vvEPA
     United States
     Environmental Protection
     Agency
                                    Best Practices for Identifying
                                    Reference Condition in
                                    Mid-Atlantic  Streams
REFERENCE CONDITION CONCEPT

The Clean Water Act (CWA) poses significant
challenges to states and tribes charged with
evaluating whether aquatic resources under their
management achieve the biological integrity
objective and the "protection and propagation" goals.
One of the critical challenges is the development of
a standard or benchmark by which to judge whether
particular water bodies are in accordance with the
CWA objective and goals. The concept of a reference
condition and its implementation form the foundation
on which to make such judgments (Stoddard et al.
2006a)

Reference conditions have been applied at site-
specific and regional scales. Regional reference
condition, described here, is recommended to support
biological criteria. Biological criteria are used to
                                                    detect deviation from reference condition to determine
                                                    whether water bodies meet their water quality
                                                    standards. The Biological Condition Gradient (BCG) is
                                                    a scientific narrative model for interpreting biological
                                                    response to increasing effects of stress on aquatic
                                                    ecosystems. The BCG describes how attributes of aquatic
                                                    ecosystems change in response to increasing levels of
                                                    human disturbance (Fig. 1, Davies and Jackson 2006).

                                                    States in the Mid-Atlantic region have developed and
                                                    implemented the concept of reference condition in a
                                                    variety of ways to meet their individual needs, without
                                                    comprehensive guidance from EPA.This brochure
                                                    offers examples from these states as case studies in the
                                                    application of the reference condition concept in water
                                                    resource management.
                 Level of Exposure to Stressors
                                                              Natural structure, functional, and taxonomic
                                                              integrity is preserved
                                                              Structure & function similar to natural community
                                                              with some additional taxa & biomass; ecosystem
                                                              level functions are fully maintained
                                                              Evident changes in structure due to loss of some
                                                              rare native taxa, shifts in relative abundance;
                                                              ecosystem fnctions fully maintained

                                                              Moderate changes in stucture due to replacement
                                                              of sensiti ve ubiquitous taxa by more tolerant taxa;
                                                              ecosystem functions largely maintained

                                                              Sensitive taxa markedly diminished conspicuously
                                                              unbalanced distribution of major taxonomic groups;
                                                              ecosystem function shows reduced complexity &
                                                              redundancy

                                                              Extreme changes in structure and ecosystem
                                                              function; wholesale changes in taxonomic
                                                              composition; extreme alterations from normal
                                                              densities
    Watershed, habitat, flow regime and
    water chemistry as naturally occurs.
                                    Chemistry, habitat, and/or flow regime
                                    severely altered from natural conditions.
Figure 1. The Biological Condition Gradient (BCG) is a tool for developing more precise aquatic life uses. The BCG displays six
positions of biological condition along a stressor-response curve, with Position 1 exhibiting the least stress and highest quality
condition, and Position 6 representing the greatest stress and lowest quality. (Modified from EPA 2005)

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TYPES OF REFERENCE CONDITION
The term reference condition can have multiple meanings.Therefore, consistent and specified
definitions of reference condition can greatly enhance collaboration among states and the transfer
of best practice technology and expertise. In all cases, reference condition is the benchmark
against which changes in current biological conditions are evaluated.The following definitions
distinguish among four specified types of reference condition:

   Reference Condition for Biological Integrity, RC(BI): the natural biological condition of
   a water body, undisturbed by human activity. As a conceptual aid, it is useful to think of an
   absolute "natural" or pristine condition that could exist in the absence of all historical and
   current human disturbances. This definition recognizes the need for a reference condition term
   reserved for"naturalness"or"biological integrity"even though we might only approximate it in
   most parts of the world.

                                                Minimally Disturbed Condition, MDC: the
                                                biological condition found in water bodies in
                                                landscapes with minimum human disturbance.
                                                Places that meet the criteria for RC(BI) are
                                                rare or impossible to find. Human activity is
                                                present throughout the global ecosystem,
                                                affecting remote systems through long-range
                                                atmospheric transport and deposition of
                                                pollutants onto pristine landscapes. Biological
                                                integrity in places with a low amount of human
                                                activity might not be significantly affected.The
                                                phrase"minimally disturbed condition"describes
                            photo credit: wayne Davis    the biological condition  in places with a minimal
Figure 2. A Mid-Atlantic stream displaying physical        amount of human disturbance.
attributes suggestive of minimally disturbed condition.
   Least Disturbed Condition, LDC: in altered landscapes, the biological condition found in
   water bodies with the least amount of human disturbance compared to similar water bodies
   in the region of interest. There is a clear need to describe the best condition of water bodies in
   landscapes that have been moderately to heavily disturbed by human activities. Definitions like
   "minimally disturbed condition" are of little practical use in these situations. Therefore, the phrase
   "least disturbed condition" has been applied to describe the condition in water bodies that are the
   least disturbed in a landscape altered by significant human activity. LDC should not be used as a
   benchmark for biological integrity. Further, in certain severely altered landscapes, LDC may not
   even be useful as a benchmark for meeting CWA aquatic life use protection and propagation goals.

