EPA/600/R-04/168 

October 2004 

A Review of Biological Assessment Tools and Biocriteria for Streams and 

Rivers in New England States 

Alicia D. Shelton 

SoBran, Inc. 

Karen A. Blocksom 

National Exposure Research Laboratory 

U.S. Environmental Protection Agency 

National Exposure Research Laboratory 

26 West Martin Luther King Drive 

Cincinnati, OH 45268 
NOTICE 

The research described in this document has been funded by the United States 

Environmental Protection Agency under contract 68D01048 to SoBran, Inc. It has been 

subjected to Agency peer and administrative review and approved for publication as an EPA 

document. 

Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. 

The correct citation for this document is: 

Shelton, A.D., and K.A. Blocksom. 2004. A Review of Biological Assessment Tools and 

Biocriteria for Streams and Rivers in New England States. EPA/600/R-04/168. U.S. 

Environmental Protection Agency, Cincinnati, Ohio. 

Cover photos by (clockwise from upper left): New Hampshire DES Biomonitoring Program; 

Hilary Snook, USEPA Region 1; Richard Levey, Vermont DEC; NHDES Biomonitoring 

Program. 

i 
ACKNOWLEDGMENTS 

This report relied on the generous assistance of state personnel in providing materials on 

bioassessment tools and biocriteria and reviewing draft chapters for each state. Thanks go to 

Ernest Pizzuto, Jr. of Connecticut DEP, Susan P. Davies of Maine DEP, Arthur S. Johnson of 

Massachusetts DEP, David Neils of New Hampshire DES, Connie Carey of Rhode Island DEM, 

and Doug Burnham, Richard Levey, and Richard Langdon of Vermont DEC. 

Peter Nolan of U.S. EPA Region 1, Wayne Davis of the U.S. EPA Office of Environmental 

Information, and Bradley Autrey of the U.S. EPA Office of Research and Development all 

provided valuable comments and suggestions on a draft of this document. Eric O’Neal of 

SoBran, Inc. provided assistance in creating some of the maps in this report. Michael T. Barbour 

of Tetra Tech, Inc. provided the suggestion for the general layout of the document. 

ii 
TABLE OF CONTENTS 

LIST OF FIGURES AND TABLES.......................................................................................... vii

ACRONYMS AND COMMON TERMS.................................................................................... x

1 INTRODUCTION..............................................................................................................1-1

1.1 Purpose of the Document............................................................................................. 1-1 

1.2 Rational for Bioassessment Programs ......................................................................... 1-1 

1.2.1 Designated Uses...................................................................................................... 1-3 

1.2.2 Water Quality Criteria for Aquatic Life Use .......................................................... 1-3 

1.2.3 Anti-degradation Policies........................................................................................ 1-4 

1.2.4 Guidance Documents .............................................................................................. 1-4 

1.2.5 Biological Monitoring Programs ............................................................................ 1-5 

1.2.6 Bioindicator Organisms .......................................................................................... 1-5 

1.2.7 305 (b) Report and 303 (d) List .............................................................................. 1-6 

1.3 Literature Cited ............................................................................................................ 1-7

2 CONNECTICUT................................................................................................................2-1

2.1 Introduction.................................................................................................................. 2-1 

2.2 Key Elements of the Biological Assessment Approach .............................................. 2-1 

2.2.1 Index Period and/or Temporal Conditions.............................................................. 2-1 

2.2.2 Monitoring Program Survey Approach .................................................................. 2-3 

2.2.3 Indicator Assemblages ............................................................................................ 2-3 

2.2.4 Reference Condition ............................................................................................... 2-4 

2.3 Field and Laboratory Protocols.................................................................................... 2-5 

2.3.1 Macroinvertebrate Protocols ................................................................................... 2-5 

2.3.1.1 Field Methods .................................................................................................. 2-5 

2.3.1.2 Laboratory Methods......................................................................................... 2-5 

2.3.2 Periphyton Protocols............................................................................................... 2-5 

2.3.2.1 Field Methods .................................................................................................. 2-5 

2.3.2.1.1 Quantitative Periphyton Sampling............................................................. 2-5 

2.3.2.1.2 Rapid Periphyton Survey ........................................................................... 2-6 

2.3.2.2 Laboratory Methods......................................................................................... 2-6 

2.3.2.2.1 Chlorophyll a ............................................................................................. 2-6 

2.3.2.2.2 Algal Identification and Density................................................................ 2-7 

2.3.2.2.3 Biomass and Biovolume Determination .................................................... 2-7 

2.3.3 Fish Protocol ........................................................................................................... 2-7 

2.4 Data Management/Quality ........................................................................................... 2-7 

2.5 Analysis of Biological Data ......................................................................................... 2-8 

2.5.1 Macroinvertebrate Data .......................................................................................... 2-8 

2.5.2 Periphyton Data ...................................................................................................... 2-9 

2.5.3 Fish Data .................................................................................................................2-9

2.5.4 Summary: Determining ALU Support.................................................................. 2-10 

2.6 Literature Cited .......................................................................................................... 2-11 

iii 
2.7 Resources ................................................................................................................... 2-12 

3 MAINE................................................................................................................................ 3-1

3.1 Introduction.................................................................................................................. 3-1 

3.2 Key Elements of the Biological Assessment Approach .............................................. 3-3 

3.2.1 Index Period and/or Temporal Conditions.............................................................. 3-3 

3.2.2 Monitoring Program Survey Approach/Natural Classification of Water Bodies ... 3-4 

3.2.3 Indicator Assemblage.............................................................................................. 3-4 

3.2.4 Reference Condition (Establishing a priori Groups).............................................. 3-4 

3.3 Field and Laboratory Protocols.................................................................................... 3-5 

3.3.1 Macroinvertebrate Protocols ................................................................................... 3-5 

3.3.1.1 Field Methods .................................................................................................. 3-5 

3.3.1.2 Laboratory Methods......................................................................................... 3-6 

3.3.1.2.1 Subsampling and Identification ................................................................. 3-6 

3.3.1.2.2 Chironomidae Identification and Subsampling ......................................... 3-7 

3.4 Data Management/Quality ........................................................................................... 3-7 

3.5 Analysis of Biological Data ......................................................................................... 3-7 

3.6 Literature Cited .......................................................................................................... 3-17 

3.7 Resources ................................................................................................................... 3-20 

4 MASSACHUSETTS .......................................................................................................... 4-1

4.1 Introduction.................................................................................................................. 4-1 

4.2 Key Elements of the Biological Assessment Approach .............................................. 4-2 

4.2.1 Index Period and/or Temporal Conditions.............................................................. 4-2 

4.2.2 Monitoring Program Survey Approach .................................................................. 4-2 

4.2.3 Natural Classification of Water Bodies .................................................................. 4-3 

4.2.4 Indicator Assemblages ............................................................................................ 4-3 

4.2.5 Reference Condition ............................................................................................... 4-3 

4.3 Field and Laboratory Protocols.................................................................................... 4-4 

4.3.1 Macroinvertebrate Protocols ................................................................................... 4-4 

4.3.1.1 Field Methods .................................................................................................. 4-4 

4.3.1.1.1 Kick Sampling ........................................................................................... 4-6 

4.3.1.1.2 Rock Basket Sampling............................................................................... 4-6 

4.3.1.1.3 Hester-Dendy Multi-plate Sampling.......................................................... 4-6 

4.3.1.2 Laboratory Methods......................................................................................... 4-7 

4.3.1.2.1 Processing of Kick Net and Rock Basket Samples.................................... 4-7 

4.3.1.2.2 Processing of Hester-Dendy Multi-plate Samples ..................................... 4-7 

4.3.1.2.3 Taxonomic Identification........................................................................... 4-8 

4.3.1.2.4 Oligochaeta and Chironomidae Identification ........................................... 4-8 

4.3.2 Periphyton Protocols............................................................................................... 4-8 

4.3.2.1 Field Methods .................................................................................................. 4-8 

4.3.2.1.1 Algal Abundance and Identification .......................................................... 4-8 

4.3.2.1.2 Biomass...................................................................................................... 4-9 

4.3.2.1.3 Chlorophyll a ............................................................................................. 4-9 

4.3.2.1.4 Percent coverage calculation......................................................................4-9 

iv 
4.3.2.1.5 Biomass Determination............................................................................ 4-10 

4.3.2.1.6 Chlorophyll a Determination ................................................................... 4-11 

4.3.3 Fish Protocols........................................................................................................4-11 

4.4 Data Management/Quality ......................................................................................... 4-12 

4.5 Analysis of Biological Data ....................................................................................... 4-12 

4.5.1 Macroinvertebrate Data ........................................................................................ 4-12 

4.5.2 Algal Data ............................................................................................................. 4-15 

4.5.3 Fish Data ............................................................................................................... 4-15 

4.5.4 Summary: Determining ALU Support.................................................................. 4-15 

4.6 Literature Cited .......................................................................................................... 4-17 

4.6.1 Resources .............................................................................................................. 4-20 

5 NEW HAMPSHIRE .......................................................................................................... 5-1

5.1 Introduction.................................................................................................................. 5-1 

