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Data Series 286

Data Series 286

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Study Methods

Fish Sampling and Field Processing

Adult common carp (greater than 300 mm total length) were collected seven times from May 1999 through May 2000 using boat-mounted, electrofishing gear (Model SR-16, Smith-Root, Inc.) at Las Vegas Bay and Overton Arm in Lake Mead (fig. 1). Samples were collected in coves, bays, and along the shoreline in the two sampling areas usually at depths of less than 3 m. Due to equipment failure of the electroshocking boat during July 1999, fish were collected using a gill net (mesh size, 8 cm), which was set in the early morning and retrieved 4 hours later. All fish were held alive in an aerated live well or a netted cage submerged on site for less than 2 hours between the time of capture and time of processing. Nine to 16 male and female common carp were selected at each site for processing. Following blunt force trauma to the head, both total length and fork length were measured to the nearest millimeter and body weighed to the nearest decagram. A 5-mL syringe with a 20-gage needle was used to collect blood from the caudal vein for Vtg analysis. Blood was transferred to heparinized Vaccu-tubes and centrifuged at approximately 1,000 × gravity for 10–15 minutes to separate the plasma. Plasma then was placed into cryovials, which were immediately placed on dry ice and stored at -80°C until the plasma was ready for analysis. Gonads were carefully dissected and weighed to the nearest 0.1 g. A small section (about 5 g) from one testis or ovary was dissected from a point one-third of its length starting from the anterior end, fixed in NoTox fixative (Earth Safe Industries, Inc., Belle Mead, N.J.), and sent to the USGS Texas Cooperative Fish and Wildlife Research Unit in Lubbock, Tex. for further processing and histological evaluation. After several weeks in NoTox fixative, tissues were transferred to 70 percent ethanol for further processing.

The other testis was removed intact, rinsed with calcium-free Hank’s balanced salt solution (HBSS) containing 10 percent streptomycin/penicillin by volume (HBSS-SP), and stored in 500-mL sterile containers filled with HBSS-SP. These containers were packed in a cooler with blue ice and shipped overnight at 4°C to the USGS National Wetlands Research Center in Lafayette, La. for sperm-quality analysis. The remaining fish carcass was wrapped in aluminum foil, double bagged, and frozen on dry ice and then sent to the USGS National Water Quality Laboratory in Lakewood, Colo. for organic chemical analysis. The gonadosomatic index (GSI) and condition factor (K) were determined according to the formulas 100 × (gonad weight/body weight) (Schmitt and Dethloff, 2000) and body weight/( fork length) 3 (Carlander, 1969), respectively.

Analytical Chemistry

Individual whole body common carp, minus one gonad in the case of males, were thawed at room temperature and then thoroughly homogenized with a Hobart meat grinder (Model No. 4146) at the USGS National Water Quality Laboratory in Lakewood, Colo. Samples were prepared for analysis using a method described by Leiker and others (1995) and were treated as follows: A 10-g aliquot of homogenate was added to 100 g of anhydrous sodium sulfate. The sodium sulfate and tissue mixture was frozen at –20°C. While frozen, the mixture was homogenized into a free-flowing powder using a blender and then quantitatively transferred into a fritted disk glass Soxhlet extraction thimble. Surrogate standards (1 ng each) consisting of decafluorobiphenyl; α-HCH-d6: p,p’-DDT-d8; and nonachlorobiphenyl were added to the sample prior to extraction. The tissue/sodium sulfate mixture was extracted overnight with a Soxhlet apparatus using dichloromethane. The extract was concentrated in a Kuderna-Danish (K−D) apparatus to a volume of 5.0 mL. A 1-mL aliquot was removed for lipid determination. Two Waters Envirogel (Milford, MA) gel permeation chromatographic (GPC) preparative columns (19 × 150 mm and 19 × 300 mm) were used to remove lipid material from a 2-mL aliquot of the extract. The mobile phase was dichloromethane and the flow rate was 5 mL/min. The fraction from 14 to 30 minutes was collected for analysis in a clean K-D unit and concentrated to 5 mL. The sample then was exchanged into hexane, and the extract was re-concentrated to about 1 mL.

