Chemosphere 61 (2005) 323–331

www.elsevier.com/locate/chemosphere

Levels of PCDDs and PCDFs in Korean river sedimentsand their detection by biomarkers

Keon Sang Ryoo a,*, Seong-Oon Ko a, Yong Pyo Hong a, Jong-Ha Choi a,Sunghwan Cho b, Yonggyun Kim b, Yeon Jae Bae c

a Department of Applied Chemistry, Andong National University, Andong 760-749, Koreab School of Bioresource Sciences, Andong National University, Andong 760-749, Korea

c Department of Biology, Seoul Women�s University, Seoul 139-774, Korea

Received 11 October 2004; received in revised form 3 February 2005; accepted 24 February 2005

Available online 21 April 2005

Abstract

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are a group of toxic halogenated aryl

hydrocarbons inducing various physiological disorders against biological organisms. Here, we investigated their levels

in sediment samples taken from 12 different rivers in Korea. The levels of PCDD/PCDFs in sediment samples were

expressed as concentrations and international TEQ values. Among 17 PCDD/PCDFs selected as target compounds

in this study, the 1,2,3,4,7,8-HxCDD and OCDD were found in all river sediments with significant variation in various

congener profiles of PCDD/PCDFs in sediments. PCDD/PCDFs could be monitored by sensitive biomarkers using

insect immune system. Out of 12 river sediment samples, the biomarkers reported four spots (up, middle, and down

Singil sites and Ansan) as putative contamination areas. When comparing both chemical and biological monitoring

results, two methods agreed three spots of Singil as contamination areas (above 10 ppt levels) as well as six river sed-

iment samples as relatively less-contaminated areas, but differed in the results in Ansan and Miho, probably due to re-

lative non-specificity of biomarkers. Despite some disparity between bio- and chemical monitoring results, the

biomarkers can be recommended as a device warning the contamination of dioxins in the environment because of a

fast and inexpensive detection method.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: PCDDs; PCDFs; Biomarker; Spodoptera exigua; Sediment; River; Korea

1. Introduction

The presence of polychlorinated dibenzo-p-dioxins

(PCDDs) and polychlorinated dibenzofurans (PCDFs)

0045-6535/$ - see front matter � 2005 Elsevier Ltd. All rights reserv

doi:10.1016/j.chemosphere.2005.02.093

* Corresponding author. Tel.: +82 54 820 5453; fax: +82 54

823 1628.

E-mail address: ksr@andong.ac.kr (K.S. Ryoo).

in the environment is strongly related with a number of

sources including waste combustion and metal industry,

as well as their formation in the manufacture of various

chlorinated chemicals (Safe, 1990; Alcock and Jones,

1996; Fiedler, 1999). Due to low water solubility and vol-

atility of PCDDs/PCDFs, they undergo so long-range

atmospheric transport and can be deposited into aquatic

systems, especially the sediments as a sink, where they act

as a long-term source of release of these pollutants into

the aquatic food chain (Gobas and Mackay, 1987;

ed.

324 K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331

Connolly and Pedersen, 1988; Thomann, 1989). More-

over, their chemical stability affords significant biomag-

nification through a food chain (Kang et al., 2002).

These chemicals are a group of highly toxic, extre-

mely anthropogenic compounds that give a potential

health hazard to humans and other organisms. In verte-

brates, PCDDs/PCDFs cause a broad spectrum of phys-

iological disorders including weight loss, reproductive

failure, thymic atrophy, hepatotoxicity, several types of

dermal lesion, and immunotoxicity (Safe, 1990; Mocar-

elli et al., 1996; Luebke et al., 2001). The molecular

mechanism of the toxicities has been illustrated with a

TCDD model binding to aryl hydrocarbon (Ah) recep-

tor via induction of 7-ethoxyresorufin-O-deethylase

(EROD). However, though invertebrates have their

own Ah receptors, the TCDD toxicity is limited proba-

bly due to lack of specific binding of the receptors to the

toxic ligand (Butler et al., 2001). The resistance of the

aquatic invertebrates against PCDDs/PCDFs allows

them to accumulate relatively high concentrations,

which may be transferred to sensitive vertebrates via

food webs (West et al., 1997).

