Form 3
Annual Report
LaMer, Ehime University
Date (12, March, 2019)
To Director of LaMer
Principle Investigator :Dede Falahudin

Affiliation

Research:Centerfor

Oceanography,
Indonesian Institute of
Science (LIPI)
Position:

Researcher

Name in print:

Dede Falahudin

Include the report on the result of the project/meeting in a separate sheet.
1. Project / Meeting title
Analysis of persistent organic pollutants in coelacanths (Latimeria
chalumnae) from Indonesia
2. Members of project / meeting
Name
Affiliation
PositionContribution partPI
Dede Falahudin
ResearchCenterfor Researcher Analysis samples andOceanography,
write a reportIndonesian
InstituteofScience (LIPI)
Members
Agus Sudaryanto
Agency for the Assessment Researcher Prepared proposal ofandApplicationofproject
Technology (BPPT)
LaMer Faculty memberin charge
Shin Takahashi
CMES and Graduate School Professor
Prepared for a reportofAgriculture,
Ehime
University
3. Contents (please write in separate sheet, A4-size, within 5 pages including figures
and tables. Itemize “Title, members’ names and affiliations, aim, procedure, result,publication/conference presentation, perspectives in future”).
This report will be opened to the public through LaMer website and printed matter.
Please send additional products, i.e., abstract book, reprint, if available to
LaMer Office of Research Support Division,
Ehime University, Bunkyo 2-5, Matsuyama, 790-8577 Japan
E-mail: lamer@stu.ehime-u.ac.jp
Tel and Fax: +81-89-927-8187

1 / 102 / 10

Project Report
Analysis of Persistent Organic Pollutants in the Coelacanths(Latimeria chalumnae) from Indonesia
Dede Falahudin1*, Frensly Demianus Hukom1, Zainal Arifin1, Dirhamsyah1, Agus
Sudaryanto2*, Hoang Quoc Anh4, Isao Watanabe4, Shin Takahashi3,4*
1

Research Center for Oceanography, Indonesian Institute of Sciences, Jl Pasir Putih 1, Ancol

Timur, Jakarta Utara, Indonesia
2

Agency for the Assessment and Application of Technology (BPPT), Jl MH.Thamrin 8, Jakarta

Pusat, Indonesia
3

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,

Matsuyama 790-8577, Japan
4

Center of Advanced Technology for the Environment (CATE), Graduate School of Agriculture,

7 Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan*Member of project stated in the proposal
1. Introduction
During the last few decades, contamination by anthropogenic chemicals such as
persistent organic pollutants (POPs) has spread all over the world. As evidence, they weredetected in various environmental components and biota including those far from humanactivities. Particularly, research efforts on field observations and numerical models of global
fate of POPs have revealed oceanic water bodies to be a global reservoir and final sink forthese toxic contaminants that undergo transport from emission sources and partition between
air and water and scavenge to deep-sea layers by various biogeochemical and geophysicalprocesses (Takahashi et al., 2014). Various classes of POPs such as organochlorine
pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers(PBDEs), and other brominated flame retardants (BFRs) have been detected in deep-seaorganisms from different parts of the world (de Brito et al., 2002, Lee et al., 1997, Takahashi
et al., 1998, Jamieson et al., 2017).
There were several reasons on investigation of persistent organic pollutants in the
deep-sea organisms such as to know their fate, transport and the specific accumulation
characteristics these toxicants. Afterward, we will able to measure their transport alongoceanic system and observe deep-sea environment as final location for these persistentcontaminants. Further, to implement and evaluate the effectiveness of international
agreements to protect the marine environment from the deleterious effects of POPs,interdisciplinary approaches including studies on biogeochemical and geophysical processes
in the ocean as well as field observations are required to delineate the global and regional

