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Water: Bioassessment

Chapter 2: Application Of Rapid Bioassessment Protocols (RBPs)

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2.1 A FRAMEWORK FOR IMPLEMENTING THE RAPID BIOASSESSMENT PROTOCOLS

The Rapid Bioassessment Protocols advocate an integrated assessment, comparing habitat (e.g., physical structure, flow regime), water quality and biological measures with empirically defined reference conditions (via actual reference sites, historical data, and/or modeling or extrapolation). Reference conditions are best established through systematic monitoring of actual sites that represent the natural range of variation in "minimally" disturbed water chemistry, habitat, and biological conditions (Gibson et al. 1996). Of these 3 components of ecological integrity, ambient water chemistry may be the most difficult to characterize because of the complex array of possible constituents (natural and otherwise) that affect it. The implementation framework is enhanced by the development of an empirical relationship between habitat quality and biological condition that is refined for a given region. As additional information is obtained from systematic monitoring of potentially impacted and site-specific control sites, the predictive power of the empirical relationship is enhanced. Once the relationship between habitat and biological potential is understood, water quality impacts can be objectively discriminated from habitat effects, and control and rehabilitation efforts can be focused on the most important source of impairment.

2.2 CHRONOLOGY OF TECHNICAL GUIDANCE

A substantial scientific foundation was required before the USEPA could endorse a bioassessment approach that was applicable on a national basis and that served the purpose of addressing impacts to surface waters from multiple stressors (see Stribling et al. 1996a). Dr. James Karr is credited for his innovative thinking and research in the mid-1970's and early 1980's that provided the formula for developing bioassessment strategies to address issues mandated by the Clean Water Act. The USEPA convened a few key workshops and conferences during a period from the mid-1970's to mid-1980's to provide an initial forum to discuss aspects of the role of biological indicators and assessment to the integrity of surface water. These workshops and conferences were attended by National scientific authorities who contributed immensely to the current bioassessment approaches advocated by the USEPA. The early RBPs benefitted from these activities, which fostered attention to biological assessment approaches. The RBPs embraced the multimetric approach described in the IBI (see Karr 1981, Karr et al. 1986) and facilitated the implementation of bioassessment into monitoring programs across the country.

Since the publication of the original RBPs in 1989, U.S. Environmental Protection Agency (USEPA) has produced substantial guidance and documentation on both bioassessment strategies and implementation policy on biological surveys and criteria for water resource programs. Much of this effort was facilitated by key scientific researchers who argued that bioassessment was crucial to the underpinnings of the Clean Water Act. The work of these researchers that led to these USEPA documents resulted in the national trend of adapting biological assessment and monitoring approaches for detecting problems, evaluating Best Management Practices (BMPs) for mitigation of nonpoint source impacts, and monitoring ecological health over time. The chronology of the crucial USEPA guidance, since the mid-1980's, relevant to bioassessment in streams and rivers is presented in Table 2-1. (See Chapter 11 [Literature Cited] for EPA document numbers.)

Table 2-1. Chronology of USEPA bioassessment guidance (relevant to streams and rivers).

Year Document Title Relationship to Bioassessment Citation
1987 Surface Water Monitoring: A Framework for Change USEPA calls for efficacious methods to assess and determine the ecological health of the nation's surface waters. USEPA 1987
1988 Proceedings of the First National Workshop on Biological Criteria (Lincolnwood, Illinois) USEPA brings together agency biologists and "basic" researchers to establish a framework for the initial development of biological criteria and associated biosurvey methods. USEPA 1988
1989 Rapid Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and Fish The initial development of cost-effective methods in response to the mandate by USEPA (1987), which are to provide biological data on a national scale to address the goals of the Clean Water Act. Plafkin et al. 1989
1989 Regionalization as a Tool for Managing Environmental Resources USEPA develops the concept of ecoregions and partitions the contiguous U.S. into homogeneous regions of ecological similarity, providing a basis for establishment of regional reference conditions. Gallant et al. 1989
1990 Second National Symposium on Water Quality Assessment: Meeting Summary USEPA holds a series of National Water Quality Symposia. In this second symposium, biological monitoring is introduced as an effective means to evaluating the quality of water resources. USEPA 1990a
1990 Biological Criteria: National Program Guidance for Surface Waters The concept of biological criteria is described for implementation into state water quality programs. The use of biocriteria for evaluating attainment of "aquatic life use" is discussed. USEPA 1990b
1990 Macroinvertebrate Field and Laboratory Methods for Evaluating the Biological Integrity of Surface Waters This USEPA document is a compilation of the current "state-of-the-art" field and laboratory methods used for surveying benthic macroinvertebrates in all surface waters (i.e., streams, rivers, lakes, and estuaries). Klemm et al. 1990
1991 Biological Criteria: State Development and Implementation Efforts The status of biocriteria and bioassessment programs as of 1990 is summarized here. USEPA 1991a
1991 Biological Criteria Guide to Technical Literature A limited literature survey of relevant research papers and studies is compiled for use by state water resource agencies. USEPA 1991b
1991 Technical Support Document for Water Quality­Based Toxics Control USEPA describes the approach for implementing water quality-based toxics control of the nation's surface waters, and discusses the value of integrating three monitoring tools, i.e., chemical analyses, toxicity testing, and biological surveys. USEPA 1991c
1991 Biological Criteria: Research and Regulation, Proceedings of the Symposium This national symposium focuses on the efficacy of implementing biocriteria in all surface waters, and the proceedings documents the varied applicable approaches to bioassessments. USEPA 1991d
1991 Report of the Ecoregions Subcommittee of the Ecological Processes and Effects Committee The SAB (Science Advisory Board) reports favorably that the use of ecoregions is a useful framework for assessing regional fauna and flora. Ecoregions become more widely viewed as a basis for establishing regional reference conditions. USEPA 1991e
1991 Guidance for the Implementation of Water Quality­Based Decisions: The TMDL Process The establishment of the TMDL (total maximum daily loads) process for cumulative impacts (nonpoint and point sources) supports the need for more effective monitoring tools, including biological and habitat assessments. USEPA 1991f
1991 Design Report for EMAP, the Environmental Monitoring and Assessment Program USEPA's Environmental Monitoring and Assessment Program (EMAP) is designed as a rigorous national program for assessing the ecological status of the nation's surface waters. Overton et al. 1991
1992 Procedures for Initiating Narrative Biological Criteria A discussion of the concept and rationale for establishing narrative expressions of biocriteria is presented in this USEPA document. Gibson 1992
1992 Ambient Water-Quality Monitoring in the U.S. First Year Review, Evaluation, and Recommendations Provide first-year summary of task force efforts to develop and recommend framework and approach for improving water resource quality monitoring. ITFM 1992
1993 Fish Field and Laboratory Methods for Evaluating the Biological Integrity of Surface Waters A compilation of the current "state-of-the-art" field and laboratory methods used for surveying the fish assemblage and assessing fish health is presented in this document. Klemm et al. 1993
1994 Surface Waters and Region 3 Regional Environmental Monitoring and Assessment Program: 1994 Pilot Field Operations and Methods Manual for Streams USEPA focuses its EMAP program on streams and wadeable rivers and initiates an approach in a pilot study in the Mid-Atlantic Appalachian mountains. Klemm and Lazorchak 1994
1994 Watershed Protection: TMDL Note #2, Bioassessment and TMDLs USEPA describes the value and application of bioassessment to the TMDL process. USEPA 1994a
1994 Report of the Interagency Biological Methods Workshop Summary and results of workshop designed to coordinate monitoring methods among multiple objectives and states. [Sponsored by the USGS] Gurtz and Muir 1994
1995 Generic Quality Assurance Project Plan Guidance for Programs Using Community Level Biological Assessment in Wadeable Streams and Rivers USEPA develops guidance for quality assurance and quality control for biological survey programs. USEPA 1995a
1995 The Strategy for Improving Water Quality Monitoring in the United States: Final Report of the Intergovernmental Task Force on Monitoring Water Quality An Intergovernmental Task Force (ITFM) comprised of several federal and state agencies draft a monitoring strategy intended to provide a cohesive approach for data gathering, integration, and interpretation. ITFM 1995a
1995 The Strategy for Improving Water Quality Monitoring in the United States: Final Report of the Intergovernmental Task Force on Monitoring Water Quality, Technical Appendices Various issue papers are compiled in these technical appendices associated with ITFM's final report. ITFM 1995b
1995 Environmental Monitoring and Assessment Program Surface Waters: Field Operations and Methods for Measuring the Ecological Condition of Wadeable Streams A revision and update of the 1994 Methods Manual for EMAP. Klemm and Lazorchak 1995
1996 Biological Assessment Methods, Biocriteria, and Biological Indicators: Bibliography of Selected Technical, Policy, and Regulatory Literature USEPA compiles a comprehensive literature survey of pertinent research papers and studies for biological assessment methods. This document is expanded and updated from USEPA 1991b. Stribling et al. 1996a
1996 Summary of State Biological Assessment Programs for Wadeable Streams and Rivers The status of bioassessment and biocriteria programs in state water resource programs is summarized in this document, providing an update of USEPA 1991a. Davis et al. 1996
1996 Biological Criteria: Technical Guidance for Streams and Small Rivers Technical guidance for development of biocriteria for streams and wadeable rivers is provided as a follow-up to the Program Guidance (USEPA 1990b). This technical guidance serves as a framework for developing guidance for other surface water types. Gibson et al. 1996
1996 The Volunteer Monitor's Guide to Quality Assurance Project Plans USEPA develops guidance for quality assurance for citizen monitoring programs. USEPA 1996a
1996 Nonpoint Source Monitoring and Evaluation Guide USEPA describes how biological survey methods are used in nonpoint-source investigations, and explains the value of biological and habitat assessment to evaluating BMP implementation and identifying impairment. USEPA 1996b
1996 Biological Criteria: Technical Guidance for Survey Design and Statistical Evaluation of Biosurvey Data USEPA describes and define different statistical approaches for biological data analysis and development of biocriteria. Reckhow and Warren-Hicks 1996
1997 Estuarine/Near Coastal Marine Waters Bioassessment and Biocriteria Technical Guidance USEPA provides technical guidance on biological assessment methods and biocriteria development for estuarine and near coastal waters. USEPA 1997a
1997 Volunteer Stream Monitoring: A Methods Manual USEPA provides guidance for citizen monitoring groups to use biological and habitat assessment methods for monitoring streams. These methods are based in part on the RBPs. USEPA 1997b
1997 Guidelines for Preparation of Comprehensive State Water Quality Assessments (305[b] reports) USEPA provides guidelines for states for preparing 305(b) reports to Congress. USEPA 1997c
1997 Biological Monitoring and Assessment: Using Multimetric Indexes Effectively An explanation of the value, use, and scientific principles associated with using a multimetric approach to bioassessment is provided by Drs. Karr and Chu. Karr and Chu 1999
1998 Lake and Reservoir Bioassessment and Biocriteria Technical Guidance Document USEPA provides technical guidance on biological assessment methods and biocriteria development for lakes and reservoirs. USEPA 1998
1998 Environmental Monitoring and Assessment Program Surface Waters: Field Operations and Methods for Measuring the Ecological Condition of Wadeable Streams A revision and update of the 1995 Methods Manual for EMAP. Lazorchak et al. 1998
2.3 PROGRAMMATIC APPLICATIONS OF BIOLOGICAL DATA

States (and tribes to a certain extent) are responsible for identifying water quality problems, especially those waters needing Total Maximum Daily Loads (TMDLs), and evaluating the effectiveness of point and nonpoint source water quality controls. The biological monitoring protocols presented in this guidance document will strengthen a state's monitoring program if other bioassessment and monitoring techniques are not already in place. An effective and thorough biological monitoring program can help to improve reporting (e.g., 305(b) reporting), increase the effectiveness of pollution prevention efforts, and document the progress of mitigation efforts. This section provides suggestions for the application of biological monitoring to wadeable streams and rivers through existing state programs.

2.3.1 CWA Section 305(b)--Water Quality Assessment

Section 305(b) establishes a process for reporting information about the quality of the Nation's water resources (USEPA 1997c, USEPA 1994b). States, the District of Columbia, territories, some tribes, and certain River Basin Commissions have developed programs to monitor surface and ground waters and to report the current status of water quality biennially to USEPA. This information is compiled into a biennial National Water Quality Inventory report to Congress.

Use of biological assessment in section 305(b) reports helps to define an understandable endpoint of relevance to society--the biological integrity of waterbodies. Many of the better-known and widely reported pollution cleanup success stories have involved the recovery or reappearance of valued sport fish and other pollution-intolerant species to systems from which they had disappeared (USEPA 1980). Improved coverage of biological integrity issues, based on monitoring protocols with clear bioassessment endpoints, will make the section 305(b) reports more accessible and meaningful to many segments of the public.

Biological monitoring provides data that augment several of the section 305(b) reporting requirements. In particular, the following assessment activities and reporting requirements are enhanced through the use of biological monitoring information:

  • Determine the status of the water resource (Are the designated/beneficial and aquatic life uses being met?).
  • Evaluate the causes of degraded water resources and the relative contributions of pollution sources.
  • Report on the activities underway to assess and restore water resource integrity.
  • Determine the effectiveness of control and mitigation programs.
  • Measure the success of watershed management plans.
2.3.2 CWA Section 319--Nonpoint Source Assessment

The 1987 Water Quality Act Amendments to the Clean Water Act (CWA) added section 319, which established a national program to assess and control nonpoint source (NPS) pollution. Under this program, states are asked to assess their NPS pollution problems and submit these assessments to USEPA. The assessments include a list of "navigable waters within the state which, without additional action to control nonpoint source of pollution, cannot reasonably be expected to attain or maintain applicable water quality standards or the goals and requirements of this Act." Other activities under the section 319 process require the identification of categories and subcategories of NPS pollution that contribute to the impairment of waters, descriptions of the procedures for identifying and implementing BMPs, control measures for reducing NPS pollution, and descriptions of state and local programs used to abate NPS pollution. Based on the assessments, states have prepared nonpoint source management programs.

Assessment of biological condition is the most effective means of evaluating cumulative impacts from nonpoint sources, which may involve habitat degradation, chemical contamination, or water withdrawal (Karr 1991). Biological assessment techniques can improve evaluations of nonpoint source pollution controls (or the combined effectiveness of current point and nonpoint source controls) by comparing biological indicators before and after implementation of controls. Likewise, biological attributes can be used to measure site-specific ecosystem response to remediation or mitigation activities aimed at reducing nonpoint source pollution impacts or response to pollution prevention activities.

2.3.3 Watershed Protection Approach

Since 1991, USEPA has been promoting the Watershed Protection Approach (WPA) as a framework for meeting the Nation's remaining water resource challenges (USEPA 1994c). USEPA's Office of Water has taken steps to reorient and coordinate point source, nonpoint source, surface waters, wetlands, coastal, ground water, and drinking water programs in support of the watershed approach. USEPA has also promoted multi-organizational, multi-objective watershed management projects across the Nation.

The watershed approach is an integrated, inclusive strategy for more effectively protecting and managing surface water and ground water resources and achieving broader environmental protection objectives using the naturally defined hydrologic unit (the watershed) as the integrating management unit. Thus, for a given watershed, the approach encompasses not only the water resource, such as a stream, river, lake, estuary, or aquifer, but all the land from which water drains to the resource. The watershed approach places emphasis on all aspects of water resource quality--physical (e.g., temperature, flow, mixing, habitat); chemical (e.g., conventional and toxic pollutants such as nutrients and pesticides); and biological (e.g., health and integrity of biotic communities, biodiversity).

As states develop their Watershed Protection Approach (WPA), biological assessment and monitoring offer a means of conducting comprehensive evaluations of ecological status and improvements from restoration/rehabilitation activities. Biological assessment integrates the condition of the watershed from tributaries to mainstem through the exposure/response of indigenous aquatic communities.

2.3.4 CWA Section 303(d)--The TMDL Process

The technical backbone of the WPA is the TMDL process. A total maximum daily load (TMDL) is a tool used to achieve applicable water quality standards. The TMDL process quantifies the loading capacity of a waterbody for a given stressor and ultimately provides a quantitative scheme for allocating loadings (or external inputs) among pollutant sources (USEPA 1994a). In doing so, the TMDL quantifies the relationships among sources, stressors, recommended controls, and water quality conditions. For example, a TMDL might mathematically show how a specified percent reduction of a pollutant is necessary to reach the pollutant concentration reflected in a water quality standard.

Section 303(d) of the CWA requires each state to establish, in accordance with its priority rankings, the total maximum daily load for each waterbody or reach identified by the state as failing to meet, or not expected to meet, water quality standards after imposition of technology-based controls. In addition, TMDLs are vital elements of a growing number of state programs. For example, as more permits incorporate water quality-based effluent limits, TMDLs are becoming an increasingly important component of the point-source control program.

TMDLs are suitable for nonchemical as well as chemical stressors (USEPA 1994a). These include all stressors that contribute to the failure to meet water quality standards, as well as any stressor that presently threatens but does not yet impair water quality. TMDLs are applicable to waterbodies impacted by both point and nonpoint sources. Some stressors, such as sediment deposition or physical alteration of instream habitat, might not clearly fit traditional concepts associated with chemical stressors and loadings. For these nonchemical stressors, it might sometimes be difficult to develop TMDLs because of limitations in the data or in the technical methods for analysis and modeling. In the case of nonpoint source TMDLs, another difficulty arises in that the CWA does not provide well-defined support for regulatory control actions as it does for point source controls, and controls based on another statutory authority might be necessary.

Biological assessments and criteria address the cumulative impacts of all stressors, especially habitat degradation, and chemical contamination, which result in a loss of biological diversity. Biological information can help provide an ecologically based assessment of the status of a waterbody and as such can be used to decide which waterbodies need TMDLs (USEPA 1997c) and aid in the ranking process by targeting waters for TMDL development with a more accurate link between bioassessment and ecological integrity.

Finally, the TMDL process is a geographically-based approach to preparing load and wasteload allocations for sources of stress that might impact waterbody integrity. The geographic nature of this process will be complemented and enhanced if ecological regionalization is applied as part of the bioassessment activities. Specifically, similarities among ecosystems can be grouped into homogeneous classes of streams and rivers that provides a geographic framework for more efficient aquatic resource management.

2.3.5 CWA Section 402--NPDES Permits and Individual Control Strategies

All point sources of wastewater must obtain a National Pollutant Discharge Elimination System (NPDES) permit (or state equivalent), which regulates the facility's discharge of pollutants. The approach to controlling and eliminating water pollution is focused on the pollutants determined to be harmful to receiving waters and on the sources of such pollutants. Authority for issuing NPDES permits is established under Section 402 of the CWA (USEPA 1989).

Point sources are generally divided into two types--industrial and municipal. Nationwide, there are approximately 50,000 industrial sources, which include commercial and manufacturing facilities. Municipal sources, also known as publicly owned treatment works (POTWs), number about 15,700 nationwide. Wastewater from municipal sources results from domestic wastewater discharged to POTWs, as well as the "indirect" discharge of industrial wastes to sewers. In addition, stormwater may be discrete or diffuse, but is also covered by NPDES permitting regulations.

USEPA does not recommend the use of biological survey data as the basis for deriving an effluent limit for an NPDES permit (USEPA 1994d). Unlike chemical-specific water quality analyses, biological data do not measure the concentrations or levels of chemical stressors. Instead, they directly measure the impacts of any and all stressors on the resident aquatic biota. Where appropriate, biological assessment can be used within the NPDES process (USEPA 1994d) to obtain information on the status of a waterbody where point sources might cause, or contribute to, a water quality problem. In conjunction with chemical water quality and whole-effluent toxicity data, biological data can be used to detect previously unmeasured chemical water quality problems and to evaluate the effectiveness of implemented controls.

Some states have already demonstrated the usefulness of biological data to indicate the need for additional or more stringent permit limits (e.g., sole-source discharge into a stream where there is no significant nonpoint source discharge, habitat degradation, or atmospheric deposition) (USEPA 1994d). In these situations, the biological findings triggered additional investigations to establish the cause-and-effect relationship and to determine the appropriate limits. In this manner, biological data support regulatory evaluations and decision making. Biological data can also be useful in monitoring highly variable or diffuse sources of pollution that are treated as point sources such as wet-weather discharges and stormwater runoff (USEPA 1994d). Traditional chemical water quality monitoring is usually only minimally informative for these types of point source pollution, and a biological survey of their impact might be critical to effectively evaluate these discharges and associated treatment measures.

2.3.6 Ecological Risk Assessment

Risk assessment is a scientific process that includes stressor identification, receptor characterization and endpoint selection, stress-response assessment, and risk characterization ( USEPA 1992, Suter et al. 1993). Risk management is a decision-making process that involves all the human-health and ecological assessment results, considered with political, legal, economic, and ethical values, to develop and enforce environmental standards, criteria, and regulations ( Maughan 1993). Risk assessment can be performed on an on-site basis or can be geographically-based (i.e., watershed or regional scale), and it can be used to assess human health risks or to identify ecological impairments. In early 1997, a report prepared by a Presidential/Congressional Commission on risk enlarged the context of risk to include ecological as well as public health risks ( Karr and Chu 1997).

Biological monitoring is the essential foundation of ecological risk assessment because it measures present biological conditions -- not just chemical contamination -- and provides the means to compare them with the conditions expected in the absence of humans ( Karr and Chu 1997). Results of regional bioassessment studies can be used in watershed ecological risk assessments to develop broad scale (geographic) empirical models of biological responses to stressors. Such models can then be used, in combination with exposure information, to predict risk due to stressors or to alternative management actions. Risks to biological resources are characterized, and sources of stress can be prioritized. Watershed risk managers can and should use such results for critical management decisions.

2.3.7 USEPA Water Quality Criteria and Standards

The water quality standards program, as envisioned in Section 303(c) of the Clean Water Act, is a joint effort between the states and USEPA. The states have primary responsibility for setting, reviewing, revising, and enforcing water quality standards. USEPA develops regulations, policies, and guidance to help states implement the program and oversees states' activities to ensure that their adopted standards are consistent with the requirements of the CWA and relevant water quality standards regulations (40 CFR Part 131). USEPA has authority to review and approve or disapprove state standards and, where necessary, to promulgate federal water quality standards.

A water quality standard defines the goals of a waterbody, or a portion thereof, by designating the use or uses to be made of the water, setting criteria necessary to protect those uses, and preventing degradation of water quality through antidegradation provisions. States adopt water quality standards to protect public health or welfare, enhance the quality of water, and protect biological integrity.

Chemical, physical, or biological stressors impact the biological characteristics of an aquatic ecosystem (Gibson et al. 1996). For example, chemical stressors can result in impaired functioning or loss of a sensitive species and a change in community structure. Ultimately, the number and intensity of all stressors within an ecosystem will be evidenced by a change in the condition and function of the biotic community. The interactions among chemical, physical, and biological stressors and their cumulative impacts emphasize the need to directly detect and assess the biota as indicators of actual water resource impairments.

Sections 303 and 304 of the CWA require states to protect biological integrity as part of their water quality standards. This can be accomplished, in part, through the development and use of biological criteria. As part of a state or tribal water quality standards program, biological criteria can provide scientifically sound and detailed descriptions of the designated aquatic life use for a specific waterbody or segment. They fulfill an important assessment function in water quality-based programs by establishing the biological benchmarks for (1) directly measuring the condition of the aquatic biota, (2) determining water quality goals and setting priorities, and (3) evaluating the effectiveness of implemented controls and management actions.

Biological criteria for aquatic systems provide an evaluation benchmark for direct assessment of the condition of the biota that live either part or all of their lives in aquatic systems (Gibson et al. 1996) by describing (in narrative or numeric criteria) the expected biological condition of a minimally impaired aquatic community (USEPA 1990b). They can be used to define ecosystem rehabilitation goals and assessment endpoints. Biological criteria supplement traditional measurements (for example, as backup for hard-to-detect chemical problems) and will be particularly useful in assessing impairment due to nonpoint source pollution and nonchemical (e.g., physical and biological) stressors. Thus, biological criteria fulfill a function missing from USEPA's traditionally chemical-oriented approach to pollution control and abatement ( USEPA 1994d).

Biological criteria can also be used to refine the aquatic life use classifications for a state. Each state develops its own designated use classification system based on the generic uses cited in the CWA, including protection and propagation of fish, shellfish, and wildlife. States frequently develop subcategories to refine and clarify designated use classes when several surface waters with distinct characteristics fit within the same use class or when waters do not fit well into any single category. As data are collected from biosurveys to develop a biological criteria program, analysis may reveal unique and consistent differences between aquatic communities that inhabit different waters with the same designated use. Therefore, measurable biological attributes can be used to refine aquatic life use or to separate 1 class of aquatic life into 2 or more subclasses. For example, Ohio has established an exceptional warmwater use class to include all unique waters (i.e., not representative of regional streams and different from their standard warmwater class).  

 


 


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