The need for scientifically defensible water quality standards for nonpoint source pollution control continues to be a pressing environmental issue. The probability of impact at differing levels of nonpoint source pollution was determined using the biological response of instream organisms empirically obtained from a statistical survey. A conditional probability analysis was used to calculate a biological threshold of impact as a function of the likelihood of exceeding a given value of pollution metric for a specified geographic area. Uncertainty and natural variability were inherently incorporated into the analysis through the use of data from a probabilistic survey. Data from wadable streams in the mid-Atlantic area of the U.S. were used to demonstrate the approach. Benthic macroinvertebrate community index values (EPT taxa richness) were used to identify impacted stream communities. Percent fines in substrate (silt/clay fraction, > 0.06 mm) were used as a surrogate indicator for sedimentation. Thresholds of impact due to sedimentation were identified by three different techniques, and were in the range of 12 to 15 percent fines. These values were consistent with existing literature from laboratory and field studies on the impact of sediments on aquatic life in freshwater streams. All results were different from values determined from current regulatory guidance. Finally, it was illustrated how these thresholds could be used to develop criterion for protection of aquatic life in streams.