Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan

Chemosphere xxx (2014) xxx–xxx

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Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan Pei-Hsin Chou a,⇑, Tong-Cun Liu a, Fung-Chi Ko b,c, Mong-Wei Liao a, Hsiao-Mei Yeh a, Tse-Han Yang a, Chun-Ting Wu a, Chien-Hsun Chen a, Tsung-Ya Tsai a a b c

Department of Environmental Engineering, National Cheng Kung University, Tainan, Taiwan National Museum of Marine Biology and Aquarium, Pingtung, Taiwan Institute of Marine Biodiversity and Evolutionary Biology, National Dong Hwa University, Pingtung, Taiwan

h i g h l i g h t s  AhR agonist contaminants were frequently detected in rivers in Southern Taiwan.  Genotoxicity was often found in dry-season samples collected from Erren River.  AhR agonist activity and genotoxicity were caused by different contaminants.  PAHs were minor contributors to the AhR agonist activity elicited by sediment.  Bioassay analysis is useful in providing combined toxicity in environmental samples.

a r t i c l e

i n f o

Article history: Received 28 August 2013 Received in revised form 2 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: Aryl hydrocarbon receptor agonists Genotoxicants Bioassays Sediment Polycyclic aromatic hydrocarbons

a b s t r a c t Water and sediment samples from river systems located in Southern Taiwan were investigated for the presence of aryl hydrocarbon receptor (AhR) agonists and genotoxicants by a combination of recombinant cell assays and gas chromatography–mass spectrometry analysis. AhR agonist activity and genotoxic response were frequently detected in samples collected during different seasons. In particular, dry-season water and sediment samples from Erren River showed strong AhR agonist activity (201–1423 ng L 1 and 1374–5631 ng g 1 b-naphthoflavone equivalents) and high genotoxic potential. Although no significant correlation was found between AhR agonist activity and genotoxicity, potential genotoxicants in sample extracts were suggested to be causative agents for yeast growth inhibition in the AhR-responsive reporter gene assay. After high performance liquid chromatography fractionation, AhR agonist candidates were detected in several fractions of Erren River water and sediment extracts, while possible genotoxicants were only found in water extracts. In addition, polycyclic aromatic hydrocarbons, the typical contaminants showing high AhR binding affinity, were only minor contributors to the AhR agonist activity detected in Erren River sediment extracts. Our findings displayed the usefulness of bioassays in evaluating the extent of environmental contamination, which may be helpful in reducing the chances of falsenegative results obtained from chemical analysis of conventional contaminants. Further research will be undertaken to identify major candidates for xenobiotic AhR agonists and genotoxicants to better protect the aquatic environments in Taiwan. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction A variety of anthropogenic pollutants are frequently discharged into the aquatic environment via human activities. Among them, hydrophobic organic contaminants receive much attention owing to their ubiquitous distribution and many adverse effects, such as persistence, bioaccumulation, carcinogenicity, or endocrine disruption (Muir and Howard, 2006; Bull et al., 2011; Söffker and Tyler, ⇑ Corresponding author. Tel.: +886 6 2757575x65840. E-mail address: [email protected] (P.-H. Chou).

2012). Though their environmentally relevant concentrations may be low, these contaminants pose problems because they may elicit additive or synergistic toxicity. Thus, it is important to assess the combined toxicity of multiple contaminants and to identify major toxicants in environmental samples to protect human health and the environment. In recent years, in vivo animal testing and in vitro recombinant cell bioassays have been extensively used to evaluate the toxicity of various environmental samples. Invertebrates, fish, and bioluminescent bacteria Vibrio fischeri have been the major species used in aquatic toxicity tests since late 1980s (Brack, 2003; Eom et al.,

0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

Please cite this article in press as: Chou, P.-H., et al. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

