4. Research Report
Title of Research Project:
Assessment of the Occurrence, Distribution and Ecological Risk of Bisphenol Analogues in
the Surface Water from Citarum Watersheds
Project Members:• Dr. Agus Sudaryanto (PI)Research Center for Environmental and Clean Technology, National Research andInnovation Agency – BRIN, Kawasan Puspiptek Gedung 720, Tangerang Selatan 15314,
Indonesia• Dr. Dwindrata B Aviantara (Member)Research Center for Environmental and Clean Technology, National Research andInnovation Agency – BRIN, Kawasan Puspiptek Gedung 720, Tangerang Selatan 15314,Indonesia• Fuzi Suciati
Research Center for Environmental and Clean Technology, National Research andInnovation Agency – BRIN, Kawasan Puspiptek Gedung 720, Tangerang Selatan 15314,
Indonesia• Dr. Budi Kurniawan
Research Center for Environmental and Clean Technology, National Research andInnovation Agency – BRIN, Kawasan Puspiptek Gedung 720, Tangerang Selatan 15314,
Indonesia• Dr. Rumi TanoueCenter for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,
Matsuyama 790-8577, Japan• Prof. Tatsuya Kunisue (Member)Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,
Matsuyama 790-8577, JapanResearch Background and Purposes:
Water contamination by endocrine disrupting bisphenol-A (2,2-bis(4-hydroxyphenyl) propane,BPA) has been a matter of global concern. This is because of BPA has shown varying
different adverse effects, including to fish (Suzuki et al., 2003), rats (Liu et al., 2019) andhuman health (Rochester, 2013). Therefore, in response to the adverse effects of BPA,
several countries and regions, including China (Jin and Zhu, 2016), San Francisco (Chen etal., 2016), Canada, and the European Union (Negev et al., 2018), have issued policies
banning BPA in products. However, in order to meet the market demand for products, various
alternatives to BPA were widely used in industrial production (Zheng et al., 2019), such asbisphenol-S (4,4′-sulfonyldiphenol; BPS), which is widely used in the manufacture of epoxy
resins, bisphenol-E (bis(4-hydroxyphenyl) ethane, BPE) for cyanate resin, bisphenol-F(bis(4-hydroxypheny)methane; BPF) for epoxy resin reinforced with nano polyanilines, andbisphenol-AF (4,4′-(hexafluoroisopropylidene) diphenol, BPAF) and bisphenol-Z (4,4′(cyclohexane-1,1-diyl) diphenol, BPZ) for the manufacture of epoxy resins and polycarbonate
plastics. Indeed, recent studies have shown that BPS, BPF, and other BPA analogues werewidely detected in the water environment, and the pollution levels have an increasing trend(Chen et al., 2016). In addition, current research studies have indicated that BPS, BPAF, andBPF can be accumulated by aquatic organisms, and pose a serious threat to the wholeecosystem as BPA (Yan et al., 2017). Research is still needed to better elucidate theenvironmental sources, distribution, and fate of bisphenol analogues (BPs).
The Citarum is the largest river in the West Java, Indonesia stretches for 297 km across 13
regencies/cities with its upstream in Bandung Regency and empties into the Java Sea, atMuara Gembong, Bekasi Regency (Figure 1). Every day, the Citarum watersheds receives
no less than 20,000 tons of waste and 340,000 tons of wastewater from more 2,000 factories.
Thus, the Citarum watershed is experiencing pollution and environmental damage due tohigh domestic and industrial activities on the riverbanks. Pollution to the Citarum River is dueto includes industrial pollution, agricultural waste, livestock waste, fishery waste, and both
domestic wastewater and domestic waste (West Java Province task force for pollution controland damage to the Citarum watershed, 2018). As the result, aquatic environment of Citarum
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is under threat. For example, since 2002, lead, aluminum, manganese, and iron in theCitarum has never met the water quality standards of Government Regulation No. 82 of 2001
and levels of lead reached 1,000 times worse than the U.S. standards for drinking water(Blacksmith Institute, 2013). Citarum River considered as the “world’s most polluted river”
and thus government paid attention to restore the ecosystem through “Citarum Harum
National Project”. Despite improving visual environment of Citarum River after "Citarum
Harum" was launched at 2018, there is limited or no available information on trace toxic
contaminants including bisphenol analogues in Citarum watershed.
The purpose of this study is to determine bisphenol analogues in surface waters of Citarum
Watershed in order: (a) to understand their occurrence, distribution and sources, and (b) toestimate their potential health risk to aquatic ecosystems.
Methods
Sampling and Sample
The study combined of sampling survey to acquire the samples and chemicals analysis atlaboratory. Sampling locations were selected along Citarum River generally according towater quality monitoring point from the Ministry of Environment and Forestry of Republic
Indonesia started from upstream (upper), middle to downstream (lower) Citarum. This rivercrosses to several city and regency such in Bandung, Purwakarta, Kerawang, and Bekasi(Figure 1). Samples are surface river water consisting of a total of 61 sampling sites along
the main Citarum River and its tributaries (Figure 1). The sampling program was conductedduring 29 October – 2 November 2022 and 1 - 20 June 2023. Surface water were sampledusing a stainless steel containers. The surface water then transferred to 100 ml of bottle
sample, preserved with keep in cool box, and transferred to Center for Marine Environmental
Studies (CMES), Ehime University, Japan for chemicals analysis.