   Best Potential Condition, BPC: the highest possible biological condition deemed achievable
   through the implementation of best management practices and other rehabilitation activities
   that can be undertaken in a given landscape given social and economic considerations. In some
   circumstances, a condition could be achieved that is better than the least disturbed condition (i.e.,
   better than the condition at the best sites) with implementation of the best available practices
   to remove or minimize stressors. Even though the biological potential might approach biological
   integrity if the stressors are removed, societal/economic constraints typically mean that a condition
   is achieved that differs from biological integrity. The term "best potential condition" describes
   this condition, where the biological expectations are set somewhere between the least disturbed
   condition and biological integrity.

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APPROACHES TO DERIVING REFERENCE CONDITION
Ideally, every water body segment will have its own reference condition. In order to describe reference
condition in a way that will not change over time due to further human activity, several general methods have
been developed through practical experience. These methods include sampling the biota at sites with little
or no indication of stressors associated with human disturbance (i.e., minimally disturbed reference sites).
In altered landscapes, where such sites are few or absent, reference conditions are determined through a
combination of methods: (1) sampling biota from least disturbed sites (reference sites), (2) interpreting
historical records to deduce which biological characteristics occurred at times with substantially less human
disturbance, (3) developing models that incorporate the best ecological knowledge, and (4) using best
professional judgment.
   1. Reference Sites
   The selection and characterization of reference
   sites that are minimally or not disturbed by human
   activities have been the basis for defining reference
   conditions that approximate biological integrity.
   However, reference sites have also been identified
   as "the best of what's left" and as such are used to
   estimate a  least disturbed condition. The approach
   used to select reference sites may be similar, regardless
   of whether the sites are classified as minimally
   disturbed or least disturbed. The process and
   considerations involved in selecting reference sites is
   discussed in further detail in the following pages.

   2. Historical Reconstruction
   The role of historical reconstruction is to use available
   data to describe a range of water body or riparian
   conditions that existed at an earlier time. Historical
   reconstruction estimates a minimally disturbed
   condition rather than a least disturbed condition.
   Benefits of this approach include the following:

   •  Improving the characterization obtained from
      reference sites;
   •  Needing to be generated only once;
   •  Providing a permanent benchmark;
   •  Allowing for a more cost-effective approach than
      extensive sampling; and,
   •  Providing motivation to stakeholders as a vision of
      desirable conditions.

   3. Empirical modeling
   When the number of representative reference sites
   is low and historical information is not sufficient
   to reconstruct reference condition, predictive
   modeling can be used to construct and calibrate a
   model reference condition.This approach effectively
   leverages a smaller number of sites from the region
   or water body type than is needed for the typical
   spatially intensive reference site approach. However,
   it does require reliable data from representative sites.
Absent such data, this approach reverts to a best
professional judgment approach with its inherent
shortcomings of subjectivity. This approach is
limited also by the data used in the model, and
therefore, inferences beyond those data must be
undertaken with great caution.

Predictive modeling approaches that show promise
include the following:

 •   Extension of reference site results from
    adjacent regions or similar water body types;
 •   Application of stressor-biotic assemblage
    interactions identified in restoration
    experiments; and,
 •   Inclusion of extirpated species or exclusion of
    nonnative species.
                            Photo credit: Morris Perot
 Figure 3. Biologists conducting a physical habitat
 assessment at an unnamed tributary in Frederick
 County, MD.