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2007; Ocampo-Duque et al., 2008). Mutagenic, genotoxic, and endocrine disrupting activities of river and sediment samples have also been successfully detected by a variety of bioassays using engineered cells or microorganisms (Nukaya et al., 1997; Ono et al., 2000; Kawanishi et al., 2004; Macova et al., 2011). Compared to in vivo tests, the time- and cost-efficiency of in vitro assays make them convenient tools for preliminary screening of potential polluted sites. In addition, using in vitro assays in combination with chemical fractionation has been suggested to be a promising technique for isolating target toxicant candidates in environmental samples (Lukasewycz and Durhan, 1992; Reemtsma, 2001; Hewitt and Marvin, 2005; Chou et al., 2007). In the present study, we aimed to investigate the occurrence and the combined toxicity of organic contaminants showing aryl hydrocarbon receptor (AhR) agonist activity or genotoxicity in several river systems located in Southern Taiwan. AhR is a ligand-activated transcription factor that mediates the toxic effects of numerous organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorinated dibenzo-p-dioxins (Denison and Heath-Pagliuso, 1998; Denison and Nagy, 2003; Bock and Köhle, 2006). AhR binding affinity of environmental pollutants may be considered as an indication of potential toxicity since many organic contaminants induce a broad spectrum of toxic responses via AhR activation. Genotoxic compounds are potential DNA-reactive agents that may damage DNA integrity. Several PAHs and their metabolites have been considered as possible genotoxicants, and one of the most famous examples is benzo[a]pyrene-7,8-diol9,10 epoxide, a highly reactive electrophilic metabolite of benzo[a]pyrene that binds covalently to nucleic acids and forms DNA adducts (Bartsch, 1996; Alexandrov et al., 2010). During the past decade, various organic contaminants have been detected in the aquatic environments in Taiwan, such as PAHs, halogenated compounds, and pharmaceuticals, while the combined toxicity remains unknown (Hung et al., 2006; Peng et al., 2007; Doong et al., 2008; Huang et al., 2008; Lin and Tsai, 2009). To examine the toxic effects elicited by various xenobiotic AhR agonists or genotoxic compounds, we collected water and sediment samples from three river systems in Southern Taiwan during different seasons, and analyzed the AhR agonist activity and genotoxicity of these samples using in vitro bioassays. In addition, samples were fractionated using high performance liquid chromatography (HPLC) to isolate active factions, and PAH concentrations were analyzed by gas chromatography–mass spectrometry (GC–MS) to evaluate their contribution to the combined toxicity. Our results revealed that AhR agonists and genotoxicants were frequently detected in the river systems in Southern Taiwan. However, PAH concentrations were unable to reflect the combined toxicity of sample extracts. Further monitoring and water quality improvement is necessary to protect the aquatic environments in Taiwan.

YS

3 4

1 2

5

Taiwan 1

ER AGD 3

2 1

4

3 2 7

6 5

Fig. 1. Sampling locations in YS River, ER River, and AGD River.

fold higher in wet seasons) owing to the difference in seasonal precipitation. To compare the AhR agonist activity and genotoxicity, dry- and wet-season samples were collected in December 2010 (dry season, ER), May 2011 (wet season, YS), July 2011 (wet season, AGD, ER), and December 2011 (dry season, YS, AGD). Sediment samples were also taken at ER River in September 2010 (wet season) and February 2011 (dry season) for PAH analysis. 2.2. Water and SS sample extraction Water grab samples were filtered and extracted within 24 h after collection. Hydrophobic organic compounds were extracted from 1 L of each river water sample by solid phase extraction using Sep-PakÒ Plus Environmental C18 Cartridge (Waters, USA) following filtration through 0.60 lm pre-weighed glass fiber filters (Advantec, Japan). Each cartridge was washed with water and eluted with 3 mL of methanol and 1 mL of dimethyl sulfoxide (DMSO) after extraction. The eluents were evaporated to dryness in a centrifugal vacuum concentrator (EYELA, Japan), and the extracted compounds were redissolved in 1 mL of DMSO to obtain a 1000-fold concentrated extract (concentration factor = 1000). Filters containing SS of each river water sample were dried in a 105 °C oven before extraction. Hydrophobic organic compounds in each SS sample were extracted with 30 mL of anhydrous sodium sulfate-added hexane:acetone (1:1, v:v) solution by ultrasonic extraction for 60 min, and were further extracted by 15-min ultrasonic extraction for another three times with 5 mL of hexane:acetone (1:1, v:v) solution, 5 mL of hexane, and 5 mL of hexane. Supernatants of each extraction were combined, evaporated to dryness, and redissolved in 1 mL of DMSO to obtain a 1000-fold concentrated extract. The 1000-fold concentrated extract was further diluted using DMSO to obtain a dilution series for bioassay analysis. 2.3. Sediment extraction

2. Materials and methods 2.1. Sample collection River water, suspended solids (SS), and sediment (Sed) grab samples were taken at three river systems located in Southern Taiwan, including Yanshuei River (5 sites, YS1-YS5, sediment sample of YS4 was not available), Erren River (7 sites, ER1-ER7), and Agondian River (3 sites, AGD1-AGD3) (Fig. 1). The area is populated with around 1 million residents, and major pollution sources include domestic wastewater, swine wastewater, and industrial wastewater from electroplating factories, metal finishing industries, and etc. (Table S1, Supplementary material). Flow rates of the three river systems varied greatly during dry- and wet seasons (6.5–8.5-