Figure 1. Sampling locations of surface water at Ciliwung River and its tributaries fromupstream to downstream.
Chemicals Analysis
Ten commonly used BPs such as BPA, BPAF, BPF, BPS, BPB, BPC, BPC12, BPE, BPZ andTBBPA were selected as target analytes. The target of bisphenol analogues in this study
include ten commonly used BPs such as bisphenol-A (2,2-bis(4-hydroxyphenyl) propane;
BPA), bisphenol-AF (4,4′-(hexafluoroisopropylidene) diphenol, BPAF), bisphenol-F (bis(4hydroxypheny)methane; BPF), bisphenol-S (4,4′-sulfonyldiphenol; BPS), bisphenol-B (4,4'(1-methylpropylidene)bisphenol; BPB), bisphenol-C (4,4′-isopropylidenedi-o-cresol; BPC);
bisphenol-C12(4,4'-(dichlorovinylidene)diphenol;
BPC12),bisphenol-E(bis(4hydroxyphenyl) ethane, BPE), bisphenol-Z (4,4′-(cyclohexane-1,1-diyl) diphenol, BPZ), andTetrabromobisphenol A [4,4’-isopropylidenebis(2,6- dibromophenol)] (TBBPA). The
bisphenol analogues analysis were conducted at Laboratory of Environmental Chemistry and4/7

Ecotoxicology, Center for Marine Environmental Studies (CMES), Ehime University, Japan.
Surface water samples were analysed according to the method published elsewhere11) .
Briefly, extraction of the sample was carried out by filtering water samples (20 mL), addedwith internal standard (IS) and then inserted into the Oasis HLB Plus Light cartridge as asolid phase extraction for the clean-up procedure. The target BPs retained in the cartridge
was 5 / 9 eluted with a solution of methanol/MTBE (3 mL, by volume ratio 7:3), and the eluate
was concentrated to 0.2 mL under a stream of N2. For identification and quantification of BPs,
the residue was dissolved in acetonitrile/methanol/Milli-Q (1 mL, volume ratio 1:2:7).
Identification and quantification of target BPs was carried out using a Prominence Ultra-Fast
Liquid Chromatograph equipped with a mass spectrometer. This equipment operates inpositive and negative electrospray ionization (ESI) mode with dual reaction monitoring(MRM).
Results
Occurrence, Distribution and Sources
A total of 61 surface water samples collected along Citarum River and its watersheds
comprising three segments of upper, middle and lower Citarum River has been analyzed forten bisphenol analogous (BPs). Figure 1 shows the concentration of BPs analogous insurface water from main Citarum River and its watersheds from upper part to lower Citarum.
BPs were detected at 35 locations which accounted 57% of the total locations (61 locations).
Among the 10 BPs, only 2 BPs i.e BPA and BPS were generally detected at relatively in wide
range of Citarum River, 1 BPs was detected in only 1 location i.e TBBPA and the rest BPs
were not detected in any locations. BPS (57% of the locations) showed a more detection
frequency than BPA (43%). Among the detected BPs, BPA had the highest level in surface
water with a concentration up to 5800 ng/L, whereas maximum concentration of BPS was4100 ng/L. The detectable TBBPA in one location was 19 ng/L.

