Oral Presentations

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Society of Environmental Toxicology and Chemistry Asia-Pacific 2018 Conference

Society of Environmental Toxicology and Chemistry Asia-Pacific 2018 Conference

Oral Presentations September 17(Mon) ~ 19(Wed)

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SETAC AP 2018

Society of Environmental Toxicology and Chemistry Asia-Pacific 2018 Conference

The 11th Society of Environmental Toxicology and Chemistry - Asia Pacific 2018

Session 1-1 Multigenerational effect of thermal stress on life-history traits, oxidative stress and cellular energy allocation in Daphnia magna Hyungjoon IM1*, Joorim NA2, Jinho JUNG3 Korea University Summary

Fig. 2: Multigenerational effect of elevated temperature (25°C) on (a) growth and (b) reproduction of D. magna for 21 days. Values are presented as mean ± standard deviation (n = 12).

Influence of temperature on zooplanktons such as Daphnia magna is concerned mainly due to the expected environmental variability from climate change. The purpose of this study was to evaluate the effects of elevated temperature on oxidative stress responses, life-history traits and cellular energy allocation of Daphnia magna for multiple generations. Compared to those reared in 20°C, daphnids in 25 °C showed reduction in body length (growth) and anti-oxidative enzyme activities, while fecundity (reproduction) and intrinsic rate of population growth drastically increased for the early generations. However, in later generations, anti- oxidative enzyme activities and fecundity were recovered back to level of control. These results may indicate an adaptation process or a transgenerational effect on D. magna adjusting survival strategy throughout generations by giving up some traits to cope up with the extra energy demand for other traits.

Fig. 3: Multigenerational effect of elevated temperature (25°C) on (a) total energy reserve and (b) content ratio of the energy reserve of D. magna for 21 days. Values are presented as mean ± standard deviation (n = 3, p ≤ 0.05).

Fig. 1: Schematic experimental design for multigenerational study of D. magna in thermal stress

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The 11th Society of Environmental Toxicology and Chemistry - Asia Pacific 2018

developing embryos by analyzing their hepatic transcriptome with next generation sequencing (NGS). In

Session 1-2

addition, we revealed the sex differences in effects by comparing the gene expression profiles.

Effect of prenatal bisphenol a exposure on the hepatic transcriptome in chicken embryos: understanding of sexspecific effects

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Fertilized chicken (Gallus gallus domesticus) eggs were divided into five groups; control (corn oil), BPA-L (BPA 10 ng/g egg), BPA-M (BPA 1,000 ng/g egg), BPA-H (BPA 100,000 ng/g egg), and E2 (17 β-estradiol 10 ng/ g egg). On the 2nd incubation day, test chemicals were injected into the air sac. On the 21st incubation day,

Shohei ITO1 *, Midori LIDA1,2, Hoa Thanh NGUYEN1, Tetsuro AGUSA1,3, Masashi HIRANO1, 1

2. Materials and Methods

the liver and other tissues were collected by the dissection of embryos. In order to detect embryonic death,

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Mari OCHIAI , Eun-Young KIM , Hisato IWATA 1

eggs were observed on the incubation day 7, 14, and 21.

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Sex determination of embryos was conducted by amplifying the CHD1W and CHD1Z on the chicken Z and

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W chromosomes [6]. Genomic DNA used in PCR was extracted from muscle or liver samples.

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For the NGS analysis, RNA solutions were extracted from liver samples (n=3-4/treatment group). In order

Center for Marine Environmental Studies (CMES), Ehime University, Japan Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Japan Faculty of Environmental and Symbiotic Science, Prefectural University of Kumamoto, Japan

Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University,

Korea

to identify the differentially expressed genes (DEGs) by chemical treatment, gene FPKM (Fragments Per Kilobase of exon per Million mapped fragments) were statistically analyzed with cuffdiff. To predict the high-risk diseases and their mechanisms, the phenotype, gene ontology (GO), and

Summary

transcription factor enrichment analyses of the DEGs by BPA- or E2-treatment were carried out by R,

There is a growing concern about the toxic effects of bisphenol A (BPA) on developing embryos,

Comparative Toxicogenomics Database (CTD), TFactS, and Enrichr.

because the early life stage is sensitive to chemical exposure. This study investigated the effect of in ovo BPA exposure on chicken embryos by hepatic transcriptome analysis. In addition, we

3. Results and Discussion

revealed the sex differences in the effect on the transcriptome. To predict high-risk diseases

Survival rates were significantly decreased in the BPA-H, suggesting that prenatal exposure to BPA exerted

and their mechanisms, the phenotype, gene ontology (GO), and transcription factor enrichment

the developmental toxicity in avian embryos. Data analyses of NGS indicated that BPA affected gene

analyses were carried out. The transcriptome analyses indicated that BPA affected cell cycle-

expression profiles and in particular males in the BPA-M had the largest number of DEGs. Comparison of

related genes, which may lead to liver cirrhosis. Differential effects on the transcriptome between

DEGs between male and female embryos unveiled that males had more DEGs than females in BPA-M and

males and females were confirmed notably in high dose (100,000 ng/g egg) of BPA-treated

BPA-H, suggesting that males are more sensitive to BPA than females. Phenotype enrichment analysis

embryos. Cell cycle-related genes were up-regulated in males but down-regulated in females.

suggested that the liver cirrhosis may be induced by prenatal BPA exposure in developing embryos (Table 1).

These results suggest that BPA may cause developmental toxicity and its mode of action may be

GO enrichment analysis predicted the effects on the cell cycle in the BPA-treated embryos, suggesting that

different between males and females.

the prenatally disrupted cell cycle may trigger the liver cirrhosis during the postnatal period. Transcription factor enrichment analyses intimated that activation of MYC, TP53, and E2F by BPA exposure could be critical molecular initiating events in the adverse outcome pathway. Table 1. High-risk diseases identified by disease enrichment analysis of DEGs in BPA-H chicken embryos.

1. Introduction Bisphenol A (BPA) has long been used for the production of polycarbonates and epoxy resins. Because of its widespread and extensive usage, BPA has been detected in the environment such as river water, soil and wild animals including birds [1–3]. Exposure to BPA elicites various toxic effects such as endocrine disruption, developmental abnormalities in animals [4,5]. There is a growing concern about the toxic effects of BPA in developing organisms because the early life stage is highly sensitive to chemical exposure. However, there is less information regarding the effects of BPA on avian embryos. We thus performed in

ovo BPA injection test of chicken eggs and investigated the effects of BPA and their mechanisms on the

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Group

Disease Name Liver Cirrhosis, Experimental Polycystic Ovary Syndrome BPA-H_Male Thrombocytopenia Colonic Neoplasms Liver Cirrhosis, Experimental BPA-H_Female Myocardial Ischemia Heart Failure

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Related-DEGs 44 13 5 13 49 14 10

ALL1 516 80 12 95 516 116 69

p-value 1.8.E-04 1.9.E-04 6.2.E-04 8.3.E-04 2.5.E-13 1.0.E-05 4.8.E-05

FDR2 2.1.E-02 2.1.E-02 4.7.E-02 4.7.E-02 8.2.E-11 1.1.E-03 3.2.E-03

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Group

Disease Name Carcinoma, Hepatocellular Reperfusion Injury Carcinoma Liver Cirrhosis Cholestasis, Extrahepatic BPA-H_Female Infarction, Middle Cerebral Artery Leukemia, Myeloid, Acute Status Epilepticus Neurotoxicity Syndromes Hypertension 1

Society of Environmental Toxicology and Chemistry Asia-Pacific 2018 Conference

The 11th Society of Environmental Toxicology and Chemistry - Asia Pacific 2018

Related-DEGs 15 9 12 9 5

ALL1 158 58 115 63 17

p-value 6.6.E-05 7.1.E-05 1.6.E-04 1.3.E-04 2.5.E-04

FDR2 3.4.E-03 3.4.E-03 5.3.E-03 5.3.E-03 7.5.E-03

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3.8.E-04

9.8.E-03

9 7 4 10

81 53 17 120

6.9.E-04 1.1.E-03 2.1.E-03 2.7.E-03

1.6.E-02 2.4.E-02 4.2.E-02 5.0.E-02

Session 1-3 Differences in POPs and transcriptomes of wild and hatchery-reared baltic salmons Mirella KANERVA1 *, Nguyen Minh TUE2, Tatsuya KUNISUE2, Kristiina A M VUORI3, Hisato IWATA1 1

Ehime University, CMES, Lab. of Environmental Toxicology, Japan

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Ehime University, CMES, Lab. of Environmental Chemistry, Japan

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University of Turku, Department of Biology, Division of Physiology and Genetics, Finland

Summary

The number of genes related to the disease in the reference genome dataset of the chicken 2 False Discovery Rate

Hepatic persistent organic pollutants (POPs) levels and transcriptome profiles were compared between wild and hatchery-reared Baltic salmons, caught from three different areas of the Baltic

4. References

Sea. Differences between areas were more prominent than differences between wild and hatchery-

[1] Flint S, Markle T, Thompson S, Wallace E. 2012. Bisphenol A exposure, effects, and policy: A wildlife

reared fish, both in the POPs and transcriptomes. Cell cycle and apoptosis related KEGG pathways were enriched in wild fish, while lipid metabolism related pathways were enriched in hatchery-

perspective. J. Environ. Manage. 104:19–34. [2] Renz L, Volz C, Michanowicz D, Ferrar K, Christian C, Lenzner D, El-Hefnawy T. 2013. A study of parabens and bisphenol A in surface water and fish brain tissue from the Greater Pittsburgh Area. Ecotoxicology.

reared salmons. Non-dioxin-like PCBs and PBDEs had the most associations with transcriptome profiles. Keywords: salmon, wild, hatchery, POPs, transcriptome.

22:632–641. [3] Staniszewska M, Falkowska L, Grabowski P, Kwaśniak J, Mudrak-Cegiołka S, Reindl AR, Sokołowski A, Szumiło E, Zgrundo A. 2014. Bisphenol A, 4-tert-Octylphenol, and 4-Nonylphenol in The Gulf of Gdańsk

1. Introduction

(Southern Baltic). Arch. Environ. Contam. Toxicol. 67:335–347.

The increase of temperature in the Baltic Sea has been higher than in any other seas in the world during

[4] Lu B, Zhan P. 2010. The effects of bisphenol A on sex hormone levels of F0 female rats and F1 male rats

the last decades (Belkin, 2009) and it has been considered to be the most polluted sea in the world (HELCOM, 2010). Organisms living in the Baltic Sea have to cope with multiple stressors caused by human activities

during weaning period. Sect. Title Toxicol. 92:1729–1733. [5] Berg C, Halldin K, Brunström B. 2001. Effects of bisphenol A and tetrabromobisphenol A on sex organ

besides the challenges brought by a shallow and brackish environment. The marine survival rates of both wild and hatchery-reared Baltic salmon (Salmo salar), as well as other

development in quail and chicken embryos. Environ. Toxicol. Chem. 20:2836–2840. [6] Fridolfsson A-K, Ellegren H. 1999. A Simple and Universal Method for Molecular Sexing of Non- Ratite

fish species are declining and some of the reasons reported for this are increased temperature and changes in the food web composition (Peyronnet et al. 2008). Lower survival is especially a problem with

Birds. J. Avian Biol. 30:116–121.

reared fish, and is probably due to their lower adaptation capacity. The aim of this study is to investigate the molecular level differences in wild and hatchery-reared salmon to elucidate the effects of origin and environment. 2. Materials and Methods 2.1 Salmon samples Salmon samples were collected from the Baltic Sea in the end of the years 2007 and 2008. The fish were caught from Baltic main basin (BMB), Bothnian Sea (BS) and Gulf of Finland (GoF). A total of six or seven liver samples (some pooled) from wild and reared salmons for each area were used for transcriptomic and

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POPs analyses. The origin and the river stock were determined with DNA microsatellites (Vuori et al. 2012).

When comparing the hatchery-reared fish, lipid metabolism related pathways were the most enriched in all of the comparisons.

2.2. POPs, transcriptome and statistical analyses

Transcription factor analyses showed that cell cycle and apoptosis and lipid metabolism related

The POPs analyses of liver samples were preformed following the method of Tue et al., (2010, 2014).

transcription factors were the most abundant in all of the different comparisons.

Concentrations of PCBs and PBDEs were determined using GC-qMS, and organochlorine pesticides were measured using GC-MS/MS.

3.2. Connections between POPs and transcriptome

RNA was isolated using the Direct-zolTM RNA MiniPrep kit (Zymo Research). The transcriptome was

Co-inertia analysis (Fig 1) showed high correlation in the variances of the POPs and transcriptome data

analyzed by next generation sequencing, performed by Hokkaido System Sciences (HSS) using Illumina

(RV=0.74, p=0.048).

HiSeq 2500.

The FAMT analysis revealed the highest number of genes to be associated with non-dioxin-like PCBs and

Differentially expressed genes (DEG) among three areas and between wild and hatchery-reared fish

PBDEs, although PBDE concentrations were not statistically different between areas. Most of the genes,

were analyzed by HSS, using Salmo salar genome as a reference, BLAST was done against the zebrafish

associated with both contaminants, were connected to amino acid metabolism related pathways.

(Danio rerio). Differences in POPs levels were analyzed with ANOVA and Kruskal-Wallis, using SPSS. The

To conclude, the differences in transcriptome between areas were more prominent than those between

associations between contaminants and transcriptome were analyzed using co-inertia analysis (ade4

wild and hatchery-reared salmons, suggesting that POPs contamination may be one of factors that affect

package) and factor analysis for multiple testing (FAMT package) with R. Enrichment analyses (KEGG) were

the transciptome profiles.

done using ClueGO plug-in for Cytoscape. Transcription factor analyses were done with TFactS. 4. References 3. Results and discussion

Belkin, I. M., 2009. Rapid warming of Large Marine Ecosystems. Prog. in Ocean. 81(1-4)207-213.

3.1. POPs levels and transcriptome profiles

HELCOM, 2010. Hazardous substances in the Baltic Sea – An integrated thematic assessment of hazardous

Comparison of POPs levels between wild and hatchery-reared salmons indicated that there was a statistical

substances in the Baltic Sea.

difference only in the GoF for dioxin-like and non-dioxin-like PCBs. Comparison among three areas showed

Peyronnet, A., Friedland, K. D., Maoileidigh, N. O., 2008. Different ocean and climate factors control the

that the concentrations of dioxin-like PCBs were significantly higher in GoF than in BMB or BS and the non-

marine survival of wild and hatchery Atlantic salmon Salmo salar in the north-east Atlantic Ocean. Jour. of

dioxin like PCBs were higher in BS fish than in GoF or BMB. Chlordane levels were significantly higher in BS

Fish Biol. 73(4)945-962.

fish than in GoF and BMB, whil

Tue, N.M., Sudaryanto, A., Minh, T.B., Isobe, T., Takahashi, S., Viet, P.H., Tanabe, S., 2010. Accumulation of

T showed an opposite trend.

BS had the highest number of DEGs when comparing wild and hatchery-reared salmons, followed by

polychlorinated biphenyls and brominated flame retardants in breast milk from women living in Vietnamese

GoF and BMB. Nine transcripts were common to all areas and were related to metabolism and genetic

e- waste recycling sites. Sci Total Environ. 408(9):2155-62.

information processing. In comparisons between different

Tue, N.M., Katsura, K., Suzuki, G., Tuyen le, H., Takasuga, T., Takahashi, S., Viet, P.H., Tanabe, S., 2014.

areas within wild fish, BMB vs GoF had the most DEGs,

Dioxin-related compounds in breast milk of women from Vietnamese e-waste recycling sites: levels, toxic

followed by BMB vs. BS and BS vs. GoF. Within hatchery-

equivalents and relevance of non-dietary exposure. Ecotoxicol Environ Saf. 106:220-5.

reared fish, BMB vs. GoF had the highest number of DEGs,

Vuori, K., Kiljunen, M., Kanerva, M., Koljonen, M. L., Nikinmaa, M., 2012. Stock-specific variation of trophic

followed by BS vs. GoF and BMB vs. BS.

position, diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations

For comparisons of wild vs. hatchery-reared salmons,

in the Baltic Sea. Jour. of Fish Biol. 81(6)1815-1833.

lipid metabolism related pathways were enriched in both BS and GoF, no enriched pathways were found in BMB. When comparing wild salmons, the enriched pathways were mainly related to cell growth and death and signal transduction in BMB vs. BS or GoF, while in the comparison between BS vs. GoF the enriched pathways were related to lipid metabolism.

