Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan

Marine Pollution Bulletin xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan Nien-Hsin Kao a,⇑, Ming-Chien Su b, Jheng-Rong Fan b, Chih-Chun Yen b a b

Department of Environmental Engineering, Kun Shan University, No. 195, Kun-Da Road, Yun Kan District, Tainan City 710, Taiwan Department of Natural Resources and Environmental Studies, National Dong Hwa University. No. 1, Sec. 2, Da Hsueh Rd., Shoufeng, Hualien 97401, Taiwan

a r t i c l e

i n f o

Article history: Received 20 April 2015 Revised 28 May 2015 Accepted 29 May 2015 Available online xxxx Keywords: Sediment Lubricant Bicyclic sesquiterpanes Terpanes Steranes PAHs

a b s t r a c t Three fishing harbors were investigated to study the polycyclic aromatic hydrocarbons in the sediments and trace possible anthropogenic sources by identification of cyclic terpenoid biomarkers. Seventeen terpanes, 10 steranes and 10 bicyclic sesquiterpanes in the marine diesel and the three kinds of lubricants that are mainly used by fishing boats were identified and quantified. Eighteen biomarker diagnostic ratios are suggested and the correlation coefficients among the lubricants and sediment samples have the R2 value greater than 0.73. Analyzed 16 PAHs in the sediment shows non-normal distributions and the Kruskal Wallis Test shows the significant differences (p value smaller than 0.05) with the greatest variability in benzo[g,h,i]perylene which more than 84% of the effective size (E.S.) is accounted. X-ray Photoelectron Spectroscopy (XPS) analysis was applied and the Kruskal Wallis Test shows a significant difference (p value smaller than 0.05) among certain atoms with the effective size greater than 60%. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Persistent organic pollutants (POPs), polycyclic aromatic hydrocarbons (PAHs) and biomarkers are the viable indication organic compounds in the invisible surface and may be harmful to the biota of the sedimentary environment (Lee et al., 2013; Solé et al., 2013). Generally, PAHs are found in fossil oils, coal and tar deposits, and are produced due to incomplete combustion, oil spills, burning, engine combustion and many other activities. Generated PAHs then can be transported into the environment by wet or dry deposition as well as by daily human operations. The distribution of PAHs and petroleum biomarker compounds in the marine sediments of rivers, estuaries, coastlines and sea beds have been commonly investigated (Boonyatumanond et al., 2006; Gong et al., 2014; Grice et al., 2009; Keshavarzifard et al., 2015; Page et al., 1999; Pauzi Zakaria et al., 2001; da Silva and Bicego, 2010; Tolosa et al., 2004; Trolio et al., 1999; Wang et al., 2011). Researchers have reported that the investigated PAHs in the marine sediments and the analyzed concentrations (in lg/kg) were up to 8399, 51,261, 434 and 11,557 lk/kg in the coastal areas of Thailand, along the river mouths close to Kaohsiung harbor (Taiwan), the coasts of Sicily (Italy) and the Gulf of Gela (Italy), ⇑ Corresponding author. E-mail addresses: [email protected] (N.-H. Kao), [email protected] (M.-C. Su), [email protected] (J.-R. Fan), [email protected] (C.-C. Yen).

respectively (Boonyatumanond et al., 2006; Dong et al., 2012; Frenna et al., 2013; Orecchio et al., 2010). Yunker et al. (2002) reported the application of the various ratios of principal mass 178, 202, 228 and 276 parent PAHs to infer and relate the investigated possible sources of PAHs. Moreover, researchers also reported the application of fingerprinting or diagnostic ratios to identify whether the investigated PAHs were generated from pyrolytic, petroleum or digenetic sources (Barreca et al., 2014; Frenna et al., 2013; Orecchio, 2010; Orecchio et al., 2010; Yunker et al., 2002). Generally, oil spill incidents and related contamination were frequently found in the various environments, while unknown pollutants in contaminated soils or sediments were mostly hard to detect or identify. Thus, other than the PAHs, there are also various kinds of biomarker compounds that can be used to identify the sources of pollutants. Biomarkers are a group of complex organic compounds and are mostly found in petroleum sources such as rock, crude oil and bitumen. Five groups of biomarkers, including the sesquiterpanes, terpanes, steranes, adamantanes and diamantanes, are generally used in the forensic chemical analyses to identify the possible sources of petroleum pollutants. Comparisons of the diagnostic ratios of detected PAHs and biomarker compounds have also been commonly applied in investigating weathering effects and possible sources of spills, in which terpanes and steranes have been commonly investigated. (Aboul-Kassim and Simoneit, 1995; Bieger et al., 1996; Chandru et al., 2008; Mulabagal et al., 2013; Wang et al., 2001; Yim et al.,

http://dx.doi.org/10.1016/j.marpolbul.2015.05.070 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

2

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

2011; Yunker et al., 2002; Zakaria et al., 2000). Our previous study also showed that C2-naphthalenes, adamantanes and bicyclic sesquiterpanes can be used to distinguish various kinds of local gasoline and diesel oil products (Kao et al., 2015). There are not many commercially available external standards to identify biomarkers. Therefore, the application of a retention index (RI or Kováts Index), relative response factors (RRF) and diagnostic ratios were the possible methods to identify biomarker compounds or investigate a spill site (Boehm et al., 1997; Guan et al., 1989; Kao et al., 2015; Lubeck and Sutton, 1983; Lucero et al., 2009; Stout et al., 2001; Wang and Fingas, 1997, 2003; Yang et al., 2006). Other than that, the application of GCFID and/or GCMS accompanied by biomarker identification have been the major methods to identify possible constituents in crude oil, petroleum products or spill sites (Stout et al., 2001, 2005; Wang et al., 2005, 2013; Yang et al., 2006, 2009, 2012). Similarly, biomarker parameters and calculated ratios among biomarkers were also used to identify oil spills in the sediment or to access the maturity of sediment and crude oil (Asif et al., 2009; Barakat et al., 2002). Among the characteristics of crude oil, mousse and tar ball samples, researchers reported the detected hopanes and steranes by GC/MS m/z 191 and 217. Mulabagal et al., 2013 reported the calculated ratios of 18a(H)-22,29,30-trisnorneohopane/17a (H)-22,29,30-trisnorhopane and 17a(H),21b(H)-30-nor-hopane/17 a(H),21b(H)-hopane were relatively unaffected by weathering effects. Researchers also reported that 17a(H),21b(H)-hopane was very resistant to biodegradation and can be used to study weathering effects (Mulabagal et al., 2013; Prince et al., 1994). Wang et al. (2001) reported a biomarker degradation study in the case of a 24 year old oil spill site in the Strait of Magellan. They concluded that even the most refractory biomarker compounds were also degraded to some extent after long term exposure in the environment (Wang et al., 2001). The reviewed studies were almost all predominantly conducted in investigating crude oil related spills in the ocean, coastlines or sediments of great open bodies of water. In this study, based on field investigations, we have focused on marine diesel and the three other kinds of lubricants that are mostly used by fishing boats in Taiwan. Thus, in this study, PAHs and the three categories of biomarkers, the bicyclic sesquiterpane, terpane and sterane compounds, were investigated in the oil products and sediments of the harbors. The objectives of the study are to develop a set of suitable biomarkers and forensic chemical procedures for investigating the oil related pollutants in the sediments of three fishing harbors and identify the possible sources of these pollutants.