3 / 10fate of POPs (Takahashi et al., 2014).
2. Aim of Study
The aim of this study is to determine the occurrence of POPs and related compounds,
comprising PCBs, PBDEs, and selected novel brominated flame retardants (NBFRs) indifferent tissues of coelacanth (Latimeria menadoensis) obtained from Indonesia.
3. Analytical Procedures
Samples. Three tissue samples (muscle, liver, and ovary) were sampled from the specimenof female sea king fish (Indonesian Coelacanth, Latimeria menadoensis) with CCC No. 299
that caught by gill net on November 5, 2014 around Gangga Island, North Sulawesi. This
specimen was had a body length of 130cm and weight of around 22kg.
Chemical analysis. Tissue samples were freeze-dried and grinded into fine powder by usingmortar and pestle. The sample (2 to 3 g) was transferred into a 50mL tube and subsequently
extracted with 20mL acetone, 20mL a mixture of acetone/hexane (1:1, v/v) and 20mL hexane
by using a homogenizer (T 25 Digital Ultra-Turrax®; IKA Japan K.K.). The crude extract wasconcentrated and solvent-exchanged into hexane. A portion of extract corresponding to 1 g ofsample was used for BFR and PCB analysis. Before purification, the extract was spiked withsurrogate standards of PBDEs (monofluorinated FBDE-15, -99, -183, -208, and13

C12-BDE-209) and PCBs (Congener, 13C12-PCB-1, -3, -4, -8, -15, -19, -28, -52, -54, -70, -77,

-81, -95, -101, -104, -105, -114, -118, -123, -126, -138, -153, -155, -157, -167, -169, -170,-180, -188, -189, -202, -205, -208, and -209). The extract was cleaned up by passing througha multilayer silica gel column and an activated silica gel column with elution solvents as
mixtures of 10% and 5% dichloromethane in hexane, respectively. The eluate wasconcentrated and spiked with internal standards of PBDEs (FBDE-154) and PCBs
(13C12-PCB-9, -37, -79, -111, -162, -194, and -206) before GC/MS quantification. Chemicals
and solvents used in this study were reagent grade for the determination of PCBs andpurchased from Wako Pure Chemical Industries, Ltd.
Instrumental analysis for BFRs. Thirty eight PBDEs (di- to decabrominated congeners,
including BDE-7, -10, -15, -17, -28, -30, -47, -49/71, -66, -77, -85, -99, -100, -119, -126, -138,-139, -140, -153, -154, -156/169, -171, -180, -183, -184, -191, -196, -197, -201, -203, -204,-205, -206, -207, -208, -209) and four NBFRs (such as PBEB, BB-153, BTBPE, DBDPE)
were quantified using a gas chromatograph connected to a quadrupole mass spectrometer(GCMS-QP2010 Ultra, Shimadzu). BFRs were separated on a fused-silica capillary column(DB-5ht, 15 m × 0.25 mm × 0.1 μm, Agilent Technologies). Helium was used as carrier gas.
Temperature of injection port was 260oC. Initial column oven temperature was 135 oC for 1
min, increased to 215 oC (10 oC min-1), to 275 oC (5 oC min-1), to 295 oC (20 oC min-1, held 0.5min) and finally raised at 20 oC min-1 to 310 oC and held for 4 min. The mass spectrometer4 / 10was operated in electron capture negative ionization (ECNI) mode. Methane was used asmoderating gas. The temperature of the interface and ion source was 310 oC and 250 oC,
respectively. Twelve ions were selectively monitored, including: m/z = 79.0/81.0 and
158.8/160.8 (Br- and HBr2-, for all compounds); 406.6/408.6 and 486.5/488.5 (C6HBr4O- andC6Br5O-, for hepta- to decaBDEs); 426.5/428.5 (C6FBr4O-, for FBDE-208); and 496.6/498.5(13C6Br5O-, for 13C12-BDE-209).
Instrumental analysis for PCBs. PCBs (209 mono- to decachlorinated congeners) were
quantified using a 6890N gas chromatograph (Agilent Technologies) connected to a
JMS-800D high resolution mass spectrometer (JEOL). The separation was performed on a
HT8-PCB capillary column (60 m length × 0.25 mm internal diameter × 0.25 μm film thickness,
Kanto Chemical). Helium was used as carrier gas at a constant flow of 1 mL min –1. Inlet
temperature was 280 oC. A sample volume of 1 μL was injected in splitless injection mode.
Column oven temperature was programmed from 120 oC, increased to 180 oC (20 oC min–1),
to 260 oC (2 oC min–1), and ramped to 300 oC (5 oC min–1, hold 4 min). Mass spectrometer
was operated in positive electron ionization (EI) mode at a resolution of ≥ 10,000 at 10%
valley. Ionization energy was 38 eV and acceleration voltage was 10 kV. Temperature ofinterface and ion source was 280 oC. Data were acquired in selected ion monitoring (SIM)mode using two molecular ions for each native and 13C12-PCB congener.
Lipid content. Sub samples (1mL) of extract were added to the pre-weighed porcelain
crucible and record the weight. After that, the solvent was evaporated and the porcelains
were re-weighed again to calculate total lipid content (%) for each sample.
Quality assurance and quality control (QA/QC). All methods for PBDEs and PCBs
analysis were validated by the replicate analysis of standards and samples, spiking surrogate
standard and analysis of SRM (Table 1).
Table 1. Parameter of QC/QA analysis of coelacanth samples
Parameter