The extract was purified further and divided into two fractions by passing it through a column dry packed (from top to bottom) with 5 g of 8.5 percent water-deactivated neutral alumina, 3 g of 2 percent water-deactivated silica, and 0.5 cm of granular sodium sulfate. The column was pre-rinsed with 50 mL of hexane and the rinse discarded. The sample extract was applied to the column and eluted with 30 mL of hexane (fraction 1). The column then was eluted with 25 mL of 50 percent (v/v) acetone in hexane (fraction 2). Each fraction was collected in a 35-mL K-D receiver, concentrated to a volume of 5 mL, and then concentrated further under a gentle stream of filtered ultrapure nitrogen at ambient temperature to a volume of 0.5 mL. The extract was transferred quantitatively, with hexane rinses, to a 1-mL gas chromatography (GC) vial. The volume of the extract was adjusted to 0.5 mL. The GC vial was sealed with a Teflon®-lined cap and stored at –20°C until analysis. Just prior to analysis, injection standards consisting of 4,4´-dibromooctafluorobiphenyl (25 ng, resulting in a concentration of 50 pg/µL in the final extract) and naphthalene-d8 (1 µg, resulting in a concentration of 2 ng/µL in the final extract) were added to each sample as injection standards.

Authentic standards of methyl triclosan and its higher chlorinated forms were not available. A commercial triclosan standard was derivatized with ethereal diazomethane according to the procedure of Fales and others (1973). Ethereal diazomethane produced from N-methyl-N’-nitrosoguanidine (97 percent) was allowed to react with triclosan overnight at an ambient temperature in a sealed conical-shaped reaction vial. Based on gas chromatography/mass spectrometry (GC/MS) analysis, the purity of the derivatized triclosan is estimated to be greater than 95 percent. The molecular structure of the derivatized product was established by capillary gas chromatography under low- and high-resolution electron ionization (EI) mass spectrometry. Chromatographic and mass spectral conditions for confirming the structure of the derivative methyl triclosan and methyl triclosan in the sample extract were identical. The response of 4,4´-dibromooctafluorobiphenyl was used to estimate the concentration of the higher chlorinated analogues of methyl triclosan during the electron-capture negative ion (ECNI) analysis and derivatized triclosan was used to estimate the concentration of methyl triclosan during the EI analysis. Reporting limits for compounds detected in common carp from Lake Mead are given in table 1. Reported values less than these concentrations are estimated and based on the lowest point of the calibration curve of the compound.

Quality assurance of analytical methods was further validated by analyzing certified chlorinated organic compounds in cod liver oil from the National Institute of Standards and Technology (SRM 1588) (table 2).

Gas Chromatograph-Mass Spectrometry Analyses

The capillary gas chromatographic low-resolution electron-capture negative ion mass spectral (GC/ECNIMS) analyses were carried out on a Hewlett Packard 5890 gas chromatograph interfaced to Hewlett Packard 5989A mass spectrometer at the USGS National Water Quality Laboratory in Lakewood, Colo. Chromatographic separations were achieved on a 60 m × 0.25 mm inside diameter, 0.25 µm film thickness, 5 percent phenyl-95 percent dimethylpolysiloxane fused-silica capillary column (FSCC) using the following temperature program: initial oven temperature was held at 50°C for 3 minutes, oven temperature was ramped to 150°C at 20°C per minute, from 150°C to 275°C at 2°C per minute, from 275°C to 300°C at 20°C per minute and held for 20 min at 300°C. Splitless injections of 2 µL of each sample extract were made. The carrier gas was helium with a linear velocity of 25 cm/s. The injection port temperature was 250°C, and the transfer line temperature was 275°C. The GC/ECNIMS analyses were conducted under the following conditions: modifying gas, methane; source temperature, 125°C; quadrupole temperature, 110°C. Instrument was repetitively scanned from 150 to 600 Daltons with a cycle time of 1.29 seconds per scan; emission current, 300 µA; and electron energy, 200 eV. Perfluorotributyl amine (FC-43) was used for mass axis calibration and tuning. Source pressure (2 × 10-4 torr) was adjusted to maximize m/z 633 while minimizing m/z 452 with methane (modifying gas), and then tuned to maximize m/z 452.