2. Experimental methods 2.1. Studied area There are over 190 listed fishing harbors on the main island of Taiwan with only 10 of them located on the eastern coast of our studied counties (Fisheries Agency, Council of Agriculture, Executive Yuan, Taiwan, 2014). In Taiwan, the Northeast monsoon season generally occurs around October through April and during that period fishing activities are generally reduced. Thus, based on our available schedule, seasonal (winter and summer) sampling was conducted in the three studied harbors (Hua-lien (HL; 23°590 5500 N, 121°380 2300 E), Cheng-kung (CK; 23°50 4600 N, 121°220 5900 E) and Fu-gang (FG; 22°470 3100 N, 121°110 4100 E) which are all located on the eastern coast. Sediments in the preparation and maintenance area for the fishing boats in the harbors were sampled over the period of the last two consecutive years. According to the last published open records, the fishery production ratio in the selected HL and CK fishing harbors was 88% of

the 2 listed harbors and 75% of the total listed 8 harbors in the area, respectively (Fisheries Agency, Council of Agriculture, Executive Yuan, Taiwan, 2013). Moreover, to this day, CK harbor is still the largest fishing harbor in these two counties. Although small, FG fishing harbor was selected for background comparison due to its main purpose of embarking and disembarking ferry passengers for the purpose of tourism instead of fisheries. 2.2. Materials All chemicals were of high-performance liquid chromatography purity grade without any further purification. External calibration standards were used including 16 PAHs (Mixture #6 PAHs, Chem Service Inc., USA) and 10 C2-naphthalenes (2-ethylnaphthalene, 1-ethylnaphthalene, 1,2-dimethylnaphthalene. 1,3-dimethylnaphthalene, 1,4-dimethylnaphthalene, 1,5-dimethylnaphthalene, 1,6-dimethylnaphthalene, 1,8-dimethylnaphthalene, 2,6-dimethylnaphthalene, 2,7-dimethylnaphthalene; AccuStandard, Inc. USA). Internal standards included 2-fluorobiphenyl (Aldrich, USA), acenaphthene-d10 (SUPELCO, USA), cis-decalin, 17a(H),21 b(H)-hopane, 17b(H), 21a(H)-30-nor-hopane, 17ß(H), 21a(H)-hopane and 17b(H), 21b(H)-hopane (Chiron AS, Norway). Normal alkanes (C10–C25 and C16–C44, ULTRA SCIENTIFIC, USA) were used as the calibration compounds for the biomarker quantification. Marine diesel (MD) was collected from the major oil dispatch center of the CPC Corporation in Hualien County which is responsible for supplying the whole area of eastern Taiwan. Three kinds of the most widely used lubricant oils were purchased from a CPC gas station which is located in the HL harbor. It should be noted that there is only the CPC gas station which is in the studied harbors while the purchased 4-Liter container lubricant products are sold around Taiwan. These lubricants are named CF, CG4 and R68 with their product numbers LB 54295, LB 51940 and LA60343, respectively. 2.3. Sample preparation Sixteen USEPA listed PAHs, 10 bicyclic sesquiterpanes, 17 terpanes, 10 steranes and applied internal standards were examined in the study as listed in Table 1. Sediments were collected by using a sediment core sampler (Kajak–Brinkhurst corer). Sediment samples were studied based on their dry weight quantity and the surrogate standard was added into the freeze dried samples before the extraction process. Sample preparation was based on our laboratory protocol, that is the sediment samples were freeze dried (50 °C, 50 mmTorr, Kingmech FD6-12P-D) and meshed (No. 10 mesh) before extraction. PAH extraction methods were automated Soxhlet extraction (NIEA M193.00C), the silica gel cleanup method (NIEA M183.01C), and the analysis of semi-volatile organic compounds by GC/MS (NIEA M731.01C) (Environmental Analysis Laboratory, EPA, Taiwan, 2014). An accurate 10 g of each sediment sample was spiked with 2-fluorobiphenyl as the surrogate standard using a three-stage auto extraction (Gerhardt, SOX 416) conducted with solvents (n-hexane and acetone). The extracted sample solution was concentrated by a nitrogen purge concentration workstation (Caliper, Turbovap II) and concentrated to 1 mL. Silica gel was used for the cleanup process, which was rinsed with n-hexane, and then wrapped with aluminum foil and dried for 20 min in a 400 °C oven for pre-treatment. An appropriate amount of the pre-treated silica gel was dried at 130 °C for 16 h before it was put into the cleanup column, which was filled with 12 g of silica gel topped with 2 g of anhydrous sodium sulfate for the sample clean up. Hexane and mixed hexane/dichloromethane (8/1, v/v) were used as the elute solvents. The cleaned solution was then concentrated again and refreshed with cyclohexane and concentrated to 2 mL for analysis. Fresh marine diesel (MD) oil sample

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

3

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx Table 1 Studied terpane, sterane, bicyclic sesquiterpane compounds and applied internal standards.

a b c

Terpanes (at m/z 191)a

Abbreviation

Formula

RIc

Bicyclic sesquiterpanesb

Abbreviation

Formula

Ion peak

C23 tricyclic terpane C24 tricyclic terpane C24 tetracyclic terpane 18a(H),21b(H)-22,29,30-trisnorhopane 17a(H),21b(H)-22,29,30-trisnorhopane 17a(H),21b(H)-30-nor-hopane 18a(H),21b(H)-30-nor-neohopane 17ß(H),21a(H)-30-nor-hopane 17a(H),21b(H)-hopane 17a(H)-30-nor-29-homohopane 17ß(H),21a(H)-hopane 22S-17a(H),21b(H)-30-homohopane 22S-17a(H),21b(H)-30-homohopane 22S-17a(H),21b(H)-30,31-bishomohopane 22R-17a(H),21b(H)-30,31-bishomohopane 22S-17a(H),21b(H)-30,31,32-trishomohopane 22R-17a(H),21b(H)-30,31,32-trishomohopane Sterane (at m/z 218)a C21 5a(H),14b(H),17b(H)-sterane C22 5a(H),14b(H),17b(H)-sterane 20R-5a(H),14b(H),17b(H)-cholestane 20S-5a(H),14b(H),17b(H)-cholestane 20R-5a(H),14b(H),17b(H)-ergostane 20S-5a(H),14b(H),17b(H)-ergostane 20S-5a(H),14a(H),17a(H)-stigmastane 20R-5a(H),14b(H),17b(H)-stigmastane 20S-5a(H),14b(H),17b(H)-stigmastane 20R-5a(H),14a(H),17a(H)-stigmastane

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R

C23H42 C24H44 C24H42 C27H46 C27H46 C29H50 C29H50 C29H50 C30H52 C30H52 C30H52 C31H54 C31H54 C32H56 C32H56 C33H58 C33H58

2312 2365 2562 2896 2937 3071 3077 3119 3156 3164 3196 3244 3254 3309 3324 3396 3422

C4-decalin C14 sesquiterpane C15 sesquiterpane C15 sesquiterpane 8b(H)-drimane C15 sesquiterpane C16 sesquiterpane C16 sesquiterpane C16 sesquiterpane 8b(H)-homodrimane Internal standards 17b(H),21b(H)-hopane 17ß(H),21a(H)-30-nor-hopane 17ß(H),21a(H)-hopane 17a(H),21b(H)-hopane cis-Decalin Acenaphthene-d10 Acenaphthene-d10

BS1 BS2 BS3 BS4 BS5 BS6 BS7 BS8 BS9 BS10

C14H26 C14H26 C15H28 C15H28 C15H28 C15H28 C16H30 C16H30 C16H30 C16H30

179 179 193 193 123 123 123 193 193 123

1359 1374 1445 1484 1494 1509 1524 1571 1576 1582

C30H52 C29H50 C30H52 C30H52 C10H18 C12D10 C12D10

191 191 191 191 138 164 164

3279a 3119a 3196a 3156a 1115b 1594b 1483a

S21 S22 C27bbR C27bbS C28bbR C28bbS C29S C29bbR C29bbS C29R

C21H36 C22H38 C27H48 C27H48 C28H50 C28H50 C29H52 C29H52 C29H52 C29H52

2226 2340 2830 2839 2935 2942 3001 3019 3024 3062

M29 M30 H30 d10 d10

RI

Analyzed by using HP-5MS column. Analyzed by using SPB-1701 column. Calculated retention index (RI).