Sediment grab samples were obtained with a stainless-steel Ekman dredge (Wildlife Supply Company, USA). After collection, 1 g of each sediment sample was dried in a hood for 48–72 h, and was ground and passed through a 20 mesh sieve. Hydrophobic organic compounds in each air-dried sediment sample were extracted with 10 mL of anhydrous sodium sulfate-added hexane:acetone (1:1, v:v) solution by shaking extraction at 200 rpm for 24 h. Supernatants were collected after centrifugation, evaporated to dryness, and redissolved in 1 mL of DMSO to obtain a concentrated sample extract (1000 mg Sed equivalent mL 1 DMSO). The concentrated extract was also diluted using DMSO to obtain a dilution series for bioassay analysis. Sediment samples collected at ER River were also extracted using Soxhlet apparatus for GC–MS analysis. One gram of each

Please cite this article in press as: Chou, P.-H., et al. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

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sample was homogenized and freeze-dried with anhydrous sodium sulfate before Soxhlet extraction with hexane:acetone (1:1, v:v) solution for 24 h. The extract was concentrated to less than 5 mL by rotary evaporation, and interference was removed by fractionating the solution through an alumina oxide column (4 g, 6% deactivated with Milli-Q water) with hexane. Desulfurization was also carried out by adding activated copper (Kuo et al., 2012). After purification steps, the extract was concentrated to approximately 1 mL using a purified nitrogen stream before GC–MS analysis. 2.4. AhR agonist activity evaluated with a yeast-based reporter gene assay AhR agonist activity of each sample was measured by a yeastbased reporter gene assay using the recombinant YCM3 strain developed by Dr. Charles A. Miller III, Tulane University, USA (Miller III, 1999). The AhR signaling assay was carried out as described (Chou et al., 2007) with several modifications. The yeast was grown in a synthetic glucose medium (2% glucose, lacking tryptophan) at 30 °C, and then 2 lL of a positive control (b-naphthoflavone, b-NF), a negative control (DMSO), or a sample extract was mixed with 193 lL of a synthetic galactose medium (2% galactose, lacking tryptophan) and 5 lL of the overnight yeast culture in a 96-well microplate (carried out in triplicate). The mixture was subsequently incubated at 30 °C for another 18 h. After incubation, cell density was determined by reading the optical density at 595 nm (OD595) using a microplate reader (Bio-Rad, USA), and then 10 lL of the cell suspension was transferred into a new microplate and mixed with 140 lL of Z-buffer and 50 lL of o-nitrophenyl-b-Dgalactopyranoside solution (4 mg mL 1 in Z-buffer) thoroughly. The optical density at 405 nm (OD405) was measured after incubating at 37 °C for 60 min. AhR agonist activity was calculated using the quotient of OD405 and OD595 of each sample, and was shown as fold induction of DMSO (FI of DMSO = (OD405/OD595)SAMPLE/ (OD405/OD595)DMSO), which indicates the relative AhR agonist activity of a tested sample to DMSO (solvent blank). AhR agonist activity of each sample was also converted to b-NF equivalent concentration (b-NF EQ) using the concentration-activity curves of b-NF. 2.5. Genotoxicity evaluated with rec assay Genotoxicity of each sample extract was measured using the DNA repair-deficient Bacillus subtilis Rec assay. Rec assay utilizes two strains, including the recombination proficient strain (Rec+) H17 (recE+, uvrABC+), and a derivative strain M45 (recE , uvrABC+) as the recombination deficient strain (Rec ). The M45 is extremely sensitive to genotoxic agents such as c-rays, ultraviolet rays, and pharmaceuticals (Matsui et al., 1992). The assay procedure was carried out as described (Takigami et al., 2002) with adequate modification. In brief, 2 lL of a positive control (4-nitroquinoline-1-oxide), a negative control (DMSO), and a sample was each mixed with 98 lL of LB-broth and 100 lL of a bacterial culture of either Rec+ or Rec strain (OD595 = 0.02) in a 96-well microplate (carried out in triplicate). OD595 was measured by a microplate reader before incubation (OD595,0h), and then the mixture was incubated at 37 °C for 5 h (OD595,5h) using a microplate shaker. After incubation, OD595 was measured again to calculate the survival rates (%) of Rec+ and Rec strains (OD 595,5h OD 595,0h ) SAMPLE /(OD 595,5h OD 595,0h ) DMSO  100%). Genotoxicity was represented as R50, which is the quotient of median inhibitory concentrations (IC50, i.e. survival rate (%) = 50%) of Rec+ and Rec strains (R50 = (IC50)Rec+/(IC50)Rec ). Genotoxic response was confirmed when R50 of a test sample was higher than 1.5 (Oksuzoglu et al., 2008).

