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Comparing dioxin-like polychlorinated biphenyls in most consumed fish species of the Caspian Sea
Journal Pre-proof Comparing dioxin-like polychlorinated biphenyls in most consumed fish species of the Caspian Sea Ayub Ebadi Fathabad, Khadijeh Jafari, Hossein Tajik, Maryam Behmanesh, Nabi Shariatifar, Sepideh Sadat Mirahmadi, Gea Oliveri Conti, Mohammad Miri PII:
S0013-9351(19)30675-9
DOI:
https://doi.org/10.1016/j.envres.2019.108878
Reference:
YENRS 108878
To appear in:
Environmental Research
Received Date: 20 August 2019 Revised Date:
28 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Fathabad, A.E., Jafari, K., Tajik, H., Behmanesh, M., Shariatifar, N., Mirahmadi, S.S., Conti, G.O., Miri, M., Comparing dioxin-like polychlorinated biphenyls in most consumed fish species of the Caspian Sea, Environmental Research (2019), doi: https://doi.org/10.1016/ j.envres.2019.108878. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Comparing dioxin-like polychlorinated biphenyls in most consumed
1
fish species of the Caspian Sea
2
Ayub Ebadi Fathabad 1, Khadijeh Jafari 2, Hossein Tajik 1, Maryam Behmanesh3, Nabi
3
Shariatifar 4, Sepideh Sadat Mirahmadi 5, Gea Oliveri Conti6, Mohammad Miri 7*
4
1- Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, 5
6
Urmia, Iran.
2- Department of Environmental Health Engineering, Faculty of Health, Hormozgan University of 7 Medical Sciences & Health Services, Bandar Abbas, Iran.
8
3- Nutrition and Food Sciences Research Center, Tehran Medical Sciences, Islamic Azad University, 9
10
Tehran, Iran.
4- Department of Environmental Health Engineering, Faculty of Health, Tehran University of Medical 11
12
Sciences, Tehran, Iran.
5- Department of Food Hygiene and safety, Faculty of Health, Zanjan University of Medical Sciences & 13
14
Health Services, Zanjan, Iran.
6- Environmental and Food Hygiene Laboratory, Department of Medical, Surgical Sciences and 15 Advanced Technologies “G.F. Ingrassia” , University of Catania, Catania, Italy.
16
7- Cellular and Molecular Research Center, Department of Environmental Health, School of Public 17 Health, Sabzevar University of Medical Sciences & Health Services, Sabzevar, Iran.
18 19
Corresponding authors:
20
1- Mohammad Miri. School of Public Health, Sabzevar University of Medical Sciences, PO Box 319, Sabzevar, Iran Email: [email protected], [email protected]
21 22 23
2- Hossein Tajik: Department of Food Hygiene and Quality Control, Faculty of 24 Veterinary Medicine, Urmia University, Urmia, Iran.
1
25
Abstract
26
Among polychlorinated biphenyls (PCBs), dioxin-like PCBs (DL-PCBs) are of the 27 most concern for human health. In this study, the levels of 12 DL-PCBs congeners 28 were measured in 125 fish samples of the Caspian Sea, Iran. Five fish species 29 (Oncorhynchus mykiss, Vimba vimba, Cyprinus carpio, Rutilus frisii kutum and 30 Chelon saliens) were collected from 5 coastal cities of the Caspian Sea (25 samples 31 per each city). Duncan's multi-scope test was used to compare the mean of DL-PCBs 32 in different fish species and different cities. Probabilistic risk of exposure to DL- 33 PCBs and sensitivity analysis were assessed using Monte Carlo simulation approach. 34 The average (standard deviation) of DL-PCBs in fish samples ranged from 232 (16) 35 to 1156 (14) pg/g lipids. The total maximum concentration was detected in Cyprinus 36 carpio from Bandar Anzali, the minimum in Vimba vimba from Chalos. In all 37 samples, non-carcinogenic risk of exposure to DL-PCBs was in safe level (Hazard 38 Quotient < 1). In contrast, the lifetime cancer risk estimated for Bandar Anzali, 39 Bandar Torkaman, and Rasht exceeded the threshold value of 1×10-6 suggested by 40 United States Environmental Protection Agency. Sensitivity analysis indicated that 41 the concentration of DL-PCBs and exposure frequency were the most effective 42 parameters in increasing carcinogenic risk.
43
Keywords: Polychlorinated Biphenyls, health risk assessment, fish, Caspian Sea
44
45
1. Introduction
46
2
Fish consumption is highly recommended in human diet due to its content of 47 polyunsaturated fatty acids (n-3) (PUFA) that play an important role in the 48 maintenance of human health (Trocino et al., 2012). However, the seafood 49 consumption is the most important source for human exposure to persistent organic 50 pollutants (POPs) and other toxic materials (Dórea, 2008). Moreover, using fish in 51 waters pollution monitoring programs has been widely recommended (Avigliano et 52 al., 2016).
53
The most hazardous groups of POPs include polychlorinated dibenzodioxins 54 (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls 55 (PCBs) (Fu et al., 2018; Pacheco Ferreira, 2018; USEPA., 2000). PCBs are a group 56 of 209 PCB-congeners with different substitution patterns with high persistence and 57 capable to bioaccumulation. PCBs are classified into two groups: dioxin like-PCBs 58 (DL-PCBs, 12 congeners) and unlike dioxin PCBs or non-dioxin like PCBs (NDL- 59 PCBs) (Fu et al., 2018; Lüth et al., 2018). DL-PCBs include four non-ortho 60 congeners (77, 81, 126, and 169) and eight mono-ortho PCB congeners (Nakatani et 61 al., 2011; Pacheco Ferreira, 2018). Review studies on the topic show the presence of 62 different DL-PCBs congeners in water, soil, air, and wildlife (Salamova et al., 2013). 63 Previous studies reported that DL-PCBs concentration in fish depends on several 64 factors such as: species, environmental conditions (Storelli, 2008), type or origin of 65 feed used in farmed fish (Conti et al., 2015; Trocino et al., 2009), and ecological 66 niche occupied by wild fishes (Custódio et al., 2011).
67
Exposure to DL-PCBs can lead to carcinogenic and immunological effects but also 68 endocrine and fertility dysfunctions (Rizzi et al., 2017).
3
69
Research on Cancer (UNIARC) classified PCB 126 congener as carcinogenic for 70 humans (group 1) (IARC., 2009; Nakatani et al., 2011), but recently, all PCBs (DL- 71 PCBs and NDL-PCBs) have been identified as carcinogenic for humans (Barone et 72 al., 2018; Habibullah-Al-Mamun et al., 2019; IARC, 2016; Lauby-Secretan et al., 73 2013). The Joint FAO/WHO Expert Committee on Food Additives, recommends a 74 provisional tolerable monthly intake (PTMI) of 70 pg/kg body weight/month to 75 reduce the human exposure to the DL-PCBs through diet (Cao et al., 2018).
76
So far, several studies focused on the presence of several classes of POPs in fishes 77 caught from the Caspian Sea (Agusa et al., 2004; Anan et al., 2005; Dadar et al., 78 2016; Ebadi and Shokrzadeh, 2006; Hosseini et al., 2008; Kajiwara et al., 2002; 79 Kajiwara et al., 2003; Kajiwara et al., 2008; Katuli et al., 2014; Manavi et al., 2018; 80 Manavi and Mazumder, 2018; Mashroofeh et al., 2013). However, within the group 81 of POPs studies addressing DL-PCBs in fish in this area are missing in literature.
82
The Caspian Sea has a strategic position; it is rich in oil and gas reserves and 83 represents a source of income for commercial fishing and shrimp fishing of 84 neighboring countries. Therefore, this study aimed to evaluate the level of DL-PCBs 85 in different fish species from different areas of the Caspian Sea, Iran. Also, 86 carcinogenic and non-carcinogenic risk of exposure to these pollutants were assessed 87 as well as sensitivity analyses were carried out using the Monte Carlo simulation 88 technique.
89
2. Material and methods
90
2.1.Sampling location and fish collection
91
4
This study was carried out in March 2018 in five coastal cities (Bandar Anzali, 92 Chalous, Rasht, Astara and Bandar Torkaman) of the Caspian Sea, Iran (Figure 1). 93 The Caspian Sea is in the geographical location of longitude from 48.75° to 53.92° 94 and latitude from 36.46° to 47.10°, with an about 420 000 km2 area.
