Metabolites of chlorinated syringaldehydes in fish bile as biomarkers of exposure to bleached eucalypt pulp effluents

Article abstract of DOI:10.1006/eesa.1996.0032

Metabolites of chlorinated phenolic compounds in fish bile have been found to be sensitive biomarkers of bleached pulp mill effluent exposure. Chlorinated syringaldehydes are largely unstudied chlorophenolics found in bleached hardwood effluent. Sand flathead (Platycephalus bassensis), Australian marine fish, were exposed to 100% chlorine dioxide-bleached eucalypt pulp effluent at concentrations of 0.5, 2, and 8% (v/v) for 4 days. Metabolites of 2-chloro-syringaldehyde (2-CSA), the predominant chlorophenolic in this effluent, were measured in the bile. The major metabolite was the conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy- benzylalcohol (2-CB-OH), the reduced product of 2-CSA. 2-CB-OH was found in all fish exposed to diluted effluent and was concentrated in the bile over 1000 times above 2-CSA levels in the effluent. A separate experiment examined the metabolic fate of 2,6-dichlorosyringaldehyde (2,6-DCSA), which is one of the major chlorophenolics in chlorine-bleached eucalypt pulp effluent. Sand flathead were exposed to 2,6-DCSA by intraperitoneal injection at 15 mg/kg or through the water to 0.5, 2, or 8 μg/liter for 4 days. Analysis of the bile revealed the major metabolite of 2,6-DCSA to be the conjugate of 2,6- dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol, which was found in all exposed fish and was concentrated in the bile over 20,000 times above 2,6-DCSA exposure levels. Results reveal that the analysis of metabolites of chlorinated syringaldehydes in fish bile can provide a biomarker of bleached hardwood effluent exposure that is sensitive to low levels of exposure, specific to certain bleaching sequences, and correlates well with exposure concentrations.

Full text of DOI:10.1006/eesa.1996.0032

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 33, 253–260 (1996)
ARTICLE NO. 0032
Metabolites of Chlorinated Syringaldehydes in Fish Bile as Biomarkers
of Exposure to Bleached Eucalypt Pulp Effluents
1
CAROLYN M. BRUMLEY, VICTORIA S. HARITOS, JORMA T. AHOKAS, AND DOUGLAS A. HOLDWAY
Key Centre for Applied and Nutritional Toxicology, RMIT-University, GPO Box 2476V, Melbourne, 3001 Australia
Received July 5, 1995
Chlorophenolic compounds are produced during the
bleaching process in pulp mills by the chlorine degradation
of lignin and by chlorination of phenolic residues (McLeay,
1987). Chlorophenolics from bleached pulp effluents include
the moderately hydrophobic chlorinated phenols, guaiacols,
catechols, and vanillins found in the effluents of bleached
soft- and hardwood pulp, as well as chlorinated syringols
and syringaldehydes that are found only in bleached hard-
Metabolites of chlorinated phenolic compounds in fish bile have
been found to be sensitive biomarkers of bleached pulp mill effluent
exposure. Chlorinated syringaldehydes are largely unstudied chlo-
rophenolics found in bleached hardwood effluent. Sand flathead
(Platycephalus bassensis), Australian marine fish, were exposed to
100% chlorine dioxide-bleached eucalypt pulp effluent at concen-
trations of 0.5, 2, and 8% (v/v) for 4 days. Metabolites of 2-chloro-
syringaldehyde (2-CSA), the predominant chlorophenolic in this
effluent, were measured in the bile. The major metabolite was the
conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-
¨
wood pulp effluent (Kringstad and Lindstrom, 1984). Fish
exposed to chlorophenolics have been found to rapidly me-
CB-OH), the reduced product of 2-CSA. 2-CB-OH was found in tabolize them to polar conjugates, which are then excreted
all fish exposed to diluted effluent and was concentrated in the
bile over 1000 times above 2-CSA levels in the effluent. A separate
experiment examined the metabolic fate of 2,6-dichlorosyringalde-
hyde (2,6-DCSA), which is one of the major chlorophenolics in
chlorine-bleached eucalypt pulp effluent. Sand flathead were ex-
posed to 2,6-DCSA by intraperitoneal injection at 15 mg/kg or
primarily in the bile and urine (de Bethizy and Hayes, 1989;
Kasper and Henton, 1980). Because the concentration of
certain conjugated phenolic compounds in fish bile can be
very much greater than their concentration in the surrounding
water, Lech et al. (1973) suggested that the quantification
of metabolites in the bile can provide a marker of exposure
to very low, and even fluctuating, levels of aquatic pollutants.
through the water to 0.5, 2, or 8 mg/liter for 4 days. Analysis of
the bile revealed the major metabolite of 2,6-DCSA to be the conju-
gate of 2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol, which
was found in all exposed fish and was concentrated in the bile
over 20,000 times above 2,6-DCSA exposure levels. Results reveal
that the analysis of metabolites of chlorinated syringaldehydes in
fish bile can provide a biomarker of bleached hardwood effluent
exposure that is sensitive to low levels of exposure, specific to
certain bleaching sequences, and correlates well with exposure
concentrations. ᭧ 1996 Academic Press, Inc.
