Data Parameters and Units:
FIELD NUMBER, SITE, SPECIES [cobia, red snapper, greater amberjack, conger eel, yellowedge grouper], Volume of bile injected (µL), Naphthalene Equivalents (ng/g bile, wet wt.), Phenanthrene Equivalents (ng/g bile, wet wt.), Benzo[a]pyrene Equivalents (ng/g bile, wet wt.), Nap and Phen combined. The following is a list of sample sites and their location (Sample Site Name, Latitude, Longitude): PISCES 10-20, 29.4525, -88.6525; PISCES 10-40, 29.2355, -88.5522; PISCES 11-100, 29.0373, -88.7373; PISCES 11-40, 29.1093, -88.8760; PISCES 12-20, 28.8512, -89.4845; PISCES 12-40, 28.8241, -89.5074; PISCES 13-100, 28.0236, -90.6736; PISCES 13-20, 28.4734, -90.5568; PISCES 13-40, 28.2178, -90.5845
Methods:
Frozen bile samples collected from five species sampled in 2011 were shipped to the Northwest Fisheries Science Center (NWFSC) in Seattle, WA and were stored at -20°C until analysis. Thirty-four individual fish bile samples from 2011 (several species) and 15 samples from 2012 (red snapper) were analyzed for metabolites of PAHs using a high-performance liquid chromatography/fluorescence (HPLC-F) method (Krahn et al. 1984; Krahn et al. 2005). This method results in the determination of the concentrations of classes of PAH metabolites fluorescing in the regions typified by NPH, PHN and benzo[a]pyrene (BaP). Bile was injected directly onto a Waters © high-performance liquid chromatography/fluorescence system equipped with a C-18 reverse-phase column (Phenomenex Synergi Hydro©). The PAH metabolites were eluted with a linear gradient from 100% water (containing a trace amount of acetic acid) to 100% methanol at a flow of 1.0 mL/min. Chromatograms were recorded at the following wavelength pairs: (1) 292/335 nm where many 2-3 benzene ring aromatic compounds (e.g., NPH) fluoresce, (2) 260/380 nm where several 3-4 ring compounds (e.g., PHN) fluoresce and (3) 380/430 nm where 4-5 ring compounds (e.g., BaP) fluoresce. Peaks eluting after nine minutes were integrated and the areas of these peaks were summed. The concentrations of fluorescent PAHs in the bile samples of the marine fish were determined using NPH, PHN, BaP as external standards and converting the fluorescence response of bile to PHN (ng PHN equivalents g-1 bile), NHP (ng NPH equivalents g-1 bile) or BaP (ng BaP equivalents g-1 bile) equivalents. To ensure that the HPLC/fluorescence system was operating properly, a NPH/PHN/BaP calibration standard was analyzed five times to obtain a relative standard deviation < 15% for each PAH. As part of the laboratory quality assurance (QA) plan, two QA samples, consisting of a method blank and a fish bile control sample (bile of Atlantic salmon, Salmo salar, exposed to 25 µg/mL of Monterey crude oil for 48 hours), were analyzed with the fish bile samples. Three methods blanks revealed no traces of PAH metabolites, and the calibration standard was reproduced within the standard deviation of the source compound. Sampling in 2011 occurred from July-August, using three chartered commercial longline fishing vessels. The depth distribution of sampling was 15-195 m along 15 transects from nearshore to offshore. The maximum depth of the survey coincided with the maximum depth distribution of red snapper, the species most often reported with skin lesions. For selected specimens we determined PAH levels in muscle and liver tissues and PAH metabolites in bile using standard methods. Sampling stations were located at nominal depths of 18, 37, 73, 110, 146 and 183 m along 15 transects extending from north of the Dry Tortugas Islands, to offshore from Terrebonne Bay, Louisiana. Some additional stations were located between transects, and for some transects with steep bathymetric slopes, we reduced the number of stations fished. The longitudinal scope of the study was set to encompass the majority of the area where surface oil concentrations during the DWH event were greatest, and also to include the West Florida Shelf region that had no observed surface oiling from the spill although we cannot rule out upwelling of dissolved DWH hydrocarbons there) to compare with the heavily-impacted NGM. At each pre-determined sampling location, the vessel captain searched for suitable habitat for target species including primarily snappers