5.2 Key Elements of the Biological Assessment Approach .............................................. 5-3 

5.2.1 Index Period and/or Temporal Conditions.............................................................. 5-3 

5.2.2 Monitoring Program Survey Approach .................................................................. 5-3 

5.2.3 Natural Classification of Water Bodies .................................................................. 5-3 

5.2.4 Indicator Assemblages ............................................................................................ 5-3 

5.2.5 Reference Condition ............................................................................................... 5-3 

5.3 Field and Laboratory Protocols.................................................................................... 5-5 

5.3.1 Macroinvertebrates Protocols ................................................................................. 5-5 

5.3.1.1 Field Methods .................................................................................................. 5-5 

5.3.1.2 Laboratory Methods......................................................................................... 5-5 

5.3.2 Fish Protocol ........................................................................................................... 5-6 

5.4 Data Management/Quality ........................................................................................... 5-6 

5.5 Analysis of Biological Data ......................................................................................... 5-6 

5.5.1 Macroinvertebrate Data .......................................................................................... 5-6 

5.5.2 Fish Data .................................................................................................................5-7

5.6 Summary: Determining ALU Support......................................................................... 5-8 

5.7 Literature Cited ............................................................................................................ 5-9

5.8 Resources ................................................................................................................... 5-10 

6 RHODE ISLAND............................................................................................................... 6-1

6.1 Introduction.................................................................................................................. 6-1 

6.2 Key Elements of the Biological Assessment Approach .............................................. 6-2 

6.2.1 Index Period and/or Temporal Conditions.............................................................. 6-2 

6.2.2 Monitoring Program Survey Approach .................................................................. 6-2 

6.2.3 Natural Classification of Water Bodies .................................................................. 6-2 

6.2.4 Indicator Assemblages ............................................................................................ 6-2 

6.2.5 Reference Condition ............................................................................................... 6-2 

6.3 Field and Laboratory Protocols.................................................................................... 6-3 

6.3.1 Macroinvertebrate Protocols ................................................................................... 6-3 

6.3.1.1 Field Methods .................................................................................................. 6-3 

6.3.1.2 Laboratory Methods......................................................................................... 6-4 

v 
6.4 Data Management/Quality ........................................................................................... 6-4 

6.5 Analysis of Biological Data ......................................................................................... 6-5 

6.5.1 Macroinvertebrate Data .......................................................................................... 6-5 

6.6 Summary: Determining ALU Support......................................................................... 6-6 

6.7 Literature Cited ............................................................................................................ 6-9

6.8 Resources ..................................................................................................................... 6-9 

7 VERMONT.........................................................................................................................7-1

7.1 Introduction.................................................................................................................. 7-1 

7.2 Key Elements of the Biological Assessment Approach .............................................. 7-3 

7.2.1 Index Period and/or Temporal Conditions.............................................................. 7-3 

7.2.2 Monitoring Program Survey Approach .................................................................. 7-3 

7.2.3 Natural Classification of Water Bodies .................................................................. 7-3 

7.2.4 Indicator Assemblages ............................................................................................ 7-3 

7.2.5 Reference condition ................................................................................................ 7-3 

7.3 Field and Laboratory Protocols.................................................................................... 7-5 

7.3.1 Macroinvertebrate Protocols ................................................................................... 7-5 

7.3.1.1 Field Methods .................................................................................................. 7-5 

7.3.1.2 Laboratory Methods......................................................................................... 7-5 

7.3.2 Fish Protocols..........................................................................................................7-6 

7.3.2.1 Field Methods .................................................................................................. 7-6 

7.4 Data Management/Quality ........................................................................................... 7-6 

7.5 Analysis of Biological Data ......................................................................................... 7-7 

7.5.1 Macroinvertebrate Data .......................................................................................... 7-7 

7.5.2 Fish Data .................................................................................................................7-9

7.6 Summary: Determining ALU Support....................................................................... 7-10 

7.7 Literature Cited .......................................................................................................... 7-14 

7.8 Resources ................................................................................................................... 7-15 

8 SUMMARY ........................................................................................................................ 8-1

8.1 Comparison Across States ........................................................................................... 8-1 

8.2 Literature Cited ............................................................................................................ 8-8

APPENDIX A: PROCESS AND CRITERIA FOR THE ASSIGNMENT OF 

BIOLOGIST'S CLASSIFICATION ....................................................................................... A-1

vi 
LIST OF FIGURES AND TABLES 

FIGURES 

Figure 2-1. Major Connecticut basins sampled for the biological monitoring program using the 

rotating basin strategy. ................................................................................................................. 2-4 

Figure 3-1. Maine’s narrative aquatic life standards with the human disturbance and biological 

condition gradients (Taken from Courtemanch 2003)................................................................. 3-3 

Figure 3-2. Map of basins sampled by MDEP (2002). ............................................................... 3-5 

Figure 3-3. Maine tiered uses based on measurable ecological values (taken from Courtemanch 

2003). ........................................................................................................................................... 3-9 

Figure 3-4. Process of calculating model variables and association values using linear 

discriminant models (taken from MDEP 2003)......................................................................... 3-18 

Figure 3-5. Process for determining attainment class using association values (modified from

MDEP 2003). ............................................................................................................................. 3-19 

Figure 4-1. Massachusetts 5-Year Basin Rotation Strategy (taken from the Massachusetts 

Department of Environmental Protection website 

www.mass.gov/dep/brp/wm/files/cyclemap6.jpg). ....................................................................... 4-4 

Figure 4-2. Level III and Level IV Ecoregions of Massachusetts (taken from Griffith et al. 1994, 

http://www.epa.gov/wed/pages/ecoregions/mactri_eco.htm). ..................................................... 4-5 

Figure 5-1. Major New Hampshire basins and the northern and southern bioregion boundaries 

used for macroinvertebrate sampling (indicated by the red line). ............................................... 5-4 

Figure 6-1. Level IV Omernik subecoregions and reference streams used in RI DEM’s biological 

monitoring program. .................................................................................................................... 6-3 

TABLES 

Table 1-1. Contact information for bioassessment programs in New England states. ............... 1-2 

Table 2-1. Connecticut water quality standard classes. .............................................................. 2-2 

Table 2-2. Metrics and scoring ranges used in RBP III determinations of the level of biological 

impact based on benthic macroinvertebrates (based on Plafkin et al. (1989)). ........................... 2-8 

vii 
Table 2-3. Aquatic life use support categories and the criteria used for making decisions (taken 

from Table 2 in CT DEP 2002a)................................................................................................ 2-10 

Table 3-1. Water quality classification system for rivers and streams in Maine. (M.R.S.A. Title 

38 Article 4-A § 464-465)............................................................................................................ 3-2 

Table 3-2. Methods for the calculation of variables and measures of community structure used in 

linear discriminant models (from Davies and Tsomides, 2002). ............................................... 3-10 

Table 3-3. Coefficients for the First Stage Model (from MDEP 2003). ................................... 3-14 

Table 3-4. Coefficients for the Final Classification Models (AA/A, B, and C) (MDEP 2003).3-14 

Table 4-1. Massachusetts attainment classes with management strategy and narrative biologic 

and habitat criteria as stated in 314 CMR 4.00 (2000). ............................................................... 4-2 

Table 4-2. Methods for the calculations of metrics and scoring ranges used in RBP II 

determinations of level of biological impact (Plafkin 1989; Nuzzo 2003)................................ 4-12 

Table 4-3. Methods for the calculations of metrics and scoring ranges used in RBP III 

determinations of level of biological impact (Plafkin 1989; Nuzzo 2003)................................ 4-14 

Table 4-4. Biological, toxicological, and chemical parameters that are used collectively to 

determine ALUS. Attainment is assigned based on a “weight of evidence” evaluation. (MA DEP 

2003) (Numerical criteria for dissolved oxygen, pH, and temperature can be found in 314 CMR 

4.00 (MA DEP 2000). MA DEP uses the recommended limits published by EPA pursuant to 

Section 304(a) of the Federal Act for Toxic Pollutant Criteria). ............................................... 4-16 

Table 5-1. NH DES water quality classes and the defined designated uses for each class. 