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4. Best Professional Judgment
Best professional judgment (BPJ) should
be incorporated into all decisions; however,
comparisons with a stressor gradient are also
needed to accurately identify the position of the
reference sites. The natural variability in biological
attributes for a certain biological condition (e.g.,
biological integrity) dictates that biological data
alone should not be used to develop reference
conditions. Although BPJ is a critical part of
biological assessments, great care must be taken
to ensure that the development of reference
conditions and selection of reference sites are well
documented and include objective procedures that
can be reproduced easily by others.
    SELECTING REFERENCE SITES

    Selecting reference sites involves applying
    screening criteria to a set of sites from the region
    or water body type of interest. These criteria will
    identify sites that are most likely to be minimally
    or least disturbed by human activities. The result
    is a set of candidate reference sites, qualified as
    such because depending upon the protectiveness
    that the screening criteria represents, some of
    the sites selected may not represent the desired
    reference condition  (Fig. 4).

    Criteria useful for screening sites include stressors,
    indicators of stressor sources, and indicators
    along the pathway from source to exposure. The
    goal  is to evaluate as many of these indicators as
    is practical, efficient, and relevant to the water
    body type and region of interest. Only those sites
    that meet the criteria of minimal disturbance or
    those that are the least disturbed among the set
    of sites are considered candidate reference sites.

    Applying Reference Screening Criteria
    Screening criteria should apply to potential
    reference sites at three levels: landscape or
    watershed, reach or riparian corridor, and
    site. While watershed and reach scale analysis
    using maps and/or CIS technology can identify
    candidate reference areas, there are many human
    activities that can only be revealed by collecting
    data  at the site level. However, it is essential that
    the final list of candidate sites identified through
    this screening be evaluated against an objective set of stressor thresholds obtained from sampling
    data. Brief descriptions of the Mid-Atlantic Highlands Streams Assessment and the Maryland
    Biological Stream Survey illustrate approaches to reference site selection using objective criteria.

    Having too few reference sites to analyze each region or water body type is a common constraint
    in indicator development and may lead to relaxation of reference site criteria to obtain more sites.
           Developing Reference Condition
                Using Reference Sites
                      ALL SITES
            Applying Initial Screening Criteria
            Important consideration: Coarseness of filter
             CANDIDATE REFERENCE SITES
        Evaluating Quality and Representativeness
        Important consideration: Readjustment of filter's codrseness
                   REFERENCE SITES
                 for Reference Condition
            Applying Final Screening Criteria
     Importdnt consideration: Codrseness of filter; Reference site vdridPility
                ?EFERENCE CONDITI
                     for Biocriteria
   Figure 4. This schematic shows the steps involved in
   developing reference condition using reference sites. At
   each step, it is important to consider the coarseness of
   the filter, which influences the confidence that the quality
   of the reference sites accurately represents the intended
   quality of the reference condition.

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Prior to the Mid-Atlantic Highlands Streams
Assessment (MAHSA; Davis and Scott 2000), a
precedent for using objective, abiotic criteria for
identifying reference sites in the region had not
yet been set. MAHSA biologists anticipated that
the probabililty-based design would generate
too few candidate reference sites, and therefore
asked local resource managers to "hand-pick"
sites in order to augment the spectrum of
conditions. All sites sampled-including those
selected via BPJ-were subjected to  objective,
abiotic criteria (below) representing potential
disturbance activities including acid rain,
acid mine drainage, agriculture, and general
development. Sites were considered candidate
reference sites if they met these criteria:

 •  acid neutralizing capacity (ANC)  > 50 ueq/L
   (pH of about 6)
 •  sulfate (SO4) < 400 ueq/L
 •  total phosphorus (P) < 20 ug/L
 •  total nitrogen (N) < 750 ug/L
 •  chloride (CIO < 100 ueq/L
 •  mean Rapid Bioassessment Protocol (RBP)
   habitat score > 15

Application of the objective screening filter to
the 58 BPJ sites resulted in only 15 sites (26%)
meeting the reference site criteria. This study
established the need to support the application
of objective, abiotic screening criteria for
candidate reference sites regardless of the
method used to select sites.
The Maryland Biological Stream Survey (MBSS)
is an example of a probability-based stream
monitoring program that successfully uses
objective criteria for sampled sites to identify
reference conditions (Klauda et al. 1998).
Because the program samples approximately
300 sites per year, data are available from
all regions and stream types. No screening
was done to narrow the range of candidate
reference sites, so all sampled sites were
evaluated using the following criteria of water
chemistry, physical habitat, and land use stress
(Roth et al. 1998). To develop the Maryland fish
index of biological integrity (IBI), the MBSS
used these criteria to  identify 152 of the total
1098 sites (13.8%) as reference sites:

 •  pH >6 or  blackwater stream (pH < 6 and
   DOC >8 mg/L)
 •  ANC >50  ueq/L
 •  dissolved oxygen >.4 ppm
 •  nitrate <300 ueq/L (4.2 mg/L)
 •  urban land use ^20% of the catchment area
   (draining to the site)
 •  forest land use >;25% of the catchment area
 •  remoteness rating = optimal or suboptimal
   (>1 Don 0-20scale)
 •  aesthetics rating = optimal or suboptimal
 •  instream  habitat rating = optimal or
   suboptimal
 •  riparian buffer width >.15 m
 •  no channelization
 •  no point source discharges
The development of a fish IBI for MAHSA (Davis and Scott 2000) addressed this situation by evaluating metric
performance against three different reference definitions: (1) least restrictive criteria based on chemical
thresholds and the mean RBP habitat score (producing 46 reference sites with good geographic coverage); (2)
moderately restrictive criteria based on chemical criteria, watershed land use, road density, and quantitative
habitat filters (producing 23 reference sites with good geographic coverage); and (3) most restrictive criteria
based on the moderately restrictive criteria plus the watershed condition class (Bryce et al. 1999; producing
12 reference sites with limited geographic coverage). If less restrictive criteria are used to define reference
sites, then the lower quality may result in conditions that correspond to lower positions along the Biological
Condition Gradient (e.g., Position 3 rather than 2, in Fig. 1).

Evaluating Quality and Representativeness
Because reference criteria vary among  water quality monitoring programs, it is essential to evaluate the
selected reference sites for whether they are truly minimally disturbed and whether they are representative of
the water bodies of interest.

The Maryland Biological Stream Survey used a set of water chemistry, physical habitat, and land use reference
criteria that produced reference sites with considerably higher benthic macroinvertebrate IBIs than all sites

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sampled and were also higher than the original MBSS set
of reference sites (see dashed line in Fig. 5). Subsequently,
the reference criteria used in the original MBSS IBIs
were reviewed to identify changes that would result in
greater confidence that the new reference sites could
be defined as "minimally disturbed." Based on analysis of
urban effects on stream condition (V0lstad et al. 2003),
the presence of original reference sites with relatively
high levels of urban land (i.e., 5% to 20%) indicated that
not all reference sites were minimally disturbed; instead,
many were  impaired.Therefore, the MBSS changed the
minimum allowable forested land use from >25% to
>35% of the catchment area, maximum allowable urban
land use from ^20% to ^5%, and minimum allowable
riparian buffer from 15m to 30m. These changes in land
use and riparian width thresholds resulted in a smaller
     proportion of stream sites meeting the reference site
     criteria. Using the original reference site criteria, 152
     of the 1098 Round One sites (14%) were designated as
     reference sites. Using the new criteria, 196 of the total
     2508 sites (8%) were designated as reference. These new
     reference sites were of higher quality (dotted line in Fig.
     5) than sites meeting the original reference criteria. This
     result is consistent with greater confidence that the sites
     are minimally disturbed.

     As the criteria for selecting reference sites are
     tightened to ensure minimally disturbed sites are
     chosen, it is important that these sites still represent
     the range of natural factors likely to control the
     biotic composition  in the region of interest. For
     example, stream size, gradient, and elevation can be
       SETTING BENCHMARKS FOR REFERENCE CONDITION

       The most common approach for using reference sites to set attainment benchmarks is to select a percentile
       of the reference site index scores below which is considered degraded. As previously discussed, the selec-
       tion of the percentile should consider the coarseness, or restrictiveness, of the screening criteria. In the Mid-
       Atlantic Region, West Virginia sets a benchmark at the 5th percentile of the reference sites; Maryland uses
       the 9th percentile; Virginia uses the 10th percentile; and EPA's Mid-Atlantic Integrated Assessment (MAIA)
       uses the 25th percentile. In addition to percentile selection, the West Virginia Department of Environmental
       Protection (WVDEP), the Maryland Department of the Environment (MDE), and the Virginia Department of
       Environmental Quality (VDEQ) also include separate measures of index precision surrounding the bench-
       mark in  order to capture uncertainty for regulatory program use. These programs demonstrate innovative
       approaches for setting attainment benchmarks, as well as recommended practices for defining and imple-
       menting reference condition.
            WVSCI Scoring Criteria
                   >68.0
                 Unimpaired
                > 60.6 to 68
                "Gray Zone"
     Figure 6. The West Virginia Stream
     Condition Index (WVSCI) has a precision
     estimate of 7.4 units. Therefore, setting
     the benchmark at a score of 68 (the 5th
     percentile) effectively creates  a "gray zone"
     between 68 and 60.6. (WVDEP 2006)
West Virginia Benchmark
West Virginia DEP set a low benchmark (at the 5th percen-
tile) because their screening criteria included a secondary
review of the candidate reference sites that passed the ini-
tial objective screening criteria (akin to the evaluation step
in Fig. 4). Using BPJ in the secondary review, the number of
sites dropped from 349 candidate reference sites to 216 ref-
erence sites (Southerland 2006).The BPJ included a review
of each candidate site for its proximity to upstream point
source discharges and  an evaluation of anthropogenic ac-
tivities and disturbances near the candidate sites. For their
stream condition index (SCI), WVDEP (2006) determined a
precision estimate of 7.4 out of 100 units. This uncertainty
in the index itself sets the 5th percentile benchmark of 68
effectively to 60.6, the range between those values being  in
a "gray zone" (Fig. 6).

