Stream Chemistry

Stream chemistry conditions were measured from samples collected at each of the 30 study sites during the spring (February 19–March 4, 2003) and summer (June 30–July 16, 2003). Nine of the sites were sampled up to four additional times between October 2002 and September 2003 (Cates Creek, Black Creek, Richlands Creek at Schenk Forest, Hare Snipe Creek, Beaverdam Creek, Pigeon Creek, Camp Creek, Bolin Creek, and Morgan Creek; fig. 2). One site, Swift Creek near Apex (02087580), was sampled monthly. Water temperature, air temperature, pH, specific conductance, and dissolved oxygen were measured in the field at the time of sampling. Water samples were collected at equal-width increments across the stream channel and processed on site in accordance with standard USGS protocols (Wilde and others, 1999; Wilde and others, 2002). Water samples were analyzed at the USGS National Water-Quality Laboratory (NWQL) in Denver, Colorado, for concentrations of chloride and sulfate (Fishman and Friedman, 1989), nutrients (Fishman, 1993), dissolved and suspended carbon (Brenton and Arnett, 1993; Zimmermann and others, 1997), and pesticides (Sandstrom and others, 2001). Samples were analyzed for suspended-sediment concentration at the USGS Kentucky Sediment Laboratory (Guy, 1969).

The NWQL has established two detection limit values—a lower method detection limit, which is set to avoid a false negative reading (not detecting a compound when it actually is present), and a higher reporting limit to avoid a false positive reading (detecting a compound when it actually is not present). If a compound is identified at a concentration between these two limits, the result is noted with an “e” to indicate that the concentration has been estimated (Childress and others, 1999). The estimated values are greater than zero but are known with less confidence than values above the reporting limit. Values also may be noted as estimated when the detected concentration is outside of the calibration range for the instrument, when the average recovery for the analyte in quality-assurance samples is less than 60 percent, or when the analyte is regularly detected in laboratory blank samples. Estimated concentrations must be interpreted with caution. Values reported with a less than symbol (<) were not detected at the lower method-detection limit, and are presented as less than the (higher) reporting limit (Childress and others, 1999).

Pesticide data were summarized using several methods. The number of detections and total concentrations of different pesticide groups, such as insecticides and herbicides, were compiled. In addition, a pesticide toxicity index (PTI) was developed. The PTI combines information on exposure of aquatic biota to pesticides with toxicity estimates for multiple pesticides in each sample and produces a relative index value for a sample or stream (Munn and others, 2006). The PTI value is computed for each sample of streamwater by summing the toxicity quotients for all pesticides detected in the sample. The toxicity quotient is the measured concentration of a pesticide in a stream sample divided by its median toxicity concentration from bioassays, such as LC₅₀ or EC₅₀. Separate PTI values were computed for fish, cladocerans, and benthic invertebrates in this report by using median toxicity concentrations from Munn and others (2006).

Semipermeable Membrane Devices

To examine concentrations of hydrophobic organic compounds over time, semipermeable membrane devices (SPMDs) were placed at each site for a period of approximately 6 weeks during April and May 2003. SPMDs are passive samplers that concentrate trace levels of hydrophobic organic compounds in the water column. They are designed to mimic the bioaccumulation of organic compounds in the fatty tissues of aquatic organisms. Among the organic compounds that can be sequestered by the SPMDs are polychlorinated dioxins and furans, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine insecticides, and pyrethroid insecticides.

At the end of the 6-week deployment period, compound residues concentrated in the SPMDs were recovered and separated from the lipid by dialysis in an organic solvent by using methods described in Huckins and others (1990). Three assays were run on the dialysates from each site—an ultraviolet (UV) fluorescence scan (Johnson and others, 2004), a Microtox®bioassay (Johnson, 1998), and a P450RGS test (Ang and others, 2000). The UV fluorescence scan provided a semiquantitative screen for PAHs, which fluoresce under UV light. SPMD extracts were exposed to UV light at 280 nanometers, and a fluorometer was used to measure the fluorescence of the extract from each site compared with a standard curve for pyrene. The resulting estimated PAH concentration for each site is reported as the equivalent number of micrograms of pyrene in 1 milliliter of SPMD extract that would produce the same fluorescence as the sample. The Microtox® bioassay measured the light production of photo-luminescent bacteria when exposed to the SPMD residues; the biochemical pathway for light production is lowered by a wide range of compounds sequestered by the SPMDs. Results are reported as the effective concentration, in milligram per milliliter, of SPMD extract that reduces light output by 50 percent (EC50), which means the lower the number, the more toxic the extract. The P450RGS test, provides a rapid screen for aryl hydrocarbon receptor (AhR) compounds that include PCBs, PAHs, dioxins, and furans. All vertebrates produce detoxifying enzymes upon exposure to AhR compounds; the amount of enzymes produced is directly proportional to the concentration of the compounds. Quantifying one of these enzymes (the gene CYP1A1) serves as a measure of dioxin activity. The concentration of AhR compounds in the SPMD extract that induces CYP1A1 production is expressed as the amount of dioxin, in toxic equivalents (TEQs), which would induce the same response. Samples for the fluoroscan test were run in duplicate, and the Microtox® and P45ORGS assays were run in triplicate. Results are reported as mean values.

A portion of each SPMD dialysate also was sent to the NWQL for identification and quantification of the target compounds (Tom Leiker, U.S. Geological Survey, written commun., 2005). Internal standards and injection internal standards were added to the dialysates just prior to gas chromatography/mass spectrometry analysis to test for quality assurance. The dialysates were analyzed by capillary gas chromatography under two different ionization conditions. Electron-capture negative ionization was used to measure compounds like pesticides, PCBs, and brominated diphenyl ethers in the SPMD extracts. Electron ionization, the conventional method for analyzing dialysates by mass spectrometry, was used to measure compounds like alkyl phenols, polycyclic musks, and plant and fecal steroids. Mass spectra for individual target compounds and retention times from sample extracts were compared with authentic standards from the standard curve for identification. A 6-point linear calibration curve was used for quantification.

Results of the toxicity tests and chemical analyses were normalized for time of exposure, because the time of exposure has a direct effect on the concentrations in the SPMD. Values were divided by time of exposure and multiplied by 45 days. Therefore, the values reported have the appropriate units described in the respected analytical methods per 45 days of exposure. This allows values for all endpoints to be comparable between all sites.