the S9 mix, compounds
3, 1, and 2 exhibited agonist activity, with a treatment to base-
line ratio of up to 56, 22, and 11, respectively. Compound 3
also showed a response with a marked falling off at a con-
centration of 20,000 nM, because the S9 mix inhibits the
Estrogenicity of phenolic compounds in samples
The results of the yeast two-hybrid assays used to determine
the estrogenicities of test chemicals are shown in Table 3 and
in Figures 4 (hER
estrogenicity assay incorporating hER
␣
) and 5 (medER
␣
). In the yeast two-hybrid
without the rat liver
␣
S9 mix, compounds 3 and 1 exhibited agonist activity, with a
treatment to baseline ratio of 22 and 13, respectively, at a
concentration of 20,000 nM. In the presence of the S9 mix,
compound 3 also revealed suspected agonist activity, with a
treatment to baseline ratio of five at a concentration of 20,000
nM, although a reduction in the agonist response was observed
growth of the yeast cells. The positive control,
a similar tendency at the 5,000 nM level in the hER
and at the 2,500 nM level in the medER assay. The agonist
activity of compound 6 was eliminated by S9 metabolic ac-
t
SB, indicated
␣
assay
␣

2
364
Environ. Toxicol. Chem. 26, 2007
M. Terasaki et al.
Fig. 5. Agonist activity of identified compounds, 17
medaka fish (Oryzias latipes) estrogen receptor
represents the mean of three independent experiments, each with two replicates.
␤
-estradiol (E ), and trans-stilbene in yeast two-hybrid estrogenicity assay incorporating the
2
␣
(a and b) without rat liver S9 treatment and (c and d) with rat liver S9 treatment. Each point
tivation, whereas compound 2 exhibited activity after exposure
to the S9 mix. In the medER assay with the S9 mix, three
compounds gave positive responses both with and without the
S9 mix; however, in the hER assay, a positive response was
from 0.073 to 2.3%, with the highest binding affinity being
observed in compound 3 as compared to the positive control,
DES.
Inspection of the results from both yeast two-hybrid assays
and an ER-ELISA suggested that an important factor in de-
termining estrogenicity is the position of a bulk substituent
relative to a phenolic hydroxyl group. Miller et al. [31] re-
ported several phenolic chemicals that are capable of acting
as estrogenic compounds in a yeast bioassay. Common to the
majority of these compounds is an unhindered phenolic moiety
␣
␣
observed for only one compound. In addition, relative affinity
to E for these compounds ranged from 0.0028 to 0.018% in
2
the medER
assay. These results indicated that an assay system incorpo-
rating medER likely is more effective for monitoring xen-
␣ assay and from 0.0009 to 0.0015% in the hER␣
␣
oestrogens. The IC50s and RBAs to DES obtained from the
results of ER-ELISA are shown in Table 4. The binding affinity
similar to the A ring of E . Compound 3 also possesses such
2
to hER
␣
was found in compounds 3, 4, and 6 with and without
phenolic hydroxyl groups and exhibits estrogenicity in yeast
two-hybrid assays and an ER-ELISA. In addition, compounds
2, 4, and 6 to 8, each having a phenol ring with a hindered
moiety at the ortho position, exhibited no agonist or suspected
agonist activity. These results are consistent with the findings
the rat liver S9 mix. The ER-binding affinity of compound 7
was eliminated by S9 metabolic activation, whereas the chlo-
rinated compound 8 showed an ER-binding affinity after ex-
posure to the S9 mix. The RBAs for these compounds ranged