was collected and stored in a refrigerator at 4 °C for preservation before each run of analysis. During the analysis itself, marine diesel was diluted 2000 times with isooctane without any extraction and cleaning procedures. Each kind of lubricant sample was taken from the 4-Liter container before analysis. Without the step of extraction, 0.1 g of lubricants were diluted with 10 mL of hexane, sonicated for 15 min and prepared with the same concentration and cleaning procedures as the sediment sample except that 20 g of pre-conditioned silica gel was used in the cleanup column. Diesel samples for analysis of bicyclic sesquiterpane (BSs) were added with an appropriate amount of acenaphthene-d10 as the internal standard. Lubricant and sediment samples for analysis of terpane and sterane were also added with an appropriate amount of 17b(H), 21b(H)-hopane as internal standards. In the analytical procedures, all the sediment and lubricant samples were spiked with surrogate standard 2-fluorobiphenyl with the recovery percentages all between 90% and 98%. Besides that, two additional lubricant samples were spiked with acenaphthene-d10 and the recovery percentages were 109% and 118%. The calculation of the recovery ratio is based on the result of the response/concentration linear curve of the spiked surrogate or internal standard. The R2 values were all greater than 0.99 for the 11 calibration concentrations for the analytes of external 16 PAHs and 10 kinds of C2-naphthalene standards. The R2 values were all also greater than 0.99 for the five point response factors (RF) of the internal standards. The reproducibility of the studied compounds were also checked and are listed in Table 2 for the studied PAHs. 2.4. Qualitative and quantitative analysis PAHs were analyzed using a HP-5 or HP-5 ms capillary column (30 m  0.25 mm ID  0.25 lm film thickness, Agilent) during the

sampling periods. A split injection (10:1) mode was set with the oven program at 60 °C, held for 2 min, heated to 255 °C at 5 °C/min, then raised to 280 °C at 15 °C/min, held for 12 min and finally heated to 300 °C at 15 °C/min and then held again for 10 min. GC/MS (Thermo, Trace GC ultra/DSQ II) were operated at 70 eV electron impact in the selected ion monitoring (SIM) mode and a set ion source temperature of 250 °C. Concentrations of PAHs in oil samples were calculated by the results of external 16 PAHs and C2-naphthalene calibration standards. The identification of bicyclic sesquiterpanes (BSs) is based on our previous study of gasoline and diesel products; briefly, the diesel and sediment samples were analyzed by using a SPB-1701 capillary column (30 m  0.25 mm ID  0.25 lm film thickness, SUPELCO) and injected in splitless mode with a GC oven program at 60 °C, held for 4 min, heated to 280 °C at 5 °C/min and then held for 15 min. The characteristic fragment ions of BSs were determined and are confirmed at m/z 123 in the study. Identification of terpanes and steranes in the lubricant and sediment samples were conducted by using a HP-5 ms capillary column (30 m  0.25 mm ID  0.25 lm film thickness, Agilent) and injected in splitless mode with the oven program at 50 °C, held for 2 min, heated to 300 °C at 6 °C/min and then held for 15 min. The characteristic fragment ions of terpanes and steranes were determined by m/z 191 and 218, respectively. A qualitative check of terpane compounds was conducted by adding 17a(H),21b(H)hopane,17b(H),21a(H)-hopane and 17b(H),21a(H)-30-nor-hopane into the additional prepared sediment samples. Among these three internal standards, the 17a(H),21b(H)-hopane has presented the highest concentration in the studied samples; thus, a 5-point calibration line (1100–5500 lg/L, R2 > 0.99) of the compound was made to compare the calibrated concentration and quantified concentration using relative response factors (RRF). The results show that the 17b(H),21a(H)-30-nor-hopane is more suitable to be used as another internal standard for RRF quantification. The condition

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

4

a

Abbre.g Formula Base peak

Nap C10H8 128

AcNy C12H8 152

AcN C12H10 154

Fl C13H10 166

PhA C14H10 178

An C14H10 178

FlA C16H10 202

Py C16H10 202

BaA C18H12 228

Chy C18H12 228

BbF C20H12 252

BaF C20H12 252

BaP C20H12 252

IP C22H14 278

dBahA C22H12 276

BghiP C22H12 276

RPD (%)a

N.A.f

N.A.

N.A.

N.A.

8.6 ± 7.6

11.6 ± 6.0

6.7 ± 4.3

4.5 ± 4.1

8.7 ± 2.0

2.4 ± 2.1

8.6 ± 2.2

8.4 ± 4.9

7.6 ± 5.6

4.0 ± 4.3

N.A.

4.2 ± 3.1

CK, HL and FG harbors nb 29 Chi-Square 15.47 c df 7 d Asy. Sig. 0.03 ES (%)e 55.25

27 11.54 4 0.02 44.38

11 5.04 1 0.02 50.42

25 7.75 5 0.17 32.31

47 28.98 8 0.00 63.00

47 29.38 8 0.00 63.88

47 33.31 8 0.00 72.41

47 31.50 8 0.00 68.48

47 31.83 8 0.00 69.20

47 30.14 8 0.00 65.53

47 38.79 8 0.00 84.33

47 36.70 8 0.00 79.77

47 32.24 8 0.00 70.08

45 34.25 8 0.00 77.83

34 11.98 5 0.04 36.31

46 37.98 8 0.00 84.41

CK harbor n Chi-Square df Asy. Sig. ES (%)

21 11.84 3 .001 59.20

23 2.23 2 0.33 10.15

8 N.A.

18 4.20 2 0.12 24.70

32 4.33 3 0.23 13.96

32 13.28 3 0.00 42.84

32 6.64 3 0.08 21.42

32 8.25 3 0.04 26.62

32 2.89 3 0.41 9.32

32 0.82 3 0.84 2.65

32 18.27 3 0.00 58.94

32 13.70 3 0.00 44.19

32 7.53 3 0.06 24.28

32 13.09 3 0.00 42.24

30 6.44 3 0.09 22.22

32 18.73 3 0.00 60.41

HL harbor n Chi-Square df Asy. Sig. ES (%)

7 1.14 2 0.56 19.05

3 N.A.

3 N.A.

6 2.33 1 0.13 46.67

12 4.13 3 0.25 37.53

12 2.59 3 0.46 23.54

12 4.38 3 0.22 39.86

12 4.85 3 0.18 44.06

12 5.36 3 0.15 48.72

12 1.51 3 0.68 13.75

12 6.85 3 0.08 62.24

12 4.74 3 0.19 43.12

12 6.69 3 0.08 60.84

12 4.59 3 0.20 41.72

4 0.20 1 0.65 6.67

12 8.23 3 0.04 74.83

Relative percent difference (RPD) for the duplicated samples (n = 5) for quality check of GC/MS. Independent n samples in Kruskal Wallis test (compound concentrations with under detection limit were excluded). c Degree of freedom. d Asymptotic significant value. e Calculated effect size (Chi-Square/(n  1)). f RPD or Kruskal Wallis test was not calculated due to only one season was detected for the compound. g Abbreviation for the 16 PAHs in the order of Naphthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benz[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene, Indeno[1,2,3-cd]pyrene, Dibenz[a,h]anthracene and Benzo[g,h,i]perylene. b

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

Table 2 Studied 16 PAHs and statistic results of nonparametric Kruskal Wallis Test for the concentrations in the sediments.

5

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Fig. 1. Geographical location of the studied harbors and analyzed seasonal total PAHs concentrations in the sediment samples. (a) HL harbor. (b) CK harbor. (c) FG harbor. (Map: https://www.google.com.tw/maps, not to scale.)

Table 3 Analyzed atomic concentration (in %) and Kruskal Wallis test in the sediments of CK and HL harbors.

a

Element

K

O

Mg

Si

C

Al

Na

N

Ca

Fe

Cl

CK-1 CK-2 CK-3 CK-4 CK-5 CK-6 CK-7 CK-8 HL-1 HL-2 HL-3 HL-4 HL-5 HL-6

N.D. 0.60 0.61 0.81 0.83 N.D. 0.84 N.D. 0.91 0.80 1.02 1.20 0.69 N.D.

45.11 47.31 45.60 45.87 45.18 46.07 45.64 44.25 42.16 42.82 42.01 48.57 40.13 36.81

2.73 3.56 3.95 3.67 3.04 3.51 3.22 3.05 3.34 3.41 4.54 3.36 2.42 2.28

15.32 15.58 15.79 15.99 15.29 16.27 16.09 15.76 15.39 15.23 15.23 17.43 14.72 14.21

22.56 20.16 19.09 18.52 21.18 19.07 19.85 21.35 23.53 23.75 22.1 15.15 30.16 33.51

6.13 6.28 6.50 6.79 6.50 6.80 6.78 7.45 5.90 5.92 6.20 7.39 6.02 5.53

1.87 1.73 1.87 1.99 1.74 1.41 1.57 1.88 1.40 1.48 1.38 1.07 1.04 1.25

1.30 1.36 1.96 1.6 1.68 1.97 1.69 1.69 2.09 1.83 2.19 1.22 1.92 1.49

2.01 2.08 1.97 1.93 1.67 1.79 1.67 1.57 1.54 1.44 1.37 1.46 1.53 1.64

1.40 1.34 1.39 1.61 1.642 1.81 1.49 1.54 1.44 1.35 1.63 1.61 1.38 1.13

1.57 N.D. 1.28 1.22 1.28 1.31 1.15 1.46 2.28 1.97 2.33 1.55 N.D. N.D.

Kruskal Wallis test n Chi-Square df Asymp. Sig.a Effective size (%)

10 1.84 1 0.18 20.49

14 4.27 1 0.04 32.82

14 0.27 1 0.61 2.05

14 3.27 1 0.07 25.18

14 3.75 1 0.05 28.85

14 4.28 1 0.04 32.89

14 8.84 1 0.00 67.97

14 0.60 1 0.44 4.62

14 8.84 1 0.00 67.97

14 1.07 1 0.30 8.21

11 6.06 1 0.01 60.63

Asymptotic significant value.

and stability of GC/MS were regularly checked by the injection of acenaphthene-d10 and 17b(H),21b(H)-hopane standards. An additional check for the 5-point response factors (RF, R2 > 0.99) of the 17a(H),21b(H)-hopane was also conducted and the relative percentage difference (RPD) was 4.1% (n = 2).