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Figure 1: Co-inertia between POPs (blue triangle) and trancriptome (red circle) data in salmon caught from different areas of the Baltic Sea. W: wild, H: hatcheryreared.

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Society of Environmental Toxicology and Chemistry Asia-Pacific 2018 Conference

in which red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 was expressed. To estimate the risk of

Session 1-4

PAHs in Kesennuma fish, we investigated rsAHR transactivation potencies of PAHs which were detected at high concentrations in the tissue of greenlings (Hexagrammos otakii) from Kesennuma. Based on the PAHs detected in the greenling tissues, six PAHs were chosen. Relative potencies (REPs) of these PAHs

In vitro and in silico ahr assays for assessing the risk of heavy oilcontaminated marine fish

compared to the potency of benzo[α]pyrene were calculated from the dose-responses obtained with rsAHR based-in vitro reporter gene assay. In addition, binding potencies of the six PAHs to each rsAHR were

Su-Min BAK1 *, Haruhiko NAKATA2, Dong-Hee KOH3, Jean YOO1, Hisato IWATA1, Eun-Young KIM3,4

performed using in silico docking simulations.

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Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University, Japan

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Graduate School of Science and Technology, Kumamoto University, Japan

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Department of Life and Nanopharmaceutical Science and 4Department of Biology, Kyung Hee University,

Korea

2. Materials and methods 2.1 In vitro reporter gene assay Transactivation responses of six PAHs including dibenzothiophene, phenanthrene, acenaphthene, 2,3,5trimethylnaphthalene, 1-methylphenanthrene and benzo[α]pyrene (98.6~99.6 % purity) (Enzo Life Sciences, Inc) were measured with the in vitro reporter gene assays according to the method previously reported (Bak et al. 2013). The 50 % effective concentration (EC50) values and/or low observed effective concentration (LOEC) values were obtained by using GraphPad 5.0 (San Diego, CA). Benzo[α]pyrene-REP 20, 50 and 80 (B[α]

Summary

P-REP20, -REP50 and –REP80) values of a PAH were calculated as the ratio of concentration that induces

By the Great East Japan Earthquake on March 11, 2011, marine fish in Kesennuma Bay, Japan have

20, 50 and 80 % of B[α]P maximum responses relative to the concentration of the PAH that induces the

been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate

response corresponding to benzo[α]pyrene.

the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene,

2.2 In silico AHR homology modeling and docking simulation

acenaphthene and 1-methylphenanthrene) of which high residue levels have been detected in the

The homology modeling for the ligand binding domain (LBD) of each rsAHR and the in silico docking

tissue of Kesennuma fish, we attempted to evaluate the effect of PAHs on the signaling pathway

simulation (ASEDock) of each PAH with the rsAHR LBD models were conducted using the Molecular

of fish aryl hydrocarbon receptor (AHR). We measured AHR-mediated responses of these PAHs

Operating Environment (MOE) software (Chemical Computing Group Inc.). Due to low identities of the

in the red seabream (Pagrus major) AHR1- and AHR2-derived reporter gene assays (Bak et al.

template, human hypoxia-inducible factor 2α (huHIF2α, PBD: 3H7W) with rsAHRs (25.7% for rsAHR1-LBD

2013). The in vitro assay showed AHR isoform-, PAH- and dose-dependent transactivation. In silico

and 26.6% for rsAHR2-LBD), four models were constructed for each rsAHR. U-dock values (kcal/mol) were

docking simulation analysis indicated that AHR activation potency of PAHs could be predicted from

calculated based on the energy of interaction between each PAH and rsAHR- LBD model.

the AHR2 model. This study claims that our in vitro and in silico analyses may be a useful tool for 3. Results and discussions

assessing the risk in marine fish contaminated with PAHs.

We measured rsAHR-mediated responses of PAHs in the in vitro reporter gene assay (Fig. 1). The results showed rsAHR isoform-, PAH- and dose-dependent transactivation responses except 1methylphenanthrene that showed no activation for both rsAHRs. When comparing REP values, benzo[α] pyrene showed over 500-fold higher potencies for the activation of rsAHRs than other PAHs (data not

1. Introduction

shown). This suggests that benzo[α]pyrene is the most contributor to AHR activation in Kesennuma fish.

By the aftermath of the Great East Japan Earthquake on March 11, 2011, the Sanriku coast in the Tohoku

In silico docking simulation analysis indicated that U_dock values of PAHs were significantly correlated with

Region was struck by a Tsunami. Furthermore, in Kesennuma, this tsunami destroyed oil storage tanks

their in vitro rsAHR2 activation potencies (Fig. 2). This suggests that the in vitro transactivation responses

and resulted in the release of approximately 11.5 million liters of heavy oil. The contamination with the heavy

were predicted from the PAHs-rsAHR2 docking models (Fig. 2). This study provided an insight into the

oil-derived polycyclic aromatic hydrocarbons (PAHs) have been monitored using bivalves and sediment

application of in vitro and in silico ligand screening systems as an alternative method for risk assessment of

samples in Kesennuma Bay (Onozato et al. 2016), and 27 PAHs were detected. However, the impact of PAHs

PAHs in fish.

pollution on marine fish from the oil spill site has not yet been investigated. Our previous study has constructed an in vitro aryl hydrocarbon receptor (AHR) reporter gene assay system

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Session 1-5 Dynamics and bioaccumulation of toxic microcystins on environmental multi-media samples in Geum River estuary, Korea Do-kyun KIM1,*, Seong-jin HONG2, Ho-Dong PARK3, Kyung-Hoon SHIN1 1

Department of Marine Sciences and Convergence Technology, Hanyang University, Korea.

2

Department of Ocean Environmental Sciences, Chungnam National University, Korea,

3

Department of Environmental Science, Faculty of Science, Shinshu University, Japan.

Fig. 1: Responses of PAHs via each rsAHR are presented as a value (%) relative to the maximum response of benzo[α] pyrene (% B[α]P max). Data are presented as mean ± standard deviation (SD) from six to eight replicated wells in two independent experiments.

Summary The objectives of this study were: i) to investigate load of MCs via dike of Geum River; ii) to determine multimedia fate of MCs in estuary; and iii) to elucidate bioaccumulation characteristics of MCs. In order to measure concentrations of MCs (MC-LR, -RR, and -YR), Surface water, suspended particles, sediments and organisms were collected from 7 stations (inside and outside of dike) in June and July 2017. In order to determine trophic position and carbon source of organisms, C and N stable isotope analyses were conducted. From inner to outer estuary in June, concentrations of dissolved and particulate MCs gradually decreased respectively. On the other hand, dissolved MCs concentrations increased after freshwater discharge. Concentrations of MCs in biota were negatively correlated with trophic positions, indicating that MCs did not seem to biomagnify in marine food web. Bioaccumulation factor of MCs showed a significant positive Fig. 2: Relationships between calculated U_dock values and LOEC (A) or B[α]P-REP values (B) obtained from in vitro rsAHR2driven reporter gene assay. The average and standard deviation were calculated from docking simulations of each PAH with three rsAHR2-LBD models (model number 1, 2, and 3).

4. References Bak, S.M., Iida, M., Hirano, M., Iwata, H., Kim, E.Y., 2013. Potencies of red seabream AHR1-and AHR2mediated transactivation by dioxins: implication of both AHRs in dioxin toxicity. Environmental Science & Technology, 47(6), 2877-2885. Onozato, M., Nishigaki, A., Okoshi, K., 2016. Polycyclic aromatic hydrocarbons in sediments and bivalves on the Pacific coast of Japan: influence of tsunami and fire. PloS one, 11(5), e0156447.

correlation with δ13C values, which seems to be associated with carbon sources of organisms.

1. Introduction Microcystins (MCs) cause toxicity (e.g., hepatotoxicity) and bioaccumulation in aquatic organisms and have been found in great concentrations in freshwater environments along with cyanobacterial blooms in recent decades (Ibelings and Chorus, 2007; Kozlowsky-Suzukia et al., 2012). The concentration of microcystin in the organisms has been reported to decrease with increasing trophic level (Larson et al., 2014), but microcystin accumulation was observed in the primary consumers which directly fed cyanobacteria (Jeon et al., 2015). Therefore, it is possible that microcystins resulting from high concentration of cyanobacteria when they are released through the estuaries, may enter the marine environment and affect the pelagic or benthic organisms. Although most studies of MCs dynamics in freshwater ecosystems have been focused, fate of MCs entering the coastal environments through rivers is largely unknown (Takahashi et al., 2014; Umehara et al., 2017). The objectives of this study were: i) to investigate load of MCs via dike of Geum River;

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ii) to determine multimedia fate of MCs in estuary; and iii) to elucidate bioaccumulation characteristics of

Larson, D., Ahlgren, G., & Willén, E. (2014). Bioaccumulation of microcystins in the food web: a field study of

MCs.

four Swedish lakes. Inland Waters, 4(1), 91-104.

2. Materials and methods

Overview of Problems Cyanotoxins Produced by Cyanobacteria and the Solutions Thereby. J. Korean Soc.

2.1. Sample collection and Analysis

Environ. Eng, 37(12), 657-667.

Bong-seok Jeon, Jisun Han, Seog-Ku Kim, Jae-Hwan Ahn, Hye-Cheol Oh, Ho-Dong Park. (2015). An

Surface water, suspended particles, and sediments were collected from 7 stations (inside and outside of

Takahashi, T., Umehara, A., & Tsutsumi, H. (2014). Diffusion of microcystins (cyanobacteria hepatotoxins)

dike) in June and July 2017. Biological samples including fishes, bivalves, gastropod, crab, and polychaete

from the reservoir of Isahaya Bay, Japan, into the marine and surrounding ecosystems as a result of large-

were collected from the estuarine areas. Concentrations of MCs (MC-LR, -RR, and -YR) were measured in

scale drainage. Marine pollution bulletin, 89(1-2), 250-258.

discharged water and marine multimedia samples using HPLC-MS/MS. In order to determine trophic

Umehara, A., Takahashi, T., Komorita, T., Orita, R., Choi, J. W., Takenaka, R., ... & Tsutsumi, H. (2017).

position and carbon source of organisms, C and N stable isotope analyses were conducted using EA-IRMS.

Widespread dispersal and bio-accumulation of toxic microcystins in benthic marine ecosystems.

Chemosphere, 167, 492-500. 3. Results and discussion 3.1. Load and multimedia fate of MCs via dike of Geum River Approximately 4,300 kg of MCs was discharged to the Geum River Estuary during the period from July to August. From inner to outer estuary in June, concentrations of dissolved and particulate MCs gradually decreased from 133.6 ng L-1 to
neutrally (48.8 ± 8.2%) > negatively (1.8 ± 0.5%) buoyant. This suggests that weathering percentages of oil affected the buoyancies of OSA. In relation to this study, test on weathered oil showed that formations of OSA reduced with increasing weathering percentages. With higher weathering percentage, OSA was mostly in the form of floating

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oil-water emulsions with air bubbles engulfed in the aggregates, or sinking oil-water-SPM aggregates

Session 11-2

(Tab. 1). Although OSA formed was highly unstable, the amount of negatively buoyant OSA increased with weathering.

Mesocosm photooxidation study of two types of petroleum

Tab. 1: The effects of weathering percentage of oil on OSA formations and their physical properties. Weathering of Density of Iranian heavy oil (g/ml) @ crude oil (%) 40 ℃ 0

Type of OSA formed

0.88

Droplet

Size of OSA (µ m) 2 L for river water) was performed using a SPE method using HLB (6 cm3,

γ-HBCD was dominant in industrial wastewater. Through treatment process, PBDEs were the most

200 mg) cartridges. The extracts were then divided in half for analyzing PBDEs, structural analogues

effectively removed. The lowest removal efficiency (10.3%) was observed for OH- and MeO-BDEs and

and TBBPA, HBCDs. The fraction for PBDEs and structural analogues were purified through silica

their concentration in effluent of human wastewater was increased at maximum 1.9 fold compared with

column. The fraction for TBBPA and HBCDs were cleaned on Florisil column. The structural analogues

influent. The estimated daily load of PBDEs was highest in sewage while that of TBBPA was highest in

of PBDEs were derivatized from the extracted PBDE fraction using the acetylation reaction of acetic

industrial wastewater.

anhydride with pyridine. Filter paper was extracted using a accelerated solvent extraction (ASE-300, Dionex). The PBDEs, OH-BDEs, and MeO-BDEs were analyzed using GC/HRMS while TBBPA and the HBCDs were analyzed using LC/MS/MS. Method limit of quantification was defined as S/N ratio of 10

1. Introduction

and were 0.005–0.25 ng/L for PBDEs, 0.2–150 pg/L for MeO- BDEs, 2.0–120 pg/L for OH-BDEs, 0.001–0.3

In the aquatic environment, WWTPs form the major source of diverse micropollutants into nearby

ng/L for TBBPA, and 0.005–0.25 ng/L for the HBCDs.

rivers or the ocean. An understanding of the phase-specific distributions and fates of environmental chemicals during wastewater treatment processes is required because strong oxidation, nitrification,

3. Results and Discussion

chlorination, and/or photochemical reactions during treatment processes can cause parent compounds

3.1. Occurrence of BFRs in wastewater Compared to River

to be transformed and degraded to form other compounds. However, some hydrophobic compounds

The concentrations of total BFRs in the influents was 1.2- to 12-fold of that in the effluents. The

such as PBDEs, which are representative BFRs, have mostly been investigated in particulate phase as

PBDE concentration in the effluent (c.f., STP) was generally similar to that in river water. The similar

sludge. To date, only a few studies have been carried out to investigate BFRs in wastewater. Compared

concentrations of PBDEs between effluent (20.8–393 pg/L) and river water (62.9–117 pg/L) showed

with PBDEs, which have been regulated since 2009, HBCDs and TBBPA have received little attention in

that the mass loading of dissolved PBDEs from the treatment plants was not negligible and could be

WWTPs. In this study, the occurrence and distribution of three kinds of most widely used BFRs, PBDEs,

a major source of PBDEs in the aquatic environment. In contrast, for TBBPA and HBCDs, four- and

HBCDs, and TBBPA, were investigated in various types of wastewater samples to investigate their

nine-fold concentrations, respectively, compared with that in river water, were found in WWTP effluent

dissolved phase distribution characteristics, removal efficiency and to assess their comparative release

that implied relative easy or fast degradation and dilution potency of those two BFRs in the water

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environment compared to PBDEs.

Session 17-3

3.2. Distribution Characteristics The mean BFR distribution patterns based on the total concentration of BFRs in the influents were compared among types of treatment plants using principal component analysis (Fig. 1). With input data that detected >60% from all types of samples, consisting of 20 PBDE congeners, 3 HBCDs and

Levels of PFAS and precursor compounds in Australian biosolids.

TBBPA fraction ratios for the four types of treatment plants (WWTP A, B, HTP and STP), two principal

1 1 1 1 1 1 1 1 1 Damien MOODIE TimothyCoggan COGGAN , SuzieReichman REICHMAN , Dayanthi NUGEGODA Bradley O. CLARKE Moodie1*,*,Timothy , Suzie , Dayanthi Nugegoda andand Bradley O. Clarke

components were extracted as PC 1 (49.0%) and PC 2 (18.2%).

1

Centre for Environmental Sustainability and Remediation, School of Science, RMIT University, Australia

Summary Per and poly-fluorinated compounds and their precursor were detected in Australian biosolids from a range of treatment plants servicing a mix of industry and residential consumers using a novel alkaline extraction technique coupled with analysis by LC-MS/MS. The PFAS with the highest mean concentration detected was for perfluorodecanoic acid (PFDA) at 11 ng/g with a maximum concentration of 40 ng/g. Precursor concentrations were highest for 6-2, 8-2 diPAP with maximum concentration of 433 ng/g and Fig. 1: PCA plot (A) and major congener distribution pattern (B) for influents from HTP, STP and WWTPs.

a mean across all sampling points of 112 ng/g, to the authors knowledge this is the first record of diPAP concentrations in Australian biosolids.