Dissolved oxygen exceedance values for aquatic life criteria are also listed (NH DES 1999 ). . 5-2 

Table 5-2. Metrics and scoring for the New Hampshire B-IBI. .................................................. 5-8 

Table 6-1. Metrics used by the Rhode Island Biomonitoring program and the methods for the 

calculation of metrics and their scoring ranges based on the RBP III (Plafkin et al. 1989, RI 

DEM 2002a, RI DEM 2002b)...................................................................................................... 6-5 

Table 6-2. Percent comparability evaluation for macroinvertebrate bioassessment scores used by 

the State of Rhode Island. ............................................................................................................ 6-7 

Table 6-3. Biological, physical and chemical criteria used to determine aquatic life use (modified 

from RI DEM 2000)..................................................................................................................... 6-7 

viii 
Table 7-1. Biological ALUS management classes and associated narrative biological criteria for 

rivers and streams in Vermont. .................................................................................................... 7-2 

Table 7-2. VT DEC macroinvertebrate metrics and methods used to calculate each of the 

metrics.......................................................................................................................................... 7-7 

Table 7-3. Macroinvertebrate assemblage biocriteria thresholds for the macroinvertebrate 

community stream categories, and associated WQ classes of Vermont (VT DEC 2004). ........ 7-11 

Table 7-4. The six metrics used in scoring the fish assemblage for the CWIBI. These streams 

must naturally support two to four native species (VT DEC 2004). ......................................... 7-12 

Table 7-5. The nine metrics used in scoring cold and warm water sites for the MWIBI. These 

streams must naturally support more than four native fish species (VT DEC 2004). ............... 7-12 

Table 7-6. All possible scores for the CWIBI and MWIBI that correspond to the VT WQS 

classification scheme (VT DEC 2004). ..................................................................................... 7-14 

Table 8-1. Comparisons of the key components of state bioassessment programs. ................... 8-2 

Table 8-2. Comparison of the macroinvertebrate metrics used by states in the New England 

Region. Color shading indicates equivalent metrics across states. ............................................. 8-6 

ix 
ACRONYMS AND COMMON TERMS 

305(b) Report: Clean Water Act (CWA) section 305(b) requires each state to submit an 

assessment report biennially to U.S. EPA on the quality of surface and ground water resources. 

The EPA then compiles the data from state 305(b) reports and submits a National Water Quality 

Report to Congress 

303(d) List: The section of the Clean Water Act that requires each state to identify waters that 

are impaired according to water quality standards. Placement of water bodies on this list requires 

the preparation of Total Maximum Daily Loads (TMDLs) that will aid in the cleanup of the 

impacted waters. 

7Q10: The lowest consecutive 7-day mean stream flow that occurs during a 10-year period 

30Q5: The lowest consecutive 30-day mean stream flow that occurs during a 5-year period. 

ABN: Ambient Biomonitoring Network [Vermont] 

AFDM: Ash Free Dry Mass 

ALU: Aquatic Life Use 

ALUS: Aquatic Life Use Support 

BASS: Biomonitoring and Aquatic Studies Section [Vermont] 

B-IBI: Benthic Index of Biotic Integrity. [New Hampshire] 

BPJ: Best Professional Judgment 

CALM: Consolidated Assessment and Listing Methodology 

CT DEP: Connecticut Department of Environmental Protection 

CWA: Clean Water Act 

CWIBI: Cold Water Index of Biotic Integrity [Vermont] 

DO: Dissolved oxygen 

EDAS: Ecological Data Assessment System: A database system developed by Tetra Tech, Inc. 

that uploads ecological data to STORET for archival and has the capability to analyze, manage 

and store data and calculate metrics. 

EMAP: Environmental Monitoring and Assessment Program 

x 
EPT: Insect orders of Ephemeroptera, Plecoptera, Trichoptera, considered sensitive benthic 

orders. 

FBI: Family Biotic Index 

GIS: Geographic Information Systems 

GWHI: Ground Water Hazard Inventory 

HBI: Hilsenhoff Biotic Index 

HDG: Human Disturbance Gradient [New Hampshire] 

HUC: Hydrologic Unit Code 

IBI: Index of Biotic Integrity 

MDEP: Maine Department of Environmental Protection 

NA: non-attainment 

MA DWM: Massachusetts Division of Watershed Management 

MA DEP: Massachusetts Department of Environmental Protection 

MHG: Medium High Gradient Streams [Vermont] 

NH DES: New Hampshire Department of Environmental Services 

MWIBI: Mixed Water Index of Biotic Integrity [Vermont] 

NEWS: New England Wadeable Streams Project 

NHLC: New Hampshire Land Cover 

NPDES: National Pollutant Discharge Elimination System 

ONRW: Outstanding National Resource Waters 

QAPP: Quality Assurance Project Plan 

RCRA: Resource Conservation Recovery Act 

RBP: Rapid Bioassessment Protocol 

RI DEM: Rhode Island Department of Environmental Management 

xi 
RPS: Rapid Periphyton Survey (RBP) 

SHG: Small High Gradient Streams [Vermont] 

SOP: Standardized Operating Procedure 

TMDL: Total Maximum Daily Load 

WBS: Water Body System- Database developed by USEPA to store information to be used in 

305(b) reporting and 303(d) listing for all assessed water bodies within a region 

WPCA: Water Pollution Control Act 

WQS: Water Quality Standards 

WWMG: Warm water medium grade streams and rivers [Vermont] 

VT DEC: Vermont Department of Environmental Conservation 

xii 
1 INTRODUCTION 

1.1 Purpose of the Document 

The primary purpose of this document is to serve as a detailed description of the 

biological assessment programs for wadeable streams and rivers within U.S. Environmental 

Protection Agency (U.S. EPA) Region 1 states (i.e., Connecticut, Maine, Massachusetts, New 

Hampshire, Rhode Island, and Vermont). Specifically, this report concentrates on the target 

assemblages (e.g., benthic macroinvertebrates, periphyton, and/or fish) and the specific methods 

used by each state to determine whether biocriteria set for aquatic life use (ALU) are met in 

wadeable streams and rivers. The information contained in this report is critical to the eventual 

use of state data in assessing water resources on a national scale because it provides the 

necessary level of detail on New England state bioassessment methodologies in a single 

document. In addition, this report serves as a valuable resource for other states, tribes, and 

municipalities, both those developing bioassessment tools and those with existing programs. 

Although every attempt has been made to represent the methods and protocols used by 

each state accurately, this document is not intended to be used as a replacement for those 

protocols and Standard Operating Procedures (SOPs) that are used and approved by the state 

agencies. Thanks to the cooperation of state scientists, all protocols and procedures were 

obtained through personal communication and via state and federal published and unpublished 

documents that are referenced within this report. Each state reviewed its respective chapter for 

technical accuracy and was given the opportunity to provide comments and changes prior to 

completion of this report. However, we recommend referring directly to state protocols before 

implementation of the described methods to ensure that the most updated and complete versions 

of protocols are used. Contact details for each of the state bioassessment programs discussed are 

provided in Table 1-1. 

1.2 Rational for Bioassessment Programs 

The modern Clean Water Act (CWA) is derived from the 1948 Federal Water Pollution 

Control Act (WPCA). After the passage of the 1972 amendments, the act became commonly 

known as the CWA and its goal was to “restore and maintain the chemical, physical and 

biological integrity of the nation’s waters so that they can ‘support the protection and 

propagation of fish, shellfish, and wildlife and recreation in and on the water’” 

(http://www.epa.gov/watertrain/cwa/). This act federally recognized the aquatic inhabitants of 

water bodies and began to set water quality standards to protect these organisms. The CWA 

amendments through 1987 outlined the guidelines by which states and tribes must use 

bioassessment programs and develop biocriteria to ensure the adherence to water quality 

standards. Specifically, Section 303(c) of the CWA requires states to have water quality 

standards (WQS) that consist of three components: 1) designated uses, 2) water quality criteria to 

protect those uses, and 3) an anti-degradation policy. States are required to review their 

standards every three years and revise them as needed to achieve the purposes of the CWA, 

including the ecological integrity objective. 

1-1 
Table 1-1. Contact information for bioassessment programs in New England states. 

State Program Contact Web Site 

Connecticut Ernest Pizzuto, Jr. 

Supervising Environmental Analyst 

Connecticut Department of Environmental 

Protection 

Address: 

79 Elm St. 

Hartford, CT 06106-5127 

Phone: 860-424-3715 

Email: ernest.pizzuto@po.state.ct.us 

http://dep.state.ct.us/ 

Maine Susan P. Davies 

Program Manager, Biologist III 

Maine Department of Environmental Protection 

Address: 

SHS 17 

Augusta, ME 04333 

Phone: 207-287-7778 

Email: susan.p.davies@maine.gov 

http://www.maine.gov/dep/ 

Massachusetts Arthur S. Johnson 

Environmental Monitoring Coordinator 

Massachusetts Department of Environmental 

Protection 

Address: 

627 Main Street 

Worcester, MA 01608 

Phone: 508-767-2873 

Email: arthur.johnson@state.ma.us 

http://www.state.ma.us/dep/ 

New 

Hampshire 

David Neils 

Biomonitoring Program Coordinator 

New Hampshire Department of Environmental 

Services 

Address: 

6 Hazen Drive 

Concord, NH 03302-0095 

Phone: 603-271-8865 

Email: dneils@des.state.nh.us 

www.des.state.nh.us/ 

1-2 
State Program Contact Web Site 

Rhode Island Connie Carey 

Environmental Scientist 

http://www.state.ri.us/dem/ 

Rhode Island Department of Environmental 

Management 

Address: 

235 Promenade Street 

Providence, RI 02908-5767 

Phone: 401-222-4700 x7239 

Email: ccarey@dem.state.ri.us 

Vermont Doug Burnham 

Biomonitoring and Aquatic Studies Section 

Chief 

http://www.anr.state.vt.us/ 

Vermont Department of Environmental 

Conservation 

Address: 

103 South Main Street-10N 

Waterbury, VT 05671 

Phone: 802-241-3784 

Email: doug.burnham@anr.state.vt.us 

1.2.1 Designated Uses 

As required by CWA 40 C.F.R. § 130.10, states, territories and tribes must specify 

appropriate beneficial uses based on the intended use and the value of the waters, and these uses 

must be achieved and protected. Designated uses may be listed as general categories (e.g., 

drinking water source, wildlife, shellfish, aquatic life, recreational, industrial), or the uses may 

consist of more specific sub-categories that may target cold water versus warm water systems or 

contain special uses that are meant to protect unique, sensitive, or valuable aquatic habitat (U.S. 