˚
Oikari and co-workers (Oikari and Ana¨s, 1985; Oikari et
al., 1984; Oikari and Kunnamo-Ojala, 1987; Oikari, 1986)
have developed techniques for measuring the concentration
of various conjugated chlorophenolic compounds in fish bile,
and these techniques have been extensively applied in field
studies of bleached pulp mill effluents to describe both the
extent of effluent plumes and the exposure of fish within
them (Balk et al., 1993; Landner et al., 1994; Lindstro¨m-
¨
Seppa and Oikari, 1990). Several studies have found signifi-
cant correlations between the concentration of chlorophe-
nolic compounds in fish bile and the distance from the efflu-
ent source (Lindstro¨m-Seppa¨ and Oikari, 1990; Oikari and
Kunnamo-Ojala, 1987; So¨derstro¨m et al., 1994).
INTRODUCTION
Bleached pulp mills produce complex effluents, the com-
position of which varies with different woodstocks and pulp-
ing, bleaching, and treatment processes. Most of the toxicity
of pulp mill effluents to aquatic organisms is attributed to
chlorate, resin and fatty acids, chlorophenolic compounds,
and other chlorinated organics (McLeay, 1987). Numerous
studies, both field and laboratory based, have documented
the effects of bleached pulp mill effluents on fish (Andersson
et al., 1987; Balk et al., 1993; Munkittrick et al., 1991).
Eucalypts are native Australian hardwoods that, among
other wood species, are used as a feedstock for the Australian
pulp and paper industry. Effluents that arise from bleached
eucalypt pulp appear typical for hardwoods; they have a
low acute toxicity (Munday et al., 1992; Stauber, 1993) and
contain few chlorinated organic compounds (Nelson et al.,
1993). Chlorinated syringaldehydes (Fig. 1) are quantita-
tively the major chlorinated phenolics found in the effluents
of bleached eucalypt pulp (Smith et al., 1993), with the
degree of chlorine substitution dependent on the proportions
1 To whom correspondence should be addressed.
253
0147-6513/96 $18.00
Copyright
᭧ 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

254
BRUMLEY ET AL.
metabolites as biomarkers of bleached eucalypt effluent ex-
posure is discussed.
METHODS
Chemicals
Syringaldehyde and tricaine methanesulfonate (MS-222)
were purchased from Sigma (St. Louis, MO). 2,4,5-Tribro-
mophenol (TBP) was purchased from Aldrich Chemical
Company (Milwaukee, WI) and tetrachloronaphthalene
(TCN) from ICN Biomedicals (CA). b-Glucuronidase/aryl
sulfatase was obtained from Boehringer-Mannheim (Ger-
many). All sovents used were of analytical grade.
FIG. 1. (Left) Structure of 2-chlorosyringaldehyde. (Right) Structure of
2,6-dichlorosyringaldehyde.
of chlorine and chlorine dioxide bleach used. Thus, 2-chloro-
syringaldehyde (2-CSA) dominates in bleached hardwood
pulp effluent when chlorine dioxide bleaching is employed
and 2,6-dichlorosyringaldehyde (2,6-DCSA) dominates
when a high proportion of molecular chlorine is used (Smith
et al., 1993).
The Effluent
Simulated bleachery effluent was supplied by the CSIRO
Division of Forest Products Laboratories in Clayton, Victoria
Australia, immediately prior to the start of the experiment.
Mature Eucalyptus sp. woodchips were pulped using a kraft
process and the resulting pulp was oxygen delignified to
Kappa number 10.4.
The eucalypt kraft–oxygen pulp was bleached using a
four-stage sequence involving 100% chlorine dioxide
bleaching (Do), alkaline extraction with sodium hydroxide
(EO), followed by two 100% chlorine dioxide bleach treat-
ments (DD) [Do(EO)DD]. Effluents from each stage were
obtained by washing and hand squeezing the bleached pulp.
On completion of the final bleaching stage, all effluents were
combined to give a total volume of 5.0 liters per 100 g oven-
dried pulp. Stocks of effluent were stored in polyethylene
Little is known of the environmental fate and effects of
chlorinated syringaldehydes in aquatic systems. Stauber et
al. (1994) found that 2-CSA was far less acutely toxic to
Nitzschia closterium, an Australian marine diatom, than
more highly chlorine-substituted phenols and guaiacols. Fish
exposed to 2-CSA by intraperitoneal (ip) injection have not
been found to exhibit acute hepatotoxicity or increases in
hepatic detoxication enzymes (Brumley et al., 1996). Haritos
et al. (1995), in a study on 2-CSA metabolism, found that
the major metabolic fate of 2-CSA in fish was reduction to
the corresponding alcohol, 2-chloro-4-hydroxy-3,5-dime-
thoxybenzylalcohol (2-CB-OH), conjugation with endoge-
nous groups, and then excretion into the bile. The metabolic
fate of 2,6-DCSA is unknown, and aside from research by
Stauber et al. (1994) on the toxicity of this compound, 2,6-
DCSA has been largely unstudied. Nevertheless, metabolites
of chlorinated syringaldehydes in fish bile offer the potential
for monitoring hardwood effluents bleached by specific pro-
cesses. Thus, metabolites of 2-CSA in bile would indicate
exposure to chlorine dioxide-bleached hardwood effluent,
and metabolites of 2,6-DCSA would indicate chlorine-
bleached hardwood effluent exposure.
