Characterization of Dioxin-like and Estrogenic Compounds in - - PowerPoint PPT Presentation

Characterization of Dioxin-like and Estrogenic Compounds in Environmental Samples from Onsan Bay and its Inland Region, Korea

JS Khim1,2, KT Lee1, DL Villeneuve2, K Kannan2, JP Giesy2, CH Koh1

1Seoul National University, Seoul, Korea 2Michigan State University, East Lansing, MI, USA

Industrial Complexes in Onsan Bay Area

D0 D1 D2 D3 D4 D5 D6 W0 W1 W2 ON1 ON2 ON3 OS1 OS2 OS3 O5 O6 O7 O8 O9 O10 O4 10 m 0 1 km Onsan Nonferrous Metal I.C. Petrochemical I.C. Yongyeon Heavy I.C.

I.C. : Industrial Complex

Onsan Bay Woihang River Onsan City Daejeong Stream

Korea

Onsan Bay Study Area

Samples were collected from 22 locations in April 1999

Sediment sampling locations in Onsan Bay

To quantify chemicals of PCBs, OC pesticides, PAHs, Alkylphenols in Onsan Bay sediments To examine cell line responses of dioxin-like and estrogenic compounds associated with sediment samples To identify chemicals showing cell line responses

Objectives

Methods

Sediment extracts were obtained from Soxhlet extraction and Florisil fractionation (3 fractions) Cell line bioassay were performed using H4IIE-luc cells for dioxin-like activities and MVLN cells for estrogenic activities Mass balance analyses were applied to evaluate chemicals showing cell line activities

GC/MS & GC/ECD Bioassay GC/MS & GC/ECD Bioassay

Extraction & Fractionation Sequence

HPLC & GC/MS Bioassay

Frozen Whole Sediment Freeze Drying Homogenization Soxhlet Extraction Cu treatment Florisil fractionation Non-polar (Hexane) PCBs, HCB, DDE Mid-polar (20% DCM in Hexane) PAHs, DDD, DDT, HCHs, CHLs Polar (50% DCM in MeOH) NP, OP, BPA Raw Extract

Bioassay

In vitro Bioassay Procedure

H4IIE-luc or MVLN cells

• cells were trypsinized
• plated at 75 K cells/well

(250 μl/well)

• dosing test samples

(2.5 μl)

• After 72 h. exposure,

culture media aspirated

• cells were rinsed with PBS
• luclite reagent added into the wells
• after 10 minutes, luciferase activity

was determined with luminometer

TCDD

E2

SC blank Test

Sample

Results and Discussions

Concentration Levels in Sediments

• 1. Concentrations of PCBs, OC pesticides, PAHs,

Alkyl phenols in sediments

• 2. Comparison of concentrations with other Korean

sediments

• 3. Potential biological effects of sediment concentrations

Cell Bioassay Activities

• 4. Dioxin-like and estrogenic activity in vitro

• 1. Concentrations of PCBs, OC pesticides,

PAHs, Alkylphenols in sediment

Concentrations of PCBs in sediments were generally less than method detection limit of 10 ng/g, dry wt Among different OC pesticides analyzed, DDT concentrations (sum of p,p’-DDD, DDE, DDT) were the greatest, ranging from <0.01 to 7.6 ng/g, dry wt PAHs were detected in all the sediment samples. Highest concentrations of 300 to 1000 ng/g, dry wt. were found in 5 sampling locations in landward region. Outer bay region showed less than 30 ng/g, dry wt of concentration High concentrations of NP, OP, and BPA were found

• nly at Daejeong stream with mean concentration of 218

ng/g, dry wt. (sum of NP, OP, BPA). Other locations showed very low concentrations of 1.0 to 10 ng/g, dry wt

Mean Concentrations of PCBs: OC pesticides: PAHs: Alkyl phenols:

D0 D1 D2 D3 D4 D5 D6 W0 W1 W2 ON1 ON2 ON3 OS1 OS2 OS3 O5 O6 O7 O8 O9 O10 O4 10 m Onsan Nonferrous Metal I.C. Petrochemical I.C. 50 ng/g dry wt Unit Onsan Bay (O) 325 ng/g dry wt Woihang River (W) Onsan South Part (OS) 218 ng/g dry wt Daejeong Stream(D) Onsan North Part (ON) PCBs Pesticides PAHs Alkylphenols

• 2. Comparison of concentrations with other

Korean sediments

Sedimentary PCBs and PAHs in Korea have been reported to range from a few ng/g to several µg/g Concentrations of PCBs and PAHs in Onsan Bay were approximately 2 to 4- fold less than those reported in

• ther industrialized coastal areas such as Kyeonggi,

Masan, Ulsan, and Yeongil Bays Concentrations of AP and BPA in sediment from Onsan Bay were similar to those in Ulsan Bay but far less than those in other bay areas

• 3. Potential biological effects of sediment

concentrations

Concentrations of total TCDD equivalents (TEQs) ranged from 0.003 - 8.86 pg/g, dry wt The TEQs estimated for selected PAHs and PCBs in Onsan sediment were at the lower end of the sediment quality guideline (SQG) of 0.014-210 pg/g, dry wt, reported for dioxin equivalents None of the locations exceeded the Effect Range Lows (ERLs) for 16 PAHs and PCBs When Threshold Effect Concentration (TEC) for total PCBs were compared, only one location (D4) exceeded the limit of 35 ng/g, dry wt

• 4. Dioxin-like and estrogenic activity in vitro

Screening of raw extracts (whole sediment extract) for dioxin-like activity with the H4IIE-luc cell bioassay revealed 17 of 22 samples had significant dioxin-like activity Further fractionation of sediment extracts indicated that mid polar and more polar fractions were responsible for the significant dioxin-like activity Estrogenic activities were low in the most samples. Only

• ne raw extract sample (D3) yielded a significant

estrogenic activity in the MVLN bioassay The low estrogenic activity was consistent with low concentrations of measured estrogenic compounds in the Onsan sediments

ON1 OS1 O4 O5 O6 O10

• 20

20 40 60 80 100

%-TCDD-max

Outer bay region (O: Onsan Bay)

H4IIE-luc cell line activity: Outer Bay Region

Response magnitude presented as percentage of the maximum response

• bserved for a 2000 pM 2,3,7,8 tetrachlorodibenzo-p-dioxin standard (%-

TCDD-max.) indicates cells exhibited an altered or “stressed” morphology RE F1 F2 F3

• 20

20 40 60 80 100 W0 W1 W2 D0 D1 D2 D3 D4 D6

%-TCDD-max

Landward Area (W: Woihang River D: Daejeong stream) Response magnitude presented as percentage of the maximum response

• bserved for a 2000 pM 2,3,7,8 tetrachlorodibenzo-p-dioxin standard (%-

TCDD-max.) indicates cells exhibited an altered or “stressed” morphology

H4IIE-luc cell line activity: Landward Area

Conclusion

The relative abundance of organic residues in sediment was in the order of PAHs ≈ APs > PCBs > OC pesticides Concentration levels of organic compounds were high in landward region and those in outer bay were relatively low Mid polar (PAHs) and more polar fractions of sediment extracts were responsible for the significant dioxin-like activity Estrogenic activities were low in the most sediment

Acknowledgement

• National Institute of Environmental Research (NIER),

Ministry of Environment, Korea (Sediment Organic Compounds Bioassay Study; SORGBIOS ’98-2000)

• Dr. MD Pons, INSERM Montpiellier France
• Colleagues from Benthos Lab at Seoul National University,

Korea and Aquatic Toxicology Laboratory at Michigan State University, USA