To assess the environmental quality, many countries

have monitored the level of PCDDs/PCDFs in various

environmental samples including soil and sediment. In

Korea, the reported levels of PCDDs/PCDFs have been

so limited to certain environmental samples such as ambi-

ent air, food, and incinerator ash (Kim et al., 2001a,b; Im

et al., 2004). Few studies concerning the PCDDs/PCDFs

levels in river sediments have been reported in Korea. In

this paper, we report and discuss the data on the

PCDD/PCDF levels found in sediment samples collected

at 12 different river sites. It also analyzed the toxic effects

of the dioxin residues on insect immune system,whichwas

used for developing sensitive biomarkers.

2. Materials and methods

2.1. Reagents

Two standard solutions containing 17 unlabeled

PCDD/PCDF compounds and 18 isotopically labeled13C12- and 37Cl4-PCDD/PCDF compounds in nonane

were supplied by Cambridge Isotope Laboratories, Inc.

(Andover, MA, USA). All solvents (hexane, dichloro-

methane, acetone, iso-octane, toluene) used in this study

were HPLC grade (J.T. Baker, Deventer, Netherlands).

Anhydrous sodium sulfate (Merck, Darmstadt, Ger-

many) was used after heating overnight at 120 �C. Silicagel and alumina (Aldrich, WI, USA), before use, were

Soxhlet extracted with dichloromethane and then acti-

vated in a foil covered glass container at 150 �C for

24 h. Concentrated sulfuric acid 95–97% (Merck) were

analytical grade reagent.

2.2. Sampling

Field collection was carried out at 12 Korean rivers

(Fig. 1) in October 2003, representing heavy industrial

area (R1–R3), moderately industrial area (R4–R7), near

capital city (R8–R11), and a well reserved reference area

(R12). R1–R3 are the sampling spots from upstream to

downstream near multi-chemical factories and were ex-

pected to accumulate toxic compounds gradually from

R1 to R3. R4–R11 are the places mixed with some fac-

tories and civilian habitats, in which R4–R7 comprised

relatively more factories, but R8–R11 did more civilian

habitats near Seoul with an increasing polluted gradi-

ent from R8 to R11. R12 is located far from chemical

factories and civilian habitats so that it seems to be

hardly contaminated. The upper 0–20 cm layer of sed-

iment was sampled, and then immediately homogenized

and sieved (sieve fraction: �2 mm). In laboratory, all

samples were air-dried at 40 �C for 24 h, ground in

an agitate ball mill. The samples were kept under the

refrigeration at 4 �C until their analysis. The prepared

samples were used for both chemical residual analysis

and biomarker analysis. The PCDD/PCDFs analysis

in samples was performed using the isotope dilution

technique, designated by EPA Method 1613 (high res-

olution GC/MS method for the determination of tet-

ra-octa chlorinated dioxins and furans). About 10 g

sample was weighed and spiked with isotopically la-

beled 37Cl4-2,3,7,8-TCDD as an internal standard be-

fore extraction. Along with each series of samples, a

complete procedure blank was also performed to check

the purity of solvents, reagents, material, etc. The

blank was spiked with an internal standard mentioned

above and analyzed exactly in the same way as the

samples.

2.3. Extraction and clean-up

Extraction was carried out by soxhlet for 24 h with

toluene. After extraction, a total of 15 13C12-labelled

PCDD/PCDF congeners, shown in Table 1, as internal

standards were added to the extract. The extract was

placed in a multi-layer silica gel column including

anhydrous Na2SO4, neutral silica gel, acid modified

silica gel, and NaOH impregnated silica gel, and then

eluted with 100 ml of n-hexane. Afterwards, the extract

was purified in a basic alumina column, by eluting with

100 ml of hexane, followed by 30 ml hexane/dichloro-

methane (1:1 v/v). A hexane/dichloromethane fraction

was fractioned with an activated carbon column con-

taining Celite and then concentrated in a Kudena-

Danish apparatus under a gentle stream of nitrogen

to 1 ml. To test the recovery, the syringe standards

such as 13C12-1,2,3,4-TCDD and 13C12-1,2,3,7,8,9-

HxCDD were added to the final concentrate before

GC/MS analysis.

Fig. 1. Map of sediment sampling locations. R1: Singil-UP, R2: Singil-MD, R3: Singil-DN, R4: Ansan, R5: Balan, R6: Miho-UP, R7:

Miho-DN, R8: Gayung, R9: Wangsuk-UP, R10: Wangsuk-DN, R11: Jungryang, and R12: Gilan.