The aim of the present study was to investigate the use
of chlorinated syringaldehyde metabolites in fish bile as bio-
markers of exposure to bleached eucalypt pulp effluent. The
Australian marine fish sand flathead (Platycephalus bas-
sensis), which have been used previously as a bioindicator
organism (Holdway et al., 1994), were exposed to chlorine
dioxide-bleached eucalypt pulp effluent and, in separate ex-
periments, to ip-injected and waterborne 2,6-DCSA. The ip
injection of 2,6-DCSA into sand flathead allowed the major
metabolic products of the compound to be identified before
much lower concentrations were used in the waterborne ex-
posure. Metabolites of 2-CSA and 2,6-DCSA present in the
drums in the dark at 4
ЊC throughout the experiment.
2,6-DCSA Synthesis
Syringaldehyde (1 g, 5.49 mmol) was dissolved in 0.5 M
NaOH (15 ml), and 0.7 ml of acetic anhydride was added.
The solution was left to stir for several hours to form the
acetate derivative of syringaldehyde. Syringaldehyde acetate
was then filtered off, rinsed with water, and dried in air
overnight.
Syringaldehyde acetate (0.5 g, 2.23 mmol) was dissolved
in 20 ml of dichloromethane (DCM), and 3.6 ml of sulfuryl
chloride was added. The solution was left to stir for 48 hr
before being washed with 15 ml of MilliQ water and ex-
tracted with ethyl acetate (three times, 40 ml). The organic
extracts were combined, dried with Na2SO4 , and the solvent
was removed under vacuum. The resulting oil was stirred
with 20 ml of 1 M NaOH for 1 hr and then acidified with
3 M H2SO4 to
Ç
pH 5. 2,6-DCSA precipitated from the solu-
tion as a white powder that was recrystallized from glacial
acetic acid.
Fish
Sand flathead were caught by hook and line from Port
bile were described and quantified. The potential use of these Phillip Bay, Australia, in September, 1993 and December,

CHLOROSYRINGALDEHYDE METABOLITES IN FISH BILE
255
1994. The mean length (
were 293.8 2.1 mm and 147.2
144), and 20% of the fish were male.
{
SE) and weight (
{
SE) of the fish (7.96–8.06), temperature (18.5–20.5
Њ
C), dissolved oxygen
{
{
3.6 g, respectively (n
Å
(
ú
90%), and conductivity (55.1–55.7 mS) were measured
twice a day during the experiment and did not vary greatly
Fish were acclimated in the laboratory in flowthrough between tanks.
seawater tubs for at least 8 days prior to being placed in the
Sampling
80-liter glass exposure aquaria (six fish per tank). During
the acclimation period, fish were fed ad libitum on fresh
shrimp twice a week. Feeding ceased 24 hr prior to the start
of each exposure.
All fish were sampled 4 days after the start of exposure.
Each fish was anesthetized in a 70 mg/liter solution of
MS222, had a blood sample taken by cardiac puncture, and
was then killed by a blow to the head. The liver was removed
and immediately frozen in liquid nitrogen. Bile was aspirated
from the gall bladder and frozen in liquid nitrogen. The
blood sample was centrifuged and the resulting serum was
decanted and frozen in liquid nitrogen.
Chlorine Dioxide-Bleached Eucalypt
Pulp Effluent Exposure
Untreated chlorine dioxide-bleached eucalypt pulp efflu-
ent was diluted with seawater in 340-liter polyethylene tubs
to concentrations of 0.5, 2, and 8% (v/v). Each effluent/
seawater concentration was fed via a Watson–Marlow peri-
staltic pump to three aquaria, such that the total flow into
Bile Extraction and Analysis
The extraction and derivatization of chlorinated com-
pounds in the bile were based on the methods of Morales
et al. (1992) with modifications from Haritos et al. (1995).
The volume of each bile sample was measured with a glass
syringe before being diluted to
Each sample was acidified with one drop of 1 M H2SO4 ,
spiked with 0.5 g of the recovery standard, TBP, and then
each aquarium was 61
turnover in 15 hr (Sprague, 1969). Control fish received
seawater alone at the same rate. Each effluent/seawater batch
{
1.2 ml/min, giving a 50% molecular
was made up daily. Temperature (15–18
ЊC), pH (7.72–
Ç1 ml with MilliQ water.