3.3. Mass Flow Analysis in STP The mass flow at each step and the change in mass load during a process were calculated from the BFR concentrations in whole wastewater to allow the fates of the BFRs at the different stages of the

1. Introduction

treatment process to be assessed (Fig. 2).

Per- and polyfluorinated compounds (PFAS) are a class of chemicals valued for their unique surfactant properties (Kissa 2001). PFAS are an environmentally persistent class of compounds owing to the strength of the fluorine carbon bond (Arvaniti et al. 2014). PFAS and their precursor compounds are used as additives to manufacturing process to help reduce surface tensions in liquids, provide stain free coatings, foaming and anti- foaming properties and their conductive properties (Kissa 2001). The disposal and breakdown of consumer and manufacturing products leads to the introduction of PFAS and its precursors to waste water treatment plants. Biosolids are the end user product for the waste water solid fraction and as such are able to accumulate PFAS adsorbed to solids during the treatment process which may pose an issue for biosolid reuse programs (Sepulvado et al. 2011). Precursor compounds including diPAPs in the WWTP also have the potential to transform into longer PFAS chain compounds following microbial degradation especially even longer chain carboxylic acids

Fig. 2: Schematic diagram of the STP, with the five sampling points marked 1–5 and the sludge marked *, and the estimated BFR mass loads (in mg/d) at each treatment step (blue arrows for HBCDs, green for TBBPA, and orange for PBDEs).

(Higgins et al. 2005; Lee et al. 2014). The aim of this study was to assess the contamination impact of waste water treatment plant (WWTP) derived PFAS and its precursors on Australian WWTP sludge especially biosolids.

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2. Materials and Methods

Lee, H, Tevlin, AG, Mabury, SA & Mabury, SA 2014, 'Fate of polyfluoroalkyl phosphate diesters and

Field collected biosolid samples from 20 different Australian treatment plants of varying size and

their metabolites in biosolids-applied soil: biodegradation and plant uptake in greenhouse and field

complexity of treatments, that cover a range of population equivalents and serve a mixed variety of

experiments', Environ Sci Technol, vol. 48, no. 1, pp. 340-9.

users were collected and analyzed for PFAS compounds using a novel alkaline extraction technique

Sepulvado, JG, Blaine, AC, Hundal, LS & Higgins, CP 2011, 'Occurrence and fate of perfluorochemicals

coupled with analysis by LC MS MS. The participating WWTP’s were sent replicate 250 mL Nalgene

in soil following the land application of municipal biosolids', Environ Sci Technol, vol. 45, no. 19, pp.

sample bottles (n=3) to collect stockpiled biosolids. The returned samples were extracted for 12 hours

8106-12.

in 10 mM MeOH/NaOH and cleaned using dispersive solid phase extraction before a final filtration prior to analysis. The final extracts for all samples were monitored for 43 PFAS compounds using isotope dilution with an optimized method for LC MS MS. A further 4 treatment plants were monitored using an extended optimized method for 52 PFAS compounds. Target PFAS in the samples were identified by comparing the retention time (tr) with the tr of standard solutions and quantification/confirmation ion transition ratios (Q/C). LC MS MS data was analyzed using Agilent Technologies MassHunter qualitative analysis software (v. B.06.00) and MassHunter quantitative analysis software (v. B.05.00). 3. Results and Discussion PFAS was detected in 100 % of the samples analyzed. Perfluorooctanoic acid (PFOA) was detected in 85% of the sample replicates and Perfluorooctanesulfonic acid (PFOS) was detected in 92% biosolid replicates. The highest concentration for PFOS in the biosolids was 27 ng/g and 21 ng/g for PFOA. The highest PFAS concentration was for perfluorodecanoic acid (PFDA) at 40 ng/g, PFDA was detected in 83% of the replicates. Precursor compounds were detected in 100% of the biosolid samples analyzed with the extended optimized method. The highest concentration among the analyzed precursor compounds was for 6-2, 8-2 diPAP at 430 ng/g. The second highest observed precursor concentration was for 6-2 diPAP at 300 ng/g. 8-2 diPAP was also detected with a maximum concentration of 77 ng/g. To the authors knowledge this is the first recorded detections of diPAP’s in Australian biosolids. The relative abundance of detected precursor compounds associated with longer chain even numbered carboxylic and sulfonic acids may be indicative of the potential for biodegradation and transformation of PFAS in Australian biosolids. This research was supported by an Australian Government Research Training Program (RTP) Scholarship. The Author acknowledges Water Research Australia Limited and Water Corporation (WA) for supporting this project. 4. References: Arvaniti, OS, Andersen, HR, Thomaidis, NS & Stasinakis, AS 2014, 'Sorption of Perfluorinated Compounds onto different types of sewage sludge and assessment of its importance during wastewater treatment', Chemosphere, vol. 111, pp. 405-11. Higgins, CP, Field, JA, Criddle, CS & Luthy, RG 2005, 'Quantitative Determination of Perfluorochemicals in Sediments and Domestic Sludge', Enironmental Science & Technology, vol. 39, no. 11, pp. 3946 -56. Kissa, E 2001, Fluorinated Surfactants and Repellents 2nd Edition, Marcel Dekker, Inc, New York.

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2. Materials and methods

Session 17-4

2.1. Chemical and analytical method Target compounds were 15 PFASs including four perfluoroalkylsulfonates and eleven

Occurrence and distribution of perfluoroalkyl substnaces in soil from different sources, South Korea

perfluoroalkylcarboxylic acids. The mixture of internal standard contained eight mass-labelled

Heejeong PARK1*, Jeong-Ki YOON2, Ji-In KIM2, Hyun-Koo KIM2, Jeong-Eun OH1,

of two recovery standards (M8PFOS [13C8 -PFOS] and M8PFOA [13C 8 - PFOA] was injected prior to

perfluoroalkyl substances (18O2 -PFHxS, 13C4 -PFOS, 13C2 -PFHxA, 13C 4 -PFOA, 13C5 -PFNA, 13C2 -PFDA, 13C2 -PFUnDA, and 13C2 -PFDoDA) was used as an internal standard, and the mixture

1

instrumental analysis and used to confirm recoveries of internal standard. Samples were extracted by

2

using ultra-sonication extraction (USE), and cleaned up with solid phase extraction (SPE) with using

Department of Civil and Environmental Engineering, Pusan National University, Republic of Korea National Institute of Environmental Research, Republic of Korea

Oasis WAX cartridges. The high performance liquid chromatography and tandem mass spectrometry

Summary

(HPLC-MS/MS) was used for the analysis of PFASs.

Perfluoroalkyl substances (PFASs) are manmade chemicals that have unique oil- and water-repelling

2.2. Sample collection

properties, enabling them to be useful in industrial and commercial applications. They exhibit long

Totally 63 soil samples were collected in August 9th to September 3rd, 2017. The samples were divided

range transport, readily adsorbing to soil system, thereby detectable levels have been reported in soil but

into two types: surface soil (n = 58) and subsurface soil (n = 5). The surface soil refers to the soil of the

limited studies on PFASs soil contamination in Korea have been investigated so far. Therefore, totally 63

upper 30-cm layer, and the subsurface soil refers to the soil collected at 30-60 cm. A total of 58 surface

soil samples were collected from various sources comprising industrial and landfill area, to investigate

soil from industrial (n = 32), landfill (n = 8), mountain (n = 8), and rural area (n = 10), and subsurface soil

PFASs in association with the land use. The total PFAS concentrations varied from 0.175 to 25.8 ng/

samples at each type of land use were collected. Each soil sample was a composite of five sub-samples

g (median = 2.43). Soil samples from industrial area showed clearly higher PFOS concentrations. In

obtained from the center and four corner of an area of 5 m x 5 m grid using a stainless steel auger after

addition, statistically significantly higher total PFAS concentrations were observed in industrial soil than

carefully removing stones and vegetation from the sampling surface. Approximately, 500 g of soil sample was collected and placed in a pre- cleaned bottle, transported to the laboratory, and stored below -20°C

those in mountain- (p < 0.01), and rural soil (p < 0.05) by using Mann-Whitney U test.

for further treatment and analysis. 3. Results and discussion

1. Introduction Perfluoroalkyl substances (PFASs) are manmade chemicals that have unique oil- and water-repelling

The total PFAS concentrations varied from 0.175-25.8 ng/g (median = 2.43). The most frequently detected

properties, enabling them to be useful in industrial and commercial applications since 1940’s (Giesy and

PFASs were PFOS, PFOA, PFNA and PFDA with the median concentrations of 0.937 ng/g, 0.298 ng/g,

Kannan, 2001; Prevedouros et al., 2006). In 2009, perfluorooctane sulfonate (PFOS) production and use

0.314 ng/g, and 0.231 ng/g, respectively. The concentration of total PFAS in soil observed in this study

were restricted under the Stockholm Convention for Persistent Organic Pollutants because exposure to

was generally lower than those in the previous study from the United States (PFOS: 5.5-125.7 ng/g dw;

them can lead to adverse human health effects (Stockholm Convention, 2009). Despite of the regulation

PFOA: 0.2-28.2 ng/g dw), and from China (Total PFASs: 41.4-220 ng/g dw) (Wen et al., 2014; Xiao et al.,

on PFASs, they have been emitted into the environments due to their widespread use either from direct

2015). However, PFOS levels were clearly higher in industrial soil, and total PFAS concentrations in

sources as well as from indirect sources. Among various environmental media, soil could act as

industrial soil was also significantly higher than those in mountain (p < 0.01), and rural soil (p < 0.05) by

an important sink for PFASs through atmospheric deposition, precipitation, and etc. (Rankin et

using Mann-Whitney U test. Meanwhile, no significant difference found in the levels between industrial-

al., 2016), and can be contaminated by land application of biosoilds, usage of AFFFs, or leaching of

and landfill soil. Higher levels were observed in industrial soil in this study seem to related with PFAS-

landfill leachate. However, relatively few studies of PFASs reported on soil, also only suspected to be

related industries such as textile, metals, electronics, and chemicals (Kim et al., 2016). Furthermore, in

contaminated area (i.e. industrial area, AFFF-impacted sites, or bio-amended soil) have been previously

addition to the industrial activities, landfill leachate is known to a secondary source of PFASs (Fuertes et

investigated (McGuire et al., 2014; Sepulvado et al., 2011; Wang et al., 2013). Therefore, there remains to

al., 2017). Among landfill soil samples, one industrial landfill soil showed the highest total concentration,

be preferentially screened PFAS in soil to understand their occurrence and fate in soil by focusing on the

with as high as 96% contribution of PFCAs to total PFASs. This result implies this industrial landfill soil samples could be affected by landfill leachate, since landfill leachate have reported to have approximately

land use.

90% contribution of PFCAs to total PFASs (Fuertes et al., 2017). Detailed discussion on the possible

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relationship between PFAS concentrations and land use will be presented in the conference.

Session 17-5

4. References Fuertes, I., Gómez-Lavín, S., Elizalde, M.P., Urtiaga, A., 2017. Perfluorinated alkyl substances (PFASs) in northern Spain municipal solid waste landfill leachates. Chemosphere 168, 399–407. Giesy, J.P., Kannan, K., 2001. Global Distribution of Perfluorooctane Sulfonate in Wildlife. Environ. Sci. Technol. 35, 1339–1342. Kim, H.-Y., Seok, H.-W., Kwon, H.-O., Choi, S.-D., Seok, K.-S., Oh, J.E., 2016. A national discharge load of perfluoroalkyl acids derived from industrial wastewater treatment plants in Korea. Sci. Total Environ.

Species and habitat-dependent accumulation and biomagnification of brominated flame retardants and their metabolites 1 1 2 2, Jeong-Eun 1OH1 Gyojin Choo CHOO In-Seok LEE *,*,In-Seok Lee , Jeong-Eun Oh 1

Department of Civil and Environmental Engineering, Pusan National University, Republic of Korea

2

Marine Environment Research Division, National Institute of Fisheries Science, Republic of Korea

563–564, 530–537. McGuire, M.E., Schaefer, C., Richards, T., Backe, W.J., Field, J.A., Houtz, E., Sedlak, D.L., Guelfo, J.L., Wunsch, A., Higgins, C.P., 2014. Evidence of Remediation-Induced Alteration of Subsurface Poly- and Perfluoroalkyl Substance Distribution at a Former Firefighter Training Area. Environ. Sci. Technol. 48, 6644–6652. Prevedouros, K., Cousins, I. t., Buck, R.C., Korzenoiwski, S.H., 2006. Sources, Fate and Transport of Perfluorocarboxylates Environ. Sci. Technol. 40, 32–44. Rankin, K., Mabury, S.A., Jenkins, T.M., Washington, J.W., 2016. A North American and global survey of perfluoroalkyl substances in surface soils: Distribution patterns and mode of occurrence. Chemosphere 161, 333–341. Sepulvado, J.G., Blaine, A.C., Hundal, L.S., Higgins, C.P., 2011. Occurrence and Fate of Perfluorochemicals in Soil Following the Land Application of Municipal Biosolids. Environ. Sci. Technol. 45, 8106–8112. Wang, P., Wang, T., Giesy, J.P., Lu, Y., 2013. Perfluorinated compounds in soils from Liaodong Bay with concentrated fluorine industry parks in China. Chemosphere 91, 751–757.

Summary In this study, we identified three representative brominated flame retardants (BFRs), comprising polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs), and tetrabromobisphenol A (TBBPA), and their potential metabolites such as methoxylated- (MeO-) and hydroxylated (OH-) BDEs in seawater, sediment, and biota from the southern part of South Korea to understand their fates, distribution, and biomagnification according to the species and habitat. The concentration of HBCDs and OH-BDEs was higher in vertebrate and in invertebrate, respectively, as species differences, while the concentration of TBBPA and MeO-BDEs was higher in pelagic fish compared to that in benthos. Finally, HBCDs, MeO-BDEs, and some of PBDEs were shown the high bioconcentration and biomagnification factors (BCF and BMF), indicating higher potential on both bioaccumulation and biomagnification.

Wen, B., Li, L., Zhang, H., Ma, Y., Shan, X.-Q., Zhang, S., 2014. Field study on the uptake and translocation of perfluoroalkyl acids (PFAAs) by wheat (Triticum aestivum L.) grown in biosolids-amended soils. Environ. Pollut. 184, 547–554. Xiao, F., Simcik, M.F., Halbach, T.R., Gulliver, J.S., 2015. Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in soils and groundwater of a U.S. metropolitan area: Migration and implications for human exposure. Water Res., Occurrence, fate, removal and assessment of emerging contaminants in water in the water cycle (from wastewater to drinking water) 72, 64–74.

1. Introduction The trophic magnification of BFRs and their metabolites have been steadily evaluated for marine biota by the relationship between their concentration and trophic level or using trophic magnification factor (TMF). Previously, it was reported that the clear linearity of HBCDs between their concentration and lipid content and the strong trophic magnification of HBCDs through lipid transfer (Xian et al., 2008). However, trophic transfer of PBDEs have not been reached on consensus results (Kelly et al., 2008; Poma et al.,

5. Acknowledgement This work was supported by a grant from the National Institute of Environment Research (NIER), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIER-201730480001).

2014), and TBBPA, MeO- and OH-BDEs have received a little attention. Moreover, although the reporting of different distributions of BFRs and their metabolites in marine environmental matrixes like sediment, seawater, and biota (Gu et al., 2017; Choo et al., 2017), relatively limited studies on their bioaccumulation depending on the species or habitat effects were performed. Therefore, in this study, we identified three representative BFRs, including PBDEs, HBCDs, and TBBPA, and their potential metabolites (MeO- and OH-BDEs) in seawater, sediment, and biota from southern part of South Korea to understand their fates, distribution, and biomagnification. Vertebrate and invertebrate were compared to investigate different

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patterns according to their species, and habitat-specific distribution was also evaluated by comparison

fishes similarly with the distribution of seawater in this study. Additionally, the concentration of MeO-

between pelagic fish and benthos (demersal fish and invertebrate). Finally, the relationship between

BDEs was statistically higher in pelagic fish rather than that in benthos, implying that the formation and

BFR concentration and trophic level of biota, TMF, and BCF were investigated and compared to evaluate

uptake of MeO-BDEs seemed to be much active in pelagic fishes living near surface water where marine

their biomagnification and bioaccumulation potential.

sponge or algae live with high intensity of sunlight (Choo et al., 2017). The details on the bioaccumulation and biomagnification potential will be discussed during the conference.