Table 1

Isotopically labeled 13C12- and37C14-internal standards used in this study

Isotopically labeled standard (ng) Number

PCDDs PCDFs

Pre-sampling Before extraction 37Cl4-2,3,7,8-TCDD 0.1 – – 1

Pre-extraction Before clean-up 13C12-2,3,7,8-TCDD 1 13C12-2,3,7,8-TCDF 1 1513C12-1,2,3,7,8-PeCDD 1 13C12-1,2,3,7,8-PeCDF 113C12-1,2,3,4,7,8-HxCDD 1 13C12-2,3,4,7,8-PeCDF 113C12-1,2,3,4,6,7,8-HpCDD 1 13C12-1,2,3,4,7,8-HxCDF 113C12-1,2,3,6,7,8-HxCDD 1 13C12-1,2,3,6,7,8-HxCDF 113C12-OCDD 2 13C12-1,2,3,7,8,9-HxCDF 1

13C12-2,3,4,6,7,8-HxCDF 113C12-1,2,3,4,6,7,8-HpCDF 113C12-1,2,3,4,7,8,9-HpCDF 1

Recovery Before analysis 13C12-1,2,3,4-TCDD 1 – 213C12-1,2,3,7,8,9-HxCDD 1

Sum 9 9 18

K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331 325

2.4. GC/MS analysis

The samples were analyzed by HRGC–HRMS in a

HP 6890 gas chromatograph (Hewlett-Packard, Wil-

mington, USA) coupled a JMS-700D (Jeol, Japan) mass

spectrometer, operating in EI ionization (32 eV) at

resolving power higher than 10000. The samples were

separated in a DB-5MS (60 m · 0.232 mm, 0.25 lm)

capillary column (J&W Scientific, Folsom, USA). The

temperature program for DB-5MS was as follows: on-

column injection of 1 ll of sample at 240 �C, initial oventemperature of 160 �C for 1 min, then increased at

326 K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331

20 �C/min to 200 �C for 2 min, then increased at 5 �C/min to 220 �C for 15 min, then increased at 5.0 �C to

235 �C for 5 min, and finally at 3 �C to 310 �C for

3 min hold-up. Helium was used as the carrier gas.

The results were expressed as concentration in pg/g

and in pg TEQ/g in order to evaluate the toxicity of sam-

ple due to dioxins. To calculate TEQ, the international

toxicity equivalent factors (ITEF) were used (Van Zorge

et al., 1989).

2.5. Biomarkers detecting dioxins

Insect immune responses were modified as different

bioassays, which were used to monitor dioxin residues.

The fifth instar larvae of the beet armyworm, Spodoptera

exigua, was used as a test insect and reared on an artifi-

cial diet of Gho et al. (1990).

For hemocyte nodulation assay by the method of

Park and Kim (2000), 105 cells of the heat-killed

(10 min at 95 �C) bacteria, Escherichia coli, along with

10 ll test solution were injected into the larval hemocoel

by 10 ll microsyringe (Hamilton, Nevada) and then

incubated for 12 h at 25 �C. After the treated larvae were

dissected by opening hemoceol, melanized and dark

nodules were counted under a stereomicroscope at 50·magnification. Even though their sizes were varied

(100–800 lm at diameter), the nodules were distinct

enough to find out their locations.

For an assay of activation of prophenoloxidase

(pPO), PO activity was measured by the method of Park

and Kim (2003). Hemolymph (10 ll) of the fifth instar

larvae was pre-incubated with 2 ll of a test solution at

25 �C for 10 min, and then added by 988 ll of 50 mM

phosphate saline containing 1 lg of laminarin and

0.01 mM L-3,4-dihydroxyphenylalanine (Sigma, St.

Louis, MO). The increased absorbance was measured

at 495 nm with 5 min interval. PO activity was expressed

as absorbance change per 5 min.

For phospholipase A2 (PLA2) assay, an indirect assay

was devised by Park and Kim (2003). Briefly, two identi-

cal hemolymph samples (�A� and �B�) were prepared. �A�sample included 10 ll of hemolymph, 1 ll of arachidonicacid (5,8,11,14-eicosatetraenoic acid, AA, Sigma) stock,

and 1 ll of a test PLA2 inhibitor. �B� sample included

10 ll of hemolymph, 1 ll of 50% ethanol, and 1 ll ofthe test PLA2 inhibitor. To these 12 ll reaction mixtures,

after 10 min, 988 ll of PO substrate solution was added.

