Appendix C. 
Derivation of the Deep-Water Biologically-Based 
Reference Curve 
Step 1. We obtained a dataset of benthic scores for the Chesapeake Bay and tidal tributaries. 
Data used comprised benthic communities sampled between 1996 and 2006, from both the 
“fixed station” and “random strata” sampling programs. Only “grand score” values - which are 
an average of replicate samples - were included from the fixed station program. For the 
purposes of deriving a deep-water biological reference curve, we restricted the dataset to only 
those samples taken in segments that contain a deep-water designated use. 
Step 2. We removed from the dataset any samples obtained from the following segments: 
PATMH, SBEMH, and CB5MH. Benthic communities in PATMH and SBEMH are widely 
understood to be impacted by chemical contaminants (pers. comm, Roberto Llanso, Versar Inc.); 
a complication that confounds the relationship between hypoxia and benthic community health in 
these areas. 
In the case of CB5MH, areas greater than 12 meters in depth - which account for 35 percent of 
the bottom surface area of CB5MH - are excluded from the benthic sampling program because 
they are assumed to be azoic or nearly azoic. For their analyses of benthic health, Llanso et al. 
2009 assume that all areas greater than 12 m in depth are degraded, and perform a post-hoc 
correction to factor this assumption into their benthic assessment. For purposes of developing a 
biological reference curve, the exclusion from sampling of such a large portion of CB5MFI calls 
into question our ability to accurately characterize the health of its deep-water benthic 
communities. 
Step 3. We obtained water quality data from the Chesapeake Bay Program Water Quality 
database for the time period 1996-2006. Using the standardized method for locating pycnocline 
boundaries (see U.S. EPA 2008), we determined the depth of the upper and lower pycnocline 
boundaries for all sampling events in this time period. 
Step 4. From this dataset, we selected the sampling event that was closest in space (at a 
minimum, within the same segment) and time (at a minimum, within the same month) to each 
benthic sampling event. 
Step 5. We then classified each benthic sample as an “open-water,” “deep-water,” or “deep- 
channel” benthic sample based on its depth relative to the upper and lower pycnocline 
boundaries of the paired water quality sampling event. Benthic samples that were taken at 
depths between the upper and lower boundaries of the pycnocline were classified as “deep¬ 
water” samples. When no lower boundary was identified, benthic samples from depths below 
the upper boundary of the pycnocline were classified as “deep-water.” Benthic samples that 
could not be paired with a pycnocline boundary were discarded. 
49 