2. Material and Method Biota were collected at the southern part of South Korea from January 26 to February 10, 2015 and

Table 1. The concentration of BFRs and their metabolites in seawater, sediment, and each species of

categorized by two types into vertebrate and invertebrate. All vertebrate was 14 fish species, and

biota.

they were separated into eight pelagic and six demersal fishes to identify the habitat effect on bioaccumulation. Invertebrate and demersal fishes were categorized as the benthos where live near surface sediment. In addition, surface seawater and sediment samples were obtained at the same site

Compound

Seawater (pg/L)

Sediment (ng/g dry wt)

PBDEs

1.04-741

MeO-BDEs OH-BDEs

where biota collected to investigate the surrounding environmental effect in their habitat. Seawater samples were loaded by an automated solid phase extraction system (SPE-DEX; Horizon Technology, Salem, NH, USA), and loaded 50 mm Atlantic C18 disks were eluted with hexane/dichloromethane (DCM) (1:1) for PBDEs and metabolites. For HBCDs and TBBPA, SDB-XC was used, then disks were extracted by an accelerated solvent extractor (ASE350; Dionex, Sunnyvale, CA, USA) with hexane/DCM (1:1). For

Vertebrate (ng/g lipid wt)

Invertebrate (ng/g lipid wt)

Pelagic fish

Demersal fish

0.332-4.79

1.04-13.8 (8.32) 2)

3.11-72.9 (8.51)

5.59-59.4

ND1)-8.18

6.15-61.5 (17.6)

0.956-8.52 (2.03) 0.182-4.65 (1.89)

0.114-0.671

ND-18.5

ND-8.76 (3.18)

0.072-0.753 (0.342) 2.48-40.7 (9.68)

1.79-65.3 (14.3)

HBCDs

27.7-195

3.63-120

5.73-60.1 (13.7)

2.45-31.3 (16.2)

ND-7.38 (4.41)

TBBPA

0.035-0.538

ND-2,794

1.31-11.3 (5.42)

ND-4.45 (0.595)

ND-8.11 (4.43)

extraction of sediment samples, an accelerated solvent extractor was used with elution of hexane/DCM

1)

(1:3) for PBDEs and metabolites, and ultrasonication extraction was used by adding hexane/DCM (1:1) for

2)

ND: not detected, ( ): median value

HBCDs and TBBPA. All biota samples were extracted by using an ultrasonication extraction method with mixture of hexane, DCM, and methyl tert-butyl ether (MTBE). Finally, all extracted samples were cleaned

4. References

up by multi-layer silica gel or florisil column. The PBDEs and MeO-BDEs were quantified by GC-HRMS,

Choo, G., Kim, D.-H., Kim, U.-J., Lee, I.-S., Oh, J.-E., 2017. PBDEs and Their Structural Analogues in

and OH-BDEs, HBCDs, and TBBPA were analyzed by LC-MS/MS.

Marine Environments: Fate and Expected Formation Mechanisms Compared with Diverse Environments. J. Hazard. Mater.

3. Results and Discussion

Gu, S.-Y., Ekpeghere, K.I., Kim, H.-Y., Lee, I.-S., Kim, D.-H., Choo, G., Oh, J.-E., 2017. Brominated

The concentrations of BFRs and their potential metabolites in seawater, sediment, and biota were

flame retardants in marine environment focused on aquaculture area: Occurrence, source and

shown in Table 1. The concentration of HBCDs was statistically higher in vertebrate compared to that

bioaccumulation. Sci. Total Environ. 601–602, 1182–1191.

in invertebrate (Mann-Whitney U-test, p10 µm.

was revealed. And the solution with particles was filtered by 10 µm and 0.8 µm polycarbonate filter paper. For determining the fragmentation rate of EPS, filtered particles were weighted. The

3.4. Fragmentation rate of EPS

filtered solution progressively became yellow in color with increasing exposure time. It implies

By the weight loss of EPS cube, the fragmentation rate was calculated. It requires approximately 4.7

that the fragmented particles are migrating into the solution.

years to lose 50% of the weight of EPS cube. And approximately 4.2 mg/cm2 of fragmented micro- and nanoparticles could be produced in 4.7 year by sunlight weathering.

2.3. Particle analysis 4. References Gigault, J., Pedrono, B., Maxit, B., Ter Halle, A., 2016. Marine plastic litter: the unanalyzed nanofraction. Environ. Sci. Nano. 3, 346-350. Figure 1. The process of collecting fragmented and produced particles from weathered EPS samples

Song, Y.K., Hong, S.H., Jang, M., Hang, G.M., Jung, S.W., Shim, W.J., 2017. Combined effects of UV

Nano- and micro-sized EPS particles were confirmed scanning electron microscope (SEM), SEM-

exposure duration and mechanical abrasion on microplastic fragmentation by polymer type. Environ.

x-ray energy dispersive spectroscopy (EDS) and fluorescence microscopy after Nile Red staining in

Sci. Technol. 51, 4368-4376.

all sized groups. The produced particles were divided by three sized groups by filtration (10 µm). The images and qualitative analysis of particles were analyzed using their average size and distribution was determined using dynamic light scattering (DLS) and nanoparticle tracking analysis. 3. Results and Discussion 3.1. Confirmation of micro- and nanoparticle The produced particles had laminar shape. On the aluminum background, the carbon based element particle was confirmed using EDS. It was approximately less than 300 nm. The carbon electron in green color implies that it is a polystyrene particle. It shows that nanoplastics were apparently produced by sunlight exposure. 3.2. Nanoparticle ( 70%. Three compounds, PFOA,

in the whole data set, there was a significant (pPFHxS>PFPeA>6:2FTS>PFHpA>PFBS>PFPeS>PFDA> PFNA>8:2FTS>PFHpS>PFDoA. The

Keywords: PFAS, wastewater, recycled water, PFOS, PFOA, PreFAS

1. Introduction Per- and polyfluoroalkyl substances (PFAS) are a class of fluorinated compounds that have found many commercial applications due to their stability and unique properties. Once in the environment, these same properties make them persistent, mobile and potentially toxic (Ahrens and Bundschuh, 2014). WWTPs are a conduit of PFAS to the environment with PFAS detected in WWTP influent, effluent and solids worldwide (Arvaniti and Stasinakis, 2015). Furthermore, treatment has been demonstrated to increase some PFAS concentrations in final effluent (Arvaniti and Stasinakis, 2015, Filipovic and Berger, 2015) due to biodegradation of compounds such as perfluorosulfonamides (FOSAs), fluorotelomer phosphate diesters (diPAPs) and fluorotelomer sulfonoates (FTSs) which are considered as PFAS precursor compounds (PreFAS) (Eriksson and Kaerrman, 2015, Houtz et al., 2016). This study quantified PFAS

298

daily discharge rate of ∑PFAS at the studied WWTPs ranged from 0.05 to 113 g day-1. At all WWTPs except three, ∑PFAS increased from influent to final effluent. Linear mixed effects analysis revealed a significant difference (p50% of the total OPFRs. TBOEP was

elucidate partitioning of OPEs among vapor phase, particulate phase and indoor dust. The indoor

the predominant compound in indoor dust (>80%).

dust was collected by sweeping floors and window sills. After sampling, PUF and GF/F samples were immediately wrapped in methanol-pre-cleaned aluminum foil at the site, sealed in polyethylene bags and shipped to the laboratory. Samples were kept in a refrigerator (- 20 °C) until analysis. 2.2. Sample Extraction, Analysis and QA/QC Prior to extraction, sampler parts and all materials that come into contact with samples were thoroughly rinsed with organic solvent. Sample extraction was performed as quickly as possible after sample collection, and were analyzed within a week. PUF plugs (for vapor phase analysis) were spiked with internal standards, and mechanically shaken for 15 min and extracted by ultrasonicaion. The GF/F filters (for particulate phase analysis) were extracted by accelerated solvent extraction (ASE-200, Dionex). The dust samples were weighed after sieving (180 µ m) and extracted as that of GF/F samples. The extracts were analyzed by HPLC-ESI- MS/MS. Procedural blanks, field blanks, travel blanks,

Fig. 1: Composition of 15 OPFRs in vapor phase, particulate phase of indoor air and indoor dust from Albany, New York State .

laboratory blanks, duplicate sample, and matrix spike samples were analyzed as a part of QA/QC and all detected amounts of OPEs in blanks were subtracted from concentrations measured in air and dust

4. References

samples.

van der Veen, I., de Boer, J. Chemosphere. 2012, 88 (10), 1119-1153.

3. Result and discussion 3.1. Concentrations of OPEs in Indoor Air The total concentrations of 15 OPEs (∑15OPEs) in bulk air (summated concentration in vapor and particulate phase) ranged between 2.96 and 635 ng/m3 (mean: 101 ng/m3). Among the investigated categories, samples collected from automobile parts shops showed the highest concentrations of OPEs in indoor bulk air at an average concentration of 258 ng/m3, followed by electronics shops (mean: 224 ng/m3), nail salon and house construction tools/chemicals/and other products shops (mean: 193 ng/m3) in that order. Indoor air samples collected from homes (mean: 109 ng/m3) and cars (mean: 59.0 ng/m3) were at least 1.7 and 3.3 fold lower than those found in other indoor environments,

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while a monitoring of organo-chlorinated pesticide and bisphenol A in marine fish samples collected

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from local market was reported by Santhi et al., (2012). Similar trend was also observed for molluscs, when most of the pollution assessment for this biota matrix from Malaysian marine and costal water

Presence of emerging organic contaminants in fish and mollusc from klang river estuary, malaysia and assessment on human health risk 1*

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2

Tuan Fauzan Tuan OMAR , Ahmad Zaharin ARIS , Fatimah Md. YUSOFF , Shuhaimi MUSTAFA 1

Department of Environmental Sciences, Faculty of Environmental Studies, Universiti Putra

Malaysia, Malaysia. 2

and Cheng et al., (2015). Based on literature search, there were no available studies assessing trace organic pollution in the mollusc from Malaysian marine and coastal environment. Therefore, this study was the first to provide information on the concentration of multiclass organic micro-pollutants ECs in fish and molluscs samples of Malaysian marine and coastal area. Risk assessment was also carried out to evaluate the effect of these compounds toward human health.

Laboratory of Halal Science Research, Halal Product Research Institute, Universiti Putra

Malaysia, Malaysia. 3

were focused only on trace elements analysis as reported by Edward et al., (2010), Yap et al., (2013)

Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, Malaysia.

2. Results and discussion Emerging pollutants were presence in most of the fish samples analysed with 10 of the compounds were detected in Arius thalassinus while 8 compounds were presence in Pennahia annea . Diclofenac was detected at the highest concentration in both fish species with the concentration of 10.76 ng g-1

Summary

in Arius thalassinus and 5.69 ng g-1 for Pennahia annea . Besides diclofenac, another compounds that

The occurrence and distribution of multiclass emerging contaminants (ECs) in fish

should be highlighted during this field assessment were bisphenol A and amoxicillin. Both compounds

and mollusc from Klang River estuary was assessed. The targeted ECs consist of phenolic endocrine disrupter, organo phosphorous pesticides, estrogenic hormones and pharmaceutically active compounds. Results showed that prevalent contamination of

were found in all fish samples analysed. Concentration of bisphenol A ranged from 0.92 ng g-1 to 5.79 ng g-1 for Arius thalassinus and 1.42 ng g-1 to 2.53 ng g-1 for Pennahia annea. As for amoxicillin, the concentration ranged from 0.32 ng g-1 to 2.13 ng

Klang River estuary by emerging pollutants with diclofenac, bisphenol A, progesterone and

g-1 in Arius thalassinus and 0.29 ng g-1 to 3.66 ng g-1 for Pennahia annea. Meanwhile for mollusc, ten

amoxicillin were predominantly detected in the fish and mollusc. Diclofenac was detected at

of the compounds were detected in the samples with the highest concentration was progesterone at

the highest concentration in fish samples, while progesterone was the highest concentration

9.57 ng g-1, followed by diclofenac (7.41 ng g-1) and primidone (3.59 ng g-1).

presence in the mollusc. Health risk assessment, calculated as hazard quotient (HQ) for

Risk assessment, calculated as the Hazard Quotient (HQ) for diclofenac were relatively higher than

three compounds, diclofenac, bisphenol A and progesterone were less than 1, suggesting the

bisphenol A and progesterone in the all the biota samples evaluated. The HQ value for diclofenac in

consumption of fish and mollusc from Klang River estuary did not pose any health risk to the population. Nonetheless, this preliminary assessment is an important finding for pollution studies in tropical coastal ecosystem and can be served as baseline database for future reference. Keywords: Emerging contaminants; endocrine disrupting compounds; Klang River estuary; fish; mollusc; health risk assessment.

Arius thalassinus was 5.739 x 10-2, Pennahia annea (3.035 x 10-2), Nerita lineata for HLM (8.82 x 10-3) and Nerita lineata for ALM (4.41 x 10-3). As for bisphenol A, the HQ value for Arius thalassinus was 3.86 x 10-3, Pennahia annea (1.69 x 10-3), Nerita lineata for HLM (4.4 x 10-4) and Nerita lineata for ALM (2.2 x 10-4). Meanwhile for progesterone, the HQ value for Arius thalassinus was 1.7 x 10-4, Pennahia annea (1.6 x 10-4), Nerita lineata for HLM (9.0 x 10-5) and Nerita lineata for ALM (1.9 x 10-4). 3. Conclusion

1. Introduction

Assessment on the occurrence and potential health risk associated with the presence of ECs in fish

Determination and monitoring of pollutants in the Malaysian commercial fish products were mostly

and mollusc samples were investigated in the present study. Wild fish samples from two different

carried out for trace metals analysis as reported by Hajeb et al., (2009), Bashir et al., (2012), Mok et al.,

species (sea catfish, Arius thalassinus and big eye croaker, Pennahia annea ), and marine gastropod

(2012) and Taweel et al., (2013). Notably, there were limited studies reported on the levels of organic

mollusc, Nerita lineata were collected from selected sampling location along Klang River estuary.

micro-pollutant ECs in fish samples particularly for phenolic endocrine disrupter and pharmaceutically

Bisphenol A, diclofenac, progesterone and amoxicillin were predominantly found in the fish and

active compounds. Several studies on the levels and occurrence of dioxins/furans and dioxin-like PCBs

mollusc samples. Diclofenac was detected at the highest concentration in sea catfish, A. thalassinus

in the Malaysian fisheries products were reported by Mohamad et al., (2013) and Leong et al., (2014)

(10.76 ng g-1) and in big eye croaker, P. annea (5.69 ng g-1), while progesterone (9.57 ng g-1) was the

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highest concentration found in the mollusc samples. Health risk assessment calculated for three

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compounds, diclofenac, bisphenol A and progesterone showed HQ value less than 1, suggesting the consumption of fish and mollusc from Klang River estuary will not pose any health risk to the consumers. Result obtained from this preliminary assessment is an important breakthrough for pollution studies in Malaysian coastal ecosystem particularly for organic micro-pollutant ECs and can be served as baseline database for future reference. References

New ways for mussels to take microplastics from the environments Huahong SHI 1*, Jiana LI 1, Qipei LI 1, Prabhu KOLANDHASAMY 2 1

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, China

2

Coastal and Marine Ecology Division, Gujarat Institute of Desert Ecology, India

[1] Bashir, F.A., Shuhaimi-Othman, M., Mazlan, A.G., Evaluation of trace metal levels in tissues of two commercial fish species in Kapar and Mersing coastal waters, Peninsular Malaysia, J. Environ. Public Health, 2012 (2012) 1-10.