Based on our experimental protocol, an initial step of quantification is the calculation of non-isothermal retention indices (RI. or Kováts retention index) by using equation (Eq. (1)). In addition, a set of relative response factors (RRF) equations (Eq. (2)) were used to quantify the 10 BS, 17 terpane and 10 sterane compounds.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

6

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx 1600

350 HL (n =13)

300

1400

250

1200 1000

μ

200 150

800 600

100

combustion

petroleum

0.60

mixed souces

combustion

1.0

grass,woodand coalcombustion

0.9 0.8

petroleum 6 0.

7

5 0.

An / 178

0.

4 0.

2 0.

3

1 0.

0.

0 0.

0.20

0.2 0.1

petroleum

0.0

0. 45

0.25

CK(Jan.2013) CK(July2013) CK(Feb.2014) CK(May2014) HL(Jan.2013) HL(July2013) HL(Feb.2014) HL(May2014) FG(May2014)

0. 40

0.30

petroleum combustion

0.3

0. 35

0.35

0.5 0.4

0. 30

0.40

0.6

0. 25

petroleum combustion

0.45

grass,wood and coal combustion

0.7

0. 20

IP / (IP + BghiP)

0.50

0. 15

0.55

FLA/ (FLA + Py)

I dB P ah A Bg hiP

(b)

(a) petroleum

Py Ba A Ch y Bb F Ba F Ba P

Na p Ac Ny Ac N

I dB P ah A Bg hiP

An Fl A

Py Ba A Ch y Bb F Ba F Ba P

0

Fl Ph A

0

Na p Ac Ny Ac N

200

Fl A

400

50

Fl Ph A An

μ

CK (n =32)

BaA / 228

(c) Fig. 2. Variation of detected PAHs concentrations in the sediment samples. (a) HL harbor. (b) CK harbor. (c) Fingerprinting identification for three harbors and four seasons.

  trðbiomarkerÞ  t rðnÞ RIbiomarker ¼ 100  n þ ðN  nÞ t rðNÞ  trðnÞ

ð1Þ

where RIbiomarker = the retention index for biomarkers. trðbiomarkerÞ ; trðNÞ ; t rðnÞ = the retention time for the biomarker, the number of carbon atoms in the larger (N) and smaller (n) n-alkanes, respectively.

C biomarker ¼

Abiomarker C IS1  AIS1 RRFIS2

!  DF

ð2Þ

IS1

where C biomarker = the quantified concentration of biomarkers (BS, terpane and sterane). Abiomarker = the chromatographic area of biomarkers. C IS1 ; AIS1 = the concentration and chromatographic area of the applied internal standards (acenaphthene-d10 for BS; 17b(H),21b(H)-hopane for terpane and sterane). DF = applied dilution factor. IS2 RRFIS2 = RF = the relative response factor between internal RFIS1 IS1

standards. RFIS1 ; RFIS2 = the average 5-point (R2 > 0.99) response factor for internal standards (acenaphthene-d10 and cis-Decalin for BS; 17b(H),21b(H)-hopane and 17b(H),21a(H)-30-nor-hopane for terpane and sterane). 3. Results and discussion 3.1. Variation of PAH distribution Fig. 1 shows the geographical location and total PAH concentrations (in lg/kg) for the three harbors in the respective sampling

points. Four seasons (January 2013, July 2013, February 2014 and May, 2014) of sediment samples were consistently collected for the eight locations within the CK harbor. Originally, three points of sediment samples were regularly collected in the first three seasons, but in the last season, three more points were added for the HL harbor. For the purpose of background comparison, three sampling points in the small FG harbor were also collected in the last season. The latest records (2013) of the local county government show that there were a total number of 298 and 245 fishing boats registered in the CK and HL harbors, respectively. Moreover, there were 117 fishing boats that weighed more than 5 tonnes in the CK harbor, compared to only 75 in the HL harbor. The previous listed yearly fishery production for CK and HL harbors were 4449 and 1710 tonnes, respectively (Fisheries Agency, Council of Agriculture, Executive Yuan, Taiwan, 2010). Based on the facts of quantity differences as described, we can recognize the results of higher PAH concentrations in CK harbor. Fig. 1 shows that CK harbor had the highest analyzed concentration, up to 6900 lg/kg. Comparably, the total PAHs in the HL and FG harbors show lower concentrations, except for the newest 2 sampling points (HL-5 and 6) of HL harbor which were collected in the last season and had analyzed concentrations of up to 7000 lg/kg. The field investigation shows that these two sampling points of HL harbor had a thicker sediment layer and an odor of anaerobic digestion was detected at the moment of sampling, which represents the fact of the long term accumulation of sediments. These two points are the dead spots located inside the harbor and dredging might be the only feasible method to clean these areas. Thus, to understand the chemical composition of the sediment surface, an X-ray Photoelectron Spectroscopy (XPS; VGS, Thermo K-Alpha) was used to scan the sediment samples. Fig. 1 also shows no observed similar pattern of concentration distributions in those sample seasons between HL and CK harbors; however it generally indicates the distribution of PAH concentrations in each sample point.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

7

Fig. 3. Peak cluster of biomarker compounds among oil and sediment samples. (a) Identified 17 terpanes by m/z 191. (b) Identified 10 steranes by m/z 218.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

8

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

3.2. Statistical inference of PAHs

Photoelectron Spectroscopy (XPS) was used to scan the sediment samples. Table 3 lists the analyzed atomic concentration (in %) of the sample from the last season and in particular, the above reported two points (HL-5 and 6) have lower values (40.13% and 36.81%) for oxygen and higher values (30.16% and 33.51%) for carbon. Again, the results might indicate the long term accumulation of pollutants and the anaerobic condition of the sediment environment. Statistically, the results of the Kruskal Wallis Test in Table 3 also show significant differences among certain atoms with sodium, cesium and chlorine atoms having an effective size greater than 60%. For each PAH compound, Fig. 2(a and b) illustrate the box plots for the total sampled points in these harbors which show that most of the 2–3 ring low molecular weights PAHs had lower concentrations (below 50 lg/kg) than other compounds. The dominant PAHs were fluoranthene (FLA) and pyrene (Py) followed by the other high molecular weights compounds, which also indicates the weathering effects for those petrogenic PAHs. Due to the high concentrations, two sampling points, HL-5 and HL-6 were not plotted in Fig. 2(a). Fig. 2(b) shows that CK harbor had the highest concentrations and confirms that fishery activity might be the major factor in the contribution of anthropogenic sources of PAHs into the sediments. Our data also indicates that the small FG harbor had the highest concentrations of fluoranthene and pyrene. Two

The statistic tests (SPSS, V. 20) were conducted by using the normality test to check whether the data is normally distributed and then use the test of equality of variances to choose a suitable method for further testing. The test results indicated that the PAH data is non-normally distributed and non-parametric; thus we used the Kruskal Wallis Test to infer the PAH data. The non-parametric independent variable tests of PAHs were conducted as shown in Table 2. All the detected PAHs in the sediment samples were tested and reported in terms of the Kruskal Wallis Test which shows a significant difference between tested PAHs when the asymptotic significant value (or p value) is smaller than 0.05. Table 2 shows that all of the PAHs, except for fluorene (Fl), have significant differences among the three harbors with the greatest variability being benzo[g,h,i]perylene (BghiP), where more than 84% of the effective size (E.S.) is accounted for by seasons or by locations. For the CK harbor, the significant differences and the high effective size of PAHs are distributed among the 5 to 6 ring high molecular weight compounds. However, there are no significant differences among compounds for the HL harbor, except for BghiP, although most of the effective size is still greater than 30%. Due to the anaerobic odor emitted from the sediment samples that was collected in the last season of HL harbor, an X-ray