EPA 1991). These designated uses are typically associated with a classification system (e.g., 

Class A waters, Class B waters, Class C waters) within each state’s WQS that categorizes each 

water body according to condition. 

1.2.2 Water Quality Criteria for Aquatic Life Use 

Water quality criteria are narrative or numeric descriptions of those conditions that 

protect designated uses. In addition, the criteria need to be scientifically consistent with the 

intended designated use and must be accurate indicators of the designated use. Although the 

U.S. EPA has published guidance criteria to protect aquatic life use (U.S. EPA 2002a), 

individual states are not required to follow them and may develop their own criteria. Guidance 

for the development of numeric criteria are published in the CWA § 104(a)(1) and may be 

modified based on the needs of the state. Currently, only narrative descriptions of criteria for 

aquatic life use support are required within state WQS by the U.S. EPA. The narrative criteria 

are simply descriptions of the conditions necessary for a water body to attain its designated use 

1-3 
(U.S. EPA 2002b), and these definitions, along with organisms that can be used to assess 

attainment, vary from state to state in U.S. EPA Region 1. 

Each state in Region 1 has established its goal for protecting waters and then defined 

aquatic life use (ALU). For example, New Hampshire statutes define waters achieving ALU as 

those waters that “provide suitable chemical and physical conditions for supporting a balanced, 

integrated and adaptive community of aquatic organisms (NH DES 1999). Aquatic life use 

support (ALUS) as defined in Rhode Island WQS is “providing suitable habitat and water quality 

for the protection, maintenance, and propagation of a viable community of aquatic life” (RI 

DEM 2000). Section 3-01 of the Vermont Water Quality Standards states the provision to, 

“establish and apply numeric biological indices to determine whether there is full support of 

aquatic biota and aquatic habitat uses” and to “establish procedures that employ standard 

sampling and analytical methods to characteristics of the biological integrity of the appropriate 

reference conditions” (State of Vermont 2000). In Massachusetts, the ALUS criteria of the 

standards 314 CMR 4.00 “must provide suitable habitat for sustaining a native, naturally diverse 

community of aquatic flora and fauna” (MA DEP 2000; 2003). Massachusetts then further 

designates two subclasses: Cold Water Fishery - capable of sustaining a year-round population of 

cold water aquatic life; and Warm Water Fishery - waters that are not capable of sustaining a 

year-round population of cold water aquatic life (MA DEP 2003). Connecticut WQS express 

that “the benthic invertebrate criteria may be utilized where appropriate for assessment of the 

biological integrity of surface waters. These criteria apply to the fauna of erosional or riffle 

habitats in lotic waters which are not subject to tidal influences” (CT DEP 2002). Connecticut 

defines biological integrity as the “ability of an aquatic ecosystem to support and maintain a 

balanced, integrated, adaptive community of organisms having a species composition, diversity, 

and functional organization comparable to that of the natural habitats of a region” (CT DEP 

2002). In Maine, the use of benthic organisms to determine the attainment of ALU is written 

directly into the standards in chapter 579 (MDEP 2003). Chapter 579 gives a detailed 

description of the use of benthic organisms and the methods used to make decisions about 

classification attainment (MDEP 2003). Furthermore, narrative standards in Maine Revised 

Statutes Annotate 38 Public Chapter 3 Article 4-A § 464 and § 465 define the biological 

narrative and numerical dissolved oxygen and bacterial standards. 

1.2.3 Anti-degradation Policies 

The anti-degradation policy (CWA 40 CFR §131.12) is a set of rules designed to protect 

high quality waters. This policy must offer a framework of decision-making if water quality 

changes occur. The U.S. EPA WQS require states to implement a three-tiered system for 

addressing anti-degradation. Tier 1 requires that water quality necessary to support existing uses 

is maintained and protected, Tier 2 states that in no case shall water quality decrease to a level 

that would interfere with the designated use, and Tier 3 maintains and protects outstanding 

national resource waters (ONRW), aiming to preserve those waters with exceptional recreational 

or ecological significance (U.S. EPA www.epa.gov/waterscience/standards/about/adeg.htm). 

1.2.4 Guidance Documents 

 To support the assessment of attainment of beneficial uses, states are responsible for 

implementing a biological monitoring strategy for the design, collection and data analysis of 

1-4 
biological data. The U.S. EPA has published technical documents and guidance documents to 

offer support for the development of state biomonitoring programs for the assessment of water 

quality for ALUS. The most current documents include the Guidance for 2004 Assessment, 

Listing and Reporting Requirements Pursuant to Sections 303(d) and 305(b) of the Clean Water 

Act; TMDL-01-03 (U.S. EPA 2003), and The Consolidated Assessment and Listing 

Methodology (CALM), Toward a Compendium of Best Practices (U.S. EPA 2002b), both of 

which provide a framework for documenting the collection and use of water quality data for 

CWA Section 305(b) reporting, determining attainment of WQS, determining stream impairment 

for CWA Section 303(d) listing, and establishing anti-degradation policies. 

This document attempts to follow the general framework provided by the CALM 

document to organize the information for each of the Region 1 states in a format that is 

conceptually easy for comparisons to be made among biomonitoring programs. 

1.2.5 Biological Monitoring Programs 

After beneficial uses are established, the criteria are set, and the anti-degradation policy is 

in place, each state then implements a monitoring program. Bioassessment programs have been 

employed by states to assess the water quality with established biocriteria for a range of 

designated uses in freshwater systems. Bioassessment is used for a number of designated uses, 

which may include drinking water, recreation, industry, wildlife, agriculture, and others, but it is 

most commonly used to evaluate aquatic life use support. In 1991, U.S. EPA policy stated the 

necessity of integrating biological surveys with toxicity and chemical-specific assessment 

methods into monitoring programs to determine the attainment or non-attainment of aquatic life 

use support (U.S. EPA 1991). As of 2001, 40 entities, including all of the Region 1 states, used 

bioassessment to determine ALUS for 305(b) reporting (U.S. EPA 2002c). 

Currently, the U.S. EPA CALM guidance suggests four categories of data that may be 

collected and integrated to determine ALUS. These four categories are: biological, habitat, 

toxicological, and physical/chemical data (U.S. EPA 2002b). Although all categories of data are 

potentially useful depending on the rigor involved in the assessment method, only biological data 

provide a direct measurement of the resident aquatic organisms that integrates the abiotic 

conditions in the water body (U.S. EPA 2002b). The CALM document advises that states use 

biological data “as a core indicator for aquatic life use determinations, as they are a unique water 

body response measurement, providing information about a water body that no other 

measurement can” (U.S. EPA 2002b). The document continues to stress that the state 

“documentation of the adequate quality and rigor of the key elements of the state’s 

bioassessment program” be provided so that the biological data can accurately assess water 

quality (U.S. EPA 2002b). 

1.2.6 Bioindicator Organisms 

Biological assessments of those organisms present in the aquatic system offer the most 

direct way to measure the condition of the biological community as a function of environmental 

stressors (Yoder and Rankin 1995). Community composition may be altered as a result of 

stresses in the system and the condition of individual organisms can show pollution impacts that 

may act as an early warning detection of degradation or provide a more reliable assessment of 

changes in the biological community over time (U.S. EPA 2002d). There are several possible 

1-5 
assemblages of organisms available for use in bioassessment. However, benthic 

macroinvertebrates, periphyton, and fish are the biological indicators suggested for use by the 

U.S. EPA in lotic environments (Barbour et al. 1999). All three assemblages are widely used in 

bioassessment, but macroinvertebrates and fish are the most common indicator organisms, with 

45 entities using two to four assemblages (U.S. EPA 2002c). Although standard methods for 

sampling each of these assemblages are suggested by the U.S. EPA, many states alter the 

methods to fit into the goals of their program, adjust for ecoregional constraints, and 

accommodate limited budgets. 

Benthic macroinvertebrates are the most commonly used assemblage. As of 2001, all 57 

of the entities with a bioassessment program in place either currently used or were developing 

macroinvertebrate indicators (U.S. EPA 2002c). Benthic macroinvertebrates are a diverse 

assemblage, consist of species exhibiting a range of pollution tolerance levels, and are abundant 

in most streams (Plafkin et al. 1989, Barbour et al. 1999). Furthermore, they often live the 

majority of their lives in direct contact with both the water and sediments and their life cycles 

may span multiple seasons, thereby showing cumulative changes. They also serve as an 

important link in the food chain (Plafkin et al. 1989), maintaining the rest of the aquatic 

community and managing algal systems. Benthic macroinvertebrates are easy and affordable to 

collect, making them extremely attractive for biological monitoring. 