PO activity was then measured by the method just des-

cribed. Comparing these three PO activities, PLA2

inhibitor was recognized in the fraction that the reduced

PO activity in the �B� sample was significantly elevated by

the addition of AA as in the �A� sample.

Apoptosis assay of insect immunocompetent cells

(hemocytes) followed the method of Cho and Kim

(2004). Briefly, the larval hemolymph was collected

and centrifuged at 200g for 10 min at 4 �C. After resus-

pending the precipitated hemocytes with Grace�s insect

cell culture medium (Sigma), 10 ll of hemocyte suspen-

sion (containing over 100 cells) was loaded on the glass

coverslip and incubated in a wet chamber at 25 �C for

30 min. The supernatant was replaced with 10 ll of testsolution and further incubated for 2 h. Apoptotic cells

were recognized by membrane blebbings or apoptotic

vesicles and counted under a phase contrast microscope

at 200· magnification.

3. Results

3.1. Levels of PCDD/PCDFs in the river sediment

The concentrations of PCDD/PCDFs in each sedi-

ment sample collected from 12 river sites, designated by

R1–R12, are summarized in Table 2, in which toxic equiv-

alent factors (TEQs) levels are also presented after con-

version to the international 2,3,7,8-TetraCDD toxicity

equivalents (ITEQ). Total concentration and TEQ level

of PCDD/PCDFs at each sampling site ranged from

0.88 to 369.21 pg/g and from 0.07 to 23.93 pg TEQ/g

dry weight sediment, respectively. There was a high cor-

relation (r = 0.9995; P < 0.0001) between total concen-

tration and TEQ values of PCDD/PCDFs. The

1,2,3,4,7,8-HxCDD and OCDD were found in all sedi-

ment samples, though OCDF was the most heavily accu-

mulated when the detection levels of all sediment samples

were combined. The OCDFwas the secondmost popular

congener, with the exception of the R4 and R12. Except

extreme level of R3, relative contamination levels can

be divided by a threshold level of 10 pg/g of total

PCDD/PCDFs concentration into the contaminated

(R1, R2, R3, R6, and R7) and the relatively less-contami-

nated (R4, R5, R8, R9, R10, R11, and R12) sediments.

3.2. Biomonitoring PCDD/PCDFs

The effect of PCDD/PCDFs on insect immune system

was analyzed using TCDD as a representative com-

pound and S. exigua as a model insect. When TCDD

was injected into the larvae, it inhibited the hemocyte

(insect immunocyte) capacity of nodule formation

against the bacteria in a dose-dependent manner (Fig.

2A). Only 1 pg was enough to significantly inhibit the

nodule formation. Nodule formation of hemocytes is

mediated by the activation of prophenoloxidase (Dula-

ray and Lackie, 1985). When the activated phenoloxi-

dase activity was measured after incubation with

different doses of TCDD, 0.1 ppb of TCDD exhibited

significant inhibition in in vitro hemolymph assay (Fig.

2B). Considering a positive or functional correlation

between PLA2 activity and phenoloxidase activity in

S. exigua (Park and Kim, 2003), PLA2 can be targeted

by TCDD. The depressed phenoloxidase activity in the

Table 2

Total concentrations of PCDD/PCDFs and their TEQ levels in 12 sediment samples (R1–R12, see details in Fig. 1) in Korea