Table 4 Analyzed concentrations of terpanes, steranes and bicyclic sesquiterpanes (BSs) in the studied oil and sediment samples. Biomarker

Lubricant (mg/kg)

Terpanes

CF

CG4

R68

24.5 10.4 44.6 171.0 164.4 863.8 194.3 60.3 739.9 73.3 62.3 500.2 379.3 265.4 175.9 151.7 93.7

276.0 98.1 399.4 483.5 475.4 1465.9 283.0 91.1 904.7 92.8 65.4 436.2 324.2 198.3 119.1 89.0 46.6

23.5 9.2 133.0 571.6 417.5 1615.7 371.3 101.9 1124.3 110.5 84.9 611.4 449.2 278.1 183.2 141.6 83.1

Total

3974.9

5848.7

6310.1

Steranes

CF

CG4

R68

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R

S21 S22 C27bbR C27bbS C28bbR C28bbS C29S C29bbR C29bbS C29R

13.1 7.0 52.2 42.3 31.2 29.0 37.6 111.0 100.7 30.3

173.5 93.6 230.5 193.4 94.1 90.9 92.5 258.0 221.1 60.3

Total

454.4

1508.0

5.6 5.4 199.1 159.9 90.5 80.4 95.2 271.0 236.0 67.7

CF (n = 2)

R68 (n = 2)

Sediment (n = 6)

7.0 7.4 6.8 9.7 7.8 6.8 5.0 5.7 7.4 7.2 8.3 8.7 7.9 6.4 5.3 2.2 3.2

CG4 (n = 2) 0.1 6.7 9.0 3.5 4.5 0.0 0.7 0.4 6.3 0.1 4.7 3.3 1.3 3.3 0.4 2.5 1.9

3.3 1.7 4.2 3.1 4.7 4.2 4.4 2.8 4.4 11.0 1.2 5.5 5.4 9.8 1.1 7.3 13.6

6.1 ± 2.4 6.5 ± 4.8 5.0 ± 2.6 3.5 ± 1.6 2.7 ± 1.6 3.0 ± 2.0 2.1 ± 2.5 3.4 ± 2.6 4.1 ± 1.0 3.3 ± 1.1 6.2 ± 3.9 4.5 ± 3.4 4.0 ± 3.7 5.9 ± 4.9 4.1 ± 2.6 8.8 ± 1.6 11.8 ± 8.7

28.6 20.8 10.7 11.7 6.1 6.9 5.6 8.2 9.3 6.1

13.1 4.0 1.2 4.1 2.2 2.3 2.6 0.3 1.1 4.0

19.1 21.5 13.5 13.8 1.2 10.6 15.1 13.1 11.2 11.7

10.7 ± 2.9 11.2 ± 3.0 7.3 ± 5.5 6.4 ± 4.2 7.3 ± 6.3 8.7 ± 4.9 3.4 ± 1.6 3.1 ± 2.4 5.2 ± 2.9 10.3 ± 7.9

1210.8

BSs

Marine diesel (mg/kg)

CK (lg/kg) (n = 16)

HL (lg/kg) (n = 6)

RPD (%) Sediment (n = 4)

BSs

Marine diesel (mg/kg)

CK (lg/kg) (n = 16)

HL (lg/kg) (n = 6)

RPD (%) Sediment (n = 4)

BS1 BS2 BS3 BS4 BS5

358.7 203.2 723 601.5 1112.5

5.2 ± 8.0 1.7 ± 3.8 80.2 ± 64.2 116.9 ± 73.3 235.3 ± 148.6

N.D. N.D. 16.8 ± 11.5 24.2 ± 15.6 49.5 ± 26.9

N.A. N.A. 3.2 ± 3.7 0.8 ± 0.9 4.3 ± 4.1

BS6 BS7 BS8 BS9 BS10

555.6 252.3 323.3 113.9 1990.4

127.2 ± 80.5 89.7 ± 51.3 187.7 ± 102.0 48.6 ± 31.5 1180.9 ± 617.0

23.8 ± 14.4 16.0 ± 10.5 41.0 ± 20.8 11.9 ± 6.5 276.8 ± 131.8

4.5 ± 5.4 2.9 ± 2.8 2.4 ± 2.0 8.8 ± 8.9 4.8 ± 0.6

6234.5

2073.4 ± 1168.1

460.2 ± 234.0

Total concentration (BS1–BS10) a

Calculated RPDa (%)

Relative percent difference (RPD).

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

9

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Total concentration ( g/kg)

70000

Terpane (Feb. 2014) Terpane (May 2014) Setrane (Feb. 2014) Sterane (May 2014)

60000 50000 40000

30000 20000 10000

HL-6

HL-5

HL-4

HL-3

HL-2

HL-1

0

CK-8

CK-7

CK-5

CK-6

CK-4

CK-3

CK-2

CK-1

0

(a) 6000

5000

CK (Feb. 2014)

CK (May 2014)

5000

4000

concentration ( g/kg)

concentration ( g/kg)

4000

3000

2000

1000

3000

2000

1000

0

C29S

HL-1 to HL-4 (May 2014) HL-5 and HL-6

HL (Feb. 2014) 3000

HL -1 to HL-4 ( g/kg)

3000 concentration ( g/kg)

16000

4000

4000

2000

1000

12000 10000

2000 8000 6000 1000 4000 2000

0

0

14000

HL-5 and HL-6 ( g/kg)

(b)

C29 β β R C29 β β S C29R

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R S21 S22 C27 β β R C27 β β S C28 β β R C28 β β S

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R S21 S22 C27 β β R C27 β β S C28 β β R C28 β β S C29S C29 β β R C29 β β S C29R

0

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R S21 S22 C27 β β R C27 β β S C28 β β R C28β β S C29S C29 β βR C29 β βS C29R

TR23 TR24 TET24 Ts Tm H29 C29Ts M29 H30 NOR30H M30 H31S H31R H32S H32R H33S H33R S21 S22 C27 β β R C27 β β S C28 β β R C28 β β S C29S C29 β β R C29 β β S C29R

0

(c) Fig. 4. Variation of terpane and sterane concentrations. (a) Total concentration for harbors and lubricants. (b) Two seasons of CK harbor. (c) Two seasons of HL harbor.

fingerprinting ratios, the fluoranthene/(fluoranthene + pyrene) versus anthracene/(anthracene + phenanthrene) and inde no[1,2,3-cd]pyrene/(indeno[1,2,3-cd]pyrene + benzo[ghi]perylene)

versus benz[a]anthracene/(benz[a]anthracene + chrysene), that is the FLA/(FLA + Py) versus (An/178) and IP/(IP + BghiP) versus (BaA/228) are calculated. These two set of comparisons will

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

10

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

differentiate the petrogenic and pyrogenic sources of the PAHs in the sediment (Yunker et al., 2002). The calculation results for these comparison ratios among the harbors are illustrated in Fig. 2(c), which distinguish the possible sources of PAHs for all the samples during all sampling seasons. Due to the high concentrations, the sampling points HL-4 to HL-6 of the HL harbor in the last season were not plotted in Fig. 2(c). For the comparison set of FLA/(FLA + Py) versus (An/178), Fig. 2(c) shows that almost all the samples in the CK harbor are located above the quadrant of petroleum and have the characteristics of pyrogenic sources.