and groupers. This involved using the ship’s fish finder to locate “hard bottom” habitat typically also showing concentrations of demersal fishes. The vessel was allowed to range up to nine km from the center line of the sampling transect in search of suitable habitat. At each station, eight km of 3.2 mm galvanized steel main line was deployed, with 322-500 baited hooks. We used 91 kg test leaders, 3.7 m long attached to #13 circle hooks, with alternating cut fish and squid as bait. At the beginning and end of the main line we deployed Star:Oddi© CDST Centi temperature/time/depth recorders to record actual bottom time, as well as bottom temperature and depth fished. The recording interval of these instruments was 5 min. At set-out and haul-back we recorded latitude and longitude, time, depth from the vessel’s depth finder, the unique numbers of the TD instruments deployed at either end of the string, and local weather conditions. Once the longline was deployed, usually the vessel steamed back to the start buoy and began haul-back. The average soak time was 2 hours 1 minute. Large sharks (e.g., ~2 m and greater) were photographed for species identification at the rail and released alive. Each fish obtained was inspected for a variety of externally-symptomatic diseases and other samples obtained. Average fish catch in 2011 was 47 per haul (range 4-240), the dominant species were red snapper, red grouper, Gulf smoothhound, and Atlantic sharpnose shark, which in aggregate accounted for about 2/3 of the total catch. In 2012 we added an additional survey transect at the Mississippi Valley, transect 16 west of the Mississippi River Delta and one west of the previous limits of our survey (transect 17). Catches in 2012 consisted primarily king snake eel, Atlantic sharpnose shark, red snapper, Gulf smoothhound, and tilefish, also comprising about 2/3 of the catch. Our catches of large pelagic species were augmented by trolling surface lures while transiting between longline stations. Troll-caught specimens were processed using methods similar to longline catches. Each fish was examined for the following: (1) presence of external skin lesions (e.g., ulcers, or other external eruptions of the integument or skin irritation unrelated to mechanical damage, (2) the presence of fin rot disease, (3) gills examined for the presence of parasites (data not reported here) and tumors, (4) body inspected for evidence of recent mechanical damage, thought to occur through trauma of the catching process or due to predators, and (4) inspection of the skin and internal organs for the presence of obvious tumors and tumor-like growths. Photographs were taken of skin lesions and other pathologies and the status of each lesion was evaluated (e.g., open bloody ulcer, closed skin contusions, healing or old injury). To obtain bile samples we dissected and punctured the gall bladder into a clear 15x45 mm vial. A foil liner was inserted between the cap and vial to prevent hydrocarbon contamination. The vial was wrapped in foil to prevent photo oxidation, and placed in a plastic bag. Bile samples were kept on ice or frozen at sea until return to the laboratory where they were maintained. Data for skin lesions for these samples can be found in dataset R1.x135.120:0002. Methods blanks and standard exposure material (Atlantic salmon tissue) were routinely done for these samples (see published paper for methods) and results are reported in the spreadsheet. Methods described in: Krahn, M.M., M.S. Myers, D.G. Burrows, and D.C Malins. 1984. Determination of xenobiotics in bile of fish from polluted waterways. Xenobiotica 14: 633-646. Krahn, M.M., G.M. Ylitalo, J. Buzitis, J.L. Bolton, C.A. Wigren, S.-L. Chan, and U. Varanasi. 1993. Analyses of petroleum-related contaminants in marine fish and sediments following the Gulf oil spill. Marine Pollution Bulletin 27: 285-292. Krahn, M.M., G.M. Ylitalo, and T.K. Collier. 2005. Analysis of bile of fish collected in coastal waters of the Gulf of Mexico potentially affected by Hurricane Katrina to determine recent exposure to polycyclic aromatic compounds (PACs). M.S. NOAA, National Marine Fisheries Service, Northwest Fisheries Science Center, Seattle, Washington.
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