3

2.6. HPLC fractionation Sample extracts showing higher AhR agonist activity or genotoxic potential were fractionated by HPLC (Hitachi L-2130, Japan) using a Shim-Pack FC-ODS column (5 lm, 150  4.6 mm, Shimadzu, Japan) equipped with a diode array detector (Hitachi L-2455, Japan). The column was eluted with a gradient condition of 10– 100% methanol in water from 0 to 20 min, and then maintained at 100% methanol for another 20 min for water sample extracts or 40 min for sediment sample extracts. The flow rate was 0.8 mL min 1, and the injection volume was 20 lL. Fractions were collected every minute from 0 to 40 min (F1–F40), evaporated to dryness, and redissolved in 20 lL of DMSO for bioassay analysis. 2.7. GC–MS analysis of PAHs PAHs in ER River sediment samples were quantified and identified using a capillary GC (Varian CP-3800, USA) and a ion trap mass analyzer (Varian 4000 MS, USA) equipped with a Factor Four VF5 ms capillary column (length 30 m, i.d. 25 mm, film thickness of the stationary phase: 0.25 lm, Varian, USA). Two lL of each sample was injected in a splitless injection. Twenty-three PAHs were measured under the selected ion monitoring mode and were identified by retention times identical to those of PAH standards. Helium was used as the carrier gas, and the flow rate was 1 mL min 1. The temperature program was set as described (Cheng et al., 2013), and the injector and capillary interface temperatures were 250 °C and 310 °C, respectively. PAHs analyzed in this study were acenapthylene (ACL), acenaphthene (ACT), fluorene (FLU), dibenzothiophene (DBT), phenanthrene (PHN), anthracene (ANT), 4,6-dimethyldibenzothiop (DMD), fluoranthene (FLT), pyrene (PYR), retene (RET), benzo[a]fluorene (BaFl), benzo[b]fluorene (BbFl), benzo[a]anthracene (BaA), chrysene (CHR), benzo[b]fluoranthene (BbFa), benzo[k]fluoranthene (BkFa), benzo[e]pyrene (BeP), benzo[a]pyrene (BaP), perylene (PER), indeno[1,2,3-c,d]pyrene (INP), dibenzo[a,h]anthracene (DahA), benzo[g,h,i]perylene (BgP), and coronene (COR). Deuterated PAHs, including d10-acenaphthene, d10-phenanthrene, d12benzo[a]anthacene, and d12-benzo[g,h,i]perylene were used as internal standards. In addition, d8-napthalene, d10-fluoranthene, d10-fluorene, and d12-perylene were added to each sediment sample as surrogates. The recoveries of the surrogates were 52 ± 21%, 96 ± 35%, 82 ± 27% and 79 ± 24% (n = 35), respectively, while PAH concentrations were not corrected for surrogate recoveries. 3. Results and discussion 3.1. AhR agonist activity in sample extracts Dry-season water extracts of ER River and AGD River, and wetseason water extracts of YS River elicited high AhR agonist activity at environmentally relevant concentrations, which were 201– 1423, 304–851, and 376–1106 ng L 1 b-NF EQ, respectively (Fig. 2A). Several SS extracts, such as wet-season SS extracts taken at ER5 (602 ng L 1 b-NF EQ) and AGD1 (973 ng L 1 b-NF EQ) showed significant activity as well. Stronger AhR agonist activity was particularly found in the dry-season sediment extracts collected at ER River and AGD River (1374–5631 and 414–2805 ng g 1 Sed dw b-NF EQ, respectively) (Fig. 2B). The highest activity was detected in the dry-season ER7 sediment extract, which was 10– 20 times higher than that found in YS River sediment extracts (225–552 ng g 1 Sed dw b-NF EQ). AhR agonist activity in YS River, ER River, and AGD River water extracts was high comparing to those detected in Japanese rivers (28–129 ng L 1 b-NF EQ) or Australian rivers (70–180 ng L 1 b-NF EQ) (Kawanishi et al.,

Please cite this article in press as: Chou, P.-H., et al. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

β-NF EQ (ngL -1)

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3000

β-NF EQ (ngg -1 Sed dw)

4

7500

2000

A

Water SS

1000 0

B

Sediment

5000 2500

**

0

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 1 2 3 D

W

D

YS

W ER

D

W AGD

Fig. 2. AhR agonist activity of (A) water and SS samples (B) sediment samples taken at YS River, ER River, and AGD River during dry (D) and wet (W) seasons (⁄: unable to collect sediment samples).