Otherwise, the samples in FG harbor and most of the samples in the HL harbor have revealed possible sources from petroleum combustion. However, in the comparison set of IP/(IP + BghiP) versus (BaA/228), Fig. 2(c) shows that most of the samples in HL harbor are distributed in the quadrants of mixed and petroleum combustion. Similarly, the samples in the CK harbor have additional marks in the quadrants of grass, wood and coal combustion. We also calculated the diagnostic ratio by using the reported total index as the sum of single indices (total index = fluoranthene/(fluoranthene + pyrene)/0.4 + anthracene/(anthracene + phenanthrene)/0.1 + benz[a]-

Fig. 5. Variation of biomarkers. (a) Seasonal BSs concentrations between harbors. (b) Normalized ratios of BSs among harbors and marine diesel. (c) Normalized ratios of terpane (divided by H29) and sterane (divided by C29bbR) among lubricants. (d) Normalized ratios of terpane and sterane between harbors.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

11

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

anthracene/(benz[a]anthracene + chrysene)/0.2 + indeno[1,2,3-cd]pyr ene/(indeno[1,2,3-cd]pyrene + benzo[ghi]perylene)/0.5) (Barreca et al., 2014; Frenna et al., 2013; Orecchio, 2010; Orecchio et al., 2010; Yunker et al., 2002). The calculated averaged total index for the four sampling seasons are 8.12 ± 1.24 (n = 32) and 8.08 ± 0.67 (n = 12) for CK and HL harbors, respectively. These two averaged total indices are greater than 4 which indicates that the sources of the investigated PAHs might be due to high temperatures or the combustion process. Otherwise, if the total index is less than 4, it indicates low temperature sources such as petroleum products. According to our previous research, diesel oil contains various kinds of high concentrations of C2-naphthalene compounds (Kao et al., 2015). To investigate the possibility of marine diesel spillage in the harbors, the analysis of C2-naphthalenes was also conducted in one season (February 2014). The result shows that total C2-naphthalene concentrations (in lg/kg) in the sediments of CK (8 points), HL (3 points) and FG (3 points) were 90, 68 and 107 lg/kg, respectively. These detected low concentrations might indicate no major oil spill incidents, but might also indicate low temperature sources of petroleum products for these harbors as shown in Fig. 2(c). This should be due to the fact that the fishing activities are still being operated and long term accumulation of oil products into the sediment is possible. The above findings suggest that both petrogenic and pyrogenic origins are another possible source of PAHs in the sediment might be due to the combustion of fossil fuels, urban runoff or others. That is the variation of PAH distribution in the sediment sample has other uncertainty factors. Thus, a further study of biomarker identification is necessary to track and relate the pollutants and their anthropogenic sources.

by the different shape of the ‘‘hump’’ clusters in the SIM mode of the MS scan. The hump is the signature of complex hydrocarbon compounds with high molecular weights and high boiling points that exist in high-range distillate petroleum products. Fig. 3(b) illustrates the 10 detected steranes at m/z 218, where the inserted figure shows low concentrations for these two compounds (S21 and S22). Moreover, the BS compounds can also be identified by m/z 123 and were eluted between n-C13 and n-C16 (boiling point range: 235–287 °C) for marine diesel and sediments. Eq. (2) was applied to quantify the biomarker BS, terpane and sterane compounds in marine diesel, lubricants and sediment samples. Table 4 lists the calculated biomarker concentrations for lubricants, marine diesel, concentrations of bicyclic sesquiterpanes (BSs) in the sediments and the respective relative percentage difference (RPD, in %) of oil and sediment samples. Due to the different research periods, not all of the biomarkers were analyzed, in which the terpanes and steranes are the samples of two of the seasons (February 2014 and May 2014), while the BSs are the samples of seasons July 2013 and February 2014. The calculated RPD shows that most of the data is under 10%, with only a few greater values appearing in the steranes of lubricants. Table 4 shows that BSs still can be detected in the sediments, which can be understood by the fact that marine diesel is the only fuel used by the fishing boats. However, the BSs with low molecular weights show obvious weathering effects. Moreover, Table 4 shows that the concentrations of terpanes are much higher than steranes among the lubricants. Fig. 4 illustrates the comparison of concentrations among sediments (in lg/kg). From the view point of total concentration, Fig. 4(a) shows that terpane is still the major biomarker in the sediment. Although we also examined the BSs for these three lubricants before, there were only a few BSs with unquantifiable peaks that existed. Similarly, with the PAH distribution, CK harbor had a higher biomarker concentration with the two sampling points (HL-5 and HL-6) having the highest concentrations, up to about 60,000 lg/kg. However, less variation in biomarkers between seasons was observed, which indicates weathering resistance for the terpane and sterane compounds in the studied period. More clearly, Fig. 4(b and c) shows the error bars for each compound with the dominant 17a(H),21b(H)-30-nor-hopane (H29) and 17a(H),21b(H)-30-hopane (H30) compounds, which are suitable to be used as major biomarkers for further study. The high concentrations of terpanes appear at the 17a(H),21b(H)-30-nor-hopane (H29) and are followed by 17a(H),21b(H)-30-hopane (H30), which indicates the input of petroleum sources which agrees with the reported high abundance

3.3. Identification of biomarkers In the study, biomarker compounds in the marine diesel and lubricants were investigated, the bicyclic sesquiterpanes, terpanes, and steranes. Thus, these three categories of biomarker compounds were analyzed to relate the anthropogenic sources of pollutants to the sediments. Table 1 lists the calculated retention index for the studied 17 terpane, 10 sterane and 10 bicyclic sesquiterpane compounds. Fig. 3(a and b) illustrate a part of the chromatographic peaks at m/z 191 for the biomarker terpanes and m/z 218 for steranes that were eluted after n-C22 (boiling point 224 °C) for the lubricant oils and sediment. Comparatively, Fig. 3(a) shows that the low molecular weight terpanes had lower concentrations of these lubricants. These three lubricants can also be distinguished

Table 5 Calculated 18 categories of diagnostic ratios (in %) for the studied lubricants and sediment samples. Lubricant

Lubricant

Diagnostic ratio

R68

CF

CG4

CK harbor CK-1

CK-2

CK-3

CK-4

CK-5

CK-6

CK-7

CK-8

HL harbor HL-1

HL-2

HL-3

TR24/TR23 Ts/(Tm + Ts) H30/H29 C29Ts/H29 NOR30H/H30 TR23/H30 TR24/H30 H31R/H31S H32R/H32S H33R/H33S C27bb/(C27 + C28 + C29)bb C28bb/(C27 + C28 + C29)bb C29bb/(C27 + C28 + C29)bb C27bb(S + R)/C29bb(S + R) C28bb(S + R)/C29bb(S + R) C27bb/H30 C28bb/H30 C29bb/H30

39.1 57.8 69.6 23.0 9.8 2.1 0.8 73.5 65.9 58.7 34.6 16.5 48.9 70.8 33.7 31.9 15.2 45.1

42.4 51.0 85.7 22.5 9.9 3.3 1.4 75.8 66.3 61.8 25.8 16.4 57.8 44.6 28.4 12.8 8.1 28.6

35.5 50.4 61.7 19.3 10.3 30.5 10.8 74.3 60.1 52.3 39.0 17.0 44.0 88.5 38.6 46.9 20.5 53.0

63.0 51.6 77.3 23.3 9.2 42.5 26.8 75.8 61.7 55.6 26.1 23.3 50.6 51.5 46.0 21.3 19.1 41.5

66.7 51.2 80.9 23.2 8.5 37.0 24.7 75.1 61.2 54.2 22.6 24.0 53.4 42.3 45.0 18.0 19.1 42.6

66.8 51.8 81.3 23.5 8.9 31.5 21.0 72.6 60.6 55.5 22.1 24.0 53.9 41.1 44.5 17.5 18.9 42.6

66.3 51.0 80.0 22.7 8.3 33.5 22.2 73.7 63.8 51.0 23.2 22.5 54.3 42.7 41.3 17.5 16.9 40.9

67.3 50.4 78.7 21.6 10.3 34.3 23.1 73.1 61.3 54.8 22.9 24.2 52.9 43.2 45.6 17.8 18.7 41.1

63.7 50.5 77.5 22.8 11.7 37.0 23.6 75.6 64.3 50.4 23.1 21.7 55.2 41.8 39.3 16.3 15.3 38.9

64.7 49.5 79.0 22.3 12.0 32.5 21.0 75.0 60.4 54.0 22.7 22.3 55.0 41.3 40.6 15.2 15.0 36.9

62.8 51.0 76.6 23.3 14.0 38.8 24.4 77.1 61.8 54.1 22.0 24.1 53.9 40.9 44.7 14.2 15.5 34.7

63.4 51.4 76.9 22.4 10.3 29.1 18.5 75.5 62.7 54.4 25.6 22.9 51.5 49.7 44.4 19.5 17.4 39.2

64.5 52.0 77.0 23.5 10.1 32.6 21.0 72.2 59.8 49.6 24.8 23.2 52.0 47.7 44.5 19.1 17.8 40.1

62.0 52.6 75.7 23.6 11.5 37.7 23.4 75.6 63.7 51.4 26.0 22.4 51.6 50.5 43.4 20.1 17.2 39.7