8.02), dissolved oxygen ( 90%), and conductivity (51 mS
ú
in the 8% effluent aquaria; 54.2–55.9 mS in the others) were
measured twice a day during the experiment and did not
vary greatly over time.
m
extracted with 3:1 methyltert-butylether:DCM (MTBE:
DCM, three times, 2 ml). The organic fractions were com-
bined and evaporated to
nitrogen. The remaining aqueous phase was incubated with
-glucuronidase/aryl sulfatase (4000/32000 units) in 0.3 M
sodium acetate buffer (2 ml), pH 5 for 24 hr. With the
glucuronide and sulfate conjugate groups cleaved, each sam-
ple was then reextracted with 3:1 MTBE/DCM (three times,
2 ml), and the organic fractions were treated as described
previously. All of the organic fractions were acetylated by
Ç
1 ml under a gentle stream of
2,6-DCSA Exposure—Injection
b
2,6-DCSA was dissolved in dimethyl sulfoxide (DMSO)
and then diluted in corn oil to give a final concentration of
DMSO of 4% (v/v). Fish were anesthetized in a solution of
MS222 (70 mg/liter) and then received an ip injection of
2,6-DCSA at 15 mg/kg and 1 ml/kg. Control fish received
a 1 ml/kg ip injection of corn oil spiked with 4% (v/v)
DMSO.
the addition of 100
pyridine and heated at 70
then washed with 0.5 M HCl (0.5 ml), and 0.5
was added as an instrument internal standard. Fractions were
m
l of acetic anhydride and 50
C for 30 min. Each fraction was
g of TCN
ml of
Њ
m
2,6-DCSA Exposure—Waterborne
2,6-DCSA was dissolved in DMSO and then diluted with analysed by gas chromatography/mass spectrometry (GC/
seawater to concentrations of 0.013, 0.052, and 0.208 mg/ MS) using a Shimadzu QP2000 (Japan). Splitless injections
liter in 20-liter stock buckets, which were made up daily. of 1
Each of these solutions was fed via a Watson–Marlow peri- column coated with 0.25
staltic pump to a mixing chamber, which fed to one aquar- sis of fish bile from the chlorine dioxide-bleached effluent
ium. Seawater flowed into the mixing chamber via Cole– study involved a temperature program of 90 C, 1 min; 10 C/
Palmer flowmeters, such that the total flow into each aquar- min, 250 C; 6 min. The temperature program for the 2,6-
ium was approximately 520 ml/min. This flow rate resulted DCSA-exposed fish was 90 C 1 min; 15 C/min, 250 C; 6
in 99% molecular replacement in 12 hr (Sprague, 1969). min. The injector port, interface, and ion source for both
Control fish received seawater alone at the same rate. Nomi- studies were maintained at 250 C. Selected ion monitoring
nal concentrations of 2,6-DCSA in the aquaria were 0.5, 2, was used for quantitation of metabolites in samples. Mass
and 8 g/liter, which approximate to the concentrations of spectral ions monitored were 216/218 for 2-CSA; 260/201
m
l were made onto a 30 m
1
0.25-mm fused silica
mm DB-1 (J&W, USA). The analy-
Њ
Њ
Њ
Њ
Њ
Њ
Ç
Њ
m
2,6-DCSA that would be found in chlorine-bleached euca- for 2-CB-OH; 250/252 for 2,6-DCSA; 217/294 for 2,6-di-
lypt pulp effluent diluted to 0.5, 2, and 8% (v/v), respectively chloro-4-hydroxy-3,5-dimethoxybenzylalcohol (2,6-DCB-
(A. Wallis, personal communication). There were two tanks OH), the reduced product of 2,6-DCSA; 266 for TCN; and
for each concentration and control. Individual aquarium pH 330/332 for TBP. The underlined ion was used for quantita-

256
BRUMLEY ET AL.
tion because it was the base peak in the mass spectrum of 2,6-DCSA Injection
the compound; the second ion was used as a confirming
The major metabolite found in the bile of fish exposed to
ion. The concentrations of 2-CB-OH and 2,6-DCB-OH were
calculated from five-point calibration curves constructed
from prepared standards in the range of 10 ppb–1 ppm. Bile
samples in which the analyte concentration measured outside
the quantitation range were diluted in 3:1 MTBE:DCM and
reanalyzed.
2,6-DCSA by ip injection was the glucuronide or sulfate
conjugate of the reduced product, 2,6-DCB-OH. Conjugated
2,6-DCB-OH was detected in the bile of all fish exposed to
2,6-DCSA, with a mean level of 277.2
mg, whereas nothing
corresponding to this chemical was detected in the bile of
control-injected fish. Unconjugated 2,6-DCB-OH was also
present in the bile of exposed fish, at a mean level of 0.576
Effluent Extraction
m
g, which was approximately 500 times lower than that in
the conjugated fraction.