Low-resolution and high-resolution capillary gas chromatographic electron ionization mass spectral (GC/EIMS) analyses were conducted using a Varian 3400 gas chromatograph interfaced to a MAT-95 high-resolution mass spectrometer. Chromatographic separations for both low- and high-resolution mass spectral analyses were described above. Low-resolution EI analysis was made under the following conditions: resolution, 1,000; electron energy, 70 eV; emission current, 100 µA; high voltage, 5 kV; and source temperature, 200°C. The instrument was scanned repetitively from 50 to 700 Daltons with a scan-rate of 1-second per decade. High-resolution EI analysis was conducted under the following conditions: resolution, 7,500 (set statically); electron energy, 70 eV; emission current, 100 µA; high voltage, 5 kV; and source temperature, 200°C. The instrument was repetitively scanned from 50 to 600 Daltons with a scan-rate of 3 seconds per decade. Perfluorokerosene was used for low- and high-resolution mass axis calibration and tuning for all EI analysis.


Vtg concentration in common carp blood plasma was determined at the University of Florida, Gainesville, with a direct enzyme-linked immunosorbent assay (ELISA). Plasma samples were diluted 1:100, 1:10,000, 1:100,000 and 1:1,000,000 with 10 mM phosphate, 150 mM NaCl, 0.02 percent azide, 10 KIU/mL aprotinin, pH 7.6 (PBSZ-AP). Carp Vtg standards (0, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 µg/mL) containing 1:200, 1:10,000, 1:100,000, and 1:1,000,000 male plasma (in PBSZ-AP) were added to account for matrix effect (Denslow and others, 1999). Samples and standards were loaded onto a 96-well ELISA plate in triplicate and stored overnight at 4ºC in a humidified container. The following day the plates were washed four times with PBSZ and then blocked with 1percent bovine serum albumin (BSA) in 10 mM tris, 150 mM NaCl, 0.05 percent tween, 0.02 percent azide, 10 KIU/mL Aprotinin, pH 7.6 (1 percent BSA/TBSTZ-AP) for 2 hours at room temperature. The plates were rewashed with PBSZ (four times) and the monoclonal antibody (mAb) was loaded into wells on each plate. The lowest dilution (1:100) was probed with 1 µg/mL of the mAb and dilutions of 1:10,000 and higher with 0.1 µg/mL. After the addition mAb, the plates were stored at 4ºC overnight in the humidified container. The following day the plates were washed and the biotinylated secondary antibody (goat anti mouse IgG-biotin) was added to each well at 1:1000 dilution in 1 percent BSA/TBSTZ-AP and incubated at room temperature for 2 hours. The plates were washed, and strepavidin-alkaline phosphatase was added at 1:1,000 dilution in 1 percent BSA/TBSTZ-AP and incubated for 2 hours at room temperature. After a final wash of the plates, the color was developed by adding 1 mg/mL p-nitro-phenyl phosphate in carbonate buffer (0.03M carbonate, 2 mM MgCl2, pH 9.6) and the color was measured using an ELISA plate reader (SpectraMax Plus384, Applied Biosystems) at 405 nm. Concentrations of the unknowns were determined from the standard curves.

The limit of detection for carp Vtg direct ELISA was 0.005 mg/mL. All assays were performed in triplicate and reported as the mean of the three measurements. The coefficient of variation was less than 10 percent for all samples analyzed. Inter- and intra-assay variability was routinely measured by analyzing controls on several plates and different runs and was determined to be less than 10 percent, and less than 5 percent, respectively.


Gonadal tissue processing was done at the USGS Texas Cooperative Fish and Wildlife Research Unit in Lubbock, Tex., according to Patiño and others (2003). Standard paraffin sections of 7 µm of ovarian and testicular tissue were prepared and stained with Weigert’s hematoxylin and eosin (Luna, 1992). Testicular sections also were stained using the Sigma HT20 iron stain kit (Sigma Chemical Co., St. Louis, Mo.) for demonstration of hemosiderin using bright field microscopy and ceroid-lipofuscin using fluorescence microscopy, and with periodic acid-Schiff’s reagent to visualize polysaccharide-rich tissue elements (Sigma 395-B). The NoTox solution did not adequately fix yolky ovarian follicles, so the exposed paraffin block tissue face (0.5-1 cm2) was soaked from overnight to several days in equal parts of glycerol and water to improve the quality of the sections. The presence of any postovulatory and atretic follicles in each ovarian section was determined by visual examination under 100× magnification. The percentage of distribution of germ cell stages was determined for each testicular section according to the general method described by Jobling and others (1996). Testicular sections were randomly placed under a 0.7-mm2 ocular grid at a total magnification of 400×, and the stage of the germ cell over each crosshair was recorded. For each sample, the total counts per stage were expressed as a percentage of the total crosshairs in the grid, including the corners. The following categories were recorded: spermatogonia, primary spermatocyte, secondary spermatocyte, spermatid, sperm, and interstitial (interlobular) tissue (Patiño and Redding, 2000).