Figure 2. Concentration of BPs analogous in surface water from main Citarum River and its
watersheds from upper part to lower Citarum.
Even though it has been replaced by other BPA analogues in recent years, BPA is still themost dominant compound in the surface water of the Citarum River. Although there is very
little information on BP use data, the results of this study show that there is common use ofBPA and BPS in several areas around the Citarum River. The distribution of BPs at eachobservation station shows that BPA and BPS were detected in the Citarum River, especiallyin the lower Citarum segment which crosses Kerawang City and Bekasi Regency, namelydetected at all stations from CR-21 to CR-35, especially in Kerawang City, Bekasi. Meanwhile,the concentration of BPs in the upper and middle segments of Citarum is much lower and is
not even detected in many locations, including in its tributaries. However, BPs were also
detected in several Citarum river tributaries, especially in rivers that cross the city of Bandung(CR-7 to CR-17). Kerawang City, Bekasi Regency and Bandung City have higher population
numbers and industrial activity, resulting in the widespread presence of BP in rivers.
Potential Health Risk to Aquatic Ecosystems.
The risk quotient (RQ) method was used to assess the ecotoxicity of the selected BPs namely
BPA and BPS which generally detected in the water bodies of Citarum River. This approachhas been used elsewhere (Wang et al. 2022). For each compound of BPs, RQi value is theproportion of the maximum measured environmental concentration (MECi) to the predictedno-effect concentration (PNECi). The total RQ (TRQ) of BPs was calculated based on the5/7

following equation.
∑RQ (TRQ)= ∑RQi = ∑MECi / PNECi
The TRQ was the sum of RQ of each separately measured contaminant (RQi ) at eachsampling site. Generally, environmental risks are classified as high risk (RQ>1.0), mediumrisk (0.1<RQ<1), and low risk (0.01<RQ). In this study, the PNEC values are obtained fromdividing the minimum toxicity value (i.e., lowest short-term EC50 for BPS and long-term
NOEC value for BPA) in the literature by an assessment factor (AF) of 1000 and 100,
respectively (Wang et al. 2022). The toxicity values of BPA and BPS used in the present
study were derived from literature published study (Yan et al., 2017).
The range of Total RQ for the detectable of BPA and BPS in Citarum River was 0.000068 –0.99 (Figure 3). The result indicates that none of the sampling site had high risk to aquaticorganism (RQ<1). At all site, only 16 % of the river water bodies had moderate risk to river
ecosystem and at locations where BPs were detected (35 sites), 29 % of them represent
moderate risk. However, at one location in CR-3-32, the TRQ was close to 1 (0.99) which aborder of the value for high risk. Among BPs, BPA was the most contributor to total RQ ofwhich the all-moderate risk was contributed by BPA than BPS.

Figure 3. Calculated of risk quotient (RQ) for BPA and BPS as well total RQ (TRQ) for BPs.
Future Challenges.
This study highlights hot spot BPs contamination in specific locations of river bodies atCitarum River. In particular, current study found BPA and BPS were the only detected BPsand indicate common use of BPA and BPS in several areas around the Citarum River. BPA
was the most contributor to total RQ of which the all-moderate risk was contributed by BPA
than BPS. However, in order to meet the market demand for products, currently and time tocome, various alternatives to BPA are widely used in industrial production. Thus, futurecontamination of BPs other than BPA may will increase. Since some BPs, i.e., BPF, BPS,and BPAF, etc., had similar estrogenic effects and anti-androgenic effects to that of BPA, thus
risks may be significantly enhanced due to their elevated combined effects of complex
estrogenic contaminants. Therefore, it is necessary to take more effective actions todecrease the discharge of BPs as well as long-term monitoring study of these compounds.
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