Untreated 100% chlorine dioxide-bleached eucalypt pulp
effluent (500 ml) was acidified to pH
Ç
2.5 with 3 M sulfuric
2,6-DCSA Waterborne
acid and extracted by shaking in a separating funnel with
3:1 MTBE:DCM (three times, 100–150 ml). The combined
organic fractions were washed with 100 ml of MilliQ water,
dried with Na2SO4 , and reduced by rotary evaporation to 10
Glucuronide- or sulfate-conjugated 2,6-DCB-OH was the
major metabolite found in the bile of fish exposed through
the water to 2,6-DCSA. This conjugated metabolite was de-
tected in the bile of all exposed fish (Table 2). The level of
conjugated 2,6-DCB-OH was significantly different between
ml. An aliquot (1 ml) was acetylated by adding 100
acetic anhydride and 50 l of pyridine and heating to 70
for 30 min. The extract was washed with 0.5 M HCl (0.5
ml), and TCN (0.5 g) was added as an instrument internal
ml of
m
ЊC
exposure groups (P
£
0.0001) and was linearly correlated
0.99; Fig. 3). Some 2,6-
with exposure concentration (r2
Å
m
standard. The acetylated effluent extract was analyzed by
GC/MS for the biliary metabolites.
DCB-OH was present in the bile as an unconjugated metabo-
lite but at levels approximately 10–20 times lower than
that of the conjugated metabolite (Table 2). The level of
unconjugated 2,6-DCB-OH was also significantly different
Statistical Analysis
across groups (P
£
0.0001). The amount of 2,6-DCB-OH
Data were assessed for homogeneity of variance using
Cochran’s c test. Heterogeneous data were log transformed.
Differences between groups were assessed by one- and two-
way analyses of variance (ANOVA) using the software pack-
age SuperANOVA. When significant differences were
found, Fisher’s protected LSD test was used to distinguish
the differences between the means.
per milliliter of bile was significantly different across all
groups and was concentrated in the bile over 20,000 times
more than 2,6-DCSA levels in the water (Table 2).
DISCUSSION
The major metabolic pathway of 2,6-DCSA (reductive
metabolism followed by conjugation) was found to be the
same as that which has been previously described for 2-CSA
(Haritos et al., 1995); however, phase I metabolism before
conjugation is not a common metabolic fate for chlorophe-
nolics. Chlorinated phenols, guaiacols, and catechols have
been demonstrated to be directly conjugated before excretion
RESULTS
Chlorine Dioxide-Bleached Eucalypt
Pulp Effluent Exposure
˚
The major metabolite found in the bile of effluent-exposed into the bile (Oikari and Ana¨s, 1985; So¨derstro¨m et al.,
fish was the glucuronide or sulfate conjugate of 2-CB-OH, 1994), although Glickman et al. (1977) found that penta-
the reduced product of 2-CSA. No unconjugated 2-CB-OH chloroanisole was demethylated to pentachlorophenol before
was present in the bile. Conjugated 2-CB-OH was found in conjugation and excretion. In the present study, the reduction
all fish exposed to diluted effluent and was concentrated in of the chlorinated syringaldehyde molecules occurred at the
the bile greater than 1,000 times over levels of 2-CSA in aldehyde functional group, which is also present in vanillins
the effluent (Table 1). Significant differences (P
in the amount of 2-CB-OH per milliliter of bile were found et al., 1980).
£
0.0001) but not in phenols, guaiacols, catechols, or syringols (Voss
between all groups of fish exposed to diluted effluent and
The high percentage of conjugated 2-CB-OH and 2,6-
the control group (Table 1). A significant linear relationship DCB-OH found in this study is in agreement with previous
(r2
Å
0.998; P
of effluent and 2-CB-OH content in the bile (Fig.2).
2-CSA was measured in the extracted effluent stock at a that over 95% of seven different chlorophenolics were pres-
concentration of 27.5 g/liter. The reduced derivative 2-CB- ent as conjugates in the bile of rainbow trout (Oncorhynchus
OH was present as a minor component at 0.062 g/liter. mykiss). Studies using 4,5,6-trichloroguaiacol have found the
£
0.001) existed between the concentration research on other chlorinated phenolics. Both Oikari and
˚
Ana¨s (1985) and Lindstro¨m-Seppa¨ and Oikari (1990) found
m
m

CHLOROSYRINGALDEHYDE METABOLITES IN FISH BILE
257
TABLE 1
Mean Concentration (n; SE) of Metabolites in the Bile of Sand Flathead after 4 Days of Exposure to 100% Chlorine Dioxide-
Bleached Eucalypt Pulp Effluent, Concentration of 2-CSA in the Effluent, and Calculated Bile Bioconcentration Factor
Effluent
concentration (%)
2-CB-OH in bile
in g (n; SE)
2-CB-OH in bile
in g/ml (n; SE)
2-CSA in effluent
in g/ml
Bile bioconcentration
factor
m
m
m
0
0.0000a
0.0000a
—
—
(17; 0.000)
(17; 0.000)
0.5
2
0.0330b
(17; 0.004)
0.2660b
(17; 0.054)
0.0001
0.0006
0.0022
1930
1400
1350
0.0945c
(17; 0.008)
0.7698c
(17; 0.110)
8
0.4427d
2.9815d
(18; 0.049)
(18; 0.258)
Note. Raw data are presented; statistical analyses were performed on log-transformed data. Results within columns without a superscript letter in
common are significantly different (P 0.05) based on Fisher’s LSD test. Bile bioconcentration factor represents the concentration of 2-CB-OH in the
£
bile over 2-CSA in the effluent; however, the metabolic ratio between 2-CSA and 2-CB-OH is expected to be 1:1. 2-CSA, 2-chlorosyringaldehyde;
2-CB-OH, 2-chloro-4-hydroxy-3,5-dimethoxybenzylalcohol.