2008; Chinathamby et al., 2013). In addition, several samples elicited stronger AhR agonist activity than those found in effluents from Australian, German, Swiss, or Chilean wastewater treatment plants (16–279, 387–741, or 21–754 ng L 1 b-NF EQ) (Alexandrov et al., 2010; Allinson et al., 2011; Stalter et al., 2011; Chamorro et al., 2012). Wastewater effluents are generally considered as major pollution sources for water contamination, and pollutant concentrations may be lower due to river water dilution. The strong AhR agonist activity in YS River, ER River, and AGD River suggested heavy pollution of AhR agonist contaminants in the aquatic environments in Southern Taiwan. Xenobiotic AhR agonists in these river systems may come from sewage effluents, swine wastewater, or industrial wastewater discharged by electroplating factories, metal finishing industries, etc. (Table S1, Supplementary material).

higher R50 was found in dry-season sediment extracts, including those collected at ER1–ER7 (R50 = 1.8–2.7), YS5 (R50 = 2.8), and AGD3 (R50 = 2.4). Dry-season ER River water extracts also showed strong genotoxicity (R50 = 1.7–2.4), whereas only wet-season ER1 and ER7 water extracts exhibited genotoxic potential (R50 = 2.0, 1.5). The relationship between genotoxicity and the corresponding yeast survival in the AhR signaling assay was examined since inhibition of yeast growth was often observed in some highly concentrated sample extracts. As shown in Fig. 3A (upper left region), strong yeast growth inhibition (survival <50%) was found in 22 out of 28 genotoxic sample extracts (R50 > 1.5), indicating that the genotoxicants present in the sample extracts could be causative agents for reduced yeast growth. Several studies have demonstrated that the genotoxicity or mutagenicity in sediment can be correlated with certain organic contaminants, such as parent and metabolically activated PAHs and polychlorinated biphenyls (Brack et al., 2005; Costa et al., 2008; Di Giorgio et al., 2011; Lübcke-von Varel et al., 2011), which are also considered as potential AhR agonists (Machala et al., 2001; Misaki et al., 2007). However, no or very low correlations could be found between the genotoxicity and the AhR agonist activity detected in either water or sediment extracts of ER River, YS River, and AGD River (Fig. 3B), suggesting that AhR agonist compounds, including PAHs and their derivatives, may not be the major contributors to the genotoxicity in samples collected from the river systems in Southern Taiwan. In addition, the Rec assay carried out in this study was performed in the absence of S9 activation, thus the contribution of genotoxic PAH metabolites may be insignificant.

3

A

2.5

3.2. Genotoxicity in sample extracts Genotoxic potential of river water and sediment extracts were evaluated by Rec assay. As shown in Table 1, significant genotoxic response was frequently detected in the sediment extracts, and

R50

1.5

Table 1 Genotoxicity (R50) of water and sediment samples taken at YS River, ER River, and AGD River during dry and wet seasons. Site

a

Water

2

1 0

50

100

Yeast Survival (%)

Sediment

3

Dry season

Wet season

Dry season

Wet season

YS1 YS2 YS3 YS4 YS5

NDa ND 1.6 >1.1b NDa

NDa >1.1b 1.0 >1.3b NDa

NDa NDa >1.4b NAc 2.8

1.7 NDa 1.2 NAc 1.6

ER1 ER2 ER3 ER4 ER5 ER6 ER7

2.4 1.7 2.2 2.0 2.0 2.3 1.8

2.0 NDa NDa NDa NDa NDa 1.5

1.8 2.7 2.2 2.3 2.3 2.1 2.5

>2.4b NDa 1.7 1.6 >1.4b 1.2 1.7

AGD1 AGD2 AGD3

1.0 NDa NDa

1.1 1.1 NDa

>1.5b >1.1b 2.4

>1.3b 1.7 2.1

No detectable bacterial inhibition, IC50,Rec+ and IC50,Rec were greater than the highest concentrations in the dilution series. b IC50,Rec+ was greater than the highest concentration in the dilution series. c Not available, unable to collect sediment samples.

B

2.5

R50

2

1.5

Sediment Water 1 0

1500

3000

4500

6000

β-NF EQ (ngL -1 or ng g-1 Sed dw) Fig. 3. Correlations among genotoxicity (R50) elicited by water (white circle) and sediment (green diamond) samples with (A) yeast survival in YCM3 assay (B) AhR agonist activity (R2 = 0.009 and 0.1467, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Chou, P.-H., et al. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

5

UV absorbance at 254 nm (mAU)

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200

A

100

0

FI of DMSO

6

ER1-D

ER3-D

ER7-D

4 2 0 0

5

10

15

20

25

30

35

40

Retention Time (min)

UV absorbance at 254 nm (mAU)

200

B

100

0 8

FI of DMSO

ER1-D

ER3-D

ER7-D

6 4 2 0 0

5

10

15

20

25

30

35

40

Retention Time (min) Fig. 4. HPLC chromatograms (UV absorption at 254 nm) and AhR agonist activity elicited by corresponding HPLC fractions of (A) dry-season ER1, ER3, ER7 water samples (concentration factor = 10) and (B) dry-season ER1, ER3, ER7 sediment samples (1000 mg Sed equivalent mL 1 DMSO).