95
Annually, 600 000 tons of different fish species are caught from this sea and 96 consumed by citizens (Tolosa et al., 2004). In this study, 5 common fish species (5 97 specimens from each species) including Oncorhynchus mykiss, Vimba vimba, 98 Cyprinus carpio, Rutilus frisii kutum, and Chelon saliens were randomly collected 99 from 5 markets for each coastal city included in our study for a total of 125 samples. 100 The selected cities covered all Iranian coastline of the Caspian Sea, and we made 101 sure that the fish specimens were collected from the Caspian Sea. All collected 102 samples were placed in cool boxes and transferred to the laboratory. In the 103 laboratory, biometers of the fishes, i.e., weight and length were recorded (Table S1 of 104 Supplemental materials). 50 g muscle tissue from each fish were sampled, wrapped 105 in aluminum foil and stored at -20°C until analysis. 2.2.Extraction of DL-PCBs
106 107
The initial DL-PCBs extraction was done according to USEPA method 1668 revision 108 A (U.S. EPA, 1999). Briefly, 50 g of fillet was homogenized by a meat grinder 109 (Moulinex, Ecully Cedex, France). The process was repeated three times for each 110 muscle sample. Then, 100 g sodium sulfate anhydrous (Na2SO4, Merck, Germany) 111 was added to each sample and homogenized at 50 °C for six h. Besides, about 50 ng 112 of internal standard PCB 209 (Sigma-Aldrich, Germany) was added to the sample to 113 monitor the PCB extraction and its recovery. The lipid extraction process was carried 114 5
out using Soxhlet Extractor (Soxhlet Extraction System B-811 (Büchi AG, Flawil, 115 Switzerland)). The extractions were performed using a mixture of hexane and 116 acetone (90:10 v/v). The process for each sample was repeated for 260 cycles (Liu et 117 al., 2016; USEPA, 1999). The concentration of lipid was determined gravimetrically. 118 1 g of extracted lipid was dissolved in 10 mL n-hexane, and this diluted extract was 119 used for further analyses. All extracts were purified using silica gel multi-layer 120 absorbent columns (see Figure S1 of Supplemental materials). In this study, the 121 cleanup columns used were similar to the Japanese industrial standard method based 122 on the EPA1613 method (Masunaga et al., 2001). The silicates were initially 123 activated and then dried at 200 °C for 24 h. After that, silica was washed with 60 mL 124 n-hexane to remove contaminants from the silica. To prepare silica gel, the 125 inactivated silica was mixed with sulfuric acid 98% (40% w/w) (Akutsu et al., 2005; 126 USEPA, 1999). The column layers composition for chromatography was similar to 127 those reported by Masunaga et al. (2006) and Aries et al. (2001) (Aries et al., 2006; 128 Masunaga et al., 2001).
129
Briefly, the first layer was a mixture of silica gel (3 g) with silver nitrate (AgNO3, 130 10% w/w, Merck, Germany), which eliminated sulfur compounds from the sample. 131 The next layer was made of silica (6 g) with sulfuric acid (H2SO4, 22%), for the 132 removal of lipid layers and elimination of its oxidation metabolites from the sample. 133 The third layer was constituted by 44% H2SO4, which was used to remove the 134 remaining lipid impurities and hydrolyzed fats in the sample. The fourth layer was 135 made of a mixture of silica gel (3 g) and potassium hydroxide (KOH-2%, Merck, 136 Germany), that eliminated the possible acids and acidic compounds. Finally, The 137
6
DL-PCBs were eluted through the column by 50 mL n-hexane (HPLC grade) and 138 concentrated using a rotary evaporator at 40 °C to reach a final volume of 1 ml for its 139 injection into HRGC/HRMS (Barone et al., 2018; USEPA, 1999). After passing the 140 sample through the column, the column was washed with further solvent (60 mL of 141 n-hexane) to remove the remaining PCBs in the column.
142
2.3.Analysis of DL-PCBs
143
The analyses of DL-PCBs in the fillets samples were done according to USEPA 144 method (USEPA, 1999). The analysis of samples was carried out at the Laboratory of 145 Tehran
University of
Medical
Sciences
using a
High
Resolution
Gas 146
Chromatography (Agilent 6890 Series, Agilent Technologies, USA) coupled with 147 High
Resolution
Mass
Spectrometer
AutoSpec
Ultima
NT–HRGC/HRMS 148
(Micromass, USA), equipped with the HP-5MS 30 m × 0.25 mm × 0.25 µm column 149 (Agilent Technologies) and helium as carrier gas. In this study 12 congeners of DL- 150 PCBs (PCB 77, PCB 81, PCB 105, PCB 114, PCB118, PCB 123, PCB 126, PCB 151 156, PCB 157, PCB 167, PCB 169 and PCB 189) (more details are described in 152 Table S2 of Supplemental materials) were determined and reported as pg/g fat 153 (Trocino et al., 2009; Van den Berg et al., 2006). The detector calibration (as linear 154 response range) was determined by injecting standard dilutions under actual 155 conditions to HRGC/HRMS. An appropriate amount (from 1 to 10 µL) of the extract 156 was injected to HRGC/HRMS, and the congeners and their concentrations were 157 determined (Akutsu et al., 2005; U.S. EPA, 1999). For all samples, a HRMS single- 158 ion monitoring (SIM) method (>10000 resolution) was applied (Masunaga et al., 159 2001). Two daughters ions (for natural and marked DL-PCBs) were considered to 160
7
determine the best ratio of signal to noise (S/N). The response factors were measured 161 by injecting 1 µL of standard solution into the HRGC/HRMS. Other MS detector 162 condition was 1 mL/min as a flow rate, injector in splitless mode, and injector 163 temperature at 290 ºC. Oven temperature program was 90 ºC (isothermal: 2 min and 164 first rate at 5 ºC/m) to 280 ºC (isothermal: 3min).
165
2.4.Quality control and recovery
166
Quantification of DL-PCBs was done based on multi-level calibration curves, and we 167 found a good linearity (R2 > 0.99) for the standards range which had the same 168 concentrations range of the samples. The standard curve of 10 concentrations of DL- 169 PCBs ranged from 1 to 1000 µg/l (Sigma-Aldrich, Germany). DL-PCBs 170 concentrations were measured based on a peak-by-peak height comparison and a 171 calibration curve of each certified PCB congener. All PCBs had a peak height at the 172 range of calibration curve, and the samples with higher peak were diluted. The 173 efficiency of the residue analysis method used to measure the DL-PCBs in the fish 174 samples was tested based on recovery (%). The mean recoveries were between 98% 175 and 110% for all compounds tested.
176
Blank sample analysis was carried out after injecting every 10 samples to calibrate 177 the system and results indicated that all blank samples showed no detectable 178 concentrations or traces of PCBs (Salihovic et al., 2012).
Limits of quantification 179
(LOQ) for all DL-PCBs ranged between 0.03 to 0.09 pg/g fat. 2.5.Toxic equivalents (TEQs)
180 181
The toxicity level of the DL-PCBs was considered based on the most toxic known 182 between the dioxin compounds, namely 2,3,7,8 tetrachloro-dibenzo-dioxin (TCDD), 183
8
as the toxic scale of 1 for it and the toxicity of other DL-PCBs was compared with it 184 (Table S3 of Supplemental Materials) (Su et al., 2012; Turyk et al., 2006; Xu et al., 185 2006). The total toxic equivalents (TEQs) were calculated as follows (Van den Berg 186 et al., 2006):
187
TEQs = ∑ (Ci × TEFi)
(1)
188
Where Ci is the concentration of DL-PCB congener i; TEFi is the toxic equivalent 189 factor of congener i.
190
2.6.Health Risk Assessment
191
Monte Carlo simulation method was applied to determine the carcinogenic and non- 192 carcinogenic risk caused by exposure to DL-PCBs through the consumption of fish in 193 all cities of the studied area. Sensitivity analysis was done based on effective 194 variables on risk assessment such as concentration (C) of DL-PCBs, exposure 195 frequency (EF), body weight (BW), the averaging time (AT) and the maximum 196 allowable daily consumption of contaminated fish (FCR).