degree of conjugation to range from 70 to 100% (Oikari et tion of their reduced metabolites in bile might be expected
al., 1988; Wachtmeister et al., 1991).
to be strong. However, such linearity should also hold in
The linear relationship between the concentration of chlo- field situations of sand flathead living within an effluent
rophenolics in fish bile and their concentration in receiving plume, because sand flathead are a relatively nonmigratory
waters, or distance from effluent outfall, is well documented marine fish (Dix and Martin, 1975).
(Landner et al., 1994; Oikari and Kunnamo-Ojala, 1987;
The degree of bioconcentration of a xenobiotic in fish
Oikari, 1986; So¨derstro¨m et al., 1994). Indeed, Landner et tissue is strongly inversely correlated with its rate of clear-
al. (1994) found that the analysis of conjugated chlorophe- ance. This rate can be estimated from the log Pow value of
nolics in fish bile was the only method of analysis out of the xenobiotic. Chlorophenolic compounds are weak acids
several examined (including EOCl and chlorophenolic con- and have log Pow values in the range of 3–5 (Xie et al.,
tent of sediment and suspended solids) that clearly estab- 1984). Several studies have revealed that chlorophenolics
lished a gradient of bleached kraft mill effluent compounds are not highly bioconcentrated in fish tissues, with bio-
with distance from outfall. The present study was laboratory concentration factors in the range of 5–1600 (Niimi et al.,
based, and a linear relationship between the concentration 1990; Paasivirta et al., 1985; Rogers et al., 1988). Con-
of chlorinated syringaldehydes in water and the concentra- versely, bile bioconcentration factors for fish exposed in
the laboratory to chlorinated quaiacols and phenols range
˚
between 27,000 and 260,000 (Oikari and Ana¨s, 1985; Oikari
et al., 1988; Wachtmeister et al., 1991), and field research
on bleached kraft mill effluents has found that most highly
substituted guaiacols, catechols, and phenols have bile bio-
concentration factors in the range of 10,000–100,000 (Oikari
and Holmbom, 1986; Oikari and Kunnamo-Ojala, 1987;
So¨derstro¨m et al., 1994). The high bioconcentrations factors
for bile, compared with those for muscle, indicate that fish
bile may be a more sensitive measure of low-level chloro-
phenolic exposure than tissue measurement. Indeed, the high
bile bioconcentration factors found in this study infer that
detectable levels of chlorosyringaldehyde metabolites would
be present in the bile at exposure concentrations as low as
5 ng/liter. However, the process of concentration in the bile
is not only dependent on the exposure concentration, but also
on the metabolic capability of the liver to produce conjugated
metabolites.
FIG. 2. Relationship between the amount (mean
2-chloro-4-hydroxy-3,5-dimethoxybenzylalcohol (2-CB-OH) in the bile of
sand flathead and dilutions of 100% chlorine dioxide-bleached eucalypt
{
SE) of the metabolite
effluent after 4 days of exposure. The equation of the line is y
Å
0.055x
/
0.003; r2
Å
0.99; n 4; P 0.001.
Å
£

258
BRUMLEY ET AL.
TABLE 2
Mean Concentration (n; SE) of Metabolites in the Bile of Sand Flathead after 4 Days of Exposure to 2,6-DCSA,
Concentration of 2,6-DCSA in the Water, and Calculated Bile Bioconcentration Factor
2,6-DCSA
exposure
concentration
Unconjugated 2,6-DCB-OH
in g (n; SE)
Conjugated 2,6-DCB-OH
in g (n; SE)
Total 2,6-DCB-OH
in g/ml (n; SE)
Bile bioconcentration
factor
(
m
g/liter)
m
m
m
0
0.000a
0.000a
0.000a
—
(11; 0.000)
(11; 0.000)
(11; 0.000)
0.5
2
0.214b
(12; 0.133)
2.158b
(12; 0.338)
13.481b
(12; 1.472)
26,960
20,070
23,100
0.496b
(12; 0.391)
5.419c
(12; 0.717)
40.146c
(12; 5.073)
8
1.481c
32.832d
184.762d
(12; 0.723)
(12; 9.338)
(12; 33.477)
Note. Raw data are presented; statistical analyses were performed on log-transformed data. Results within columns without a superscript letter in
common are significantly different (P 0.05) based on Fisher’s LSD test. Bile bioconcentration factor represents the concentration of 2,6-DCB-OH in
£
the bile over 2,6-DCSA exposure concentration; however, the metabolic ratio between 2,6-DCSA and 2,6-DCB-OH is expected to be 1:1. 2,6-DCSA,
2,6-dichlorosyringaldehyde; 2,6-DCB-OH, 2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol.