PBDEs

PCBs

Results of SRM Mussel NIST 2974a

Accuracy 57–101%

Accuracy 55–98%

Results of CRM Seabass NMIJ 7404a–

Accuracy 71–95%

Results of duplicate samples

RSD < 15%

RSD < 15%

Recoveries of surrogate standards

70–120%60–110%0.01–0.1 ng/g wet wt.

0.003–0.3 pg/g wet wt.

Method detection limits
4. Results and discussion

Concentration of BFRs in Coelacanth. Percentages of lipid content varied in different
tissue of coelacanth, with liver tissue having higher percentage of lipid content (71.79%) thanmuscle (10.57%) and ovary (1.40%). These results agree with other study that deep-sea fish
have high percentage of lipid on their liver where the processes of metabolism and storage ofxenobiotic compounds occurred (Covaci et al., 2020). Therefore, on this report, concentration

5 / 10

of PBDEs and NFBRs were normalized with lipid content and reported on lipid weight, l.w.,
basis. In general, concentration of Σ9PBDE in coelacanth ranged from 0.93 to 1.94 ng/g l.w.
with average of 1.5 ng/g l.w (Table 2). Variation of residue value of PBDE in different tissue of
coelacanth was influenced by several factor including physical and chemical properties ofcongeners, biotransformation processes, metabolism and also permeability of membrane(Ma et al., 2013).
Due to different on the number of PBDEs congener determined, sample types, and themethods of samples analyzed, comparison of PBDEs concentrations to other study werelimited and quite challenging. However, previous study reported that composition of PBDE
congener in biota is similar over the world with the most detected are BDE-47, BDE-99,
BDE-100, BDE-153, and BDE-154, which all of congener was as flammable agent on thecommercial penta-BDE mixture consumer products (Covaci et al., 2020). Therefore, tounderstand the status of PBDEs pollution in coelacanth from Indonesia, residue levels of
PBDE found in the present study were compared only limited on congener basis including
BDE-17, -28, -47 and BDE-99 (Table 3). The sum of Σ4PBDE (1.36 ng/g l.w.) in muscle tissueof coelacanth from this study were slightly higher than the level reported for deep-sea fishes
from NW Mediterranean (Koenig et al., 2013) and the Sulu Sea (Ramu et al., 2013), except
species Bathygadus sp and Lamprogrammus niger. In contrast, the result from this study was
lower than Σ3PBDE from two Mediterranean deep-sea fishes Trachyrinchus trachyrinchus
and Coelorhynchus coelorynchus (Covaci et al., 2020).
Table 2. Concentrations of PBDEs and NBFRs in different tissue samples of coelacanth (ngg−1 lipid weight) collected Indonesia
Compound