197 198
2.6.1. Estimated daily intake
199
Estimated daily intake (EDI) of DL-PCBs (ng/kg/day) by fish consumption was 200 calculated using the following equation:
EDI =
× × ×
201
(2)
×
202
Where, FCR is set to 25.5 g/day for Iranian people (Miri et al., 2017b), BW is set to 203 70 ± 10 kg for Iranian adults (Miri et al., 2017b), EF is based on day/year, which 204 calculated based on the rate of fish consumption for Iranian inhabitants (minimum, 205 9
once per week, on average twice and maximum three meals per week), ED is the 206 exposure duration in terms of years (70 years) and AT is the averaging time in days 207 (AT= EF × ED).
208
2.6.2. Hazard quotient
209
Hazard quotient (HQ) of the non-carcinogenic risk related to the consumption of fish 210 contaminated with DL-PCBs was calculated as follows (Saha et al., 2016): =
(3)
211
212
RFD is the oral reference dose set to 20 ng/kg/day for PCBs in this study (Quadroni 213 and Bettinetti, 2017).
214
In our study, the effect of cooking on the concentration of DL-PCBs was not 215 considered, and the HQ was calculated based on pollutant concentration in row 216 samples (Saha et al., 2016).
217
2.6.3. Carcinogenic risk
218
The lifetime carcinogenic risk (LTCR) through exposure to DL-PCBs was 219 determined using the cancer slope factor (CSF) (2×10−6 ng/kg/day) provided by the 220 United States Environmental Protection Agency (USEPA) with the following 221 equation (Saha et al., 2016; Shaheen et al., 2016):
=
× × × × × ! "# ×
%$× 10()
222
(4)
223
Where CF is conversion factor (0.001 pg/ng). Other parameters are same as equation 224 2.
225
10
2.7.Statistical analysis
226
Data analysis was carried out using a completely randomized design in a factorial 227 experiment in three replications. Descriptive statistics were analyzed by Stata 228 software version 15 (Stata Corp LP, College Station, Texas). Normality of data was 229 tested based on Shapiro-Wilk. Duncan multi-scope test was used to compare mean of 230 DL-PCBs in different fish species and different cities. A significant level of 0.05 was 231 performed for all analyses. To simulate Monte Carlo technique and perform 232 sensitivity analyses, Crystal Ball software (version 11.1.1.1, Oracle, Lnc. USA) with 233 100 000 trails was used.
234 235
3. Results and discussion
236
3.1.Comparing DL-PCBs concentrations
237
A comparison of ∑DL-PCBs concentration of different fish species and different 238 sampling locations are presented in Table 1. There were significant differences in 239 ∑DL-PCBs concentration of different fish species and sampling location (p≤0.05). 240 The highest ∑DL-PCBs was in Cyprinus carpio species (1156.27 ± 14.55 pg/g fat) in 241 Bandar Anzali. The lowest concentration of ∑DL-PCBs was in Vimba vimba species 242 from Chalous, with a mean concentration of 232.43 ± 16.05 pg/g fat. Due to great 243 variation of PCBs type and number of them which were analyzed in previous studies, 244 as well as due to different measurement units and methods, comparing the results of 245 our study with previous reports is complicated and could not be reasonable (Cao et 246 al., 2018; Mezzetta et al., 2011). For example, in a study by Chao et al. (2018) in the 247 Shadong Sea of China, the average DL-PCBs was 0.887 ng/g wet weight (Cao et al., 248 11
2018). The concentration of measured PCBs in different fish species are shown in 249 Table S4 to S14 and Figure S2 (for PCB77) of Supplemental materials.
250
Overall, the mean concentration of non-ortho PCBs was lower than mono-ortho 251 PCBs. For mono-ortho PCBs, PCB118 (367.50 pg/g fat) had the highest mean 252 concentration in Cyprinus carpio and this our result is consistent with other studies 253 of Zhoushan Fishery and marine fish of Shandong in China, farmed fish, Latvian 254 Lake and fish species of Lake Victoria, Uganda (Cao et al., 2018; Ssebugere et al., 255 2014; Wang et al., 2015; Zacs et al., 2013). Moreover, all the other 7 mono-ortho 256 PCBs were detected in all fish species and Bandar Anzali had the highest mean 257 concentration. The lowest mean concentration of mono-ortho PCBs was that of PCB 258 157 (0.82 pg/g fat) in Vimba vimba from Chalous. Among non-ortho PCBs, the 259 highest mean concentration was that of PCB 77 (29.58 pg/g fat) in Cyprinus carpio 260 from Bandar Anzali. Also, the lowest concentration of non-ortho PCBs was found in 261 Vimba vimba from Chalous for PCB 169 with mean concentration of 0.02 ± 0.03 262 pg/g fat. Considering the different measurement units, both PCB 77 (46.1 pg/g ww) 263 and PCB169 (2.02 pg/g ww) concentrations recorded by Celik Cakirogullari et al. 264 (2010) were higher than this study results (Çelik Çakıroğulları et al., 2010). In the 265 study by Cao et al. (2018) PCB 77 (0.063 ng/g ww) had the highest mean 266 concentration, which is in line with our findings (Cao et al., 2018).
267
The highest concentrations were found in Cyprinus carpio from Bandar Anzali and 268 the lowest in Vimba vimba from Chalous. Previous studies indicated that 269 concentration of DL-PCBs in different fish species was affected by complex factors 270 e.g., geographical origin, environment condition and season. Moreover, fish lipid 271 12
content, living and feeding habits, and low biodegradability of DL-PCB congeners in 272 the aquatic ecosystem (such as Sea) and bioaccumulation capability of these 273 pollutants in organisms are the major reasons of contamination of fish species by 274 DL-PCBs (Blanco et al., 2013; Costopoulou et al., 2016; Jiang et al., 2005a; 275 Mezzetta et al., 2011; Sun et al., 2014). 3.2.TEQs
276
277
The descriptive statistic of calculated TEQs for different studied cities and different 278 fish species are presented in Figure 2. The mean (standard deviation (SD)) of TEQs 279 in Astara, Bandar Anzali, Bandar Torkaman, Chalous and Rasht were 0.60 (0.32), 280 0.82 (0.35), 0.73 (0.31), 0.59 (0.28) and 0.68 (0.29) pgTEQ/g fat respectively. The 281 mean (SD) of TEQs for Rutilus frisii kutum, Oncorhynchus mykiss, Cyprinus carpio, 282 Chelon saliens and Vimba vimba species were 0.90 (0.11), 0.48 (0.11), 1.08 (0.16), 283 0.73 (0.09) and 0.23 (0.08) pgTEQ/g fat, respectively. Our results indicated that the 284 level of DL-PCBs in Cyprinus carpio species was more than other species and the 285 TEQ value for this species was higher than 1. Han et al. (2018) reported that in 286 China, reported that the maximum level of TEQ (higher than 1) was also detected in 287 Cyprinus carpio in China (Han et al., 2018). Moreover, in another study carried out 288 in Italy, similar results have been reported for this fish species (Trocino et al., 2012). 289 Therefore, Cyprinus carpio species should be consumed with caution for sensitive 290 people groups (i.e., children and elderly) in these areas. In our study, the overall 291 mean (SD) of TEQs was 0.68 (0.32) pgTEQ/g fat. Considering differences in TEQs 292 calculation, our values were lower than those reported by Trocino et al. 2012 in Italy 293 for intensive inland basins and intensive sea cages (Trocino et al., 2012). In another 294 13
study by Çelik Çakıroğulları et al. 2010 in Turkey, reported that the actual level of 295 ∑WHO-PCDD/Fs-DL-PCBs-TEQ(TEFs
1998)
was
6.12 ± 0.16 pg/g
fat
(Çelik 296
Çakıroğulları et al., 2010). In the study by Valérie Vrommana et al. in 2012, reported 297 that the mean of TEQs(TEFs 1998) was 2.19 pg WHO-TEF/g fresh weight (Vromman et 298 al., 2012). However in study by Han et al. on the Chinese mitten crabs, the overall 299 mean of TEQ level was 5.7 ± 4.0 pg TEQ/g (Han et al., 2018). In a study by Cao et 300 al., 2018 in China, TEQ(TEFs 2006) value in each fish sample was ranged from 0.011 to 301 9.214 pg WHO-TEQ/g ww (Cao et al., 2018).