The advantage of using periphyton as an indicator is that growth of this assemblage is 

directly related to nutrient eutrophication and this assemblage may show adverse effects of 

herbicides or other chemicals more quickly than other organisms (Barbour et al. 1999). 

Periphyton assemblages exhibit stressor-related changes that alter species composition rapidly, 

and can shift to noxious levels of overgrowth, thereby contributing to water quality degradation 

(Stevenson et al. 1996, Stevenson and Bahls 1999). Similar to benthic macroinvertebrates, 

periphyton assemblages contain species with a wide range of pollution tolerances. Furthermore, 

they are easy to collect and identify by experienced taxonomists (Plafkin et al. 1989, Stevenson 

and Bahls 1999). As of 2001, only 20 entities were using algae as an indicator, although an 

additional 5 entities were developing algal indicators (U.S. EPA 2002c). 

Fish are another indicator of watershed health with easily identifiable species of varying 

trophic levels that respond differently to wide ranges of environmental stressors (Karr et al. 

1986, Barbour et al. 1999). Fish are advantageous indicators because they live their entire lives 

in water and their large geographical ranges can indicate the effects of stressors on a greater scale 

than either periphyton or macroinvertebrates. Fish provide information regarding the physical, 

chemical, biological and habitat condition of the watershed as a whole 

(www.epa.gov/bioindicators/html/fish.html). As of 2001, 41 entities were using fish for 

biological assessments (U.S. EPA 2002c). 

1.2.7 305 (b) Report and 303 (d) List 

Following data collection, processing, and analysis, each state is required to submit the 

results in the form of a biennial 305(b) Report on the water quality conditions and provide a 

303(d) List of Impaired Waters on April 1st of every even-numbered year (U.S. EPA 2003). The 

305(b) report must contain all the information collected from streams and rivers located within 

the state’s boundaries. The Integrated 305(b) Report must contain the following key 

components: “geographic referencing of all water resources; categorization of waters according 

to WQS attainment status; identification, prioritization and scheduling of waters needing Total 

1-6 
1.3 

Maximum Daily Loads (TMDL); identification of waters where information is not sufficient to 

determine a water’s status; and a schedule of monitoring for the next reporting cycle” (U.S. EPA 

2003). The EPA requires that all of the state’s assessed waters be placed into one of five 

categories that represent varying levels of water quality standards attainment. These five 

categories as stated in U.S. EPA (2003) are as follows: 

Category 1: All designated uses are met; 

Category 2: Some of the designated uses are met but there is insufficient data to determine if

remaining designated uses are met; 

Category 3: Insufficient data to determine whether any designated uses are met; 

Category 4: Water is impaired or threatened but a TMDL is not needed; 

Category 5: Water is impaired or threatened and a TMDL is needed. 

Those impaired streams where one or more designated uses are not attained and are 

consequently placed in Category 5 must be listed on the 303(d) list. Once placed on the 303(d) 

list, a TMDL must be prioritized and established. Within the 303(d) list, Section 130.7(b)(4) 

requires that each state also identify the pollutants that are known to be causing the impairment 

(U.S. EPA 2003). 

Literature Cited 

Barbour, M, 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. 

http://www.epa.gov/owow/monitoring/rbp/wp61pdf/rbp.pdf 

(CT DEP) Connecticut Department of Environmental Protection. 2002a. Water Quality 

Standards. Connecticut Department of Environmental Protection, Hartford, CT. 

http://dep.state.ct.us/wtr/wq/wqs.pdf 

Karr, J.R., K.D. Fausch, P.L. Angermeier, P.R. Yant, and I.J. Schlosser. 1986. Assessing 

biological integrity in running waters: A method and its rationale. Special publication 5. 

Illinois Natural History Survey. 

(MDEP) Maine Department of Environmental Protection. 2003. Classification Attainment 

Evaluation Using Biological Criteria for Rivers and Streams. Rules of Maine State 

Government Agencies, 06 096 Chapter 579. 

http://www.maine.gov/sos/cec/rcn/apa/06/096/096c579.doc 

Maine Revised Statutes Annotate. Title 38, Chapter 3: Protection and Improvement of Waters. 

Sections 464 and 465. http://janus.state.me.us/legis/statutes/38/title38ch3sec0.html 

(MA DEP) Massachusetts Department of Environmental Protection. 2003. Massachusetts Year 

2002 Integrated List of Waters: Part 1, Context and Rationale for Assessing and 

Reporting the Quality of Massachusetts Surface Waters. CN:125.1. Massachusetts 

1-7 
Department of Environmental Protection, Bureau of Resource Protection, Division of 

Watershed Management. http://www.mass.gov/dep/brp/wm/files/2002-il1.pdf 

MA DEP. 2000. 314 CMR 4.00, Massachusetts Surface Water Quality Standards. Massachusetts 

Department of Environmental Protection, Division of Water Pollution Control. 

http://www.mass.gov/dep/bwp/iww/files/314cmr4.htm 

(NH DES) New Hampshire Department of Environmental Services. 1999. State of New 

Hampshire Surface Water Quality Regulations, Chapter 1700. New Hampshire 

Department of Environmental Proteciton, Concord, NH. 

http://www.des.state.nh.us/wmb/env-ws1700.pdf 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA/444/4-89-001. U.S. Environmental Protection Agency, Washington DC. 

(RI DEM) Rhode Island Department of Environmental Management. 2000. Water Quality 

Regulations, Regulation EVM 112-88.97-1. Rhode Island Department of Environmental 

Management, Office of Water Resources. 

http://www.state.ri.us/dem/pubs/regs/REGS/WATER/h20qlty.pdf 

Stevenson, R.J., and L.L. Bahls. 1999. Periphyton protocols. Pages 6-1 to 6-22 in M.T. 

Barbour, J. Gerritsen, and B.D. Snyder, and J.B. Stribling (editors). Rapid Bioassessment 

Protocols for Use in Wadeable Streams and Rivers: Periphyton, Benthic 

Macroinvertebrates, and Fish, Second Edition. EPA 841-B-99-002. United States 

Environmental Protection Agency, Washington, D.C. 

Stevenson, R.J., M.L. Bothwell, and R.L. Lowe. 1996. Algal Ecology: Freshwater Benthic 

Ecosystems. Academic Press, New York. 

(U.S. EPA) U.S. Environmental Protection Agency. 1991. Policy on the Use of Biological 

Assessments and Criteria in the Water Quality Program. U.S. Environmental Protection 

Agency, Office of Water, Washington, D.C. 

http://www.epa.gov/bioindicators/pdf/bioass_policy.pdf 

U.S. EPA. 2002a. National Recommended Water Quality Criteria. EPA-822-R-02-047. U.S. 

Environmental Protection Agency, Office of Water, Washington D.C. 

http://www.epa.gov/waterscience/pc/revcom.pdf 

U.S. EPA. 2002b. Consolidated Assessment and Listing Methodology, Toward a Compendium 

of Best Practices, 1st Edition. U.S. Environmental Protection Agency, Office of Water, 

Washington, D.C. http://www.epa.gov/owow/monitoring/calm.html 

U.S. EPA. 2002c. Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

1-8 
U.S. EPA. 2002d. Biological Assessments and Criteria: Crucial Components of Water Quality 

Programs. EPA 822-F-02-006. U.S. Environmental Protection Agency, Office of Water, 

Washington D.C. http://www.epa.gov/ost/biocriteria/technical/brochure.pdf 

U.S. EPA. 2003. Guidance for 2004 Assessment, Listing and Reporting Requirements Pursuant 

to Sections 303(d) and 305(b) of the Clean Water Act; TMDL-01-03. U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. 

http://www.epa.gov/owow/tmdl/tmdl0103/2004rpt_guidance.pdf 

State of Vermont. 2000. Vermont Water Quality Standards. State of Vermont, Water 

Resources Board, Montpelier, VT. http://www.state.vt.us/wtrboard/july2000wqs.htm 

Yoder, C.O., and E.T. Rankin. 1995. Biological criteria program development and 

implementation in Ohio. Pages 109-144 in W.S. Davis and T.P Simon (editors). 

Biological Assessment and Criteria: Tools for Water Resource Planning and Decision 

Making. Lewis Publishers, Boca Raton, FL. 

1-9 
2 CONNECTICUT 

This document was prepared using documents written by the State of 

Connecticut and via Personal Communication with the State 

Supervising Environmental Analyst. Any questions concerning 

bioassessment methods should be directed to: 

Ernest Pizzuto, Jr., Supervising Environmental Analyst 

Connecticut Department of Environmental Protection (CT DEP) 

79 Elm Street 

Hartford, CT 06106-5127 

Phone: (860) 424-3715; Fax ((860) 424-4055 

Email: Ernest.Pizzuto@po.state.ct.us 

2.1 Introduction 

The CT DEP Bureau of Water Management has used the benthic macroinvertebrate 

assemblage to assess the biological integrity of surface waters since the mid-1970’s and began 

using fish assemblage data in 1999 in cooperation with the CT DEP Inland Fisheries Division. 