Fecundity and Follicle Size

Fecundity and follicle size-frequency distributions were determined according to Patiño and others (2003) at the Texas Cooperative Fish and Wildlife Research Unit in Lubbock, Tex. A small fragment of ovarian tissue containing about 50–100 follicles was randomly dissected off the fixed tissue and the diameters of all vitellogenic and fully grown follicles were measured under a stereoscope at the Texas Cooperative Fish and Wildlife Research Unit. The follicle size-frequency distribution was determined for all female fish collected. Early vitellogenic follicles were identified by their light yellow-brown color as opposed to the bright white appearance of the small previtellogenic follicles. Most follicles had a slight ovoid shape so their diameter was expressed as the average of the long and short diameter. Fecundity estimates were determined for female common carp collected in January and March 2000 prior to spawning. A larger ovarian section weighing 400–500 mg was dissected from the fixed ovarian tissue (stored in 70 percent ethanol), briefly blotted, weighed, rehydrated in water for 2 hours, and briefly blotted and reweighed. All follicles with yolk were then counted and the rehydrated weight was used to express follicle counts per gram of ovary. The total number of follicles with yolk in each female was then estimated by multiplying the number of follicles per gram of ovary by the total weight of both ovaries and expressed both as total fecundity (follicles per fish) and normalized fecundity (follicles per kilogram of fish).

Sperm Quality

Upon arrival at the USGS National Wetland Research Center in Lafayette, La., the condition of each testis was noted, and milt was removed from the collecting duct at the posterior end of the testis. Sperm motilities in undiluted milt were estimated by thorough mixing of 0.5 µL milt with 20 µL distilled water and by viewing under darkfield microscopy at 100× magnification (Jenkins and Tiersch, 1997). Triplicate readings were made per investigator, two investigators examined each sample, and the average percent (to the nearest 5 percent) of total motile cells was recorded. Milt was diluted to 1 × 106 cells/mL in HBSS in 250 µL aliquots and viability and mitochondrial function analyses were performed according to modifications of Garner and others (1994) and Segovia and others (2000) with Live/Dead Sperm Viability Kit and rhodamine 123 (Molecular Probes, Eugene, OR).

The viability assay is based on the simultaneous determination of live and dead cells using the membrane-permeant nucleic acid stain SYBR-14 (5 µL of 1 µM stock), and the conventional dead-cell stain propidium iodide (PI; 2.5 µL of 2.4 mM stock) to 250 µL sample. Ten minutes following the addition of SYBR-14 and incubation at 24°C in the dark, PI was added and tubes incubated 10 min more prior to flow cytometry (FCM). The mitochondrial function assay is based on mitochondrial membrane potential, whereby 2.5 µL of rhodamine (0.13 µM) was added to 250 µL sample and incubated similarly with PI as in the viability assay.

Quantitative assessments of fluorescent-stained sperm were made using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA) equipped with an argon laser emitting at 488 nm. The instrument had been calibrated with AUTOComp software (BDIS) and Calibrite beads (BDIS) prior to each session. Fluorescence ranges included FL1 (525 nm band pass filter) for green fluorescence, FL2 (575 nm band pass filter) for red fluorescence, and FL3 (635 nm band pass filter) for far red fluorescence. For the three assays, data from approximately 104 cells or nuclei per sample in triplicate were acquired using LYSIS II software (BDIS). For viability analyses, instrument settings were forward scatter threshold at 48, scale at E01, side scatter at 483, FL1 at 412, FL2 at 376; and FL3 at 521. Density plots of FL1 (green fluorescence; live spermatozoa) and red fluorescence (FL3; dead spermatozoa) were generated. For mitochondrial function, instrument settings were forward scatter threshold at 52, scale at E01, side scatter at 414, FL1 at 712, FL2 at 505, and FL3 at 549. Density plots of FL1 (green fluorescence; functional mitochondrial with high membrane potential) and red fluorescence (FL2; dead spermatozoa) were generated.

For sperm counts, a Makler counting chamber (MidAtlantic Diagnostics, Mt. Laurel, NJ) was used by placing a 25 uL drop of a 1:400 cell dilution, and 10 squares were counted. Two samples per fish were counted in triplicate. The count formula is cells/mL= count × 400 × 1 × 106.

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