302
303
3.3.Health risk assessment
304
In the present study, the health risk of exposure to ∑DL-PCBs was estimated based 305 on mean concentration of these pollutants in different fish species in each Caspian 306 coastal city. The results of the EDI (ng/kg/day) and non-carcinogenic (HQ) risk of 307 exposure to DL-PCBs via fish consumption in different cities are presented in Table 308 2. As shown, the highest mean value of EDI was 3.23E-1 ng/kg/day in fishes from 309 Bandar Anzali, and the lowest EDI value was observed in Chalous fishes with an 310 average of 2.11 E-1 ng/kg/day. The highest mean of HQ was observed in fishes from 311 Bandar Anzali (1.62 E-2); instead the lowest mean of HQ value was in Chalous 312 samples (1.06 E -2). According to the results, in all cities, the HQ was less than 1, 313 indicating that the mean concentration of ∑DL-PCBs in different studied areas were 314 at a safe level. Zhao et al. (2012) reported that HQs that HQ related to exposure to 315 DL-PCBs in both low and high fish consumption groups in China were in safe level 316 (Zhao et al., 2012). Similar results were reported by Jiang et al. (2005b) (Jiang et al., 317 14
2005b). Previous studies in Iran, reported that the non-carcinogenic risk for several 318 heavy metals via fish consumption were in safe level (HQ<1), which is consistent 319 with the present study (Ebadi Fathabad et al., 2019; Miri et al., 2017a). While, in a 320 study by Ebadi Fathabad et al., 2018, in Tehran's market, reported that, the HQ for 321 heavy metals in various types of natural juices and canned foods was higher than safe 322 level (Fathabad et al., 2018).
323
The results of lifetime carcinogenic risk (LTCR) of exposure to ∑DL-PCBs via 324 consumption of fish in different parts of the studied area are presented in Figure 3. 325 As shown, the 95th percentile value for LTCR for Bandar Anzali, Bandar Torkaman 326 and Rasht exceeded the threshold value of 1.00 E-6 suggested by USEPA (EPA, 327 2001). The highest mean value of LTCR was observed in Bandar Anzali (6.46 E-7), 328 and the lowest LTCR was observed in Chalous (4.23 E-7). Xia et al. (2012) reported 329 that LTCR in all low and high fish consumption groups was higher than safe level 330 (Xia et al., 2012). Otherwise, previous studies carried out in Iran showed also 331 indicated that the carcinogenic risk of exposure to heavy metals via consumption of 332 fish is at a safe level (Fathabad et al., 2018; Miri et al., 2017a).
333
In this study, sensitivity analysis was performed to determine the most effective 334 variable in increasing the carcinogenic risk through DL-PCBs using Monte Carlo 335 simulations. Figure 4 indicates sensitivity analyses of LTCR for exposure to ∑DL- 336 PCBs in different coastal cities. According to Figure 4, the concentration of DL- 337 PCBs (C) was the most effective variable in increasing the carcinogenic risk 338 (contribution to variance ranged from 39.4 to 50.9 %). The other effective parameters 339 in increasing the health risk for consumers was exposure frequency (EF) and per 340 15
capita fish consumption (FCR), respectively. Increased body weight (BW) and 341 averaging time (AT) of exposure (day) had an inverse relationship with carcinogenic 342 risk (contribution to variance ranged from -8.7 to -10.9 % and -18 to -23.9% 343 respectively). Previous studies indicated that the concentration of the pollutant was 344 an effective factor in increasing the health risk, which is consistent with the results of 345 this study (Fallahzadeh et al., 2018; Miri et al., 2018).
346 347
4- Conclusion
348
The average TEQs of DL-PCBs in fish samples from the Iranian coastline of Caspian 349 Sea was lower than previous reports. The highest level of DL-PCBs was observed in 350 Cyprinus carpio from Bandar Anzali, and the lowest levels of DL-PCBs were related 351 to Vimba vimba from Chalous. The results of the health risk assessment indicated 352 that carcinogenic risks of exposure to ∑DL-PCBs via consumption of fish in Bandar 353 Anzali, Bandar Torkaman, and Rasht were higher than the safe level. The sensitivity 354 analysis indicated, C and EF as the most effective parameters in increasing the health 355 risk. The DL-PCBs in fish samples of different studied areas should be monitored 356 continuously in the future. More attention is needed to protect the marine 357 environment by the authorities and decision-makers to prevent seawater 358 contaminations. Further studies, on the other fish species and more frequent sampling 359 in different seasons, are recommended to find out the reliability of the results of this 360 study.
361
362
Acknowledgment
363 16
The authors thank from Urmia University and Tehran University of Medical 364 Sciences. Mohammad Miri is supported by the Sabzevar University of Medical 365 Sciences.
366 367 368 369 370
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505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540
541
542
543
544
20
545
546
547
Figure captions
548
Figure 1. Geographical location of the Caspian Sea and studied cities.
549
Figure 2. Calculated toxic equivalents (TEQs) of ∑DL-PCBs based on studied 550 cities and fish species.
551
Figure 3. Lifetime carcinogenic risk (LTCR) for intake ∑DL-PCBs via
552
consuming fish in different studied cities.
553
Figure 4. Sensitivity analyses for lifetime carcinogenic risk of exposure to DL- 554 PCBs via consumption of different fishes in different cities (C: concentration of 555 DL-PCBs; EF: exposure frequency, AT: averaging time in days, BW: body 556 weight; FCR: fish consumption rate).
21
557
Table 1. Comparison of ∑DL-PCBs concentration (pg/g fat) in different fish species and different cities
Fish species/City
Bandar Torkaman
Chalous
Rasht
Bandar Anzali
Astara
Rutilus frisii kutum
923.34 ± 20.48 Bb
688.92 ± 11.85 Eb
842.31 ± 7.28 Cb
986.41 ± 10.70 Ab
783.22 ± 11.17 Db
Chelon saliens
836.74 ± 17.73 Bc
586.10 ± 11.36 Ec
746.11 ± 18.28 Cc
884.04 ± 12.91 Ac
710.37 ± 19.78 Dc
Cyprinus carpioio
1033.60 ± 17.89 Ba
799.51 ± 10.98 Ea
945.32 ± 16.15 Ce
1156.27 ± 14.55
857.96 ± 14.79 Da
Vimba vimba
407.76 ± 17.75 Be
232.43 ± 16.05 Ee
366.27 ± 15.73 Ce
462.64 ± 17.26 Ae
313.69 ± 12.42 De
Oncorhynchus
671.84 ± 25.70 Bd
438.33 ± 18.31 Ed
580.36 ± 15.66 Cd
714.44 ± 12.95 Ad
509.59 ± 49.75 Dd
mykiss *The different small letters indicate a significant difference in the columns and different large letters in the row indicating a significant difference in the row (p≤0.05).
22
Table 2. Estimated daily intake (EDI) and hazard quotient (HQ) for exposure to DL-PCBs via consumption of fish in different cities Percentile 5th
Mean
Median
Standard deviation
Percentile 95th
EDI
1.02 E-1
2.46 E -1
2.21 E-1
1.18 E -1
4.74 E-1
HQ
5.11 E -3
1.23 E -2
1.11 E -2
5.91 E -3
2.37 E -2
EDI
1.43E-1
3.23E-1
2.94E-1
1.48E-1
6.01E-1
HQ
7.14 E -3
1.62 E -2
1.47 E -2
7.38 E -3
3.00 E -2
EDI
1.30E-1
2.95E-1
2.66E-1
1.35E-1
5.49E-1
HQ
6.52E-3
1.48E-2
1.34E-2
6.67E-3
2.74E-2
EDI
8.26E-2
2.11E-1
1.87E-1
1.11E-1
4.21E-1
HQ
4.13E-3
1.06E-2
9.35E-3
5.54E-3
2.10E-2
EDI
1.15E-1
2.68E-1
2.43E-1
1.25E-1
5.10E-1
HQ
5.77E-3
1.34E-2
1.21E-2
6.27E-3
2.55E-2
Cities Astara
Bandar Anzali
Bandar Torkaman
Chalous
Rasht
23
Highlights •
DL-PCBs concentrations in five fish species of the Caspian Sea were investigated.