Summary

[2] Cheng, W.H., Yap, C.K., Potential human health risks from toxic metals via mangrove snail

The presence of microplastics in organisms is of concern because of their ecological risks and

consumption and their ecological risk assessments in the habitat sediment from Peninsular Malaysia,

the potential hazard to human health. Up to date, ingestion has been widely accepted as the sole

Chemosphere, 135 (2015) 156-165

way for the animals to uptake microplastics. Nevertheless, microplastics have also been

[3] Edward, F.B., Yap, C.K., Ismail, A., Bioaccumulation and distribution of heavy metals (Cd, Cu, Fe,

found in some organs which are not involved in the process of ingestion. In the present study,

Ni, Pb and Zn) in the different tissues of chicoreus capucinus lamarck (Mollusca: Muricidae) collected

we collected mussels from the fishery farms, conducted exposure/clearance experiments and

from Sungai Janggut, Kuala Langat, Malaysia, Environ. Asia 3 (2010) 65-71.

analyzed the accumulation of microplastics in specific organ of mussels. Our results strongly

[4] Hajeb, P., Jinap, S., Ismail, A., Fatimah, A.B., Jamilah, B., Abdul Rahim, M., Assessment of mercury

suggest that adherence rather than ingestion led to the accumulation of microplastics in those

level in commonly consumed marine fishes in Malaysia, Food Control, 20 (2009) 79–84.

organs which are not involved in ingestion process. In addition, microbeads even fused into the

[5] Leong, Y.-H., Gan, C.-Y., Majid, M.I.A, Dioxin-like polychlorinated biphenyls, polychlorinated dibenzo-

byssus of mussels. To our best knowledge, it is the first time to propose that adherence and

p-dioxins, and polychlorinated dibenzofurans in seafood samples from Malaysia: Estimated human

fusion are novel ways for animals to take microplastics beyond ingestion. This new finding makes

intake and associated risks, Arch. Environ. Contam. Toxicol. 67 (2014) 21–28.

us rethink about the bioavailability, accumulation and toxicity of microplastics to aquatic animals.

[6] Mok, W.J., Senoo, S., Itoh, T., Tsukamasa, Y., Kawasaki, K., Ando, M., Assessment of concentrations

Keywords: microplastics, ingestion, adherence, fusion, bioavailability

of toxic elements in aquaculture food products in Malaysia, Food Chemistry 133 (2012) 1326–1332. [7] Mohamad, A., Azlan, A., Razman, M.R., Ramli, N.A., Latiff, A.A., Polychlorinated biphenyls (PCBs) concentration in demersal fish and shellfish from West Coast of Peninsular Malaysia, J. Food Agric.

1. Introduction

Environ. 11 (2013) 1094-1098.

In the assessment of ecological risks associated with microplastics, one of major issues is their

[8] Santhi, V. A., Hairin, T., Mustafa, A. M.. Simultaneous determination of organochlorine pesticides

availability to marine organisms. Microplastics have a similar size range to planktonic organisms and

and bisphenol A in edible marine biota by GC-MS. Chemosphere, 86(10) (2012) 1066–1071.

other suspended particles due to their small dimensions, thereby making them readily be taken as

[9] Taweel, A., Shuhaimi-Othman, M., Ahmad, A.K., Assessment of heavy metals in tilapia fish

the food by many aquatic animals (Cole et al., 2013). It seemed that ingestion would be the sole

(Oreochromis niloticus) from the Langat River and Engineering Lake in Bangi, Malaysia, and evaluation

way for animals to uptake microplastics from the surrounding environment. However, in recent

of the health risk from tilapia consumption, Ecotoxicol. Environ. Saf. 93 (2013) 45–51.

laboratory exposure experiments, microplastics have also been found in other specific organs other

[10] Yap, C.K., Cheng, W.H. Distributions of heavy metal concentrations in different tissues of the

than stomach and intestine. For instance, microbeads are not only found in the gills of mussels

mangrove snail Nerita lineata, Sains Malays. 42 (2013) 597-603.

and crabs but also on the surface of foot of zooplanktons (Setala et al., 2016; Watts et al., 2016; Wegner et al., 2012). It indicates that animals have alternative ways to uptake and accumulate microplastics beyond ingestion.

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2. Results and Discussion

5. References

In our current studies, we collected mussels from the fishery farms, conducted exposure/clearance

Cole, M., Lindeque, P., Fileman, E., Halsband, C., Goodhead, R., Moger, J., Galloway, T.S., 2013.

experiments and analyzed the accumulation of microplastics in specific organs of mussels. Results

Microplastic ingestion by zooplankton. Environ. Sci. Technol. 47, 6646-6655.

showed that microplastics could accumulated in mantle and foot of mussels which were not directly

Kolandhasamy, P., Su, L., Li, J., Qu, X., Jabeen, K., Shi, H., 2018. Adherence of microplastics to soft

involved in the ingestion process (Kolandhasamy et al., 2018). Furthermore, microplastics could also

tissue of mussels: A novel way to uptake microplastics beyond ingestion. Sci. Total Environ. 610-611,

adhere to and even fuse into the byssus of mussels, which were observed and identified using

635-640. Setala, O., Norkko, J., Lehtiniemi, M., 2016. Feeding type affects microplastic ingestion in a

SEM analysis (Fig. 1). It suggested that microplastics could be transferred into the body of organism

coastal invertebrate community. Mar. Pollut. Bull. 102, 95-101.

by other ways in addition to ingestion.

Watts, A.J., Urbina, M.A., Goodhead, R., Moger, J., Lewis, C., Galloway, T.S., 2016. Effect of Microplastic on the Gills of the Shore Crab Carcinus maenas . Environ. Sci. Technol. 50, 5364-5369. Wegner, A., Besseling, E., Foekema, E.M., Kamermans, P., Koelmans, A.A., 2012. Effects of nanopolystyrene on the feeding behavior of the blue mussel (Mytilus edulis L .). Environ. Toxicol. Chem. 31, 2490-2497.

Fig. 1. Adherence and fusion of microplastics in byssus of mussels. The morphology of byssus of mussels (A); Fusion (A-B) and adherence (C-G) of microplastics were observed.

3. Conclusion Based on results we found in the mussels, we propose that adherence and fusion are novel ways for animals to take microplastics beyond ingestion. 4. Acknowledgement This work was supported by the Natural Science Foundation of China (41571467) and grants from National Key Research and Development Programs (2016YFC1402204).

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2. Materials and methods To investigate the impact of MPs on marine ecosystem, microalgae (Chlorella saccharophila) and bivalve Manila clam (Venerupis philippinarum) were used as test species. Two test MPs had 53-63 (small sphere,

Polyethylene microplastic impacts on microalgae and bivalve in marine ecosystem

SS) and 180-212 (large sphere, LS) μm diameter, respectively. C. saccharophila was exposed to two MPs

Yooeun CHAE*, Dasom KIM, Youn-Joo AN

photosynthetic activities were analyzed by flow cytometry and chlorophyll fluorimeter. V. philippinarum was

Department of Environmental Health Science, Konkuk University, Republic of Korea

exposed to two MPs for 24 and 48 hours and the morphologies were analyzed after H&E staining.

on the end-over-end rotator for 6 days and cell size, cell granularity, cell autofluorescence, and

3. Results and discussion Summary

3.1 MP impacts on microalgae

In these days, plastic wastes have become emerging pollutants in marine environments and

After exposed to MPs, cell size, granularity, and autofluorescence were rarely affected by MPs. However, the

many researchers are focusing on their impacts and effects in ecosystem. Among these plastic

photosynthetic activities of C. saccharophila were slightly affected by MPs exposure. These results indicate

wastes, microplastics (MP, < 5mm) have been highlighted because of their characteristics like

that plastic pollution in marine ecosystem can cause adverse effects on microalgae and these effects lead

ubiquitousness, large surface/volume ratio, edible size to microfauna, and difficulties to remove.

to disturbance of marine ecosystem via food chain. Because microalgae is primary producer and important

In this study, we conducted lab scale experiments to investigate the impacts of polyethylene

food resource of microfauna, the impacts of plastic pollutions on microalgae in marine ecosystem can be

(PE) MPs with different sizes on two marine organisms, microalgae and bivalve. Each organism

serious problem.

was exposed to PE MPs for 6 days and 24 and 48 hours, respectively, and their growth and -fluorescence were rarely affected by MPs, but photosynthetic activities slightly changed. On the

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affected by MPs and these effects can cause the disturbance of marine ecosystem in various ways.

1. Introduction Plastic pollution is one of the emerging environmental issues worldwide (Thompson et al., 2009; Rochman et al., 2013), and the interests about plastic pollutions and their impacts on ecosystems are increasing (Gross, 2013). There have been various researches about plastic pollutions and the effects of plastic debris on the organisms in aquatic environments (Chae and An, 2017) but their effects have not been clearly revealed yet. Among these plastic wastes, microplastics (MP, < 5mm) have been highlighted because of their

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Consequentially, marine organisms can be

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characteristics like ubiquitousness, large surface/volume ratio, edible size to microfauna, and difficulties to remove. Many researchers have tried to quantify the amount of plastic debris in marine environments and to investigate the effects of these MPs in marine ecosystems. In this study, we also tried to evaluate the impacts of MPs on marine microalgae and bivalve using polyethylene (PE) MPs.

Fig. 1 (A) Cell size, (B) cell granularity, (C) cell autofluorescence, and (D) photosynthetic activity of C. saccharophila exposed to small MP and large MP for 6 days

3.2 MP impacts on bivalve The organs of V. philippinarum were affected by MPs exposure for 24 and 48 hours. They were damaged by MPs and (A)these mean MPs can cause physical impacts (B)on bivalve and induce physiological disorder in organisms

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Toxicological effects and histological observation of polyethylene microplastics in mytilus galloprovincialis Jin soo CHOI1*, Kyungil PARK2, June-woo PARK1 1

Korea Institute of Toxicology Gyeongnam Department of Environmental Toxicology and Chemistry,

Republic of Korea 2

Fig. 2 (A) Gill and (B) intestine of V. philippinarum after exposure to small MP and large MP for 24 hours, respectively (Scale bar = (A) 100 and (B) 200 μm)

Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National

University, Republic of Korea

4. References Chae, Y., An, Y.J., 2017. Effects of micro-and nanoplastics on aquatic ecosystems: Current research trends

Summary

and perspectives. Mar. Pollut. Bull. 124, 624-632.

We examined the toxic effect of M. galloprovincialis when exposed to polyethylene microplastics of

Gross, M., 2013. Plastic waste is all at sea. Curr. Biol. 23, R135–R137.

63 μm in size at concentrations of 0.5 μg/L (environmental concentration) and 50 mg/L (maximum

Rochman, C.M., Hoh, E., Hentschel, B.T., Kaye, S., 2013. Long-term field measurement of sorption of

concentration) for 21 days. There were no mortalities (LC50 > 50 mg/L) during the exposure

organic contaminants to five types of plastic pellets: implications for plastic marine debris. Environ. Sci.

period, and there was no significant difference in the growth changes observed in the shell hight,

Technol. 47,

shell length, and weight. The ALP test showed that the VTG level was slightly increased at the

1646–1654.

highest concentration (50 mg/L) as compared to the control group. Microplastics was found only

Thompson, R.C., Swan, S.H., Moore, C.J., Vom Saal, F.S., 2009. Our plastic age. Philos. Trans. R. Soc. Lond.

in mussels exposed to 50 mg/L concentration and accumulated in gill, stomach, stilos capsule,

Ser. B Biol. Sci. 364, 1973–1976.

secondary duct and intestine of mussels. Hemocyte infiltration and digestive tubular atrophy are found in the surrounding organs where microplastics accumulate, suggesting that there is a physiological disorder due to microplastics. The effects of polyethylene microplastics on marine mussels Mytilus galloprovincialis were investigated. Mussels are commonly found on coasts around the world, and because of their filtration feeding, they have biologic characteristics suitable for ingestion and concentration of microplastics and are widely used for marine environmental monitoring and polluted organisms (Avio et al. 2017). A number of microplastic toxicity studies have been conducted on mussels, but most have investigated toxic effects in vivo accumulation, recently, physiological responses using cellular or histological methods (von Moos et al., 2012; Avio et al., 2015; Santana et al., 2018). In this study, we examined the toxic effect of M. galloprovincialis when exposed to polyethylene microplastics of 63 μm in size at concentrations of 0.5 μg/L (environmental concentration) and 50 mg/L (maximum concentration) for 21 days. There were no mortalities (LC50 > 50 mg/L) during the exposure period, and there was no significant difference in the growth changes observed in the shell hight, shell length, and weight. The ALP test showed that the VTG level was slightly increased at the highest concentration (50 mg/L) as compared to the control group. Microplastics

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was found only in mussels exposed to 50 mg/L concentration and accumulated in gill, stomach, stilos capsule, secondary duct and intestine of mussels. Hemocyte infiltration and digestive tubular atrophy are found in the surrounding organs where microplastics accumulate, suggesting that there is a physiological disorder due to microplastics. This study will provide a broad understanding of the impact of microplastics on mussels in laboratory environments.

Identifiacation of mechanisms of that drive toxicity of HDPE microplastics: C. elegans transcription factors RNAi library screening approach Youngho KIM1*, Seungki LEE2, Inhee CHOI2 and Jinhee CHOI1,

References

1

School of Environmental Engineering, University of Seoul, Republic of Korea

2

Deaprtment of Life Science, University of Seoul, Republic of Korea

Avio, C.G., Cardelli, L.R., Gorbi, S., Pellegrini, D., Regoli, F., 2017. Plastics and microplastics in the oceans: From emerging pollutants to emerged threat. Mar Environ Res. 128, 2-11. Avio, C.G., Gorbi, S., Milan. M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L.,

Summary

Regoli, F., 2015. Pollutants bioavailability and toxicological risk from micriplastics to marine mussels.