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

12

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Fig. 6. Regression results of the diagnostic ratios among lubricants and harbors. (a1–a3) CK harbor (n = 8). (b1–b3) HL harbor (n = 3).

of C29ab and C30ab-hopanes in oil (da Silva and Bicego, 2010). Moreover, the calculated ratios of 18a(H)-22,29,30-trisnorneohop ane (Ts)/17a(H)-22,29,30-trisnorhopane (Tm) for the lubricants and sediments are 114.2 ± 19.7 (n = 3) and 104.9 ± 3.6 (n = 11), respectively. This indicates the resistance of weathering effects. Otherwise, the indication compound of weathering effects, 17a(H)-30-nor-29-homohopane (NOR30H), has the lower ratio between the compounds, which is verified by the calculated ratio of 17a(H),21b(H)-30-nor-hopane/17a(H),21b(H)-hopane for the lubricants and sediments being 10.0 ± 0.2 (n = 3) and 10.4 ± 1.7 (n = 11), respectively. The detected stereoisomers in the lubricants and sediments also reflect the thermo stability of ab and ba-hopanes. Additionally, Table 4 shows that the 8 four-ring (C27–C29) sterane homologues detected in the sediments had the higher concentrations among steranes and can be related to the lubricants. 3.4. Confirmation of lubricants Normalization and diagnostic ratios are used to relate the studied oil products and sediments. Fig. 5(a) illustrates the seasonal comparison of BSs between CK and HL harbors which shows similar curvatures among the sampling points for each respective

harbor, but presents differently in two seasons. The results indicate the high concentration of BSs in CK harbor and irregular distribution of sample sites and seasons as described in the PAH distribution above. However, BS10 had the highest concentration among all samples; thus after normalization, Fig. 5(b) shows similar curvatures among the BS ratios. The results indicate that marine diesel is one of the anthropogenic sources along with the weathering effects for the low molecular weights BSs in the sedimentary environment. Fig. 5(c) shows the original distribution ratios of lubricants, which indicates a differentiable concentration characteristic among the lubricants. Fig. 5(d) shows similar normalized ratio curvatures between CK and HL harbors in two sampling seasons. The figure indicates that despite the differences in concentration among harbors, seasons and the long distance (123 km) between the two harbors, the distributions of terpane and sterane biomarkers have an analogous response to the weathering effects. Fig. 5(c and d) indicates that, once these refined end products have diffused into the sediment environment, there will be a persistent effect in the biota. To relate the possible lubricants and pollutants with each sampling point and harbor, 18 possible diagnostic ratios are suggested and calculated as shown in Table 5. The table uses

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

one season (February, 2014) as an example of sediment samples, which indicates ratios with greater differences. When considering all of the diagnostic ratios, Fig. 6 shows the regression results among lubricants and harbors. The correlation coefficient R2 indicates the trend of the possible distribution source of lubricants in the sediment in which CG4 has a higher R2 value up to 0.82. Based on the field investigation, these three lubricants are the major applied oil products in these harbors, and the figures confirm these facts and raises the degree of certainty of tracking the sources of pollutants. 4. Conclusions A chemical forensic study of the PAHs and biomarkers in the fishing harbors was conducted. Statistical analysis of the PAHs data in the sediment of the three harbors shows non-normal distributions of concentrations among seasons. The fingerprinting comparison ratios indicates that both petrogenic and pyrogenic origin are the possible sources of PAHs and the variation of PAH distribution in the sediment sample has other uncertainty factors. Moreover, the biomarker identification can be used to track and relate the target pollutants and their anthropogenic sources. The characteristic of the three lubricants can also be identified by using suggested 18 categories of diagnostic ratios. The application of X-ray Photoelectron Spectroscopy (XPS) and statistical analysis can also be used to indicate a sign of long term accumulated pollution. The detected C2-naphthalenes and the comparison of analyzed BSs, terpanes and steranes confirmed that the studied petroleum products are anthropogenic sources due to the long term operation of these harbors. Despite the distance and geographical locations of the three harbors, the results indicate the similar trends of the concentration distributions of biomarkers. It should be noted that there are only two major crude oil refinery companies in Taiwan and the studied products are produced by the oil company dominating a major part of the market. Moreover, the results and the analyzed diagnostic ratios can be applied in investigating a fresh or long term contamination site either in soil or sediment in any other area and the reported data could be a major source of information for further study. Acknowledgments This study was funded by a research project supported by the Taiwan EPA. The views or opinions expressed in this article are those of the writers and should not be construed as opinions of the Taiwan EPA. The mention of trade names, vendor names, or commercial products does not constitute endorsement or recommendation by the Taiwan EPA. References Aboul-Kassim, T.A., Simoneit, B.R., 1995. Petroleum hydrocarbon fingerprinting and sediment transport assessed by molecular biomarker and multivariate statistical analyses in the Eastern Harbour of Alexandria, Egypt. Mar. Pollut. Bull. 30 (1), 63–73. Asif, M., Alexander, R., Fazeelat, T., Pierce, K., 2009. Geosynthesis of dibenzothiophene and alkyl dibenzothiophenes in crude oils and sediments by carbon catalysis. Org. Geochem. 40 (8), 895–901. Barakat, A.O., Mostafa, A.R., Qian, Y., Kennicutt Ii, M.C., 2002. Application of Petroleum Hydrocarbon Chemical Fingerprinting in Oil Spill Investigations–– Gulf of Suez, Egypt. Spill Sci. Technol. Bull. 7 (5), 229–239. Barreca, S., Bastone, S., Caponetti, E., Martino, D.F.C., Orecchio, S., 2014. Determination of selected polyaromatic hydrocarbons by gas chromatography–mass spectrometry for the analysis of wood to establish the cause of sinking of an old vessel (Scauri wreck) by fire. Microchem. J. 117, 116– 121. Bieger, T., Hellou, J., Abrajano Jr., T.A., 1996. Petroleum biomarkers as tracers of lubricating oil contamination. Mar. Pollut. Bull. 32 (3), 270–274. Boehm, P.D., Douglas, G.S., Burns, W.A., Mankiewicz, P.J., Page, D.S., Bence, A.E., 1997. Application of petroleum hydrocarbon chemical fingerprinting and