•
The highest DL-PCBs concentration was observed in Cyprinus carpio species.
•
LTCR for Bandar Anzali, Bandar Torkaman, and Rasht exceeded the threshold value suggested by USEPA (1×10-6).
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0013-9351(19)30675-9
DOI:
https://doi.org/10.1016/j.envres.2019.108878
Reference:
YENRS 108878
To appear in:
Environmental Research
Received Date: 20 August 2019 Revised Date:
28 October 2019
Accepted Date: 28 October 2019
Please cite this article as: Fathabad, A.E., Jafari, K., Tajik, H., Behmanesh, M., Shariatifar, N., Mirahmadi, S.S., Conti, G.O., Miri, M., Comparing dioxin-like polychlorinated biphenyls in most consumed fish species of the Caspian Sea, Environmental Research (2019), doi: https://doi.org/10.1016/ j.envres.2019.108878. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Comparing dioxin-like polychlorinated biphenyls in most consumed
1
fish species of the Caspian Sea
2
Ayub Ebadi Fathabad 1, Khadijeh Jafari 2, Hossein Tajik 1, Maryam Behmanesh3, Nabi
3
Shariatifar 4, Sepideh Sadat Mirahmadi 5, Gea Oliveri Conti6, Mohammad Miri 7*
4
1- Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, 5
6
Urmia, Iran.
2- Department of Environmental Health Engineering, Faculty of Health, Hormozgan University of 7 Medical Sciences & Health Services, Bandar Abbas, Iran.
8
3- Nutrition and Food Sciences Research Center, Tehran Medical Sciences, Islamic Azad University, 9
10
Tehran, Iran.
4- Department of Environmental Health Engineering, Faculty of Health, Tehran University of Medical 11
12
Sciences, Tehran, Iran.
5- Department of Food Hygiene and safety, Faculty of Health, Zanjan University of Medical Sciences & 13
14
Health Services, Zanjan, Iran.
6- Environmental and Food Hygiene Laboratory, Department of Medical, Surgical Sciences and 15 Advanced Technologies “G.F. Ingrassia” , University of Catania, Catania, Italy.
16
7- Cellular and Molecular Research Center, Department of Environmental Health, School of Public 17 Health, Sabzevar University of Medical Sciences & Health Services, Sabzevar, Iran.
18 19
Corresponding authors:
20
1- Mohammad Miri. School of Public Health, Sabzevar University of Medical Sciences, PO Box 319, Sabzevar, Iran Email: [email protected], [email protected]
21 22 23
2- Hossein Tajik: Department of Food Hygiene and Quality Control, Faculty of 24 Veterinary Medicine, Urmia University, Urmia, Iran.
1
25
Abstract
26
Among polychlorinated biphenyls (PCBs), dioxin-like PCBs (DL-PCBs) are of the 27 most concern for human health. In this study, the levels of 12 DL-PCBs congeners 28 were measured in 125 fish samples of the Caspian Sea, Iran. Five fish species 29 (Oncorhynchus mykiss, Vimba vimba, Cyprinus carpio, Rutilus frisii kutum and 30 Chelon saliens) were collected from 5 coastal cities of the Caspian Sea (25 samples 31 per each city). Duncan's multi-scope test was used to compare the mean of DL-PCBs 32 in different fish species and different cities. Probabilistic risk of exposure to DL- 33 PCBs and sensitivity analysis were assessed using Monte Carlo simulation approach. 34 The average (standard deviation) of DL-PCBs in fish samples ranged from 232 (16) 35 to 1156 (14) pg/g lipids. The total maximum concentration was detected in Cyprinus 36 carpio from Bandar Anzali, the minimum in Vimba vimba from Chalos. In all 37 samples, non-carcinogenic risk of exposure to DL-PCBs was in safe level (Hazard 38 Quotient < 1). In contrast, the lifetime cancer risk estimated for Bandar Anzali, 39 Bandar Torkaman, and Rasht exceeded the threshold value of 1×10-6 suggested by 40 United States Environmental Protection Agency. Sensitivity analysis indicated that 41 the concentration of DL-PCBs and exposure frequency were the most effective 42 parameters in increasing carcinogenic risk.
43
Keywords: Polychlorinated Biphenyls, health risk assessment, fish, Caspian Sea
44
45
1. Introduction
46
2
Fish consumption is highly recommended in human diet due to its content of 47 polyunsaturated fatty acids (n-3) (PUFA) that play an important role in the 48 maintenance of human health (Trocino et al., 2012). However, the seafood 49 consumption is the most important source for human exposure to persistent organic 50 pollutants (POPs) and other toxic materials (Dórea, 2008). Moreover, using fish in 51 waters pollution monitoring programs has been widely recommended (Avigliano et 52 al., 2016).
53
The most hazardous groups of POPs include polychlorinated dibenzodioxins 54 (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls 55 (PCBs) (Fu et al., 2018; Pacheco Ferreira, 2018; USEPA., 2000). PCBs are a group 56 of 209 PCB-congeners with different substitution patterns with high persistence and 57 capable to bioaccumulation. PCBs are classified into two groups: dioxin like-PCBs 58 (DL-PCBs, 12 congeners) and unlike dioxin PCBs or non-dioxin like PCBs (NDL- 59 PCBs) (Fu et al., 2018; Lüth et al., 2018). DL-PCBs include four non-ortho 60 congeners (77, 81, 126, and 169) and eight mono-ortho PCB congeners (Nakatani et 61 al., 2011; Pacheco Ferreira, 2018). Review studies on the topic show the presence of 62 different DL-PCBs congeners in water, soil, air, and wildlife (Salamova et al., 2013). 63 Previous studies reported that DL-PCBs concentration in fish depends on several 64 factors such as: species, environmental conditions (Storelli, 2008), type or origin of 65 feed used in farmed fish (Conti et al., 2015; Trocino et al., 2009), and ecological 66 niche occupied by wild fishes (Custódio et al., 2011).
67
Exposure to DL-PCBs can lead to carcinogenic and immunological effects but also 68 endocrine and fertility dysfunctions (Rizzi et al., 2017).
3
69
Research on Cancer (UNIARC) classified PCB 126 congener as carcinogenic for 70 humans (group 1) (IARC., 2009; Nakatani et al., 2011), but recently, all PCBs (DL- 71 PCBs and NDL-PCBs) have been identified as carcinogenic for humans (Barone et 72 al., 2018; Habibullah-Al-Mamun et al., 2019; IARC, 2016; Lauby-Secretan et al., 73 2013). The Joint FAO/WHO Expert Committee on Food Additives, recommends a 74 provisional tolerable monthly intake (PTMI) of 70 pg/kg body weight/month to 75 reduce the human exposure to the DL-PCBs through diet (Cao et al., 2018).
76
So far, several studies focused on the presence of several classes of POPs in fishes 77 caught from the Caspian Sea (Agusa et al., 2004; Anan et al., 2005; Dadar et al., 78 2016; Ebadi and Shokrzadeh, 2006; Hosseini et al., 2008; Kajiwara et al., 2002; 79 Kajiwara et al., 2003; Kajiwara et al., 2008; Katuli et al., 2014; Manavi et al., 2018; 80 Manavi and Mazumder, 2018; Mashroofeh et al., 2013). However, within the group 81 of POPs studies addressing DL-PCBs in fish in this area are missing in literature.
82
The Caspian Sea has a strategic position; it is rich in oil and gas reserves and 83 represents a source of income for commercial fishing and shrimp fishing of 84 neighboring countries. Therefore, this study aimed to evaluate the level of DL-PCBs 85 in different fish species from different areas of the Caspian Sea, Iran. Also, 86 carcinogenic and non-carcinogenic risk of exposure to these pollutants were assessed 87 as well as sensitivity analyses were carried out using the Monte Carlo simulation 88 technique.
89
2. Material and methods
90
2.1.Sampling location and fish collection
91
4
This study was carried out in March 2018 in five coastal cities (Bandar Anzali, 92 Chalous, Rasht, Astara and Bandar Torkaman) of the Caspian Sea, Iran (Figure 1). 93 The Caspian Sea is in the geographical location of longitude from 48.75° to 53.92° 94 and latitude from 36.46° to 47.10°, with an about 420 000 km2 area.