High-density polyethylene (HDPE), one of the plastics, is commonly used. The contamination

Environ Pollut.

of aquatic system with HDPE-microplastics (MPs) is an emerging as a global environmental

198, 211-222.

concern. However, the mechanism of toxicity of HDPE on aquatic species and human health

Santana, M.F.M., Moreira, F.T., Pereira, C.D.S., Abessa, D.M.S., Turra, A., 2018. Continuous exposure to

is not fully understood. Our strategy was to begin with the nematode C. elegans transcription

microplastics does not cause physiological effects in the cultivated mussel perna perna. Arch Environ

factors (TFs) RNAi library screening test to gain insight into toxicity- related pathways and the

Contam Toxicol. 74, 594-604.

selected pathways were confirmed in C. elegans and zebrafish. To develop this, we treated HDPE

Von Moos, N., Burkhardt-Holm, P., Köhler A., 2012. Uptake and effects of microplastics on cells and

MPs to Age-synchronized RNAi-fed worms, reproduction of RNAi-fed worms was subsequently

tissue of the blue mussel Mytilus edulis L. after an experimental exposure. Environ Sci Technol. 46,

investigated by counting the number of off-springs using complex object parametric analyzer

11327-11335.

and sorter (COPAS). Bioinformatics analysis was subsequently conducted on those TFs using KEGG and REACTOME database for toxicity pathway identification. C. elegans TFs RNAi screening with HDPE revealed the nucleotide excision repair (NER) and TGF-β signaling pathway as being significantly altered by HDPE exposure. The expression of related pathway genes was confirmed in C. elegans after exposure to HDPE. The same experiment was conducted in zebrafish and the gene expression patterns were similar to what was seen in C. elegans. Overall, the study suggests that HDPE may cause bulk damage to DNA and activation of the NER system and regulates cell cycle regulation through TGF-β signaling pathway. Our results also suggest that these conserved mechanism of toxicity may also appear in the human by HDPE exposure Keywords: Microplastics, High density polyethylene, C. elegans transcription factors RNAi library, Zebrafsih

1. Introduction MPs can be classified into various types according to monomer structure such as polyamide (PA), polyethylene (PE), polypropylene (PP), polystyrene (PS), polypropylene (PP), and polyvinyl chloride (PVC). MPs are induced their toxicity according to size, shape and chemical composition. Previously, it was reported that PS induced inflammation in the gut of zebrafish, ingestion of PE results in increased

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Daphnia magna mortality and various types of MPs induced acute toxicity in grass shrimp. However,

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regulation were popped up.

the mechanism of toxicity of MP on aquatic species and human health is not fully understood. In this study, we screened the toxic mechanism of MPs on C. elegans using HDPE. HDPE is commonly used in beverage bottles, personal care product bottle and grocery bags. HDPE is found in aquatic and terrestrial environment and is expected to take in the range of hundreds of years until fully degradation. To understand mechanism of toxicity of HDPE, C.elegans TF RNAi screening was performed followed by confirmation with C.elegans and zebrafish models. 2. Materials and Methods

C. elegans exposed to 0, 100, 200, 500 and 1000 mg/L HDPE MPs in K-media for 72 h. Reproduction and Lifespan were investigated. Based on this results, C. elegans RNAi screening was conducted using 384 Transcription factors RNAi library. Age-synchronized RNAi fed worms were treated with HDPE MPs, and reproduction of RNAi fed worms was subsequently investigated by counting the number of off-springs using complex object parametric analyzer and sorter (COPAS). Bioinformatics analysis was subsequently conducted on those TFs using KEGG and REACTOME database for toxicity pathway identification. To verify this, the expression of related pathway genes was confirmed in C.

elegans after exposure to HDPE. The same experiment was conducted in zebrafish To thoroughly investigate the mechanisms of that drive toxicity of HDPE microplastics, Pathway analysis was conducted by KEGG mapper and REACTOME pathway browser using the TFs RNAi screen data of cut

Table 1. List of RNAi related to HDPE toxicity

off 20 % of top or bottom. We further analyzed gene expression level to identify whether the candidate mechanisms were relevant in C. elegans as well as in zebrafish model. 2.1. HDPE decreased lifespan and fecundity of C. elegans First, to investigate whether HDPE induce toxicity in C. elegans, we investigated lifespan and fecundity at 100, 200, 500 and 1000 mg/L HDPE concentration. Exposure to graded concentrations of HPDE caused a dose- dependent decrease in the number of offspring. After 10 days, the average life span showed a survival curves and average life span decreased significantly under the long-term exposure. (Figure 1) 2.2. C. elegans transcription factors RNAi screening reveals potential toxicity pathways of HDPE

Figure 1. Effect of HDPE on reproduction and lifespan in C. elegans

To investigate the mechanism in HPDE-induced toxicity, we performed high-throughput C. elegans reproduction assay using the 384 transcription factors RNAi bacterial library based on rrf-3(pk1426) mutant. HDPE was treated at 200 mg/L, which leads to an approximately 30% decrease in reproduction for C. elegans (Figure 1). Among 384 TFs, we showed 19 RNAi candidates that caused rescued or exacerbated reproductive toxicity at more than 2-fold compared than EV-fed worm (Table 1). To further understand the mechanisms of toxicity, we conducted pathway analysis based on Table 2 using the KEGG mapper and REACTOME software. As a result, Nucleotide excision repair system, TGF-β signaling pathway and cell cycle

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Figure 2. Effect of HDPE on NER pathway in C. elegans

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2.3 Nucleotide excision repair system is activated to HDPE exposure To verify candidate mechanisms of toxicity, the number of offspring was analysed in NERdeficient C. elegans exposed to 100, 200, 500 and 1000 mg/L HDPE-MPs. The number of offspring of T16H12.4 RNAi-fed worms was reduced than EV RNAi-fed worms at all concentrations. Expression of the NER pathway genes was investigated in C. elegans exposed to 100, 200, 500 and 1000 mg/L of HDPE-MPs. As a results, Increased NER gene expression level was observed at all concentrations (Figure 3C), suggest that HDPE-MPs induced the NER system (Figure 2). 2.4. TGF-beta signaling pathway is involved in HDPE-induced toxicity

Figure 4. Effect of HDPE on NER pathway and TGF-β signaling in Zebrafish

The number of offspring was also analysed in TGF-β-deficient C. elegans exposed to 100, 200, 500 and 1000 mg/L HDPE-MPs. The number of offspring of efl-1 RNAi-fed worms was reduced than

3. Conclusion

EV RNAi-fed worms at all concentrations. Expression of the TGF-β signaling pathway genes was

In this study, the mechanism of toxicity of HDPE-MPs was identified in an unbiased way, using

investigated in C. elegans exposed to

C. elegans RNAi screening, which was later extrapolated to a zebrafish model. Using two

100, 200, 500 and 1000 mg/L of HDPE-MPs. As a results, Increased TGF-β gene expression level

complementary model organisms has the potential to reveal a much more detailed view of the

was observed at all concentrations, except daf-3 (Figure 3). These results suggest that HDPE-MPs

mechanism of toxicity than when used individually. Thus, simultaneous use of complementary

alter the TGF-β signaling pathway (Figure 3).

models is a prudent way to diagnose chemical toxicity and plan for chemical management. Our study also suggest that HDPE-MPs alter NER pathway and TGF-beta signaling. Further research is needed to identify whether these alteration lead to the possibility of disease outcome in human. Acknowledgement: This work was supported by a grant from the Korean Ministry of Environment through ‘Environmental Health R&D Program’ (2017001370001). 4. References Wright, S. L.; Kelly, F. J., Plastic and Human Health: A Micro Issue? Environ Sci Technol 2017, 51, (12), 6634-6647.

Figure 3. Effect of HDPE on NER pathway in C. elegans

Ivleva, N. P.; Wiesheu, A. C.; Niessner, R., Microplastic in Aquatic Ecosystems. Angew Chem Int 2.5 Alteration of NER pathway and TGF-beta signaling by HDPE exposure is conserved in zebrafish model

Edit 2017, 56, (7), 1720-1739.

We next investigated whether the toxicity mechanism identified in the C. elegans model is conserved in other model organisms, such as zebrafish. The NER pathway and TGF-β signaling

Cozar, A.; Echevarria, F.; Gonzalez-Gordillo, J. I.; Irigoien, X.; Ubeda, B.; Hernandez-Leon, S.;

genes identified in C. elegans are equivalent to those in zebrafish, such as NER(xpc, xpa, gtf2g2,

Palma, A. T.; Navarro, S.; Garcia-de-Lomas, J.; Ruiz, A.; Fernandez-de-Puelles, M. L.; Duarte, C. M.,

and ercc) and TGF-β(tgfb2b, tgfbr2b, smad4a and e2f5) were investigated in zebrafish following

Plastic debris in the open ocean. P Natl Acad Sci USA 2014, 111, (28), 10239-10244.

exposure to HDPE-MPs. A similar trend was also observed with the HDPE-MPs (Figure. 4). Although a clear exposure dose-response was not observed, this result suggests that alteration of

Napper, I. E.; Bakir, A.; Rowland, S. J.; Thompson, R. C., Characterisation, quantity and sorptive

NER pathway and TGF-beta signaling by HDPE-MPs exposure is conserved in zebrafish model.

properties of microplastics extracted from cosmetics. Mar Pollut Bull 2015, 99, (1-2), 178-185.

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column and bottom water were collected and analyzed at eight semi-enclosed bays and coastal areas (41 stations and 123 samples) in South Korea in from July to August, 2016. Along with this insitu observation data and microplastic abundance data reported in the literature and bioassay data

Ecological risk assessment of microplastics in salt water envionment

available in literature, preliminary ecological risk assessment was conducted.

Won Joon SHIM1,2*, Young Kyoung SONG1,2, Soeun EO1,2, Sang Hee HONG1,2

2. Materials and methods

1

Korea Institute of Ocean Science and Technology, Republic of Korea

Horizontal and vertical distribution and composition of microplastics were determined in seawater in

2

Korea University of Science and Technology, Republic of Korea

along the coast of South Korea in July and August, 2016. Each 100 L of top 20 cm of surface water by grab sampling and mid-water column and bottom water by submerged pump sampling at a station was filtered through a 20-μm mesh net. The volume-reduced water samples were filtered through a 5

Summary

μm filter paper and all plastic like particles on the filter papers were identified by spectroscopy using

Microplastic in surface water, water column and bottom water of Korean coastal waters were

micro-FTIR.

determined down to 20 mm in size. Along with this in-situ observation data and microplastic abundance data reported in the world and bioassay data available in literature, preliminary

Microplastic abundance in seawater reported in literature and laboratory bioassay of particle toxicity

ecological risk assessment was conducted. The highest microplastic abundance observed in

including both micro- and nano-sized plastic particles and fibers were collected.

saltwater environment is about three orders of magnitude lower than the most sensitive toxic effects reported for water flea. However, microplastic abundance in seawater is likely to increase

3. Results and discussion

with current increasing rate of plastic production. Precautionary approach is highly required

3.1. Spatiotemporal distribution and characteristic of microplastic

to reduce input or standing stock of plastic debris. In addition, big uncertainty still exists for

The microplastic abundance was in the range of 10-3,480 particle/m3. The mean abundance of

assessment of ecological risk due to limited taxa in bioassay especially saltwater species,

plastics from surface waters (1,778±1,161 particle/m3) was significantly higher (Kruskal-Wallis test;

limited chronic toxicity data, and data based on non-standardized bioassays. It is highly

p < 0.05) than those in middle and bottom (433±339 and 403±444 particle/m3). Fragment type of

recommended to conduct harmonized and well-designed microplastic monitoring and bioassay

microplastics was dominant (83%) and it was followed by fibers. The microplastics less than 300 μm in

for ecological risk assessment.

size accounted for 86% except for fiber type, while in fibers, size < 300 μm only accounted for 29%.

Keywords: Microplastics; exposure and effect analysis; characterization of ecological risk The mean abundance of microplastics in seawater reported worldwide ranged from 4.8 ´ 10-6 particle/ m3 in eastern equatorial Pacific to 8.6 ´ 103 particle/m3 off the Swedish coast, except for a surface 1. Introduction

microlayer concentration of 1.6 ´ 104 particle/m3, showing a maximum difference of nine orders of

Ecological risk assessment of microplastics is based on the exposure as well as effect analysis.

magnitude. The median of the 70 mean values was 8.9 ´ 10-2 particle/m3, and 45% of studies reported

However, due to wide size spectrum of microplastics, there has been a mismatch of microplastic

mean abundances between 0.01 and 10 particle/m3.

size in monitoring and laboratory toxicity tests. Majority of monitoring studies in water have reported microplastic abundance larger than 300 mm using neuston nets, while bioassays have been conducted

3.2. Ecological risk assessment

in laboratories with microplastic generally less than 300 mm in size and many cases well below 1

The environmental levels of >20 μm microplastics in Korean coastal waters were compared with

mm. Furthermore, there is another mismatch of habitats for monitoring and organism exposure.

the exposure levels causing adverse biological effects by particles in laboratory toxicity test including

Most of monitoring studies have focused on top 20 cm of surface water in offshore and open oceans

the reported values in the literature. Ecological risk was assessed preliminarily with currently

where light plastics are floating and accumulating, while large number of species is expected to be

available exposure and effect data. When only lethal concentration (LCx) or effect concentration (ECx)

exposed in water column and benthic habitats rather than sea surface in coastal areas. In addition,

concentrations of microplastics were used for effect analysis, the lowest toxic level reported so far in

there has been a lack of studies for assessing environmental risk of microplastics to support decision

the literature including both acute and chronic toxicity of microplastic particle was 5,500 particles/L

making. To overcome these knowledge gaps, microplastic down to 20 mm in surface as well as mid-

of polyester fiber (280±50 mm) with LC10 for waterflea (Ceriodaphnia dubia) (Ziajahromi et al., 2017).

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This level was three orders of magnitude higher than the highest concentration (3.48 particles/L) found

Session31-1

in this study. So far environmental levels in coastal areas of South Korea and the microplastic levels reported in seawater in the world (see review Shim et al., 2018) are below acute and chronic toxic effect level (n/L) by over 1,000 times. Although different sizes and different shapes still make assessing ecological risk difficult, current levels of detectable microplastics of 0.02-5 mm in seawater are unlikely to cause an effect by ingestion. However, microplastic abundance in environments including seawater

Potential AhR-active chemicals in oil components determined using effect-directed analysis: A review

is likely to increase with current increasing rate of plastic production. Precautionary approach is highly

Seongjin HONG1,*, Junghyun LEE2, Un-Hyuk YIM3, Jong Seong KHIM2

required to reduce input or standing stock of plastic debris which can be a source of microplastic by

1

Department of Ocean Environmental Sciences, Chungnam National University, Republic of Korea

weathering process. In addition, big uncertainty still exists for assessment of ecological risk due to

2

School of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul National

limited taxa in bioassay especially saltwater species, limited chronic toxicity data, and data based on non-standardized bioassays. It is highly recommended to conduct harmonized and well-designed

University, Republic of Korea 3

Oil and POPs Research Group, Korea Institute of Ocean Science and Technology (KIOST), Republic of Korea

microplastic monitoring and bioassay for ecological risk assessment 4. References

Summary

Ziajahromi S, A Kumar, PA Neale, FDL Leusch. 2017. Impact of microplastic beads and fibers on

Crude oil consists of a complex mixture of hydrocarbons with varying properties that can

waterflea (Ceriodaphnia dubia) survival, growth, and reproduction: implications of single and mixture

cause severe ecosystem damage, when they are released into the ocean. In the present review,

exposures. Environmetnal Science and Technology. 51:13397-13406.

studies on identification of major aryl hydrocarbon receptor (AhR)-active chemicals in crude oil components using effect-directed analysis (EDA) were collected and summarized the key

Shim WJ, SH Hong, S Eo. 2018. Marine Microplastics: Abundance, Distribution and Composion. In:

findings. Among the thousands of chemicals in crude oil, alkyl-substituted 3-

Microplastic Contamination in Aquatic Environments . Ed. Zeng E., Elsevier.

4-ring polycyclic aromatic hydrocarbons (PAHs) and sulfur and nitrogen contained heteroaromatics were found to be the major AhR agonists. Those chemicals are resistant to oil weathering and appeared to be persistent in the environments. Further studies on identification of the unknown toxic chemicals in oil components would be needed using non-targeted screening in the near future. EDA approach will be useful for prediction of toxicities of residual oil in the environments and designation of priority monitoring chemicals in oil spilled area. Keywords: effect-directed analysis, crude oil, PAHs, alkyl-PAHs, Aryl hydrocarbon receptor

1. Effect-Directed Analysis EDA has become a powerful tool for identification of key toxicant(s) in environmental mixtures such as crude oil. EDA is based on a combination of biotesting, fractionation, and chemical analysis (Fig. 1), which facilitates identification of key toxicants in environmental samples (Hong et al., 2006). It is very important to identify the major toxic substances in the monitoring and management of oil-contaminated sites. Many researchers have attempted to identify major toxic substances in the oil component through EDA approach over the past decade. However, identifying toxic causative agents among the various components of oil seems to be challenging due to their great complexity and mixture toxicity. In this review, we highlighted the current status of our understandings on major toxic chemicals in oil components using EDA, particularly focusing on the AhR-active compounds.

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Bioassay & endpoint AhR activity (CAFLUX, H1G1-flu)

Sample type Organic extracts of worm, sediments, and crude oils Crude and weathered oils

Fractionation

Dosing

SARA & RPHPLC (log Kow)

EtOH

Open column & HPLC

AhR activity (H4IIE-luc)

Crude oil

AhR activity (CALUX, H4IIE-luc)

Porewater from crude oiled and weathered oiled sediment Oiled sediment Crude oil

AhR activity (CALUX, H4IIE-luc)

Fig. 1: Scheme of effect-directed analysis (EDA) of crude oil and oil-contaminated sediments (modified from Hong et al., 2016).