The benthic macroinvertebrate assemblage is assessed based on the Rapid Bioassessment 

Protocol (RBP) III Single Habitat method (Plafkin et al. 1989, Barbour et al. 1999), and an index 

modified from Plafkin et al. (1989) is used to determine the level of ALUS (i.e., Full Support, 

Threatened, Partial Support, Not Supporting). Connecticut WQS state that “the benthic 

invertebrate criteria may be utilized where appropriate for assessment of the biological integrity 

of surface waters. The criteria apply to the fauna of erosional or riffle habitats in lotic waters 

which are not subject to tidal influences” (CT DEP 2002a). In addition to the biological 

component, habitat, aquatic toxicity, sediment, and ambient chemical and physical data collected 

by the Connecticut Ambient Biological Monitoring Program are used to determine compliance 

with State WQS (Table 2-1) and are ultimately used to report on the ALUS under section 305(b) 

and 303(d) of the CWA. Furthermore, the ambient monitoring program seeks to evaluate 

pollution control program effectiveness, collect data for baseline characterization and 

identification of reference conditions, assess water quality trends, evaluate ecological damage 

due to emergency pollution events, identify existing and emerging pollution problems, and 

investigate nuisance complaints (CT DEP 1999). 

2.2 Key Elements of the Biological Assessment Approach 

2.2.1 Index Period and/or Temporal Conditions 

Biological monitoring by CT DEP utilizes benthic macroinvertebrates as the primary aquatic 

assemblage for ALUS assessment purposes. Fish assemblage data have been incorporated on a 

limited basis since 1999. Based on differences in the biology of these indicator assemblages and 

logistical considerations, different index periods have been selected for their collection. Benthic 

macroinvertebrate data are collected in the late autumn (October 1-December 1). This time 

frame provides for the collection of individuals that are large enough to identify. It also 

2-1 
Table 2-1. Connecticut water quality standard classes. 

Class Management Biological Standard 

A Designated as a 

potential drinking 

water supply, fish and 

wildlife habitat, 

recreation, industrial 

supply and other 

legitimate uses, 

including navigation. 

A wide variety of macroinvertebrate taxa should 

normally be present and all functional feeding groups 

should normally be well-represented. Presence and 

productivity of aquatic species are not limited except 

by natural conditions, permitted flow regulation or 

irreversible cultural impacts. Water quality shall 

be sufficient to sustain a diverse macroinvertebrate 

community of indigenous species. Taxa within the 

orders Plecoptera (stoneflies), Ephemeroptera 

(mayflies), Coleoptera (beetles), and Trichoptera 

(caddisflies) should be well-represented. 

B Designated as a use for 

habitat for fish and 

other aquatic life and 

wildlife, recreation, 

navigation, and 

agricultural and 

industrial water supply. 

Water quality shall be sufficient to sustain a diverse 

macroinvertebrate community of indigenous species. 

All functional feeding groups and a wide variety of 

macroinvertebrate taxa shall be present; however, one 

or more may be disproportionate in abundance. 

Waters which currently support a high quality aquatic 

community shall be maintained at that high 

quality. Presence and productivity of taxa within the 

orders Plecoptera, Ephemeroptera; and pollution 

intolerant Coleoptera and Trichoptera may be limited 

due to cultural activities. Macroinvertebrate 

communities in waters impaired by cultural activities 

shall be restored to the extent practical through 

implementation of the department’s procedures for 

control of pollutant discharges to surface waters and 

through Best Management Practices (BMPs) for nonpoint sources of pollution. 

C Suitable for certain fish 

and wildlife habitat, 

recreational activities, 

industrial use, and 

other legitimate uses, 

including navigation. 

Not defined in WQS 

D May be suitable for 

bathing or other 

recreational 

purposes, certain fish 

and wildlife habitat, 

industrial uses and 

navigation. 

Not defined in WQS 

2-2 
provides the worst-case scenario for impairment of waste-receiving waters, and allows for 

conclusions of this impairment to be drawn based on the macroinvertebrate assemblage 

assessment. Fish monitoring is conducted during the summer low flow period. This is a period 

of high stress for fish assemblages in Connecticut streams, and low stream flow facilitates 

sample collection. In 2002-2003, CT DEP was funded by U.S. EPA to collect periphyton data 

for a pilot project. It was the intention of CT DEP to use the project for incorporating the 

methods developed into the ambient biological monitoring program in the future. For the pilot 

project, periphyton was collected in July and August, the period of stable flow and high 

periphyton growth rates (due to maximum ambient temperature from increased available sunlight 

and day length). 

2.2.2 Monitoring Program Survey Approach 

Water quality monitoring in Connecticut has historically employed a focused approach 

targeting major rivers and waste receiving waters. Consequently, many smaller streams 

remained unassessed. In an effort to prioritize surface water monitoring activities and increase 

monitoring coverage, a five-year rotating basin monitoring strategy was developed and 

implemented in 1997. One major drainage basin was targeted each year during the five-year 

cycle that ended in 2001. Within each basin, approximately fifty sites were sampled annually. 

Criteria used to select sites for sampling were sub-basin size, location of wastewater discharges, 

land use, and resource value. A subset of approximately 24 targeted sites was chosen each year 

to assess the fish assemblage. Additionally, an increased effort was made to incorporate data 

from volunteers, academics and municipalities. To work toward the goal of a comprehensive 

assessment, the CT DEP accepted the opportunity to participate with U.S. EPA in a two-year 

monitoring project following completion of the five-year rotating basin strategy in 2001. This 

project was conducted during 2002 and 2003 and assessed wadeable streams based on a 

statewide probabilistic design. Sample coverage included monitoring of macroinvertebrates at 

60 sites, fish at 24 sites, and periphyton at 30 sites. Water samples were collected quarterly for 

chemical analyses at the 60 sites sampled for benthos. The CT DEP is currently developing a 

Comprehensive Monitoring and Assessment Strategy to meet CWA Section 106 requirements. 

This strategy will be completed by October 2004 and will cover a ten-year period. It will include 

elements of the previous rotating basin strategy as well as a probabilistic component. 

2.2.3 Indicator Assemblages 

Currently, CT DEP primarily uses benthic macroinvertebrates as the indicator 

assemblage for biological monitoring. The CT DEP incorporates fish assemblage data using best 

professional judgment to make decisions about class attainment and is currently developing a 

Fish Index of Biotic Integrity (IBI) based on the State of Vermont model. A pilot project for 

periphyton sampling was conducted in 2002-2003, and the data were used only to supplement 

other biological data. Methods for the use of periphyton in the monitoring program are under 

development. 

2-3 
2.2.4 Reference Condition 

The CT DEP has selected reference sites to compare to test sites within each of 

Connecticut’s major basins (Figure 2-1). Those sites designated as reference have been defined 

as those that are least disturbed and minimally impacted by human influences. Furthermore, 

reference sites are selected for comparison against test sites so that the streams are within one 

stream order (± 1) and drainage areas are within one order of magnitude of one another. 

Reference sites are also used for comparison if the stream gradient is similar to that of the test 

site. The natural features of a reference site should include wadeable streams with optimal 

habitat including a hard bottom and erosional substrate (i.e., riffle habitat with cobble or gravel 

substrate). 

Figure 2-1. Major Connecticut basins sampled for the biological monitoring program 

using the rotating basin strategy. 

2-4 
2.3 Field and Laboratory Protocols 

2.3.1 Macroinvertebrate Protocols 

2.3.1.1 Field Methods 

The CT DEP uses a modified RBP Single Habitat Approach to collect macroinvertebrate 

samples (Plafkin et al. 1989, Barbour et al. 1999). After twelve riffle sampling points (“stops”) 

are chosen in a sampling reach, a rectangular kick net (9 in x 18 in) with 800-900 μm mesh, 

modified from the RBP recommended 500 μm, is placed at the bottom of each riffle with the 

opening facing upstream. An approximately 2-m2

 area of substrate upstream of the net is 

disturbed at each riffle using a kicking and stomping motion and the loosened 

macroinvertebrates are then trapped in the net. The sample is removed, the net is repositioned at 

different riffles or within the same riffle, and the process is repeated until all twelve samples are 

collected. The twelve samples are then composited into one jar, labeled and preserved with 70% 

ethanol. 

2.3.1.2 Laboratory Methods 

Benthic macroinvertebrate samples are rinsed through a #30 sieve (600 μm) to remove 

preservative and any large debris. The entire sample is then spread over a gridded tray 

containing 56 squares. Enough water is added to moisten organisms and spread the sample 

evenly. Random squares are selected to sort completely until 200 organisms (± 10%) are 

counted. After the sample is subsampled in this way, any large, rare or representative taxa are 

removed from the remaining debris. The sample is preserved with 70% ethanol. The sample is 

then identified by a qualified taxonomist using dissecting microscopes (10x-64x) to the lowest 

possible taxonomic classification using various keys and Peckarsky et al. (1990). Chironomids 

are placed in 15% KOH overnight, mounted on slides using glycerine, and then identified using a 

compound microscope. The CT DEP maintains a reference collection of taxa and any taxa 

unable to be identified by CT DEP taxonomists are verified by regional taxonomists. 