95
Annually, 600 000 tons of different fish species are caught from this sea and 96 consumed by citizens (Tolosa et al., 2004). In this study, 5 common fish species (5 97 specimens from each species) including Oncorhynchus mykiss, Vimba vimba, 98 Cyprinus carpio, Rutilus frisii kutum, and Chelon saliens were randomly collected 99 from 5 markets for each coastal city included in our study for a total of 125 samples. 100 The selected cities covered all Iranian coastline of the Caspian Sea, and we made 101 sure that the fish specimens were collected from the Caspian Sea. All collected 102 samples were placed in cool boxes and transferred to the laboratory. In the 103 laboratory, biometers of the fishes, i.e., weight and length were recorded (Table S1 of 104 Supplemental materials). 50 g muscle tissue from each fish were sampled, wrapped 105 in aluminum foil and stored at -20°C until analysis. 2.2.Extraction of DL-PCBs
106 107
The initial DL-PCBs extraction was done according to USEPA method 1668 revision 108 A (U.S. EPA, 1999). Briefly, 50 g of fillet was homogenized by a meat grinder 109 (Moulinex, Ecully Cedex, France). The process was repeated three times for each 110 muscle sample. Then, 100 g sodium sulfate anhydrous (Na2SO4, Merck, Germany) 111 was added to each sample and homogenized at 50 °C for six h. Besides, about 50 ng 112 of internal standard PCB 209 (Sigma-Aldrich, Germany) was added to the sample to 113 monitor the PCB extraction and its recovery. The lipid extraction process was carried 114 5
out using Soxhlet Extractor (Soxhlet Extraction System B-811 (Büchi AG, Flawil, 115 Switzerland)). The extractions were performed using a mixture of hexane and 116 acetone (90:10 v/v). The process for each sample was repeated for 260 cycles (Liu et 117 al., 2016; USEPA, 1999). The concentration of lipid was determined gravimetrically. 118 1 g of extracted lipid was dissolved in 10 mL n-hexane, and this diluted extract was 119 used for further analyses. All extracts were purified using silica gel multi-layer 120 absorbent columns (see Figure S1 of Supplemental materials). In this study, the 121 cleanup columns used were similar to the Japanese industrial standard method based 122 on the EPA1613 method (Masunaga et al., 2001). The silicates were initially 123 activated and then dried at 200 °C for 24 h. After that, silica was washed with 60 mL 124 n-hexane to remove contaminants from the silica. To prepare silica gel, the 125 inactivated silica was mixed with sulfuric acid 98% (40% w/w) (Akutsu et al., 2005; 126 USEPA, 1999). The column layers composition for chromatography was similar to 127 those reported by Masunaga et al. (2006) and Aries et al. (2001) (Aries et al., 2006; 128 Masunaga et al., 2001).
129
Briefly, the first layer was a mixture of silica gel (3 g) with silver nitrate (AgNO3, 130 10% w/w, Merck, Germany), which eliminated sulfur compounds from the sample. 131 The next layer was made of silica (6 g) with sulfuric acid (H2SO4, 22%), for the 132 removal of lipid layers and elimination of its oxidation metabolites from the sample. 133 The third layer was constituted by 44% H2SO4, which was used to remove the 134 remaining lipid impurities and hydrolyzed fats in the sample. The fourth layer was 135 made of a mixture of silica gel (3 g) and potassium hydroxide (KOH-2%, Merck, 136 Germany), that eliminated the possible acids and acidic compounds. Finally, The 137
6
DL-PCBs were eluted through the column by 50 mL n-hexane (HPLC grade) and 138 concentrated using a rotary evaporator at 40 °C to reach a final volume of 1 ml for its 139 injection into HRGC/HRMS (Barone et al., 2018; USEPA, 1999). After passing the 140 sample through the column, the column was washed with further solvent (60 mL of 141 n-hexane) to remove the remaining PCBs in the column.
142
2.3.Analysis of DL-PCBs
143
The analyses of DL-PCBs in the fillets samples were done according to USEPA 144 method (USEPA, 1999). The analysis of samples was carried out at the Laboratory of 145 Tehran
University of
Medical
Sciences
using a
High
Resolution
Gas 146
Chromatography (Agilent 6890 Series, Agilent Technologies, USA) coupled with 147 High
Resolution
Mass
Spectrometer
AutoSpec
Ultima
NT–HRGC/HRMS 148
(Micromass, USA), equipped with the HP-5MS 30 m × 0.25 mm × 0.25 µm column 149 (Agilent Technologies) and helium as carrier gas. In this study 12 congeners of DL- 150 PCBs (PCB 77, PCB 81, PCB 105, PCB 114, PCB118, PCB 123, PCB 126, PCB 151 156, PCB 157, PCB 167, PCB 169 and PCB 189) (more details are described in 152 Table S2 of Supplemental materials) were determined and reported as pg/g fat 153 (Trocino et al., 2009; Van den Berg et al., 2006). The detector calibration (as linear 154 response range) was determined by injecting standard dilutions under actual 155 conditions to HRGC/HRMS. An appropriate amount (from 1 to 10 µL) of the extract 156 was injected to HRGC/HRMS, and the congeners and their concentrations were 157 determined (Akutsu et al., 2005; U.S. EPA, 1999). For all samples, a HRMS single- 158 ion monitoring (SIM) method (>10000 resolution) was applied (Masunaga et al., 159 2001). Two daughters ions (for natural and marked DL-PCBs) were considered to 160
7
determine the best ratio of signal to noise (S/N). The response factors were measured 161 by injecting 1 µL of standard solution into the HRGC/HRMS. Other MS detector 162 condition was 1 mL/min as a flow rate, injector in splitless mode, and injector 163 temperature at 290 ºC. Oven temperature program was 90 ºC (isothermal: 2 min and 164 first rate at 5 ºC/m) to 280 ºC (isothermal: 3min).
165
2.4.Quality control and recovery
166
Quantification of DL-PCBs was done based on multi-level calibration curves, and we 167 found a good linearity (R2 > 0.99) for the standards range which had the same 168 concentrations range of the samples. The standard curve of 10 concentrations of DL- 169 PCBs ranged from 1 to 1000 µg/l (Sigma-Aldrich, Germany). DL-PCBs 170 concentrations were measured based on a peak-by-peak height comparison and a 171 calibration curve of each certified PCB congener. All PCBs had a peak height at the 172 range of calibration curve, and the samples with higher peak were diluted. The 173 efficiency of the residue analysis method used to measure the DL-PCBs in the fish 174 samples was tested based on recovery (%). The mean recoveries were between 98% 175 and 110% for all compounds tested.
176
Blank sample analysis was carried out after injecting every 10 samples to calibrate 177 the system and results indicated that all blank samples showed no detectable 178 concentrations or traces of PCBs (Salihovic et al., 2012).
Limits of quantification 179
(LOQ) for all DL-PCBs ranged between 0.03 to 0.09 pg/g fat. 2.5.Toxic equivalents (TEQs)
180 181
The toxicity level of the DL-PCBs was considered based on the most toxic known 182 between the dioxin compounds, namely 2,3,7,8 tetrachloro-dibenzo-dioxin (TCDD), 183
8
as the toxic scale of 1 for it and the toxicity of other DL-PCBs was compared with it 184 (Table S3 of Supplemental Materials) (Su et al., 2012; Turyk et al., 2006; Xu et al., 185 2006). The total toxic equivalents (TEQs) were calculated as follows (Van den Berg 186 et al., 2006):
187
TEQs = ∑ (Ci × TEFi)
(1)
188
Where Ci is the concentration of DL-PCB congener i; TEFi is the toxic equivalent 189 factor of congener i.
190
2.6.Health Risk Assessment
191
Monte Carlo simulation method was applied to determine the carcinogenic and non- 192 carcinogenic risk caused by exposure to DL-PCBs through the consumption of fish in 193 all cities of the studied area. Sensitivity analysis was done based on effective 194 variables on risk assessment such as concentration (C) of DL-PCBs, exposure 195 frequency (EF), body weight (BW), the averaging time (AT) and the maximum 196 allowable daily consumption of contaminated fish (FCR).