3.3. HPLC fractionation of AhR-active and genotoxic samples from ER River Water and sediment samples taken at ER1, ER3, and ER7 were fractionated by HPLC to further investigate potential AhR agonists and genotoxicants in the sample extracts. Fig. 4 shows the HPLC chromatograms (absorption wavelength: 254 nm) and the AhR agonist activity of corresponding HPLC fractions of dry-season ER River water and sediment extracts. After fractionation, higher AhR agonist activity was detected in several fractions of dry-season water extracts, including ER1-F19, ER1-F20, ER3-F19 to ER3-F22, and ER7-F19, ER7-F20, ER-F22 (Fig. 4A), while the AhR agonist activity in ER1-F21, ER1-F22, and ER7-F21 could not be calculated owing to strong yeast growth inhibition (survival = 17%, 19%, and 38%, respectively). Although several peaks in these fractions were potential xenobiotic AhR agonists, isolation of candidate compounds was difficult due to sample complexity. High AhR agonist activity was also found in the HPLC fractions of dry-season sediment extracts, such as ER1-F19 to ER1-F25, ER3F20 to ER3-F28, and ER7-F17 to ER7-F32 (Fig. 4B). The fractions eliciting higher AhR agonist activity in ER3 and ER7 sediment extracts were more hydrophobic, and the retention times of active fractions corresponded to the fractions (F26–F30) in which several AhR-active PAHs (BaA, BaP, BbFa, BbFl, BkFa, and CHR) were eluted.

PAHs and related compounds have been identified as major AhR agonists in sediment samples collected at different regions, such as Czech and German river basins, Dutch sea, or Oslo harbor (Hilscherova et al., 2001; Machala et al., 2001; Klamer et al., 2005; Grung et al., 2011; Lübcke-von Varel et al., 2011). However, some studies also reported that PAHs were only minor contributors to the AhR-mediated activity of sediment samples (Brack et al., 2005; Lübcke-von Varel et al., 2011). F21 of dry-season water extracts collected from ER1, ER3, and ER7 were confirmed to be genotoxic using the Rec assay, and the R50 values were 2.1, 2.0, and 1.5, respectively. Furthermore, F21 of wet-season ER1 and ER7 water extracts also showed lower yeast survival (48% and 18%) and higher genotoxicity (R50 = 1.8 and 3.0), suggesting that the genotoxicant candidates in this fraction may be regularly discharged into ER1 and ER7 during different seasons. On the contrary, although genotoxic fractions of ER1 and ER7 water extracts were separated by HPLC, no specific fractions of sediment extracts were found to inhibit yeast growth after fractionation. Thus, the genotoxicity detected in sediment extracts (Table 1) may be caused by the combined toxicity of contaminants since a complex mixture of chemicals may have synergistic effects. For example, a combination of phenanthrene and lindane has induced greater toxic effects on copepod reproduction (Evans and Nipper, 2007), and the acute toxicity of a mixture of PAHs on freshwater

Please cite this article in press as: Chou, P.-H., et al. Occurrence of aryl hydrocarbon receptor agonists and genotoxic compounds in the river systems in Southern Taiwan. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.052

P.-H. Chou et al. / Chemosphere xxx (2014) xxx–xxx

3000 2000

A

AhR-active PAHs AhR-inactive PAHs

1000 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Sep 10

β-NF EQ (ng g -1 Sed dw)

∑PAHs (ng g-1 Sed dw)

6

Dec 10

20000

Feb 11

β-NF EQ R50

B

Jul 11 4

15000 3

10000

R50

genotoxicity (Fig. 5B), higher PAH concentrations were detected in ER7 sediment extracts of 4 different months (998–2286 ng g 1 Sed dw). Potential input of PAHs in ER7 sediment extracts were characterized as petrogenic sources according to the isomeric ratios of ANT/(ANT + PHE) > 0.1 and FLT/(FLT + PYR) < 0.4 (Yunker et al., 2002; Ko et al., 2007). In addition, the BaA/(BaA + CHR) ratios of February and July 2011 ER1 sediment extracts are 0.31 and 0.35, and those of September and December 2010 ER1 sediment extracts are 0.57 and 0.47, suggesting different pollution sources for samples exhibiting low and high b-NF EQ10 PAHs (Supplementary mateP rial, Table S5 and Fig. S4). High PAHs and b-NF EQ10 PAHs found in September 2010 ER1 sediment extract may relate to the flushing of road dust containing higher levels of PAHs via storm water during typhoon seasons.