13

allocation techniques after the Exxon Valdez oil spill. Mar. Pollut. Bull. 34 (8), 599–613. Boonyatumanond, R., Wattayakorn, G., Togo, A., Takada, H., 2006. Distribution and origins of polycyclic aromatic hydrocarbons (PAHs) in riverine, estuarine, and marine sediments in Thailand. Mar. Pollut. Bull. 52 (8), 942–956. Chandru, K., Zakaria, M.P., Anita, S., Shahbazi, A., Sakari, M., Bahry, P.S., Mohamed, C.A.R., 2008. Characterization of alkanes, hopanes, and polycyclic aromatic hydrocarbons (PAHs) in tar-balls collected from the East Coast of Peninsular Malaysia. Mar. Pollut. Bull. 56 (5), 950–962. da Silva, D.A., Bicego, M.C., 2010. Polycyclic aromatic hydrocarbons and petroleum biomarkers in São Sebastião Channel, Brazil: assessment of petroleum contamination. Mar. Environ. Res. 69 (5), 277–286. Dong, C.-D., Chen, C.-F., Chen, C.-W., 2012. Determination of polycyclic aromatic hydrocarbons in industrial harbor sediments by GC–MS. Int. J. Environ. Res. Public Health 9 (6), 2175–2188. Environmental Analysis Laboratory, EPA, Executive Yuan, Taiwan. (accessed 14.10.14). Fisheries Agency, Council of Agriculture, Executive Yuan, Taiwan. (Old: (accessed September 2013), New: (accessed 16.03.15)). Fisheries Agency, Council of Agriculture, Executive Yuan, Taiwan. (accessed 14.10.14). Frenna, S., Mazzola, A., Orecchio, S., Tuzzolino, N., 2013. Comparison of different methods for extraction of polycyclic aromatic hydrocarbons (PAHs) from Sicilian (Italy) coastal area sediments. Environ. Monit. Assess. 185 (7), 5551– 5562. Gong, Y., Zhao, X., Cai, Z., O’Reilly, S., Hao, X., Zhao, D., 2014. A review of oil, dispersed oil and sediment interactions in the aquatic environment: influence on the fate, transport and remediation of oil spills. Mar. Pollut. Bull. 79 (1), 16– 33. Grice, K., Lu, H., Atahan, P., Asif, M., Hallmann, C., Greenwood, P., Maslen, E., Tulipani, S., Williford, K., Dodson, J., 2009. New insights into the origin of perylene in geological samples. Geochim. Cosmochim. Acta 73 (21), 6531– 6543. Guan, Y., Kiraly, J., Rijks, J., 1989. Interactive retention index database for compound identification in temperature-programmed capillary gas chromatography. J. Chromatogr., A 472, 129–143. Kao, N.-H., Su, M.-C., Fan, J.-R., Chung, Y.-Y., 2015. Identification and quantification of biomarkers and polycyclic aromatic hydrocarbons (PAHs) in an aged mixed contaminated site: from source to soil. Environ. Sci. Pollut. Res. 22 (10), 7529– 7546. Keshavarzifard, M., Zakaria, MP., Hwai, TS., Yusuff, FM., Mustafa, S., 2015. Distributions and source apportionment of sediment-associated polycyclic aromatic hydrocarbons (PAHs) and hopanes in rivers and estuaries of Peninsular Malaysia. Environ. Sci. Pollut. Res. http://dx.doi.org/10.1007/ s11356-015-4093-7 (Published online). Lee, C.-H., Lee, J.-H., Sung, C.-G., Moon, S.-D., Kang, S.-K., Lee, J.-H., Yim, U.H., Shim, W.J., Ha, S.Y., 2013. Monitoring toxicity of polycyclic aromatic hydrocarbons in intertidal sediments for five years after the Hebei Spirit oil spill in Taean, Republic of Korea. Mar. Pollut. Bull. 76 (1), 241–249. Lubeck, A., Sutton, D., 1983. Kovats retention indices of selected hydrocarbons through C10 on bonded phase fused silica capillaries. J. High Res. Chromatogr. 6 (6), 328–332. Lucero, M., Estell, R., Tellez, M., Fredrickson, E., 2009. A retention index calculator simplifies identification of plant volatile organic compounds. Phytochem. Anal. 20 (5), 378–384. Mulabagal, V., Yin, F., John, G., Hayworth, J., Clement, T., 2013. Chemical fingerprinting of petroleum biomarkers in Deepwater Horizon oil spill samples collected from Alabama shoreline. Mar. Pollut. Bull. 70 (1), 147–154. Orecchio, S., 2010. Assessment of polycyclic aromatic hydrocarbons (PAHs) in soil of a Natural Reserve (Isola delle Femmine) (Italy) located in front of a plant for the production of cement. J. Hazard. Mater. 173 (1), 358–368. Orecchio, S., Cannata, S., Culotta, L., 2010. How building an underwater pipeline connecting Libya to Sicilian coast is affecting environment: polycyclic aromatic hydrocarbons (PAHs) in sediments; monitoring the evolution of the shore approach area of the Gulf of Gela (Italy). J. Hazard. Mater. 181 (1), 647–658. Page, D., Boehm, P., Douglas, G., Bence, A., Burns, W., Mankiewicz, P., 1999. Pyrogenic polycyclic aromatic hydrocarbons in sediments record past human activity: a case study in Prince William Sound, Alaska. Mar. Pollut. Bull. 38 (4), 247–260. Pauzi Zakaria, M., Okuda, T., Takada, H., 2001. Polycyclic aromatic hydrocarbon (PAHs) and hopanes in stranded tar-balls on the coasts of Peninsular Malaysia: applications of biomarkers for identifying sources of oil pollution. Mar. Pollut. Bull. 42 (12), 1357–1366. Prince, R.C., Elmendorf, D.L., Lute, J.R., Hsu, C.S., Haith, C.E., Senius, J.D., Dechert, G.J., Douglas, G.S., Butler, E.L., 1994. 17a(H)-21b(H)-hopane as a conserved internal marker for estimating the biodegradation of crude oil. Environ. Sci. Technol. 28 (1), 142–145. Solé, M., Manzanera, M., Bartolomé, A., Tort, L., Caixach, J., 2013. Persistent organic pollutants (POPs) in sediments from fishing grounds in the NW Mediterranean: ecotoxicological implications for the benthic fish Solea sp. Mar. Pollut. Bull. 67 (1), 158–165. Stout, S.A., Uhler, A.D., McCarthy, K.J., 2001. A strategy and methodology for defensibly correlating spilled oil to source candidates. Environ. Forensics 2 (1), 87–98.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070

14

N.-H. Kao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Stout, S.A., Uhler, A.D., McCarthy, K.J., 2005. Middle distillate fuel fingerprinting using drimane-based bicyclic sesquiterpenes. Environ. Forensics 6 (3), 241–251. Tolosa, I., de Mora, S., Sheikholeslami, M.R., Villeneuve, J.-P., Bartocci, J., Cattini, C., 2004. Aliphatic and aromatic hydrocarbons in coastal Caspian Sea sediments. Mar. Pollut. Bull. 48 (1), 44–60. Trolio, R., Grice, K., Fisher, S.J., Alexander, R., Kagi, R.I., 1999. Alkylbiphenyls and alkyldiphenylmethanes as indicators of petroleum biodegradation. Org. Geochem. 30 (10), 1241–1253. Wang, Z., Fingas, M., 1997. Developments in the analysis of petroleum hydrocarbons in oils, petroleum products and oil-spill-related environmental samples by gas chromatography. J. Chromatogr., A 774 (1–2), 51–78. Wang, Z., Fingas, M.F., 2003. Development of oil hydrocarbon fingerprinting and identification techniques. Mar. Pollut. Bull. 47 (9), 423–452. Wang, Z., Fingas, M., Owens, E., Sigouin, L., Brown, C., 2001. Long-term fate and persistence of the spilled Metula oil in a marine salt marsh environment: degradation of petroleum biomarkers. J. Chromatogr., A 926 (2), 275–290. Wang, Z., Yang, C., Fingas, M., Hollebone, B., Peng, X., Hansen, A.B., Christensen, J.H., 2005. Characterization, weathering, and application of sesquiterpenes to source identification of spilled lighter petroleum products. Environ. Sci. Technol. 39 (22), 8700–8707. Wang, C., Sun, H., Chang, Y., Song, Z., Qin, X., 2011. PAHs distribution in sediments associated with gas hydrate and oil seepage from the Gulf of Mexico. Mar. Pollut. Bull. 62 (12), 2714–2723.

Wang, C., Chen, B., Zhang, B., He, S., Zhao, M., 2013. Fingerprint and weathering characteristics of crude oils after Dalian oil spill, China. Mar. Pollut. Bull. 71 (1), 64–68. Yang, C., Wang, Z., Hollebone, B., Peng, X., Fingas, M., Landriault, M., 2006. GC/MS quantitation of diamondoid compounds in crude oils and petroleum products. Environ. Forensics 7 (4), 377–390. Yang, C., Wang, Z., Hollebone, B.P., Brown, C.E., Landriault, M., 2009. Characteristics of bicyclic sesquiterpenes in crude oils and petroleum products. J. Chromatogr., A 1216 (20), 4475–4484. Yang, C., Wang, Z., Hollebone, B., Brown, C., Landriault, M., Fieldhouse, B., Yang, Z., 2012. Application of light petroleum biomarkers for forensic characterization and source identification of spilled light refined oils. Environ. Forensics 13 (4), 298–311. Yim, U.H., Ha, S.Y., An, J.G., Won, J.H., Han, G.M., Hong, S.H., Kim, M., Jung, J.-H., Shim, W.J., 2011. Fingerprint and weathering characteristics of stranded oils after the Hebei Spirit oil spill. J. Hazard. Mater. 197, 60–69. Yunker, M.B., Macdonald, R.W., Vingarzan, R., Mitchell, R.H., Goyette, D., Sylvestre, S., 2002. PAHs in the Fraser River basin: a critical appraisal of PAH ratios as indicators of PAH source and composition. Org. Geochem. 33 (4), 489–515. Zakaria, M.P., Horinouchi, A., Tsutsumi, S., Takada, H., Tanabe, S., Ismail, A., 2000. Oil pollution in the Straits of Malacca, Malaysia: application of molecular markers for source identification. Environ. Sci. Technol. 34 (7), 1189–1196.

Please cite this article in press as: Kao, N.-H., et al. Investigation of polycyclic aromatic hydrocarbons (PAHs) and cyclic terpenoid biomarkers in the sediments of fishing harbors in Taiwan. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.070