The degree of bioconcentration of a xenobiotic is also Some of this difference may be attributed to the different
correlated with its rate of uptake (Pa¨rt, 1989). For ionizable exposure media: fish were exposed to 2-CSA in effluent,
compounds, such as chlorophenolics, this rate depends on and a proportion of the 2-CSA may have adsorbed onto
the dissociation constant (pka) of the xenobiotic and its log particulate matter in the effluent and partitioned out of solu-
P
ow value. To the authors’ knowledge, pka and log Pow values tion. Perhaps more important, there are essential metabolic
for chlorosyringaldehydes have not been described; how- processes that occur between uptake from the water and
ever, the pka of 2-CSA and 2,6-DCSA may be calculated excretion that may determine the relative concentration of
using the Hammett equation (Perrin et al., 1981) as 6.80 2,6-DCSA and 2-CSA metabolites in the bile.
and 5.97, respectively. These values would indicate that in
The concentrations of 2-CSA and 2,6-DCSA used in this
seawater, in which the pH is 8.0–8.3, more 2,6-DCSA would study were based on the concentrations of these compounds
be in an ionized form than would 2-CSA, which could limit found in dilutions of untreated effluents. Chlorophenolic
uptake. In the present study, however, more metabolites of compounds have been demonstrated to be fairly resistant to
2,6-DCSA were found in the bile than 2-CSA metabolites. biological treatment (Holmbom and Lehtinen, 1980); how-
ever, depending on the type of treatment, the degree of deg-
radation can range from 0 to 95% (Gergov et al., 1988;
Kringstad et al., 1984; Lindstro¨m and Mohamed, 1988; for
review see McLeay, 1987). Chlorophenolics have also been
found to be fairly persistent in receiving waters (Oikari and
Kunnamo-Ojala, 1987; Xie et al., 1986). Because chlori-
nated syringaldehydes are rarely monitored in receiving wa-
ters, little is known of their concentration and persistency.
Morales et al. (1992) found 2,6-DCSA and 2-CSA present
in pulp mill receiving waters at concentrations up to 48 and
4.1
m
g/liter respectively, and Robinson et al., (1994) reported
g/liter for 2,6-DCSA downstream of a mill
a value of 0.006
m
employing full secondary treatment. In untreated bleached
eucalypt effluent, the levels of 2-CSA and 2,6-DCSA are in
the range of 25–100 mg/liter (Charlet and Claudio-Da-Silva,
1994; Stauber et al., 1994). A laboratory-based anaerobic
and aerobic treatment of chlorine dioxide-bleached eucalypt
pulp effluent found that 2-CSA levels fell about 66% after
FIG. 3. Relationship between the amount (mean
2,6-dichloro-4-hydroxy-3,5-dimethoxybenzylalcohol (2,6-DCB-OH) in the
bile of sand flathead and exposure concentration of 2,6-dichlorosyringalde-
{
SE) of the metabolite
hyde (2,6-DCSA) after 4 days of exposure. The equation of the line is y
Å
4.141x 0.99, n 4; P 0.001.
0
0.769; r2
Å
Å
£

CHLOROSYRINGALDEHYDE METABOLITES IN FISH BILE
259
of regional and large-scale biological effects caused by bleached pulp
mill effluents. Chemosphere 27, 631–650.
treatment to 30 mg/liter (Stauber et al., 1994). Research on
the effectiveness of aerated lagoons for removing chlorophe-
nolic compounds from bleached eucalypt effluent found that
Brumley, C. M., Haritos, V. S., Ahokas, J. T., and Holdway, D. A. (1996).
Evaluation of biomarkers of exposure to 2-chlorosyringaldehyde and sim-
ulated ECF bleached eucalypt pulp effluent. In Environmental Fate and
Effects of Pulp and Paper Mill Effluents (M. R. Servos, K. R. Munkittrick,
J. H. Carey, and G. J. Van Der Kraak, Eds.), pp 369–378. St. Lucie
Press, Boca Raton, FL.
2-CSA levels fell about 95% to 1
mg/liter after treatment
(Charlet and Claudio-Da-Silva, 1994). In the present study,
metabolites in the bile could easily be detected after 4 days
of exposure at nominal water concentrations of 0.1 mg/liter
for 2-CSA and 0.5
m
g/liter for 2,6-DCSA. Both of these Charlet, P., and Claudio-Da-Silva, E. Jr. (1994). Study of the effectiveness
of aerated lagoons for removal of conventional pollutants, chlorinated
values are well below the concentrations of chlorinated sy-
ringaldehydes found in bleached eucalypt pulp effluents after
laboratory- and field-based treatments. Chlorosyringalde-
hyde metabolites in fish bile could therefore be applied in
the field to monitor exposure to treated effluents.
phenolic compounds and acute toxicity. In Proceedings, 2nd Interna-
tional Conference on Environmental Fate and Effects of Bleached Pulp
Mill Effluents, p, 43. Vancouver, Canada.
de Bethizy, J. D., and Hayes, J. R. (1989). Metabolism: A determinant of
toxicity. In Principles and Methods of Toxicology (A. W. Hayes, Ed.),
pp. 29–58. Raven Press, New York.