2. EDA Methodology We collected and reviewed all the research papers that have successfully identified the major AhR agonists using the EDA approach. Crude oil, weathered oil, and oil-contaminated sediments were mainly analyzed in the EDA studies. The samples were fractionated into several fine fractions by use of open column chromatography (based

AhR activity (H4IIE-luc) AhR activity (H4IIE-luc)

Time (h) 6, 24

Major AhR agonist

Instrumental analysis GC-FID & GC-MSD

Reference

DMSO 0.8%

4

Alkyl-substituted three and four-ring PAHs including S and N contained heteroaromatics Three to four ring (alkyl)-PAHs such as C4-phenanthrene, C1-chrysene, and C3-chrysene Three to four ring PAHs

GC×GC −TOFMS

Radovićet al. (2014)

Silica gel column & NPHPLC (aromatic ring number) Boiling point fractions

DMSO 0.1%

72

GC-MSD

Hong et al. (2015)

DMSO 0.25%

6

HPLC & GC-FID

Jonker et al. (2006)

Silica gel column Distilled fractions

DMSO 0.1% DMSO 0.1%

72

PAHs and resins

GC-MSD

4, 72

Aromatics with > 360°C boiling points

GC-MSD

Kim et al. (2017) Lee et al. (2018)

Aromatic and resinlike chemicals with log Kow of 5–8

Vrabie et al. (2012)

Fig. 1: Scheme of effect-directed analysis (EDA) of crude oil and oil-contaminated sediments (modified from Hong et al., 2016).

4. References on chemicals polarity), reverse phase HPLC (based on log Kow), and distillation method (based on boiling

Hong, S. et al., 2016. Effect-Directed Analysis: Current Status and Future Challenges. Ocean Sci. J. 51, 413-

point). The AhR-mediated potency was measured mainly by use of a recombinant cell line such as H4IIE-

433. Hong, S. et al., 2015. Effect-directed analysis and mixture effects of AhR-active PAHs in crude oil and in

luc. Finally, identification of toxicants in highly potent fractions was determined using GC-FID, GC-MSD,

coastal

and/or GC×GC−TOFMS (Tab. 1).

sediments contaminated by the Hebei Spirit oil spill. Environ. Pollut. 199, 110-118. Jonker, M.T.O. et al., 2006. Weathering and toxicity of marine sediments contaminated with oils and

3. Potential AhR-Active Chemicals in Oil Components

polycyclic aromatic hydrocarbons. Environ. Toxicol. Chem. 25, 1345-1353.

AhR-mediated potency was mainly found in fractions of oil including aromatic compounds due to their

Kim, C. et al., 2017. Reconnaissance of dioxin-like and estrogen-like toxicities in sediments of Taean, Korea-

structural properties (Vrabie et al., 2012; Hong et al., 2015). In particular, AhR potency of the aromatic

seven years after the Hebei Spirit oil spill. Chemosphere 168, 1203-1210.

compounds (including PAHs and alkyl-PAHs) with 3-4 rings and log Kow 5-7 was relatively strong (Jonker et

Lee, I. et al., 2018. Dioxin-like and endocrine disrupting potencies of fractions of crude oil determined by use

al., 2006; Hong et al., 2015). In addition, sulfur and nitrogen contained heteroaromatics were found to have

of cell-based assays and zebrafish larvae. Chemosphere (In revision).

AhR potencies (Radović et al., 2014). Among the aromatic compounds, the AhR agonists are resistant to

Radović, J.R. et al., 2014. Chemometrics-assisted effect-directed analysis of crude and refined oil

natural oil weathering and could persist in the environments for a long time (Hong et al., 2015). Some AhR

using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Environ.

agonists occurred in resin and polar fractions of crude oil, but their AhR activities were degraded easily with

Sci. Technol. 48, 3074-3083.

the progress of oil weathering (Kim et al., 2017; Lee et al., 2018). Non-targeted screening analysis (NTSA)

Vrabie, C.M. et al., 2012. Effect-directed assessment of the bioaccumulation potential and chemical nature

was rarely utilized for identification of unknown AhR agonists in the highly potent fractions. Further studies

of Ah receptor agonists in crude and refined oils. Environ. Sci. Technol. 46, 1572-1580.

on identification of the unknown AhR agonists in oil components will be needed in the future by combined use of EDA and NTSA.

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1. Introduction

Session31-2

The oil spill has always occurred since mankind began using the oil, and it is estimated that 0.9-1.3 million tons of oil has been spilling out into the marine environment every year (NRC, 2003). Due to the

Comparative evaluation of bioremediation techniques of oil contaminated sediments for restoration of benthic ecosystem health 1*

2

1

3

1,*

Changkeun LEE , Seongjin HONG , Bong-Oh KWON , Un-Hyuk YIM , Jong Seong KHIM , 1

School of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul

National University, Republic of Korea

uncertainty and poor estimation in changes of dynamic marine environments, application of optimum purification methods would be very difficult, as exampled by the Deepwater Horizon accident case (Atlas and Hazen, 2011). In order to find the optimized purification techniques in situ, targeting oil spills on sandy shoreline, the semi-field experimental aquarium system, simulating an artificial tidal condition, has been developed. To determine the best remediation methods, we adopted RTMV (ratio to mean values) approach to assess the recovery efficiency of purification treatments by monitoring of

2

Department of Ocean Environmental Sciences, Chungnam National University, Republic of Korea

six parameters; 1) total petroleum hydrocarbons (TPHs), 2) sedimentary residual toxicity, 3) bacteria

3

Oil and POPs Research Group, Korea Institute of Ocean Science and Technology (KIOST),

community structure, 4) microalgal biomass, 5) benthic primary production, and 6) purification cost

Republic of Korea

(Cesar et al., 2009). 2. Material and methods

Summary

A total of 10 treatments (2 replicates per treatment) were constructed using combinations of five

Oil spill is one of the worst anthropogenic disasters to threaten the marine ecosystems due

bioremediation techniques (Fig. 1). The concentration of TPHs was determined using an gas

to the (in)direct adverse effects associated with varying toxic components of mixture oils. In

chromatograph equipped with a flame-ionization detector. The benthic amphipod Monocorophium

particular, oil contaminated sediments in coastal area would cause long-term ecological effects

uenoi was used to test sedimentary residual toxicity. For the microbial community analysis, DNA were

on benthic communities, with prolonged recovery in site- specific manner. While, various

extracted from sediments using a DNA Isolation Kit, and sequencing was conducted using the Illumina

bioremediation techniques have been applied to promote restoration of benthic ecosystem

MiSeq Platform, with 16S, rRNA gene amplicons. Microalgal biomass (viz., Chl-a) was measured from

health, the methodological efficiency in situ remains unclear. In this study, the performance

surface sediments (at 0-0.5 cm depth) by spectrophotometric method. Benthic primary production was

of bioremediation tools being commonly used for removal of sedimentary oils was evaluated

measured in each treatment of experimental aquariums (n = 3 per treatment) during the daytime by

in aspect of benthic community recovery. Through ninety days of a semi-field exposure test,

use of a Diving-PAM fluorometer.

efficiency in removal of sedimentary residual oils, benthic community recovery, and residual in vitro toxicity are comprehensively examined. Concentrations of total petroleum hydrocarbons and amphipod sediment toxicities for all bioremediation treatments decreased rapidly within the first 10 days. The result of sediment toxicity tests showed dose (concentration of residual oils)dependent responses for all the treatments, in general, indicating potential toxicity could be rapidly removed by any type of bioremediation tools. Of note, control treatment showed moderate recovery in terms of removal of residuals, community recovery, and residual toxicity, thus no action could be considered as one possible measure in restoration of benthic community health against oil pollution. Keywords: oil spill accident, benthic community, restoration, semi-field experiment, PAHs degrading bacteria Fig. 1: Schematic experimental condition and design. N.A represents not available.

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3. Results and discussion

Session31-3

The targeted six experimental endpoints during the purification test of oil-contaminated sediments were determined based on the RTM values of each endpoint (Fig. 2). The results suggested that the most effective bioremediation treatment was found to be the treatment for combined application of fertilizer, microbe, and multi enzyme liquid. While most of the bioremediation efficiency scores were between 7 and 8 in bioremediation treatments, the difference in the recovery efficiency of each

Situational analysis and future perspectives of the regulatory framework for oilfield chemicals in the ASEAN region

treatment did not seem to be large. The emulsifier treatment groups showed good recovery efficiency

Carlos ARIAS-BARREIRO1*, Kim JAKOBSEN2, Salmaan INAYAT-HUSSAIN1

in the most of components, but low scores on recovery of PAHs sensitive bacteria was evidenced. The

1

Product Stewardship & Toxicology Dept., Group Health, Safety & Environment, PETRONAS, Malaysia

rice straw treatment groups showed low recovery efficiency on the microalgal biomass and PAHs

2

Coastal and Marine Department, DHI Water & Environment (M) Sdn Bhd, Malaysia

sensitive bacteria, but would be advantaged in terms of bioremediation cost. While it would be also noteworthy that “no action” treatment showed good recovery efficiency score for the most of targeted endpoints. Altogether, the results suggested that relatively low concentrations of crude oil in sediments

Summary

could exhibit high recovery efficiencies with sufficient seawater circulation.

This is an opinion work which explores the current status of regulatory issues and requirements for offshore oilfield chemicals (OFCs) in the Association of Southeast Asian Nations (ASEAN) region and makes a proposal for the adoption of a unifying regulatory framework for hazard and risk assessments for OFCs to improve the protection of the marine environment. Keywords: ASEAN, marine, oilfield chemicals, chemical regulatory framework, safer alternatives.

1. Abstract With a combined coastline of 173,000 km and one of the world’s most biodiverse hotspots1, the marine environment belonging to the member countries of the Association of Southeast Asian Nations (ASEAN) provides critical ecosystem services in the region. The ASEAN is also one of the most dynamic parts of the global energy system at a time when there is no sign of abatement in the near future on the demand for the region’s offshore oil and gas reserves. Offshore drilling as well as the process of production of oil and gas require the use of oil field chemicals (OFCs), which are expected to grow in demand to approximately 5 billion USD by 2025 in the Asia Pacific region2. Efforts have been made by ASEAN working groups to protect the region’s marine environment e.g. by releasing technical guidelines proposing marine Fig. 2: Comparison of bioremediation efficiency objectives among the treatments.

4. References NRC (National Research Council), 2003. Oil in the sea III: inputs, fates, and effects. National Research Council of the National Academies. Atlas, R.M. and Hazen, T.C., 2011. Oil biodegradation and bioremediation: a tale of the two worst spills in US history. ACS Publications. Cesar, A et al., 2009. A simple approach to integrate the ecotoxicological and chemical data for the establishment of environmental risk levels. Braz. Arch. Biol. Technol. 52, 233-240.

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water quality guidelines for consideration of subsequent adoption by member states (AMSAT, AusAID, ASEAN, 2008) and releasing joint declarations pledging to continue with hazardous chemicals and waste management global strategies3. At the same time opportunities still exist to use the regional network’s influence to promote a more integrated environmental protection framework for OFC discharges. Specifically, ASEAN oil and gas regulators and operators could benefit from a holistic regulatory-driven hazard and risk framework for the selection of OFCs with better environmental performance. Given the existence of established regulatory regimes in other oil and gas producing regions, this work explores the current status and the need for the adoption of a standardised regional approach to reduce the use of highly hazardous OFCs as well as the promotion of the use of safer chemical alternatives in oil and gas drilling and production operations in the ASEAN.

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2. References

Session31-4

AMSAT, AusAID, ASEAN, 2008. ASEAN Marine Water Quality Management Guidelines and Monitoring Manual, first ed. 1

http://environment.asean.org/awgcme/ (Accessed on 01 June 2018)

2

https://www.grandviewresearch.com/press-release/global-oilfield-chemicals-market (Accessed on 01

June 2018) 3

http://asean.org/storage/2017/11/Annex-2_Joint-Declaration-HCWM-Adopted-by-AMME.pdf (01 June

2018)

A comprehensive ecological risk assessment of an accidental spill of palm stearin on the subtropical marine environment Guang-Jie ZHOU1, Racliffe Weng Seng LAI1, Ronia Chung-Tin SHAM1, Chung-Sum LAM1, Katie Wan Yee YEUNG1, Juan Carols ASTUDILLO1, Kevin King Yan HO1, Mana Man Na YUNG1, Jason Kin Chung YAU1, Kenneth Mei Yee LEUNG1,2* 1

The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong

Kong, Hong Kong, China 2

State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong, China

Summary On 3 August 2017, two container vessels collided in the Pearl River Estuary leading to release over 1,000 tonnes of palm stearin into adjacent waters. This study aimed to assess its ecological risk to marine ecosystems in subtropical Hong Kong. Samples of surface seawater, sediment and three intertidal gastropods in six locations were collected seven days and four months after the incident, subject to GC-MS analysis. Standard toxicity tests were conducted with five marine species. The results showed that seawater and sediment samples were heavily contaminated by the palm stearin after one week of the incident, and their levels significantly decreased after four months. However, levels of fatty acids in the animals generally increased over the same period. Although the palm stearin had relatively little effects on marine animals, it was toxic to microalgae species as reflected by growth inhibition. An interim criterion maximum concentration (CMC) for the palm stearin was determined as 0.389 mg/L. Overall, the palm stearin only posed a short-term risk. Keywords: Palm oil; Ecological risk assessment; Water quality criteria; Oil spill

1. Introduction Marine pollution emergency accidents are increasing in the marine environments around highly urbanized coastal cities around the world. On 3 August 2017, two container vessels collided in the Pearl River Estuary, southwest of Hong Kong, leading to release over 1,000 tonnes of palm stearin into adjacent waters. About 200 tonnes of the stearin reached south coasts of subtropical Hong Kong after two days (HKSAR 2017). Palm stearin is the solid fraction of palm oil that is produced by partial crystallization at controlled temperature (Norizzah et al. 2004). It is more variable in composition than palm olein, the liquid fraction of palm oil, especially in terms of its solid fat content. Therefore, palm stearin has more variable physical

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characteristics. Overall, vegetable oils are considered environmental friendly in comparison to mineral oil

3. Results and Discussion

because they undergo 70–100% biodegradation within 28 days; however, degradation of vegetable oils can

The results showed that seawater and sediment samples were heavily contaminated by the palm stearin

generate intermediary metabolites that could be more toxic than the oil itself (Aluyor et al. 2009). Records of

after one week of the accidental pollution occurrence, and their levels significantly decreased after four

palm oil spillages (including kernel oil) are limited to three cases, with a spill of 2500 tonnes of palm oil (mixed

months (Fig.2). However, levels of fatty acids in the animals generally showed an increasing trend over the

with 7500 tonnes of variable vegetable oils) onto a coral reef on Fanning Island in 1975 as the most severe

same period (data no shown). Although the palm stearin had relatively little effects on marine animals,

case in which the oil killed fishes, molluscs and crustaceans by asphyxiation and clogging of the digestive

it was toxic to microalgae species as reflected by growth inhibition (Fig. 3). Its toxic mechanisms on the

tract and enhanced excessive growth of green microalgae on the shore by reducing algal competitors and

microalgae may be associated with its adsorption onto microalgal cells, and reduction of light penetration to

grazers (Fingas et al. 2014; Russell and Carlson 1978).

the cells due to obstruction from the stearin and palm oil. Using all toxicity data generated from this study, an interim short- term WQG (i.e., criterion maximum concentration) for the palm stearin was determined

However, toxicity and ecological risk of palm stearin to marine organisms were still largely unknown. This

as 0.389 mg/L.

study, therefore, aimed to comprehensively investigate its contamination levels in seawater, sediment and animal samples collected from six locations along the south coasts of Hong Kong after the accidental spill;

250

0

HSYB DWB

RB

CHK

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0 TTI

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CHK

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Paracyclop ina nana

0

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Concentration (mg/L)

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Tigriopusjapon 50icus Paracyclop ina nana

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96-h Mortality (%)

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Isochrysisgalbana Tetraselm is suecica

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50 Chaetoceros gracilis Isochrysisgalbana Tetraselm is suecica

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Fig. 2: Concentrations of fatty acids in seawater and 150 sediment samples collected from the six locations within 150 150 0 Tigriopusjapon icusthe accidental spill of palm stearin. Chaetoceros gracilis and after four months Oryzias melastigma one week (August 2018) of HSYB (November DWB RB CHK TTO 2018) TTI HSYB DWB RB CHK TTO TTI RB CHK TTO TTI HSYB DWB RB CHK TTO TTI

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galbana and Tetraselmis suecica), two copepod species (Tigriopus japonicus and Paracyclopina nana), and larvae of the marine medaka (Oryzias melastigma).