MDL

Muscle

Liver

Ovary

BDE-7

0.01

< 0.01

ND

0.03

BDE-10

0.01

ND

ND

ND

BDE-15

0.01

0.09

0.17

ND

0.10

0.17

0.03

Σ3 Di-BDEs
BDE-17

0.01

0.26

0.14

0.71

BDE-28

0.01

0.18

0.13

ND

BDE-30

0.01

ND

ND

ND

0.44

0.28

0.71

Σ3Tri-BDEs
BDE-47

0.02

0.63

0.27

1.19

BDE-49/71

0.02

0.19

0.14

ND

BDE-66

0.02

ND

ND

ND

BDE-77

0.02

ND

ND

ND

0.82

0.41

1.19

ND

ND

ND

Σ4Tetra-BDEs
BDE-850.04

6 / 10BDE-99

0.03

0.29

ND

ND

BDE-100

0.03

ND

ND

ND

BDE-119

0.05

ND

ND

ND

BDE-126

0.05

ND

ND

ND

0.29

0.00

0.00

Σ5 Penta-BDEs
BDE-138

0.04

ND

0.07

ND

BDE-1390.02< 0.02

ND

ND

BDE-140

0.02

ND

ND

ND

BDE-153

0.03

ND

ND

ND

BDE-154

0.05

ND

ND

ND

BDE-156/169

0.05

ND

ND

ND

Σ7 Hexa-BDEs

0.00

0.07

0.00

Σ5 Hepta-BDEs

ND

ND

ND

Σ6 Octa-BDEs

ND

ND

ND

Σ3 Nona-BDEs

ND

ND

ND

BDE-209

ND

ND

ND

1.65

0.93

1.94

Total Σ36
PBDEs
PBEB

0.01

ND

ND

ND

BB-153

0.04

ND

ND

ND

BTBPE

0.03

ND

0.15

ND

DBDPE

0.10

ND

ND

ND

0.00

0.15

0.00

10.57

71.79

1.40

Total Σ4 NBFRs
Lipid content(%)

Congener profiles and potential sources of BFRs. The distribution pattern of PBDE
congeners in coelacanth tissue was different to the typical pattern of deep-sea organism
investigated from other location in the world, where higher congener (more than hexa-BDEs)
usually detected (Koenig et al., 2013). In addition, undetected higher congener of PBDE inthis study was potentially due to characteristics of higher PBDE that have low
bioaccumulation potential and easily to degrade into lower brominated PBDE congeners via
debromination (Letcher et al., 2014). Based on results from this study, of the 36 PBDE
congeners evaluated, only nine congeners (BDE-7, -15, -17, -28, -47, -49/71, -99 and -138)
and one (BTBPE) of four NFBR’s congener analyzed were detected in coelacanth tissues. All
detected congener mainly lower brominated congener (di- to tetra-BDEs) with BDE-17 andBDE-47 were the most detected congener up to 24% and 45%, respectively. This result
coherence with previous studies that also have observed high concentration of BDE-47 in the

7 / 10liver of Mediterranean deep-sea fish up to 50% and also in the amphipod from Mariana
Trench and the Kermadec Trench, showed predominance of BDE-47 account for 71% of the
ΣPBDE concentration (Jamieson et al., 2017).
Furthermore, to observe the origins or biodegradation capacity of coelacanth on PBDE
contamination, further analysis is needed including using ratio between BDE-99/BDE-100,
BDE-99/BDE-47, BDE-153/BDE-154 and BDE-183/BDE-154 (Ma et al., 2013).
5. Perspectives in future
We have found clear evidence of the expansion of contamination by emerging POPs
including PBDEs and NBFRs into the habitat of coelacanth. Even though the concentration
was smaller than other concentration of POPs found in deep-sea organism, presence ofPOPs accumulation in different part of coelacanth tissue would be as alarm for us to stop
release of such chemical into the sea. Furthermore, due to specific congener already found in
the coelacanth, further analysis is needed to investigate fate and sources of PBDEs in the
coelacanth fish. In addition, including biogeochemical and geophysical processes on study ofmarine contaminant in the depp-sea region are required to delineate the global and regional
fate of POPs.
Chemical analysis is continued to determine PCBs and other potential emerging
organic contaminants.
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