197 198
2.6.1. Estimated daily intake
199
Estimated daily intake (EDI) of DL-PCBs (ng/kg/day) by fish consumption was 200 calculated using the following equation:
EDI =
× × ×
201
(2)
×
202
Where, FCR is set to 25.5 g/day for Iranian people (Miri et al., 2017b), BW is set to 203 70 ± 10 kg for Iranian adults (Miri et al., 2017b), EF is based on day/year, which 204 calculated based on the rate of fish consumption for Iranian inhabitants (minimum, 205 9
once per week, on average twice and maximum three meals per week), ED is the 206 exposure duration in terms of years (70 years) and AT is the averaging time in days 207 (AT= EF × ED).
208
2.6.2. Hazard quotient
209
Hazard quotient (HQ) of the non-carcinogenic risk related to the consumption of fish 210 contaminated with DL-PCBs was calculated as follows (Saha et al., 2016): =
(3)
211
212
RFD is the oral reference dose set to 20 ng/kg/day for PCBs in this study (Quadroni 213 and Bettinetti, 2017).
214
In our study, the effect of cooking on the concentration of DL-PCBs was not 215 considered, and the HQ was calculated based on pollutant concentration in row 216 samples (Saha et al., 2016).
217
2.6.3. Carcinogenic risk
218
The lifetime carcinogenic risk (LTCR) through exposure to DL-PCBs was 219 determined using the cancer slope factor (CSF) (2×10−6 ng/kg/day) provided by the 220 United States Environmental Protection Agency (USEPA) with the following 221 equation (Saha et al., 2016; Shaheen et al., 2016):
=
× × × × × ! "# ×
%$× 10()
222
(4)
223
Where CF is conversion factor (0.001 pg/ng). Other parameters are same as equation 224 2.
225
10
2.7.Statistical analysis
226
Data analysis was carried out using a completely randomized design in a factorial 227 experiment in three replications. Descriptive statistics were analyzed by Stata 228 software version 15 (Stata Corp LP, College Station, Texas). Normality of data was 229 tested based on Shapiro-Wilk. Duncan multi-scope test was used to compare mean of 230 DL-PCBs in different fish species and different cities. A significant level of 0.05 was 231 performed for all analyses. To simulate Monte Carlo technique and perform 232 sensitivity analyses, Crystal Ball software (version 11.1.1.1, Oracle, Lnc. USA) with 233 100 000 trails was used.
234 235
3. Results and discussion
236
3.1.Comparing DL-PCBs concentrations
237
A comparison of ∑DL-PCBs concentration of different fish species and different 238 sampling locations are presented in Table 1. There were significant differences in 239 ∑DL-PCBs concentration of different fish species and sampling location (p≤0.05). 240 The highest ∑DL-PCBs was in Cyprinus carpio species (1156.27 ± 14.55 pg/g fat) in 241 Bandar Anzali. The lowest concentration of ∑DL-PCBs was in Vimba vimba species 242 from Chalous, with a mean concentration of 232.43 ± 16.05 pg/g fat. Due to great 243 variation of PCBs type and number of them which were analyzed in previous studies, 244 as well as due to different measurement units and methods, comparing the results of 245 our study with previous reports is complicated and could not be reasonable (Cao et 246 al., 2018; Mezzetta et al., 2011). For example, in a study by Chao et al. (2018) in the 247 Shadong Sea of China, the average DL-PCBs was 0.887 ng/g wet weight (Cao et al., 248 11
2018). The concentration of measured PCBs in different fish species are shown in 249 Table S4 to S14 and Figure S2 (for PCB77) of Supplemental materials.
250
Overall, the mean concentration of non-ortho PCBs was lower than mono-ortho 251 PCBs. For mono-ortho PCBs, PCB118 (367.50 pg/g fat) had the highest mean 252 concentration in Cyprinus carpio and this our result is consistent with other studies 253 of Zhoushan Fishery and marine fish of Shandong in China, farmed fish, Latvian 254 Lake and fish species of Lake Victoria, Uganda (Cao et al., 2018; Ssebugere et al., 255 2014; Wang et al., 2015; Zacs et al., 2013). Moreover, all the other 7 mono-ortho 256 PCBs were detected in all fish species and Bandar Anzali had the highest mean 257 concentration. The lowest mean concentration of mono-ortho PCBs was that of PCB 258 157 (0.82 pg/g fat) in Vimba vimba from Chalous. Among non-ortho PCBs, the 259 highest mean concentration was that of PCB 77 (29.58 pg/g fat) in Cyprinus carpio 260 from Bandar Anzali. Also, the lowest concentration of non-ortho PCBs was found in 261 Vimba vimba from Chalous for PCB 169 with mean concentration of 0.02 ± 0.03 262 pg/g fat. Considering the different measurement units, both PCB 77 (46.1 pg/g ww) 263 and PCB169 (2.02 pg/g ww) concentrations recorded by Celik Cakirogullari et al. 264 (2010) were higher than this study results (Çelik Çakıroğulları et al., 2010). In the 265 study by Cao et al. (2018) PCB 77 (0.063 ng/g ww) had the highest mean 266 concentration, which is in line with our findings (Cao et al., 2018).
267
The highest concentrations were found in Cyprinus carpio from Bandar Anzali and 268 the lowest in Vimba vimba from Chalous. Previous studies indicated that 269 concentration of DL-PCBs in different fish species was affected by complex factors 270 e.g., geographical origin, environment condition and season. Moreover, fish lipid 271 12
content, living and feeding habits, and low biodegradability of DL-PCB congeners in 272 the aquatic ecosystem (such as Sea) and bioaccumulation capability of these 273 pollutants in organisms are the major reasons of contamination of fish species by 274 DL-PCBs (Blanco et al., 2013; Costopoulou et al., 2016; Jiang et al., 2005a; 275 Mezzetta et al., 2011; Sun et al., 2014). 3.2.TEQs
276
277
The descriptive statistic of calculated TEQs for different studied cities and different 278 fish species are presented in Figure 2. The mean (standard deviation (SD)) of TEQs 279 in Astara, Bandar Anzali, Bandar Torkaman, Chalous and Rasht were 0.60 (0.32), 280 0.82 (0.35), 0.73 (0.31), 0.59 (0.28) and 0.68 (0.29) pgTEQ/g fat respectively. The 281 mean (SD) of TEQs for Rutilus frisii kutum, Oncorhynchus mykiss, Cyprinus carpio, 282 Chelon saliens and Vimba vimba species were 0.90 (0.11), 0.48 (0.11), 1.08 (0.16), 283 0.73 (0.09) and 0.23 (0.08) pgTEQ/g fat, respectively. Our results indicated that the 284 level of DL-PCBs in Cyprinus carpio species was more than other species and the 285 TEQ value for this species was higher than 1. Han et al. (2018) reported that in 286 China, reported that the maximum level of TEQ (higher than 1) was also detected in 287 Cyprinus carpio in China (Han et al., 2018). Moreover, in another study carried out 288 in Italy, similar results have been reported for this fish species (Trocino et al., 2012). 289 Therefore, Cyprinus carpio species should be consumed with caution for sensitive 290 people groups (i.e., children and elderly) in these areas. In our study, the overall 291 mean (SD) of TEQs was 0.68 (0.32) pgTEQ/g fat. Considering differences in TEQs 292 calculation, our values were lower than those reported by Trocino et al. 2012 in Italy 293 for intensive inland basins and intensive sea cages (Trocino et al., 2012). In another 294 13
study by Çelik Çakıroğulları et al. 2010 in Turkey, reported that the actual level of 295 ∑WHO-PCDD/Fs-DL-PCBs-TEQ(TEFs
1998)
was
6.12 ± 0.16 pg/g
fat
(Çelik 296
Çakıroğulları et al., 2010). In the study by Valérie Vrommana et al. in 2012, reported 297 that the mean of TEQs(TEFs 1998) was 2.19 pg WHO-TEF/g fresh weight (Vromman et 298 al., 2012). However in study by Han et al. on the Chinese mitten crabs, the overall 299 mean of TEQ level was 5.7 ± 4.0 pg TEQ/g (Han et al., 2018). In a study by Cao et 300 al., 2018 in China, TEQ(TEFs 2006) value in each fish sample was ranged from 0.011 to 301 9.214 pg WHO-TEQ/g ww (Cao et al., 2018).