2

5000 0

3.5. Contributions of PAHs to the AhR agonist activity in sediment extracts

1

0

500 1000 1500 2000 2500

∑PAHs (ng g-1 Sed dw) Fig. 5. (A) Total concentrations of 23 PAHs in ER River sediment samples collected P at four different months and (B) correlations among PAHs and AhR agonist activity (green diamond) or genotoxicity (white diamond) in ER River sediment samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

or benthic organisms has also been shown to be synergistic (Verrhiest et al., 2001). 3.4. PAH concentrations in ER River sediment extracts Among the 23 PAHs analyzed in this study, FLT, PYR, BaFl, BbFl, BaA, CHR, BbFa, BkFa, BaP, and INP have exhibited significant AhR agonist activity in the yeast-based reporter gene assay (Alnafisi et al., 2007; Misaki et al., 2007; Murahashi et al., 2007). The total concentrations of PAHs (shown as RPAHs = AhR-active PAHs + AhR-inactive PAHs) in ER River sediment extracts varied from 21 to 2286 ng g 1 Sed dw (Fig. 5A), which were comparable to those detected in the sediment samples collected from other river systems in Southern Taiwan, such as Kaoping River (8–356 ng g 1 Sed dw), Donggang River (23–2534 ng g 1 Sed dw), and Jengen River (283–1227 ng g 1 Sed dw) (Doong and Lin, 2004; Hsieh et al., 2010; Chen et al., 2012). Although no correlation was found between the total PAHs and the AhR agonist activity or the

Table 2 shows the b-NF EQs of individual AhR-active PAHs and the sum of 10 AhR-active PAHs (b-NF EQ10 PAHs) detected in sediment taken at ER1 and ER7 during different months. The toxic equivalents were estimated using the toxic equivalent factors (TEFs) obtained from previous studies (Alnafisi et al., 2007; Murahashi et al., 2007). The 4- and 5-rings BbFl and BbFa were major contributors to the calculated b-NF EQ10 PAHs owing to their higher TEFs. However, the contributions of 10 AhR-active PAHs (b-NF EQ10 PAHs) to the AhR agonist activity detected by the yeast bioassay (b-NF EQBioassay) varied from 0.2% to 37% in different months. Similar to our results, numerous studies have also reported that sometimes PAHs only account for a small percentage of the AhR agonist activity detected in different environmental samples (Brack et al., 2005; Qiao et al., 2006; Louiz et al., 2008; Muusse et al., 2012). Since conventional chemical analysis of target pollutants (such as PAHs) may lead to false negative results in sediment toxicity assessment, further identification work is necessary to characterize potent xenobiotic AhR agonists in the aquatic environments in Taiwan. 4. Conclusions In the present study, AhR agonist activity and genotoxicity in water, SS, and sediment samples from river systems in Southern Taiwan were investigated using recombinant cell assays in combination with instrumental fractionation and identification.

Table 2 b-NF equivalent concentrations of PAHs detected in sediment samples taken at ER1 and ER7 at different months. PAHs

TEF

PAH Conc.  TEF = b-NF EQ (ng g September 2010

FLT 0.009a PYR 0.006a BaFl 0.4b BbFl 3.7b BaA 0.3a CHR 0.5a BbFa 2.9b BkFa 0.7a BaP 0.2a IP 0.2a b-NF EQ10 PAHs b-NF EQBioassay Contribution (%) a b c

1

Sed dw)

December 2010

February 2011

July 2011

ER1

ER7

ER1

ER7

ER1

ER7

ER1

ER7

1.0 0.7 5.9 33.4 13.0 16.6 282.3 19.8 10.5 7.3 390.6 1577.4 24.8

2.3 2.6 20.9 133.3 22.9 25.8 210.4 23.2 14.3 3.4 459.1 1239.2 37.0

0.5 0.4 6.4 26.8 4.1 7.6 75.5 4.2 2.5 1.3 129.4 2244.0 5.8

2.0 1.9 9.1 69.0 12.1 14.8 112.6 12.0 6.4 3.7 243.5 5631.2 4.3

0.1 0.06 1.1 5.3 0.5 2.0 7.5 0.6 0.4 0.3 18.0 7721.6 0.2

2.1 2.2 20.5 118.4 21.7 43.1 222.6 20.7 11.3 3.0 465.6 4045.6 11.5

0.04 0.03 0.5 3.9 0.3 0.9 7.4 0.8 NDc 0.1 13.9 1377.4 1.0

3.2 3.4 26.6 171.5 24.9 45.0 296.1 24.0 16.6 8.0 619.2 2766.4 22.4

Toxic equivalent factor (TEF) = b-NF EC50/PAH EC50 (Alnafisi et al., 2007). TEF = BaP EC50/PAH EC50  BaPTEF (Murahashi et al., 2007). Not detected.