2.3.2 Periphyton Protocols (CT DEP 2003) 

2.3.2.1 Field Methods 

Two methods were used to assess benthic algae during 2002-2003. Samples were 

collected using a modified RBP Single Habitat sampling protocol (Barbour et al. 1999) for 

natural substrates to assess algal biomass and taxonomic composition. In addition, the fieldbased Rapid Periphyton Survey (RPS) (Barbour et al. 1999) was conducted. 

2.3.2.1.1 Quantitative Periphyton Sampling 

At each site, a 150-m reach is selected for assessment and fifteen pieces of substrate 

(ideally rocks) with sizes of 6.4-25.6 cm diameter are collected from throughout the reach. If 

rocks are not present, then logs and large sticks are used. The fifteen pieces of substrate are 

carried to the bank, and attached algae are scraped from a 1-in diameter area using a flat spatula 

2-5 
and a toothbrush. A rinse bottle filled with deionized water is used to wash the algal material 

into a Nalgene® collection bottle. The algae from all 15 pieces of substrate are composited into a 

single sample bottle. The bottle is placed on ice and returned to the lab where 5 ml of sample is 

removed for chlorophyll a analysis. The remaining sample is then preserved with 2% formalin 

and analyzed to determine the identification and biomass of the sampled periphyton. A duplicate 

sample is collected for quality control at 10% of sites. 

2.3.2.1.2 Rapid Periphyton Survey (Barbour et al. 1999, CT DEP 2003) 

At each site, the width of the stream is estimated to establish the number of transects that 

will be laid out (5-25). Then, transects are divided into observation points (“stops”) to be 

sampled, so that 2-10 stops are sampled at each transect. A viewing bucket with a 50 dot grid is 

immersed in the stream and at each stop the observer visually estimates and records the number 

of grid points covered by moss, macroalgae and microalgae. The observer also records the 

distance from the left bank, depth, dominant substrate, canopy cover, current velocity, size of the 

average rock/substrate within 1 ft of the observation point, and the presence or absence of 

vascular plants. All data are recorded on the CT RPS Data Sheet (CT DEP 2003). 

2.3.2.2 Laboratory Methods (CT DEP 2003) 

2.3.2.2.1 Chlorophyll a (APHA 1999) 

After arriving at the lab, the 5 ml subsample removed from the composite periphyton 

sample is filtered through a 47 mm GF/F filter with a nominal pore size of 0.7 mm. The filter is 

then stored in an aluminum foil packet and frozen at -15oC until transfer to a laboratory for 

processing (Environmental Research Institute at the University of Connecticut) within three 

weeks of filtration. The pigment is extracted from the filter using an aqueous acetone MGCO3 

solution. Chlorophyll a concentration is determined using the U.S. EPA Method AERP-12 

fluorometric method. A Turner 450 Flourometer with a 1-cm light path length is used. The 

fluorometer is calibrated by using a chlorophyll a standard of known value, which is also run by 

spectrophotometric method. The fluorescence of the extract is determined and the chlorophyll a 

concentration is calculated using the following equation: 

Chla mg/m2 = (Ca)(Extract volume in L)/(Substrate area in m2 represented by filter) 

where: 

a =Concentrat mg in Chla of ion / L =

μ Chla g 

1000L L 

Chla mg Fluoromete x reading r (instrument factor ncalibratio ) = =

Extract Volume in L = always equals 0.02 L 

Substrate area filter by drepresente =

⎛

⎜

⎝

⎜

5 filtered ml volume 

total ml in volume 

⎞

⎟

⎠

⎟(total substrate area 0076035.0 m )

2-6 

C

2 
2.3.2.2.2 Algal Identification and Density (contracted to Dr. R. Jan Stevenson 

at Michigan State University) 

A Palmer Counting Cell is used to count at least 300 cells and identify soft algae to 

species or to subspecies/variety (rarely to genus), and count live diatoms using the 

Environmental Monitoring and Assessment Program (EMAP) protocol (Lazorchek et al. 2000, 

Charles et al. 2002). Next, diatom valves are acid cleaned and mounted in NAPHRAX. Then, 

600 diatom valves are identified to species or lower level in a second count. Algal densities per 

unit area of substratum and relative abundances of algal taxa are calculated as advised in the 

RBPs for algae (Barbour et al. 1999). 

2.3.2.2.3 Biomass and Biovolume Determination 

Biomass, or Ash Free Dry Mass (AFDM), is determined using Standard Method 10300C 

(APHA 1999). Biovolumes of algae are determined by measuring at least 15 cells of each taxon 

that occurs with a relative abundance greater than 5% in any sample. For rarer taxa, fewer cells 

are measured to determine their biovolume. For taxa for which all measures cannot be made or 

taxa with less than a 1% average relative abundance among all samples, literature values or 

database values may be used to determine species biovolumes. For each sample, the relative 

biovolumes of taxonomic and functional groups (as defined by algal class and growth form: 

centric diatom, pennate diatom, filamentous cyanobacteria, filamentous green algae, etc.) are 

calculated. 

2.3.3 Fish Protocol (Plafkin et al. 1989, Hagstrom et al. 1995, CT DEP 2002b) 

Fish assemblage sampling is conducted in cooperation with the CT DEP Inland Fisheries 

Division. At each site, a 150-m reach is selected for assessing the fish assemblage. The upper 

and lower boundaries of the reach are determined by natural barriers to fish movement. The CT 

DEP varies the electrofishing equipment depending on the width of the stream to be sampled. A 

single backpack is used in the smallest streams, two backpacks are used in medium-sized 

streams, one generator towed in a canoe is used in large streams, and two or three generators are 

towed in multiple canoes for the very largest streams. In all cases, the elecrofishing crew 

(minimally consisting of three people), begin at the downstream barrier and move upstream 

collecting all species affected after one pass. All fish that are greater than 3 cm in length are 

measured, identified, and the condition and any anomalies are recorded in the field before they 

are returned to the stream. Any fish that cannot be identified in the field are sent to the 

laboratory (alive or preserved in ethanol) to be identified by a senior field biologist using 

Whitworth (1996). 

Data Management/Quality 

A Microsoft Access database is used to track all sample collection, analyses, and 

resultant metadata. All data are linked to sampling trips by unique sample identifiers and each 

location is stored as a unique point and geo-referenced. All metadata are entered in the correct 

sequence to keep sample results linked with sample metadata; electronic transfer of results is 

used whenever possible to reduce transcription error. The database is linked to ArcView GIS 

2-7 

2.4 
2.5 

software to enable the mapping and graphic analysis of data. Ultimately, all data are stored in 

the U.S. EPA Storage and Retrieval database (STORET), a repository for water quality, 

biological, and physical data. 

Analysis of Biological Data 

2.5.1 Macroinvertebrate Data 

For the macroinvertebrate assemblage, RBP III thresholds as described in Table 2.2 are 

applied to seven metrics (Plafkin et al.1989) to determine metric scores. The sum of metric 

scores is then represented as a percent of the reference total score, and the test stream is placed 

into one of four impact categories: Not Impaired, Slightly Impaired, Moderately Impaired, or 

Severely Impaired (Table 2-2). The CT DEP recognizes any test stream score of less than 54% 

as not fully supporting ALU, with a gray area lying between 50-54%. The streams in the gray 

area are placed in categories on a case-by-case basis weighted on the evidence and available 

data. 

Table 2-2. Metrics and scoring ranges used in RBP III determinations of the level of 

biological impact based on benthic macroinvertebrates (based on Plafkin et al. (1989)). 

Metric Method Scoring Ranges 

6 4 2 0 

Taxa Richness (a) The total number of distinct taxa in 

a sample 

>80% 60-80% 40-59% <40% 

EPT Index (a) The number of taxa within the 

orders of Ephemeroptera, 

Plecoptera, and Trichoptera 

>90% 80-90% 70-79% <70% 

EPT/ Chironomidae 

(abundance ratio) (a) 

(Abundance of EPT organisms)/ 

(Abundance of EPT + 

Chironomidae) 

>75% 50-75% 25-49% <25% 

HBI (modified) (b) (Number of individuals in each 

taxon multiplied by its assigned 

tolerance value)/(Total number of 

organisms in sample) 

>85% 70-85% 50-69% <50% 

Scraper/Filtering 

Collector Ratio (a) 

(Number of scrapers)/(Number of 

filtering organisms) 

>50% 35-50% 20-34% <20% 

% Contribution of 

Dominant Taxon (c) 

(Number of individuals in most 

common taxon)/(Total number of 

organisms) x 100 

<20% 20-29% 30-40% >40% 

2-8 
Metric Method Scoring Ranges 

6 4 2 0 

Community Loss 

Index (d) 

A measure of the dissimilarity 

between a test site and a reference 

site (Plafkin et al. 1989). Metric 

values increase as biological 

impairment increases. Values have 

no limits. 