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major determinants of potential biotic composition.
Therefore the set of reference sites should cover
and be limited to the range of natural conditions
important in the region of interest. If the streams
represent an elevation gradient, reference sites should
also represent that elevation gradient; if streams
considered represent a range of sizes, the set of
reference sites should also represent these sizes.
                                                                              2.00    3,00     4.00     5.00
                                                        Figure 5. Cumulative distribution of stream miles with benthic
                                                        macroinvertebrate IBI scores for (1) MBSS sites sampled in
                                                        2000-2004 (solid line), (2) subset of 2000-2004 sites meet-
                                                        ing original reference criteria (dashed line), and (3) subset of
                                                        2000-2004 sites meeting new reference criteria (dotted line).
                                                        (Southerland et al. 2005)
Virginia Benchmark
Virginia DEQ (2006) sets the attainment benchmark at the
10th percentile of their reference sites. Much like West
Virginia, the precision estimate for Virginia's stream condi-
tion index (VSCI) is ±7.9, which generates a "gray zone"
around the benchmark. As a result, the 10th percentile
benchmark of 60 effectively becomes a range from 55 to
63, where sites scoring above 63 are viewed as healthy
streams and those below 55 are deemed moderately to
severely stressed.

Maryland Benchmark
The Maryland Department of Natural Resources did
not establish a specific benchmark based on a distribu-
tion of their reference sites. They maintain a categori-
cal index score rating similar to the metric scoring
procedure of 1,3 and 5, where any score above 3 is
considered acceptable. To facilitate the use of the
Maryland IBIs for the regulatory agency, a statistical
measure  of uncertainty (confidence interval) is used
to determine whether the mean of the results from
the sites sampled in a watershed is above or below the
Index of Biotic Integrity (IBI) value considered indica-
tive of satisfactory water quality (i.e., 3). Where at least
ten sites have been sampled in a watershed, watershed-
specific confidence intervals are calculated. If the
upper bound  of the confidence interval is less than 3,
that watershed is designated as not meeting water qual-
ity criteria (MDE 2004). For comparison with other
states' methods, it was determined that the MDE index
benchmark of 3 was equivalent to the 9th  percentile of
the reference sites (Southerland 2006).
Mid-Atlantic Integrated Assessment Benchmark
For a report card on the state of streams and rivers
in the Mid-Atlantic region, data was combined from
two sample surveys conducted in the region by
MAIA from 1993 to 1998 (Stoddard et al. 2006b).
MAIA established two benchmarks based on the
distribution of reference sites. The 25th percentile
value set the lower limit on "good" condition. The
1 st percentile was used as the threshold below which
values were deemed "poor."Values between the 1st
and 25th percentiles were designated as "marginal."
These classifications were deliberately used so as not
to conflict with regulatory terms used by the States
(Fig. 7).
       100
        90
               Reference Distribution
        80
o
o
C/D
X
0)
        70
        60
        50
        40
Percentiles:
o 99th
T 95th







75th
50th
25th
1
| 5th
0 -|St






Good
1
Marginal
T

Poor
i
         Figure 7. The two benchmarks set by MAIA
         established three categories of condition: poor,
         marginal and good. (Stoddard et al. 2006b)

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Office of Environmental Information
Washington DC  20460
EPA-260-F-06-002
August 2006
                                          CONTACTS
                                          Office of Environmental Information
                                          Wayne Davis
                                          davis.wayne@epa.gov

                                          Office of Water
                                          Evan Hornig
                                          hornig.evan@epa.gov

                                          Office of Research and Development
                                          John Stoddard
                                          stoddard.john@epa.gov
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  pdf
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