302
303
3.3.Health risk assessment
304
In the present study, the health risk of exposure to ∑DL-PCBs was estimated based 305 on mean concentration of these pollutants in different fish species in each Caspian 306 coastal city. The results of the EDI (ng/kg/day) and non-carcinogenic (HQ) risk of 307 exposure to DL-PCBs via fish consumption in different cities are presented in Table 308 2. As shown, the highest mean value of EDI was 3.23E-1 ng/kg/day in fishes from 309 Bandar Anzali, and the lowest EDI value was observed in Chalous fishes with an 310 average of 2.11 E-1 ng/kg/day. The highest mean of HQ was observed in fishes from 311 Bandar Anzali (1.62 E-2); instead the lowest mean of HQ value was in Chalous 312 samples (1.06 E -2). According to the results, in all cities, the HQ was less than 1, 313 indicating that the mean concentration of ∑DL-PCBs in different studied areas were 314 at a safe level. Zhao et al. (2012) reported that HQs that HQ related to exposure to 315 DL-PCBs in both low and high fish consumption groups in China were in safe level 316 (Zhao et al., 2012). Similar results were reported by Jiang et al. (2005b) (Jiang et al., 317 14
2005b). Previous studies in Iran, reported that the non-carcinogenic risk for several 318 heavy metals via fish consumption were in safe level (HQ<1), which is consistent 319 with the present study (Ebadi Fathabad et al., 2019; Miri et al., 2017a). While, in a 320 study by Ebadi Fathabad et al., 2018, in Tehran's market, reported that, the HQ for 321 heavy metals in various types of natural juices and canned foods was higher than safe 322 level (Fathabad et al., 2018).
323
The results of lifetime carcinogenic risk (LTCR) of exposure to ∑DL-PCBs via 324 consumption of fish in different parts of the studied area are presented in Figure 3. 325 As shown, the 95th percentile value for LTCR for Bandar Anzali, Bandar Torkaman 326 and Rasht exceeded the threshold value of 1.00 E-6 suggested by USEPA (EPA, 327 2001). The highest mean value of LTCR was observed in Bandar Anzali (6.46 E-7), 328 and the lowest LTCR was observed in Chalous (4.23 E-7). Xia et al. (2012) reported 329 that LTCR in all low and high fish consumption groups was higher than safe level 330 (Xia et al., 2012). Otherwise, previous studies carried out in Iran showed also 331 indicated that the carcinogenic risk of exposure to heavy metals via consumption of 332 fish is at a safe level (Fathabad et al., 2018; Miri et al., 2017a).
333
In this study, sensitivity analysis was performed to determine the most effective 334 variable in increasing the carcinogenic risk through DL-PCBs using Monte Carlo 335 simulations. Figure 4 indicates sensitivity analyses of LTCR for exposure to ∑DL- 336 PCBs in different coastal cities. According to Figure 4, the concentration of DL- 337 PCBs (C) was the most effective variable in increasing the carcinogenic risk 338 (contribution to variance ranged from 39.4 to 50.9 %). The other effective parameters 339 in increasing the health risk for consumers was exposure frequency (EF) and per 340 15
capita fish consumption (FCR), respectively. Increased body weight (BW) and 341 averaging time (AT) of exposure (day) had an inverse relationship with carcinogenic 342 risk (contribution to variance ranged from -8.7 to -10.9 % and -18 to -23.9% 343 respectively). Previous studies indicated that the concentration of the pollutant was 344 an effective factor in increasing the health risk, which is consistent with the results of 345 this study (Fallahzadeh et al., 2018; Miri et al., 2018).
346 347
4- Conclusion
348
The average TEQs of DL-PCBs in fish samples from the Iranian coastline of Caspian 349 Sea was lower than previous reports. The highest level of DL-PCBs was observed in 350 Cyprinus carpio from Bandar Anzali, and the lowest levels of DL-PCBs were related 351 to Vimba vimba from Chalous. The results of the health risk assessment indicated 352 that carcinogenic risks of exposure to ∑DL-PCBs via consumption of fish in Bandar 353 Anzali, Bandar Torkaman, and Rasht were higher than the safe level. The sensitivity 354 analysis indicated, C and EF as the most effective parameters in increasing the health 355 risk. The DL-PCBs in fish samples of different studied areas should be monitored 356 continuously in the future. More attention is needed to protect the marine 357 environment by the authorities and decision-makers to prevent seawater 358 contaminations. Further studies, on the other fish species and more frequent sampling 359 in different seasons, are recommended to find out the reliability of the results of this 360 study.
361
362
Acknowledgment
363 16
The authors thank from Urmia University and Tehran University of Medical 364 Sciences. Mohammad Miri is supported by the Sabzevar University of Medical 365 Sciences.
366 367 368 369 370
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541
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20
545
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Figure captions
548
Figure 1. Geographical location of the Caspian Sea and studied cities.
549
Figure 2. Calculated toxic equivalents (TEQs) of ∑DL-PCBs based on studied 550 cities and fish species.
551
Figure 3. Lifetime carcinogenic risk (LTCR) for intake ∑DL-PCBs via
552
consuming fish in different studied cities.
553
Figure 4. Sensitivity analyses for lifetime carcinogenic risk of exposure to DL- 554 PCBs via consumption of different fishes in different cities (C: concentration of 555 DL-PCBs; EF: exposure frequency, AT: averaging time in days, BW: body 556 weight; FCR: fish consumption rate).
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557
Table 1. Comparison of ∑DL-PCBs concentration (pg/g fat) in different fish species and different cities
Fish species/City
Bandar Torkaman
Chalous
Rasht
Bandar Anzali
Astara
Rutilus frisii kutum
923.34 ± 20.48 Bb
688.92 ± 11.85 Eb
842.31 ± 7.28 Cb
986.41 ± 10.70 Ab
783.22 ± 11.17 Db
Chelon saliens
836.74 ± 17.73 Bc
586.10 ± 11.36 Ec
746.11 ± 18.28 Cc
884.04 ± 12.91 Ac
710.37 ± 19.78 Dc
Cyprinus carpioio
1033.60 ± 17.89 Ba
799.51 ± 10.98 Ea
945.32 ± 16.15 Ce
1156.27 ± 14.55
857.96 ± 14.79 Da
Vimba vimba
407.76 ± 17.75 Be
232.43 ± 16.05 Ee
366.27 ± 15.73 Ce
462.64 ± 17.26 Ae
313.69 ± 12.42 De
Oncorhynchus
671.84 ± 25.70 Bd
438.33 ± 18.31 Ed
580.36 ± 15.66 Cd
714.44 ± 12.95 Ad
509.59 ± 49.75 Dd
mykiss *The different small letters indicate a significant difference in the columns and different large letters in the row indicating a significant difference in the row (p≤0.05).
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Table 2. Estimated daily intake (EDI) and hazard quotient (HQ) for exposure to DL-PCBs via consumption of fish in different cities Percentile 5th
Mean
Median
Standard deviation
Percentile 95th
EDI
1.02 E-1
2.46 E -1
2.21 E-1
1.18 E -1
4.74 E-1
HQ
5.11 E -3
1.23 E -2
1.11 E -2
5.91 E -3
2.37 E -2
EDI
1.43E-1
3.23E-1
2.94E-1
1.48E-1
6.01E-1
HQ
7.14 E -3
1.62 E -2
1.47 E -2
7.38 E -3
3.00 E -2
EDI
1.30E-1
2.95E-1
2.66E-1
1.35E-1
5.49E-1
HQ
6.52E-3
1.48E-2
1.34E-2
6.67E-3
2.74E-2
EDI
8.26E-2
2.11E-1
1.87E-1
1.11E-1
4.21E-1
HQ
4.13E-3
1.06E-2
9.35E-3
5.54E-3
2.10E-2
EDI
1.15E-1
2.68E-1
2.43E-1
1.25E-1
5.10E-1
HQ
5.77E-3
1.34E-2
1.21E-2
6.27E-3
2.55E-2
Cities Astara
Bandar Anzali
Bandar Torkaman
Chalous
Rasht
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Highlights •
DL-PCBs concentrations in five fish species of the Caspian Sea were investigated.
•
The highest DL-PCBs concentration was observed in Cyprinus carpio species.
•
LTCR for Bandar Anzali, Bandar Torkaman, and Rasht exceeded the threshold value suggested by USEPA (1×10-6).
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: