1. Kentucky’s comprehensive Water Monitoring and Assessment Program addresses water quality management objectives outlined in the Clean Water Act, as well as other specific water quality management objectives and goals, through a wide variety of monitoring and assessment activities. These activities have been focused primarily on 1 st through 4 th order streams that are wadeable with only water chemistry data collected at non-wadeable streams as part of the Ambient Water Quality Monitoring Program. Formal large river biological sampling and assessment methodologies used to assess aquatic life support have not been adopted for Kentucky. In 2006, the USEPA Environmental Lab in Cincinnati, OH developed large river bioassessment protocols (LR-BP) in Concepts and Approaches for Bioassessment of Non-wadeable Streams and Rivers 1. These methods were in part developed on Kentucky waters, and thus, the Kentucky Division of Water has begun developing a non-wadeable streams and rivers biological sampling program using these methods for habitat, macroinvertebrate and fish community sampling. During the initial pilot study, non-wadeable streams and rivers are being targeted based on drainage area, order, and basins to include a broad variety of streams and rivers. Currently, streams that are >150 mi 2, ≥ 5 th order, and non-wadeable for a majority of the reach are being targeted. Establishing Transects and Habitat Sampling Sampling is conducted within a 500 meter reach of the stream and consists of six transects, 100 meters apart (labeled A-F). Transect A is the most downstream transect with F being the most upstream transect (Figure 1). The latitude and longitude of each transect is recorded on the field sheet (see handout). The nature and quality of in stream habitat and local watershed features are characterized to aid in interpreting biological community results. Habitat measurements are collected at each transect (at both banks and across stream transect) and include a combination of stream morphology, riparian canopy cover, bank morphology, riparian zone, and in stream habitat measurements (See provided handouts). If the stream bank is wadeable, then field measurements for the desired parameters are collected outside of the boat(s), but if the stream bank is non-wadeable or accessible, field measurements are collected from inside the boat as best as possible. From the field measurements, the following metrics can be calculated to characterize and classify streams: % Fines% Riffle Max Depth % Sand% Run Average Wetted Width % Gravel% Pool Average Bankfull Width % CobbleAverage Thalweg Depth Large Woody Debris % BoulderAverage Depth Average Wooded Riparian Zone % BedrockAverage Bank Depth % Desired Habitat (Macroinvertebrates) % Canopy Cover In addition to field measurements taken at each transect, an overall reach assessment is conducted using a modified version of EPA’s Rapid Bioassessment Protocols 2. This site assessment does include habitat parameters that are calculated from field measurements (i.e. large woody debris, riparian vegetative zone width, etc.). In addition, there are several parameters that are estimated from the biologist(s) professional judgment (i.e. epifaunal substrate, pool substrate characterization, bottom deposition, channel alteration, thalweg substrate, bank stability, and vegetative protection). Each category is given a score, which are then combined to achieve an overall reach score. Macroinvertebrate Community Sampling Macroinvertebrates are collected at both banks of each transect (Figure 1). At each sampling zone, six sweeps are performed, each 0.5 m in length, using a 0.3 m wide D frame net (500 μm mesh). Sweeps are performed by agitating or kicking habitats, and are proportioned among available habitat (i.e. snags, cobble, aquatic vegetation, etc.) in each sampling zone. If water exceeds 1 m at the bank, sweeps are collected along the bank from the boat. All sweeps from the entire reach are composited into a single sample. The total sampling area is approximately 10.8 m 2 ((each sweep=0.3 m x 0.5m= 0.15m2) 0.15m2 per sweep x 6 sweeps per sample zone x 12 sample zones). Macroinvertebrate samples are processed in the field, removing all large debris (i.e., leaves, sticks, rocks, etc.) and picking all macroinvertebrates until the remaining debris collected can be placed in the prescribed container and preserved in 70% ethanol. All sample(s) are returned to the laboratory, logged in the appropriate log, sorted and identified to the lowest practical taxonomic level Fish Community Sampling The LR-BP method provides an unbiased, representative and quantitative sample as possible that was designed to support bioassessment and monitoring activities. It was not designed to be a qualitative measure that maximized the number of species present at a site. The electrofishing design was constructed to provide a standard field method that is reproducible. Fish community samples are collected using a 500 m paired bank methodology. For each bank, electrofishing starts in the upstream portion of the reach and proceeds downstream to the end of the reach. The boat is operated at a speed near or just above the current of the river and maneuvered in and out of shoreline habitat. All fish are collected and placed into a livewell for processing. On occasion, shallow portions of the stream reach are encountered (i.e. shoals/riffles). When this occurs, alternate electrofishing techniques are employed including the use of a handheld anode attached to the boat electrofishing unit, hand maneuvering the boat into the shallow portions or the use of a backpack electrofisher. At the end of each electrofishing run (i.e. bank), large fish and easily identified fish are identified, enumerated and released outside of the sampling zone. Small or unknown fish that require identification in the laboratory are preserved. In addition, the electrofishing settings (i.e. pulse width, percent applied and shocking seconds ) for each bank are recorded. In-situ and Water Chemistry Sampling All water chemistry samples are collected at Transect A (furthest downstream transect) at the conclusion of all other sampling. Physicochemical samples (temperature, dissolved oxygen, pH and specific conductivity) are collected at the surface, the middle of the water column, and just off the bottom with established in situ protocols using a multiparameter probe. A suite of parameters (metals, nutrients, acidity/alkalinity, and bulk) are measured from water column grab samples that include pollutants and pollutant surrogates that affect aquatic life. These samples are collected by using a depth integrating technique Biological Sampling Methods in Kentucky’s Non-wadeable Streams and Rivers A. Payne and R. Pierce Kentucky Division of Water, Frankfort, Kentucky Introduction Methods Methods (Continued) Future goals Kentucky’s non-wadeable streams and rivers biological sampling program is currently in the pilot phase. Kentucky is unique in that it has a variety of stream types from high gradient streams with shallow riffles and shoals to low gradient streams with deep pools, with a large diversity of substrate types and available habitats for biological communities. The main focus of the pilot project is to ensure that our proposed sampling methodology is applicable to all non-wadeable streams in the Commonwealth. Once it has been determined that our methodology is applicable, a dedicated non-wadeable program will be created that will focus on the development of biological assessment indices. Our long term goal for the non-wadeable biological sampling program is the incorporation of large river sites into our existing wadeable probabilistic program for use support designations and identification of overall water quality trends. 50 m Figure 1. Example of non-wadeable stream reach and macroinvertebrate sampling zone. Sampling zone extends to midpoint of river or until depth exceeds 1 m for kick samples Literature Cited 1 Flotemersch, J. E., J. B. Stribling, and M. J. Paul. 2006. Concepts and Approaches for the Bioassessment of Non-wadeable Streams and Rivers. EPA 600-R-06-127. US Environmental Protection Agency, Cincinnati, Ohio. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.132.2044&rep=rep1&type=pdf 2 Barbour, M.T., J. Gerritsen, B.D. Snyder, and J. B. Stribling. 1999. Rapid bioassessment protocols for use in streams and wadeable rivers: periphyton, benthic macroinvertebrates, and fish, second edition. EPA 841-B-99-002. U.S. Environmental Protection Agency; Office of Water, Washington, D.C.