CONCLUSION
Dix, T. G., and Martin, A. (1975). Sand flathead (Platycephalus bassensis),
an indicator species for mercury pollution in Tasmanian waters. Mar.
Pollut. Bull. 6, 142–143.
Although some studies have found a correlation between
the concentration of chlorophenolics in fish bile and deleteri-
ous physiological effects (Balk et al., 1993; Oikari et al.,
1985), no causal relationship has been established and may
never be. Nevertheless, the measurement of the concentra-
tion of chlorophenolic compounds in fish bile can provide
an estimate of internal dose and allow low or fluctuating
levels of chlorophenolics to be monitored. In addition, al-
though chlorophenolic compounds in bile can be traced to
pulp mill effluents, chlorinated syringaldehyde metabolites
can be specifically attributed to chlorine- or chlorine dioxide-
bleached hardwood effluents. Integrated biomarkers, such
as biotransformation enzymes, have not been responsive to
chlorine dioxide-bleached eucalypt effluents (Brumley et al.,
1996), 2-CSA exposure (Brumley et al., 1996), or 2,6-DCSA
exposure (C. M. Brumley, unpublished results), and are
therefore not likely to be useful for monitoring bleached
eucalypt effluents. The use of chlorinated syringaldehyde
metabolites in fish bile, along with biochemical, physiologi-
cal, or markers of effect, provides a sensitive tool for moni-
toring exposure to bleached hardwood or mixed wood efflu-
ents.
Gergov, M., Priha, M., Talka, E., and Valttila, O. (1988). Chlorinated
organic compounds in effluent treatment at kraft mills. Tappi 71, 175–
184.
Glickman, A. H., Statham, C. N., Wu, A., and Lech, J. J. (1977). Studies
on the uptake, metabolism and disposition of pentachlorophenol and
pentachloroanisole in rainbow trout. Toxicol. Appl. Pharmacol. 41, 649–
658.
Haritos, V. S., Brumley, C. M., Holdway, D. A., and Ahokas, J. T. (1995).
Metabolites of 2-chlorosyringaldehyde in fish bile: Indicator of exposure
to bleached hardwood effluent. Xenobiotica, 25, 963–971.
Holdway, D. A., Brennan, S. E., and Ahokas, J. T. (1994). Use of hepatic
MFO and blood enzyme biomarkers in sand flathead (Platycephalus bas-
sensis) as indicators of pollution in Port Phillip Bay, Australia. Mar.
Pollut. Bull. 28, 683–695.
Holmbom, B., and Lehtinen, K.-J. (1980). Acute toxicity to fish of kraft
pulp mill waste waters. Paperi ja Puu 11, 673–684.
Kasper, C. B., and Henton, D. (1980). Glucuronidation. In Enzymatic Basis
of Detoxication (W. B. Jakoby, Ed.), pp. 3–36. Academic Press, New
York.
Kringstad, K. P., and Lindstro¨m, K. (1984). Spent liquors from pulp bleach-
ing. Environ. Sci. Technol. 18, 236A–248A.
Kringstad, K. P., Stockman, L. G., and Stro¨mberg, L. M. (1984). The nature
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state of knowledge. J. Wood Chem. Technol. 4, 389–404.
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ACKNOWLEDGMENTS
mill site on the Baltic Sea Coast. Ecotoxicol. Environ. Saf. 27, 128–157.
This work was supported by the National Pulp Mills Research Program
(Phase 2) and the Australian Research Council. The authors thank Rod
Watson and Sue Brennan for their technical assistance. C.M.B. is the recipi-
ent of an Australian Postgraduate Award. Parts of this research were pre-
sented at the 15th Annual SETAC meeting, Denver, CO, October 30–
November 3, 1994.
Lech, J. J., Pepple, S. K., and Statham, C. N. (1973). Fish bile analysis: A
possible aid in monitoring water quality. Toxicol. Appl. Pharmacol. 25,
430–434.
Lindstro¨m, K., and Mohamed, M. (1988). Selective removal of chlorinated
organics from kraft mill total effluents in aerated lagoons. Nordic Pulp
Paper Res. J. 1, 26–33.
Lindstro¨m-Seppa¨, P., and Oikari, A. (1990). Biotransformation and other
toxicological and physiological responses in rainbow trout (Salmo gaird-
neri Richardson) caged in a lake receiving effluents of pulp and paper
industry. Aquat. Toxicol.16, 187–204.
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