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Experimental results revealed that contaminants showing AhR agonist activity or genotoxic potential were ubiquitously present in YS River, ER River, and AGD River. In particular, water and sediment samples collected from ER River during dry seasons elicited the highest AhR agonist activity and genotoxicity. The yeast growth inhibition in the AhR signaling assay may relate to the genotoxicants in sample extracts, whereas no correlation was found between the AhR agonist activity and the genotoxicity. In addition, PAHs were considered as minor contributors to the AhR agonist activity detected in ER River sediment samples. Our findings showed that bioassays may provide valuable information on combined toxicity, which is helpful in assessing the extent and magnitude of environmental contamination. Further identification of potential candidates for xenobiotic AhR agonists and genotoxicants should be undertaken to better protect human health and the aquatic environment in Taiwan. Acknowledgements This work was supported by the National Science Council, Taiwan (NSC 98-2221-E-006-020-MY3, NSC 100-2221-E-006037-MY2). We thank Dr. Miller from Tulane University, USA, and Dr. Matsuda from Kyoto University, Japan, for providing the recombinant strains. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.12.052. References Alexandrov, K., Rojas, M., Satarug, S., 2010. The critical DNA damage by benzo(a)pyrene in lung tissues of smokers and approaches to preventing its formation. Toxicol. Lett. 198, 63–68. Allinson, M., Shiraishi, F., Salzman, S.A., Allinson, G., 2011. In vitro assessment of retinoic acid and aryl hydrocarbon receptor activity of treated effluent from 39 wastewater-treatment plants in Victoria, Australia. Arch. Environ. Contam. Toxicol. 61, 539–546. Alnafisi, A., Hughes, J., Wang, G., Miller, C.A., 2007. Evaluating polycyclic aromatic hydrocarbons using a yeast bioassay. Environ. Toxicol. Chem. 26, 1333–1339. Bartsch, H., 1996. DNA adducts in human carcinogenesis: etiological relevance and structure–activity relationship. Mutat. Res. – Rev. Genet. 340, 67–79. Bock, K.W., Köhle, C., 2006. Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem. Pharmacol. 72, 393–404. Brack, W., 2003. Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures? Anal. Bioanal. Chem. 377, 397–407. Brack, W., Schirmer, K., Erdinger, L., Hollert, H., 2005. Effect-directed analysis of mutagens and ethoxyresorufin-O-deethylase inducers in aquatic sediments. Environ. Toxicol. Chem. 24, 2445–2458. Bull, R.J., Reckhow, D.A., Li, X., Humpage, A.R., Joll, C., Hrudey, S.E., 2011. Potential carcinogenic hazards of non-regulated disinfection by-products: haloquinones, halo-cyclopentene and cyclohexene derivatives, N-halamines, halonitriles, and heterocyclic amines. Toxicology 286, 1–19. Chamorro, S., Monsalvez, E., Piña, B., Olivares, A., Hernández, V., Becerra, J., Vidal, G., 2012. Analysis of aryl hydrocarbon receptor ligands in kraft mill effluents by a combination of yeast bioassays and CG–MS chemical determinations. J. Environ. Sci. Health A 48, 145–151. Chen, C.-W., Chen, C.-F., Dong, C.-D., Tu, Y.-T., 2012. Composition and source apportionment of PAHs in sediments at river mouths and channel in Kaohsiung Harbor, Taiwan. J. Environ. Monitor. 14, 105–115. Cheng, J.-O., Ko, F.-C., Lee, C.-L., Fang, M.-D., 2013. Air–water exchange fluxes of polycyclic aromatic hydrocarbons in the tropical coast, Taiwan. Chemosphere 90, 2614–2622. Chinathamby, K., Allinson, M., Shiraishi, F., Lopata, A., Nugegoda, D., Pettigrove, V., Allinson, G., 2013. Screening for potential effects of endocrine-disrupting chemicals in peri-urban creeks and rivers in Melbourne, Australia using mosquitofish and recombinant receptor–reporter gene assays. Environ. Sci. Pollut. Res. 20, 1831–1841. Chou, P.-H., Matsui, S., Misaki, K., Matsuda, T., 2007. Isolation and identification of xenobiotic aryl hydrocarbon receptor ligands in dyeing eastewater. Environ. Sci. Technol. 41, 652–657. Costa, P.M., Lobo, J., Caeiro, S., Martins, M., Ferreira, A.M., Caetano, M., Vale, C., DelValls, T.Á., Costa, M.H., 2008. Genotoxic damage in Solea senegalensis

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