100

Fatty acids in sediment (�g/g)

150

November C14:0 C16:0 C18:0 C18:1 C18:2 C20:4 C20:5 Others

Fatty acids in sediment (�g/g)

Standard toxicity tests were conducted with three microalgae species (Chaetoceros gracilis, Isochrysis

200

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August 150

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November C14:0 C16:0 C18:0 C18:1 C18:2 C20:4 C20:5 Others

96-h Mortality (%)

Kong (Fig.1). Fatty acids in these samples were quantified using gas chromatography-mass spectrometry.

Fatty acids in seawater (�g/L)

twice (within seven days and four months after the incident) in six locations along the south coast of Hong

Fatty acids in seawater (�g/L)

Samples of the palm stearin, surface seawater, sediment and three intertidal gastropods were collected

250

72-h Growth Inhibition (%)

thereby assess its ecological risks to local marine ecosystems. 2. Materials and Methods

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determine its toxicities to selected marine organisms; derive its interim water quality guideline (WQG) and

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1

10

0

-50

100

Concentration (mg/L)

1

10

100

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Concentration (mg/L)

Fig. 3: Concentration-response relationships of the microalgae (Left), copepods (Middle) and larvae of the marine medaka fish (Right) after acute exposure to the palm stearin.

4. Conclusion The overall results suggested that the accidental spill of the palm stearin only posed short-term risks to the Fig. 1: A picture showing the palm stearin on a sampling site (Left) and a map showing the Hong Kong Island and Lamma Island, and the sampling sites (Right): 1: HSYB, 2: DWB, 3: RB, 4: CHK, 5: OTM, 6: ITM. Samplings were conducted in August and November 2018, respectively.

coastal marine ecosystems in subtropical Hong Kong. Its risk could be effectively reduced by removal of the stearin from the coast. This study represents the world’s first comprehensive investigation on the ecological risk of the palm stearin to marine ecosystems, and the results would facilitate environmental authorities to make informed decision on handling similar spills of palm oil.

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5. References

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HKSAR (The Government of the Hong Kong Special Administrative Region), 2017. Progress of cleaning up palm oil. http://www.info.gov.hk/gia/general/201708/07/P2017080701006.htm. Accessed 22 Nov., 2017. Norizzah AR, et al. 2004. Effects of chemical interesterification on physicochemical properties of palm stearin and palm kernel olein blends. Food Chem. 86: 229-235.

Sediment quality assessment of oil contaminated envrionment in Abu Ali Island, gulf of arabian after the Persian gulf oil spill

Aluyor EO, et al. 2009. Biodegradation of vegetable oils: A review. Sci. Res. Essays 4:543-548.

Taewoo KIM1,*, Junghyun LEE1, Seo Joon YOON1, Seongjin HONG2, Ahmed A. ALLAM3,4, Wael Nabil

Fingas M, Fieldhouse B, Jokuty P. 2014. Vegetable oil spills: oil properties and behaviour. Handbook of Oil

HOZZEIN3, Abdulaziz A. AL-KHEDHAIRY3, Jong Seong KHIM1

Spill Science and Technology.

1

Spill Science, Edmonton, Alberta, Canada. p79. Russell, D.J., Carlson, B.A., 1978. Edible-oil pollution on Fanning Island.

School of Earth and Environmental Sciences & Research Institute of Oceanography, Seoul National

University, Republic of Korea 2

Department of Ocean Environmental Sciences, Chungnam National University, Republic of Korea

3

Zoology Department, College of Science, King Saud University, Saudi Arabia

4

Zoology Department, Faculty of Science, Beni-Suef University, Egypt

Summary After the Gulf War Oil Spill in 1991, there have been many investigations about determination of pollutants in the Abu Ali and its nearby areas, but lacking in toxicological studies. In this study, we determined potential toxicities associated with residual oils in sediment and marine organisms by combined use of instrumental and bioanalytical analyses. The elevated concentrations of total petroleum hydrocarbons (TPH) and alkylated- PAHs were detected in the sediment and various biological samples. The result clearly indicated that marine organisms are chronically exposed to relatively great concentrations of sedimentary residual oils even after the 25 years of oil spill. Alkylated-PAHs, weathering products of oils, seemed to more bioaccumulate in bristle worms compared to other taxa. AhR-mediated potencies (%-BaP max ) associated with sediment samples were found to be also great, indicating a high potential risk. In conclusion, our results provided useful information about the contamination status and associated environmental risks in the Abu Ali Island even after 25 years of the Gulf War Oil Spill. Keywords: Total petroleum hydrocarbons, Alkylated-PAHs, Gulf War, in vitro bioassay, bioaccumulation

1. Introduction The Gulf War Oil Spill occurred in 1991 have severely contaminated the Persian Gulf environemnt, particularly in the Abu Ali Island, Saudi Arabia, as the spilled oil from Kuwait moved southward along the coast of Saudi Arabia. Traces of oil such as asphalt mat have remained thick in the Abul Ali Island coast and could be potentially harmful to the marine organisms inhabiting hard or soft bottom of intertidal to subtidal zone. Nevertheless, there have been relatively few studies looking for the potential toxicities in sediment or bioaccumulation effects for the marine organisms in the given environment. The main objective of the present study was to assess overall contamination status in coastal sediment in heavily impacted area of

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the Abu Ali Island by the spilled oils after 25 years of the spill. The state of art in vitro bioassay technique has been applied to address the potential toxicities associated with sedimentary residual oils. In particular, the combined use of instrumental and bioanalytical analyses was practiced to identify the causative chemicals responsible for the observed biological activities in terms of mass balance. As part of the present study, the luminescent bacteria toxicity test was additionally performed for the set of sediment and biological samples to address the total toxicity associated with residual oils in the given environment. 2. Material and methods Surface sediment and various biological samples including bristle worm, mud shrimp, chiton, and crab etc. were collected from three sites in the coast of Abu Ali Island in 2017. Target analytes of total petroleum hydrocarbons (TPH), PAHs, and alkylated-PAHs were determined by the GC/MSD-SIM as described previously (Hong et al., 2012). Samples were tested for determination of potential toxicities by use of H4IIEluc bioassay and luminescent bacteria toxicity test, of which methodological details and statistics applied are described elsewhere (Hong et al., 2012; MOMAF, 2005). 3. Results and discussion Concentrations of TPH and PAHs detected in sediments and biological samples from the Abu Ali Island coast varied greatly between locations and/or samples (Fig.1). First, sediments collected from the severely contaminated area by the Gulf War Oil Spill (Site-1) showed the greatest concentrations of TPH and PAHs. Second, similar to the sedimentary data, the biological samples collected from Site-1 showed the greatest concentrations of TPH and PAHs, particularly the elevated concentrations were found in the bristle worms. The greatest concentrations of TPH and PAHs were found at Site-1, both for the sediment and most of the biological samples, but the other two locations did not show contamination gradient between the samples. Biota-sediment accumulation factor (BSAF) values calculated for the various marine organisms indicated that species-dependent variability in bioaccumulation, for example, BSAFs of PAHs and Alkylated-PAHs

Fig. 1. (A) Map showing the study area in the Abu Ali Island, (B) Concentrations of PAHs and alkylated-PAHs in sediment and biological samples, and (C) AhR-mediated activities associated with raw extracts of sediment and biological samples.

4. References Hong, S., Khim, J.S., Ryu, J., Park, J., Song, S.J., Kwon, B.O., Choi, K., Ji, K., Seo, J., Lee, S., Park, J., Lee, W., Choi, Y., Lee, K.T., Kim, C.K., Shim, W.J., Naile, J.E., Giesy, J.P., 2012. Two years after the herbei spirit oil spill: residual crude-derived hydrocarbons and potential AhR-mediated activities in coastal sediments. Environ. Sci. Technol. 46, 1406-1414. Lee, J., Hong, S., Yoon, S. J., Kwon, B. O., Ryu, J., Giesy, J. P., Ahmed A. Allam, Abdulaziz A. Al-khedhairy, Khim, J. S. (2017). Long-term changes in distributions of dioxin-like and estrogenic compounds in sediments of Lake Sihwa, Korea: Revisited mass balance. Chemosphere, 181, 767-777. MOMAF (Ministry of Maritime Affairs and Fisheries of South Korea), 2005. Establishment of integrative management system for ocean dumping., Seoul, Korea. (in Korean)

for the bristle worm and mud shrimp were one to two orders of magnitude greater than those for chiton or crab, on average. Meantime, significant AhR-mediated activities were evidenced for the extracts of all sediments and some biological samples, particularly greater AhR-activities were associated with extracts of bristle worms and crabs, which back-supported the high potentials in bioaccumulation of AhR-active PAHs compounds in the corresponding taxa. Mass balance analysis revealed that up to 40% and 43% of the biological activities observed for the extracts of sediment and bristle worms could be explained by the known concentrations of AhR-active PAHs (Lee et al., 2017). Luminescent bacteria toxicity test also indicated the general significant toxicities associated with sediment and some biological samples, thus potential toxicities relating to the environmental residues of oils in the study area remain under significant risks.

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In these SPME-GC studies WAFs of GTL products and conventional fuels were prepared at loading rates of

Session31-6

100 mg/L and stirred for up to 48 hours.

Weight of evidence assessment of the bioaccumulation potential of Gas to Liquid (GTL) Fuel 1

2*

Graham WHALE , Jonathan NAILE

3. Results and discussion • The fish tissue spiking and recovery study indicated that it would be theoretically feasible to determine BCFs for C14, C16 and C18 normal alkanes (providing their aqueous exposure concentrations could be continuously maintained and determined throughout the duration of the uptake phase of an aqueous

1

Shell Health Risk Science Team, UK

exposure BCF study). However, the study also found that extracts taken from rainbow trout obtained

2

Shell Health Risk Science Team, USA

from a local supermarket and stock fish kept at the testing laboratory contained measureable levels of n-pentadecane (n-C15) and n-heptadecane (n-C17). This created a problem because there is evidence in

1. Introduction Typically bioaccumulation potential under REACH notification requirements is assessed in an OECD 305 fish bioconcentration test on a specific chemical substance. However, GTL Fuel (“Distillates (Fischer-Tropsch), C8-C26 branched and linear”) is considered under the EU REACH definitions to be a UVCB (Unknown, of Variable Composition, or of Biological Origin) substance. Consequently it is unsutable to assess its bioaccumulation potential using standard OECD 305 tests. However, it is feasible to assess constituents of such products and these could be used in Quantitative Structure Activity Relationship (QSAR) models to predict BCFs (bioconcentration factors) of other constituents of the GTL Fuel. The European Chemicals Agency (ECHA) recommended that the constituents

the scientific literature that alkanes like n-pentadecane and n-heptadecane are naturally occurring and present in aquatic systems[4] and hence would interfere with bio-concentration studies of GTL Fuel. • In the SPME-GC study, the observed responses can be explained by their composition. GTL products are mainly comprised of linear and relatively simply branched alkanes and their response decreases with increased carbon chain length from GTL Naphtha (C4-C9) to GTL Fuel (C8-C26) due to decreased solubility. Conventional petroleum products typically contain a greater fraction of water soluble/bioavailable consituents (i.e. aromatics and highly branched alkanes) which is reflected in their peak areas and indicates that these have greater potential to bioaconcentrate in aquatic organisms when compared to their equivalent GTL counterparts.

to be used in the bioaccumulation testing should be based on the outcome of soil biodegradation testing. This could not be achieved because of analytical and REACH requirements. In effect, the sensitivity of the analytical methods was such that although no quantifiable persistent constituents could be determined in soil, it could not be verified whether some constituents remained in the soil at concentrations > 0.1% w/w of the GTL Fuel[1]. Therefore, additional studies have been undertaken to provide weight of evidence that GTL Fuel does not have significant potential to bioaccumulate. Furthermore, the justification for not undertaking any further bioaccumulation assessments of GTL Fuel is also presented and discussed. 2. Material and methods A method development and validation investigation was conducted on the recovery of GTL Fuel from whole rainbow trout at concentrations in fish tissue required to meet the bioaccumulation “B” criteria in a fish bioconcentration study. This data was used to provide an indication of the feasibility of conducting an OECD 305 study of the GTL Fuel. To provide additional reassurance that the constituents of the GTL Fuel have limited potential to bioaccumulate via aqueous exposure, solid phase micro extraction coupled with gas chromatography (SPME-GC) assessment of water accommodated fractions (WAFs) was undertaken. SPME is recognised by OSPAR[2] and ECHA guidelines[3] as a technique which can give an initial estimate of bioaccumulation potential of constituents of effluents and multi constituent substances.

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Substance GTL Naphtha GTL Kerosene GTL Fuel Conventional Kerosene Conventional Gasoil

SPME total peak area 24h 1.7 x 106 1.9 x 105 1.2 x 104 2.9 x 106 1.7 x 106

Table 1: Comparison of SPME-GC total peak area for GTL and conventional petroleum derived substances

4. Conclusions At the outset, the bioaccumulation potential of GTL Fuel was not considered to be significant as this substance is readily biodegradable. The poor water solubility of the constituents of the GTL Fuel and the complex ‘UVCB’ nature of this substance makes it unsuitable for undertaking conventional fish bioaccumulation studies via aqueous exposure. The feasibility study confirms that it would not be possible to quantify most components in fish tissue even if these met the ‘B’ criteria and concentrations of components could be maintained in water in an OECD 305 test. Furthermore, there is no evidence from soil degradation studies and analysis of solutions at the end of OECD 301 aquatic biodegradation tests that there are

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recalcitrant constituents of GTL Fuel which warrant further investigation for bioaccumulation assessment

Session32-1

in a fish dietary accumulation study. The data presented from the SPME-GC study provide weight of evidence to not only support this view but also provide reassurance that GTL Fuel or constituents thereof will not significantly bioaccumulate. The concerns regarding the need to provide additional data on the bioaccumulation potential for substances like GTL Fuel also need to be put into perspective. For example, GTL Fuel has been shown to be non toxic

Multiple effects of nickel-contaminated suspended sediment exposure on the Coral, Acropora Muricata

to mammals with an acute oral toxicity data value for rats and Japanese quail of > 5000 mg/kg and was not

Francesca GISSI1,2*, Megan L. GILLMORE1,3, Jenny L. STAUBER3, Amanda J. Reichelt-Brushett4, Lisa A.

chronically toxic to fish. Even if specific constituents did have some potential to bioaccumulate, due to their

GOLDING3, Anthony CHARITON5, Paul GREENFIELD6, Craig A. HUMPHREY7, Andrea SEVERATI7, and

low water solubilty (i.e. test substance < 1 mg/l and the majority of constituents predicted to be significantly

Dianne F. JOLLEY1

2000) would be lower than those which has been administered in prolonged repeat dose toxicity

2

CSIRO Oceans and Atmosphere, Australia

studies with GTL Fuel in rats. The latter repeated dose toxicity data is important since in the two generation

3

CSIRO Land and Water, Australia

(OECD 216) and prenatal (OECD 214) studies there were no treatment-related deaths, clinical signs of

4

Marine Ecology Research Centre, School of Environment Science and Engineering, Southern Cross

toxicity or test item-related findings on the integrity and performance of the male and female reproductive

University, Australia

systems at the maximum dosage level of 750 mg/kg/day. Therefore, in the context of risk assessment even

5

Macquarie University, Australia

if significant bioaccumualtion of GTL Fuel (or components therof) could occur there is no plausible way

6

CSIRO Data 61, North Ryde, Australia

that these would be ingested by mammals at concentrations above the No Observed Adverse Effect Level

7

The National Sea Simulator, Australian Institute of Marine Science, Australia

(NOAEL) for developmental, reproductive and systemic toxicity. It could therefore be argued that for UVCB substances like GTL Fuel, where there is sufficient data to demonstrate that the substance (and its respective constituents and degradation products) are not

Summary

persistent and have low eco/mammmalian toxicity, that the requirement to undertake any bioaccumulation

The mining and production of nickel is expected to increase in tropical Asia-Pacific. Local marine

assessments could be waived. In the context of risk assesment this waiver would be on the basis that the

ecosystems, including coral reefs are potentially at risk. We investigated the effects of nickel

substance will not pose any significant risks via secondary poisoning.

exposure (in dissolved (operationally defined as