Evaluation of dioxin-like polychlorinated biphenyls in fish of the Caspian Sea

Graphical abstract

USA), equipped with the HP-5MS 30 m Â 0.25 mm Â 0.25 mm column (Agilent Technologies) and helium as carrier gas). Meat grinder (Moulinex, Ecully Cedex, France). All of the chemical agent was from Merck, Germany. Internal standard PCB 209 (Sigma-Aldrich, Germany) and Soxhlet Extraction System B-811. Experimental design: A total of 125 samples of fish (Rutilus frisii kutum kanesky, Chelon saliens, Vimba vimba, Cyprinus carpio and Oncorhynchus mykiss) were prepared from 5 coastal areas of the Caspian Sea including Bandar Anzali, Chalous, Rasht, Astara and Bandar Torkaman (25 samples per each city). 12 DL-PCBs congeners were determine in their tissue and then the mentioned parameters above, in abstract section, were analyzed according to the EU and JECFA standards. Trial registration: No applicable Ethics: No applicable

Value of the Protocol
Exposure to DL-PCBs can lead to complications due to high resistance, toxic and bioaccumulation in humans and wildlife of DL-PCBs. Data analysis showed that the mean concentration of DL-PCBs in fish samples were in accordance with the EU and JECFA standards.
Contriling of DL-PCBs in contaminated industries and environmental health to reduse the DL-PCBs concentration in food chain is necessary.

Study area description
The Caspian Sea, in the geographical location of 40 N and 51 E, is the largest lake in the world. The average water depth is 187 m and the water volume is 78,200 km 3 . The Caspian Sea is the strategic location for many human needs and activities. Also, Caspian sea is a source of fishing and shrimp fishing for neighboring countries. Annually, 600,000 ton of fish species from this sea are hunted.

Determination of DL-PCBs concentration in fish samples from Caspian sea
Five common fish species, a total of the 125 fish samples (25 samples from each city), was randomly collected from predetermined stations of 5 location including: Bandar Anzali, Rasht, Chalous, Bandar Torkaman and Astara that placed in cold boxes with ice. In the laboratory, fishes biometrics, were recorded and the muscle tissue was separated about 50 g. Then, the samples were wrapped in aluminum foil and stored at À20 C until analysis in a dark environment [1][2][3]. The DL-PCBs extraction was in accordance to USEPA method 1668 revision A [4]. For extraction the DL-PCBs, first, the samples were crushing for three times (Moulinex, Ecully Cedex, France). In each sample, about 50 g of homogenized muscle tissue was combined with 100 g Na 2 SO 4 and then homogenized at 50 C for 6 h. In addition, they added about 50 ng of internal standard PCB 209 and using Soxhlet Extractor, the lipid extraction process was carried out. To DL-PCBs extract, hexane and acetone solvents were used in the ratio of 90:10 and about 260 times, repeated extraction [5,6]. The concentration of lipid was determined gravimetrically. 1 g of extracted lipid was dissolved in 10 mL n-hexane, and this diluted extract was used for further analyses. All extracts were purified using silica gel multi-layer absorbent        The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05). The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05).  The different small letters indicate a significant difference in the columns and different large letters indicating a significant difference in the row (p 0.05).  [7][8][9]. The silicates were initially activated [6,10]. The DL-PCBs were passed through the column filled with silica and collected. Finally, the DL-PCBs were eluted through the column by 50 mL n-hexane (HPLC grade) and concentrated using a rotary evaporator at 40 C to reach a final volume of 1 ml for its injection into HRGC/HRMS [6,11]. Also, calibration curve had a good linearity for 1-10 standards from 1À1000 mg/l (R 2 > 0.99  [12,13]. The toxicity level of the DL-PCBs based on the most toxic known of dioxin compounds, namely, 3,2,7,8 tetra chloro-di benzo-dioxin (TCDD), was considered as the toxic scale of 1 for it and the toxicity of other dioxins-like compounds (DL-PCBs) were compared with it [14][15][16].

Statistical design of experiments
Data analysis was carried out with SPSS 22 software (Duncan's multi-scope test and descriptive statistics). P-value 0.05 was considered significant. Microsoft Excel version 2016 for plotting calibration curves and basic mathematical calculations. The data presented here deals with DL-PCBs concentration in fish species according fish species and city location.