Congener Congener concentration (pg/g) of each environmental sediment samples

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

2,3,7,8-TCDD –a – 0.91 – 0.07 – – – – – – –

1,2,3,7,8-PeCDD – – 3.46 – – – – – – – – –

1,2,3,4,7,8-HxCDD 0.72 0.41 3.66 0.55 0.90 0.66 0.64 0.60 0.70 0.77 1.00 0.66

1,2,3,6,7,8-HxCDD – – 4.15 – – 0.08 – – – – – –

1,2,3,7,8,9-HxCDD – – 3.51 – – – – – – – – –

1,2,3,4,6,7,8-HpCDD 0.81 0.87 21.98 – 0.35 0.95 1.53 0.19 0.00 0.18 0.31 0.00

OCDD 5.92 3.56 50.48 0.89 1.88 7.82 5.58 0.80 1.21 0.84 2.39 0.22

2,3,7,8-TCDF 1.42 1.35 8.68 – 1.29 0.53 – 1.56 – 1.39 – –

1,2,3,7,8-PeCDF – 0.47 12.58 – 0.10 0.33 – – – – – –

2,3,4,7,8-PeCDF – – 20.65 – 0.09 0.32 0.67 – – – – –

1,2,3,4,7,8-HxCDF – 0.46 19.60 – 0.09 0.40 0.48 – – – 0.07 –

1,2,3,6,7,8-HxCDF – 0.23 19.94 – – 0.40 0.56 – – – – –

1,2,3,7,8,9-HxCDF – – 9.58 – – – – – – – – –

2,3,4,6,7,8-HxCDF 0.67 0.27 24.52 – 0.15 0.80 0.82 – – – – –

1,2,3,4,6,7,8-HpCDF 2.07 0.87 50.96 0.31 0.19 1.39 2.38 0.14 0.30 – – –

1,2,3,4,7,8,9-HpCDF 0.60 – 9.13 – – – 0.48 – 0.39 – – –

OCDF 2.53 1.51 105.42 – 0.36 2.34 3.29 0.62 1.12 0.26 0.46 –

Total (pg/g, ww) 15.92 10.00 369.21 1.75 5.47 16.02 16.43 3.91 3.72 3.44 4.23 0.88

TEQ (pg/g, ww) 0.44 0.32 23.93 0.06 0.37 0.50 0.64 0.24 0.08 0.22 0.11 0.07

a Below detection limit.

K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331 327

presence of TCDD was recovered by addition of arachi-

donic acid, a catalytic product of PLA2 (Fig. 2C). How-

ever, the depressed phenoloxidase activity by exposure

to PCBs, which can co-exist with PCDD/PCDFs and

be extracted together in environmental samples, was

not recovered by addition of arachidonic acid (data

not shown). Finally, TCDD could induce apoptosis of

the hemocytes in a dose-dependent manner (Fig. 2D).

Four susceptible biomarkers to TCDD were applied

to monitor the presence of PCDD/PCDFs in the sedi-

ment samples of 12 Korean rivers. Only four sediment

samples of R1–R4 significantly inhibited all four immune

endpoints (Fig. 3). These positive responses suggest that

the environment samples were exposed to dioxins. These

biomarker results also suggest the contamination levels,

based on the sensitivities of different biomarkers. Posi-

tive responses of R1–R4 samples in nodulation assay

indicate that these locations may be exposed to upper

ppt level of dioxin. This estimation is well supported

by both PO activity and apoptosis assays. Also, this im-

mune inhibitory action was recovered by adding arachi-

donic acid in all positive samples. This supports the

immunodepression was induced by inhibiting PLA2,

which is modulated by dioxins, not by PCBs among

major organic persistent pollutants in environment.

4. Discussion

This study reports the levels of PCDD/PCDFs in

Korean river sediments collected from 12 different loca-

tions. In no known similar reports in Korea, the total

PCDD/PCDFs levels of these Korean samples were

much lower than the levels (range of 121–814 pg/g dry

weight basis) in river sediments of the most populated

and industrialized PO city in Italy (Fattore et al.,

2002). However, the levels were varied among different

localities probably with positively correlation with

anthropogenic activities around the sample sites. The

lowest exposure levels were recorded in the sample col-

lected in the R12 (Gilan), which is known as the least

contaminated river that has not been so far affected by

direct urbanization and industrial activities. In R12, only

two congener profiles such as 1,2,3,4,7,8-HxCDD and

OCDD were detected. A moderate level was found in

R8 (Gapyung), which was relatively uncontaminated

river surrounded by mountains like R12, showing that

its natural environment has been well preserved, even

under quite often human activities near the river because

of its vicinity from a capital city, Seoul. Three rivers near

Seoul such as R9 (Wangsuk-UP), R10 (Wangsukchun-

DN), and R11 (Jungryang) had another moderate levels

of the total PCDD/PCDFs concentrations in sediments.

The substantially increased concentration of PCDD/

PCDFs and TEQ levels were observed in the sediment

samples taken at the R1 (Singil-UP), R2 (Singil-MD),

and R3 (Singil-DN). The total concentration and TEQ le-

vel of PCDD/PCDFs in each sediment increased in an

order of R1, R2, and R3, where the total concentration

of R3 was about 23–37 times higher than those of R1

and R2, and total TEQ level was 54–75 times higher. A

marked increase of total PCDD/PCDFs concentrations

TCDD (ppb)0 0.01 0.1 1 10

PO a

ctiv

ity

(Abs

at 4

05 n

m/5

min

)

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06a

a

b

c

d

TCDD (pg/larva)0 0.1 1 10 100

Num

ber o

f nod

ules

0

5

10

15

20

25

aa

b

cc

TCDD (ppb)0.1 1 10

PO a

ctiv

ity(a

bs a

t 405

nm

/5 m

in)

0.00

0.02

0.04

0.0650% ethanol10ng AA

* * *

(A)

(D)

(B)

(C)

TCDD (ppb)0 0.1 1 10 100

Apop

tosi

s (%

)

0

20

40

60

80

100

aa

b

cc

Fig. 2. Effect of 2,3,7,8-tetrachlorodobenzo-p-dioxin (TCDD) on insect immune systems of Spodoptera exigua. All experiments used

the hemolymph of fifth instar larvae of S. exigua. (A) In vivo hemocyte nodule formation in response to injection of 105 cells of

Escherichia coli along with different doses of TCDD. (B) In vitro apoptosis of hemocytes in response to different doses of TCDD. (C)

Effect of TCDD on the activation of hemolymph prophenoloxidase in response to an inducer, laminarin. The activated phenoloxidase

(PO) activity was measured by absorbance change at 495 nm for 5 min. (D) Rescue effect of arachidonic acid (AA) on the depressed PO

activity by different doses of TCDD. Each dose treatment was replicated with three times. The error bar indicated standard deviation.

In (A)–(C), different letters above the error bars indicate significant difference among means at Type I error = 0.05 (LSD test). In (D),

the asterisk indicates significant difference between two treatments in each dose of TCDD at Type I error = 0.05 (LSD test).

328 K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331

and TEQ levels of R3 has been expected since it flows

through Sihwa industrial complex which is one of the

most active industrialized zone in Korea, receiving a

direct load of industrial wastes.

Another two sediment samples were also collected

from the R6 (Miho-UP) and R7 (Miho-DN) sites adja-

cent to the Jungbu expressway and some factories. The

total PCDD/PCDFs concentrations and TEQ levels of

both samples were found to be 16.02 and 16.45 pg/g,

and 0.50 and 0.64 TEQ pg/g, respectively. In fact, the

total concentrations and TEQ levels of these samples

were expected to be equivalent or lower than R8. In con-

trast, the total concentrations and TEQ levels detected

in these samples showed about 3–4 times higher, despite

no direct sources of the PCDD/PCDFs near this river.

We speculate that the sources of PCDD/PCDFs entering

this river have been attributed to the automobile exhaust

gases (atmospheric transport and deposition).

Different congeners of PCDD/PCDFs were found in

different sediment localities. Most abundant congeners

included OCDD, OCDF, HpCDD, HpCDF, HxCDD,

and HxCDF. Generally, TCDD and TCDF are easily

accumulated in biological organisms, while OCDD

and OCDF are readily deposited in sediments due to

their low solubility in water (Muir et al., 1991). These re-

sults indicate that three localities of Singil (R1, R2 and

R3) and two localities of Miho (R6 and R7) are relatively

highly contaminated sites above 10 pg/g of total PCDD/

PCDFs.

TCDD has been regarded as less potent in inverte-

brates because of poor binding affinities of their Ah

receptor to the toxicant (Butler et al., 2001). However,

this study showed that even ppt levels of TCDD could

inhibit insect immune capacity. Insect immune system

involves both cellular and humoral reactions and exerts

their reaction after specific non-self pattern recognition

(Gillespie and Kanost, 1997). They can recognize bacte-

rial or fungal infection by binding pathogen-related sur-

face molecules such as peptidoglycan of Gram-positive

bacteria, lipopolysaccharides of Gram-negative bacteria,

and b-1,3-glycan of fungi (Yu et al., 2002). This recogni-

tion is mediated by cytokine like PSP1 or eicosanoids to

activate actual immune reactions (Clark et al., 1997;

Stanley, 2000). In humoral immune reactions, insect

can express several antibacterial peptides such as lyso-

zyme, cecropin, attacin (Boman and Hultmark, 1987).

Sediment samples

PO a

ctiv

ity

Abs

at 4

95nm

/5 m

in0.00

0.05

0.10

0.15

0.2050% EtOH100ng AA

*

R1 R2 R3 R4

* **

PO a

ctiv

ityAb

s at

495

nm

/5 m

in

0.00

0.05

0.10

0.15

0.20

0.25

CON R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

*

* **

Sediment samples(C) (D)

Apop

tosi

s (%

)

0

20

40

60

80

100

* *

* *

CON R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

Num

ber o

f nod

ules

0

5

10

15

20

25

*

* *

*

CON R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12(A) (B)

Fig. 3. Biomonitoring using insect immune endpoints against the sediment samples (R1–R12, see details in Fig. 1) with reference to a

solvent control (�CON�). All sediment and control samples were prepared by the extraction method used for measurement of PCDD/

PCDFs. The extracted samples were analyzed by four different immunological assays described in Fig. 2. Each environmental sample

was replicated with three times. The error bar indicated standard deviation. In (A)–(C), different letters above the error bars indicate

significant differences of the environmental samples in reference to the control at Type I error = 0.05 (LSD test). In (D), the asterisk

indicates significant difference between two treatments in each environmental sample at Type I error = 0.05 (LSD test).

K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331 329

While the humoral immune reactions take several hours

to express such peptides, cellular immune reactions exe-

cuted by hemocytes (insect blood cells) can be induced in

several minutes (Ratcliffe et al., 1985). These include

phagocytosis, nodule formation, and encapsulation

by hemocytes. Phenoloxidase catalyzes oxidation of

L-DOPA and produces quinine molecules, which are

toxic to pathogens and also important in nodule forma-

tion and encapsulation reactions by forming melanized

coat around pathogens (Dularay and Lackie, 1985).

The phenoloxidase should be in an inactive form, pro-

phenoloxidase, and should be tightly regulated in its

activation (Soderhall and Smith, 1986).

In this study, TCDD significantly inhibited the acti-

vation of prophenoloxidase of S. exigua in the presence

of laminarin, and inhibited hemocyte nodule formation.

These inhibitory actions of TCDD can be explained by

the positive correlation between phenoloxidase activity

and PLA2 activity in S. exigua (Park and Kim, 2003) be-

cause TCDD could inhibit PLA2. Thus, we speculate

that the inhibition of PLA2 by TCDD could not activate

prophenoloxidase, which in turn did not complete

hemocyte nodulation. The inhibitory effect of TCDD

on vertebrate immune system has been suggested by

the hypersensitivity of thymus (Vos et al., 1997), where

TCDD also induces apoptosis of T cells (Camacho

et al., 2004). In S. exigua, TCDD also induced apoptosis

of hemocytes, in which the cells formed apoptotic blebb-

ings and vesicles, typical symptoms of apoptotic cells

(Cho and Kim, 2004). Interestingly, the immune reac-

tions against TCDD was highly sensitive but different

in threshold doses for susceptibility: 0.1 ppb in pheno-

loxidase activity, 1.0 ppb in apoptosis, and 1.0 pg in

nodule formation.

The highly sensitive immune systems of S. exigua

against TCDD were used to monitor PCDD/PCDFs in

the environmental samples. Four local sediment samples

(R1, R2, R3, and R4) inhibited the immune biomarkers.

When comparing both chemical and biological monitor-

ing results, two methods agreed three spots of Singil as

contamination areas (above 10 pg/g levels) as well as

six river sediment samples as relatively less-contami-

nated areas, but differed in the results in R4 (Ansan)

and R6 (Miho-UP) and R7 (Miho-DN), probably due

to relative non-specificity of biomarkers. The immune

reactions can be affected by other compounds in the

sample extracts. Because in most cases a variety of con-

taminants may be present in environment, reliable bio-

markers should have some degree of specificity.

Alternatively, a battery of biomarkers is required to

330 K.S. Ryoo et al. / Chemosphere 61 (2005) 323–331

cross-check different classes of contaminants (Melancon,

1995). Our current test using the immune biomakers

suggests that heavy metals also give significant inhibi-

tory actions on insect immune systems (unpublished

data). So, to clarify somewhat vagueness in this study,

various environmental compounds should be tested in

their inhibitory actions against the insect immune sys-

tem. Despite some disparity between bio- and chemical

monitoring results, the biomarkers can be recommended

as a device warning the contamination of dioxins in the

environment because of a fast and inexpensive detection

method.

Acknowledgement

This study was funded as Ecotechnopia 21 project by

Korea Institute of Environmental Science and Technol-

ogy (KIEST). This support is gratefully acknowledged.

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