Phenolics in paper-recycling process water
Environ. Toxicol. Chem. 26, 2007
2365
Table 4. Estrogenicities of compounds detected in samples collected from paper-recycling process water discharge areas in an estrogen receptor
a
competitive enzyme-linked immunosorbent assay
Ϫ
S9 test
ϩS9 test
No.
Compound name
IC50 (nM)b
RBA (%)c
IC50 (nM)
RBA (%)
1
2
3
4
5
6
7
8
9
2-Naphthol
Drometrizole
Bisphenol A
Totarol
Abietic acid
2,4-Bis(1-phenylethyl)-phenol
Ͼ
Ͼ
870
37,500
37,500
Ͼ
Ͼ
12,000
11,000
37,500
37,500
Ϫ
1
1
Ϯ
Ϯ
100
3,400
2.3
Ϯ
Ϯ
1,100
1,500
1.7
1.7
ϫ
ϫ
10
10
Ϫ
2
Ϫ
24,000
8.3
ϫ
10
Ͼ
15,000
27,000
37,500
Ͼ
37,500
7,900 1,500
37,500
25,000 2,700
Ϫ
Ϫ
1
2
10 1
10 2
Ϫ
Ϯ
Ϯ
2,400
2,000
1.4
7.3
ϫ
ϫ
10
10
Ϯ
2.5
8.1
ϫ
ϫ
4,4
Ј
-Butylidenebis(6-
t
-butyl-
m
-cresol)
Ͼ
Ϫ
2,4-Bis(1-phenylethyl)-6-chlorophenol
Ͼ
Ͼ
20
37,500
37,500
Ϯ
37,500
37,500
4-Hydroxy-4
Ј
-isopro-poxydiphenyl sulfone
Ͼ
Ͼ
1,900
d
DES
t
Ϯ
0
100
SBe
Ϯ
340
1.1
a
Values are presented as the mean
Ϯ
standard deviation obtained from two independent experiments, each with two replicates.
50% inhibition concentration.
relative binding affinity (relative to diethylstilbestrol [DES]).
b
c
d
e
IC50
RBA
ϭ
ϭ
Positive control in absence of S9 preparation.
SB trans-stilbene; positive control in presence of S9 preparation.
t
ϭ
of Miller et al. [31]. Compounds 4, 6, and 7, however, exhibited
the binding affinity to hER in an ER-ELISA. The ER-ELISA
itatively in groundwater collected near hazardous waste sites
in the United States [36] and in the wastewater from the oil
shale–processing industry [37]. In addition, compound 1 has
been described as the product of the photodegradation or mi-
crobial degradation of naphthalene, a ubiquitous polycyclic
aromatic hydrocarbon contaminant [30]. Compound 2 has been
detected in sediment cores collected from the discharge areas
of chemical plant effluents [29]. On the other hand, the present
study demonstrates the occurrence of the phenolic compounds
4 and 6 to 9 in river water and sediment. To our knowledge,
the emission of these pollutants into an aquatic environment
because of the manufacture of thermal paper has not been
reported thus far. The partitioning of organic pollutants, such
as estrogenic compounds, in an aquatic environment is of fun-
damental importance in determining their transport. Some
chemicals, such as nonylphenol, BPA, and estrone, were con-
centrated in colloids and sediment [38]. Therefore, phenolics
(compounds 1 and 3) in paper-recycling process water may be
accumulated in sediment and cause the endocrine disruption
of male fish in an aquatic environment. This feminization has
been observed in flatfish and other species that are intimately
associated with sediment [39].
The present study revealed eight phenolics in the samples
collected from areas where paper-recycling process water is
discharged. In general, wildlife is exposed not to single agents
but, rather, to mixtures of multiple estrogenic compounds—
the xenoestrogens in an aquatic environment [40]. Therefore,
we are working toward the assessment of their estrogenic mix-
ture effects. A comprehensive assessment of the estrogenic
mixture effects of phenolics and their implications for aquatic
life and environmental safety may require an examination of
all the components of the phenolics, as described in the present
study. The present results lead to concerns regarding the oc-
currence of phenolic compounds originating from paper-re-
cycling process water. Although BPA (compound 3) is not
acutely toxic to either plants or animals, it has been shown to
exhibit weak estrogenic activity [28]. In addition, compound
3 is released into the environment both accidentally and
through permitted discharges, and its widespread distribution
has been a major cause of concern for regulatory agencies and
other organizations. Given this situation, compound 9 has
gradually replaced compound 3 as a developer for thermal
␣
can purely determine the ER-binding affinity of a test chemical.
Meanwhile, yeast two-hybrid assays have apparent advantages
that offer a more realistic model of animal and human cells,
which show the ligand-dependent interaction between ER and
coactivators in living cells. These results may indicate that
compounds 4, 6, and 7 are antagonist active. The estrogenicity
of hydroxy polycyclic aromatic hydrocarbons, such as com-
pound 1, has been reported previously [32], and the results of
the present study correspond to the previous data. Compound
9
possesses a noncarbon atom at the central position. The lack
of estrogenic activity observed for compound 9 is consistent
with the findings of Hashimoto et al. [33], who reported the
lack of estrogenicity for 4,4
yeast two-hybrid system [33]. Compound 5 showed no activity
in assays using both hER and medER . One of the reasons
Ј-dihydroxydiphenyl sulfide in the
␣
␣
for this may be the absence of a phenolic hydroxyl group in
the molecule.
A report on the metabolites of compound 3 was presented
by Yoshihara et al. [34]; the estrogenicity of BPA increased
several-fold after incubation with four species of liver S9 frac-
tions, including rat liver, requiring both microsomal and cy-
tosolic fractions. Metabolic activation was observed in the
extract by a solid-phase extraction method. One of the active
metabolites was confirmed to be 4-methyl-2,4-bis( p-hydrox-
yphenyl)pent-1-ene [35]. In contrast, the present results in-
dicate that the agonist activities of compounds 1 and 3 in the
medER
approximately 139 and 56%, respectively, as compared with
the EC 10 values before exposure to the S9 mix. In the
␣ assay after exposure to the S9 mix decreased by
ϫ
present study, the metabolites by S9 mix were investigated
directly using the yeast two-hybrid assays or ER-ELISA with-
out cleanup (e.g., solid-phase extraction). The differences in
the estrogenicities of metabolites may be attributed to the in-
vestigations performed with or without purification after in-
cubation with the rat liver S9 fraction.
Several of the identified compounds are known to be an-
thropogenic pollutants and, consequently, are reported in some
environmental studies. Compounds 3 and 5 are reported to be
present in the effluents of packaging board mills and paper-
recycling plants [10,12]. Compound 1 has been identified qual-

2
366
Environ. Toxicol. Chem. 26, 2007
M. Terasaki et al.
2
2
2
0. Oi F, Yanagise N, Kitahara T, Nagaoka E. 2003. Method for
producing diphenylsulfone compound. Japan Patent Office
paper in Japan. The findings suggest that compound 9 exhibits
no estrogenic activity in assays using both hER and medER␣.
␣
0
81938. Wakayama Prefecture, Tokyo, Japan.
Therefore, it appears to be a desirable material, and its oc-
currence in the environment may increase. Future studies of
the identified compounds, however, may discover hitherto un-
defined toxicological properties. To reduce the risks caused by
pollutants to aquatic organisms, it is imperative to prevent the
contamination of waste paper by thermal paper.
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