<0.5 0.5-1.5 1.6-4.0 >4.0 

CLI = a - c / b 

where: a = number of genera in 

reference sample, b = number of 

genera in test sample, c = number of 

genera common to both samples 

% Total Observed Score compared to Reference Condition 

> 83% Not Impaired 

54-79% Slightly Impaired 

21-50% Moderately Impaired 

<17% Severely Impaired 

(a) Value is converted to ratio of test to reference site *100 

(b) Value is converted to ratio of reference to test site *100 

(c) Actual percent contribution used in scoring, not ratio to reference 

(d) Uses range of values actually obtained 

2.5.2 Periphyton Data 

The CT DEP is under the process of developing algae as an indicator using probabilistic 

sampling in their 2002-2003 pilot study. The data collected were used to supplement other 

biological data collected while the methods are under development. Literature values were used 

to calculate metrics during the first year of study. However, autoecological information for 

species within Connecticut populations can be generated with data from the pilot study and then 

tested using data from the second year of the study. Metrics calculated are derived from RBP 

periphyton protocols (Barbour et al. 1999). Data that are generated from the RPS and data from 

Chlorophyll a, biomass, and species composition and abundance will be evaluated to determine 

the components that will be used in the routine ambient monitoring program. 

2.5.3 Fish Data 

Although CT DEP does not currently have a fish index, they are developing a Fish Index of 

Biotic Integrity (IBI) based on the State of Vermont model. Until the IBI is developed the CT 

DEP incorporates the results of the fish assemblage data using best professional judgment to 

make decisions about class attainment. The data collected from fish assessments are species 

composition, trophic structure and age class distribution and these measurements taken from 

sampled streams are compared to those measurements in unimpaired and impaired streams to 

make inferences about the condition of the fish assemblage. 

2-9 
2.5.4 Summary: Determining ALU Support 

Connecticut narrative criteria in WQS, the macroinvertebrate quantitative index, fish data 

(while metrics are under development), and any other supplemental physical, chemical, and other 

biological data are used to make the ALU assessment (listed in Table 2-3). These assignments 

are then outlined in the 305(b) report and any streams not in attainment are placed on the 

Connecticut 303(d) list. Table 2-3 outlines the guidelines CT DEP biologists use in conjunction 

with BPJ to determine ALUS at sites. 

Table 2-3. Aquatic life use support categories and the criteria used for making decisions 

(taken from Table 2 in CT DEP 2002a). 

ALUS Criteria/Indicators 

Fully Supporting  Benthic assemblage: bioassessment indicates assemblage is nonimpaired or slightly impaired (Plafkin et al. 1989), and meets 

narrative criteria in CT WQS; RBP III Community Score ≥ 54% 

of reference condition. 

 Fish assemblage: species composition, trophic structure, and age 

class distribution as expected for a non-impacted stream of 

similar size. 

 Conventional physical/chemical criteria not exceeded. 

 Measured toxicants do not exceed chronic toxicity criteria. 

 No record of catastrophic events (e.g., chemical spills, fish kills) 

 No evidence of flow diversion 

Threatened  Benthic assemblage: non-impaired or slightly impaired, but still 

meets narrative criteria in CT WQS; RBP III Community Score 

≥54% reference condition. 

 Fish assemblage as above, but documented trend is downward or 

conditions exist that may impact the assemblage in the future. 

 Slight exceedences of either conventional or toxicant criteria in < 

10% of samples; exceedences difficult to discern from expected 

analytical variability or error. 

 Discharge effluent constitutes >20% of stream flow. 

 Land use conditions exist that may cause impairment. 

 Flow reductions due to diversions have been observed. 

Partially 

Supporting 

 Benthic assemblage: bioassessment indicates assemblage is 

moderately impaired; RBP III Community Score 21-50% of 

reference condition. 

 Fish assemblage: species composition, trophic structure and age 

class distribution significantly less than expected for nonimpacted stream of similar size: diversity and abundance of 

intolerant species reduced; top carnivores rare; trophic structure 

skewed toward omnivory. 

 Either fish or benthic assemblage meets above conditions, but the 

other assemblage is fully supporting. 

 Conventional physical/chemical criteria exceeded in > 10% but < 

2-10 
2.6 

ALUS Criteria/Indicators 

25% of samples. 

 Measured toxicants exceed chronic criteria < 10% of samples. 

 Flow is reduced significantly during drought conditions. 

Not Supporting  Benthic assemblage: bioassessment indicates assemblage is 

severely impaired: RBP III Community Score < 17% of 

reference condition. 

 Fish assemblage: species composition, age class distribution and 

trophic structure greatly impaired in comparison to a nonimpacted or minimally impacted stream of similar size; 

assemblage dominated by highly tolerant species, omnivores and 

habitat generalists; in extreme cases, few species present and/or 

diseased fish common. 

 Conventional physical/chemical criteria exceeded in > 25% of 

samples 

 Measured toxicants exceed chronic criteria >10% of samples 

 Stream known to dry completely for significant periods. 

 Documented catastrophic event (e.g., chemical spill, fish kill) 

Not Attainable Stream completely enclosed in conduit or cleared concrete trough. 

Stream is dewatered most of the time due to an upstream 

impoundment or diversion. 

Literature Cited 

(APHA) American Public Health Association. 1999. Standard Methods for the Examination of 

Water and Wastewater, 20th edition. American Public Health Association, Washington, 

D.C. 

Barbour, M, 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. 

Charles, D.F., C. Knowles and R.S. Davis (editors). 2002. Protocols for the Analysis of Algal 

Samples Collected as Part of the U.S. Geological Survey National Water-Quality 

Assessment Program. Report No. 02-06. The Academy of Natural Sciences, 

Philadelphia, PA. 

(CT DEP) Connecticut Department of Environmental Protection. 1999. Ambient Monitoring 

Strategy for Rivers and Streams: Rotating Basin Approach. CT Department of 

Environmental Protection, Bureau of Water Management, Planning and Standards 

Division, Hartford, CT. http://dep.state.ct.us/wtr/wq/rotbasinplan.pdf 

CT DEP. 2002a. Water Quality Standards. Connecticut Department of Environmental 

Protection, Bureau of Water Management, Planning and Standards Division, Hartford, 

CT. http://dep.state.ct.us/wtr/wq/wqs.pdf 

2-11 
CT DEP. 2002b. Quality Assurance Project Plan: Ambient Biological Monitoring – Fish 

Community Structure. Prepared by Michael Beauchene, CT Department of 

Environmental Protection, Bureau of Water Management, Planning and Standards 

Division, Hartford, CT. 

CT DEP. 2003. Quality Assurance Project Plan: Ambient Biological Monitoring: Periphyton 

and Chlorophyll Concentrations. Prepared by Lisa Wahle, CT Department of 

Environmental Protection, Bureau of Water Management, Planning and Standards 

Division, Hartford, CT. 

Lazorchak, J.M., B.H. Hill, D.K. Averill, D.V. Peck, and D.J. Klemm (editors). 2000. 

Environmental Monitoring and Assessment Program-Surface Waters: Field Operations 

and Methods for Measuring the Ecological Condition of Non-wadeable Rivers and 

Streams. EPA 620/R-00/007. U.S. Environmental Protection Agency, Cincinnati, OH. 

Hagstrom, N.T., M. Humphreys, W.A. Hyatt, and W.B. Gerrish. 1995. A Survey of Connecticut 

Streams and River: Statewide Summary. F-66-R: Final Report. Connecticut Department 

of Environmental Protection, Federal Aid in Sport Fish Restoration. 

Peckarsky, B.L., P.R. Fraissinet, M.A. Penton, and D.J. Conklin, Jr. 1990. Freshwater 

Macroinvertebrates of Northeastern North America. Cornell University Press, Ithaca, 

NY. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA/444/4-89-001. U.S. Environmental Protection Agency, Washington, D.C. 

Whitworth, W. 1996. Freshwater Fishes of Connecticut. CT DEP Bulletin number 114, 2nd 

edition. State Geologic and Natural History Survey, Connecticut Department of 

Environmental Protection, Hartford, CT. 

2.7 Resources 

CT DEP Web Page http://dep.state.ct.us/index.htm. 

U.S. EPA. Biological Indicators of Watershed Health, Connecticut Webpage: 

http://www.epa.gov/bioindicators/html/state/ct-bio.html 

U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

2-12 
3 MAINE 

This document was prepared using documents written by Maine 

Department of Environmental Protection personnel. Any questions 

concerning bioassessment methods should be directed to: 

Susan P. Davies, Program Manager, Biologist III 

Maine Department of Environmental Protection 

SHS 17 

Augusta, ME 04333 

Phone: (207) 287-7778; Fax (207) 287-7191 

Email: susan.p.davies@maine.gov 

3.1 Introduction 

Maine Department of Environmental Protection (MDEP) has developed a biological 

monitoring and biocriteria program to assess water quality and ensure the adherence to ALU 

designations defined in Maine’s WQS. In 1983, MDEP began its standardized benthic 

macroinvertebrate sampling program. This program began building a database to aid in the 

development of numeric biocriteria using a discriminant model approach. The numeric 

biocriteria developed were refined on a regional scale to increase the accuracy of measuring the 

adherence of streams to aquatic life use standards. The biocriteria program was written into law 

in April 1986 with the passing of M.R.S.A. 38 Public Law Chapter 698: The Classification 

System for Maine Waters (State of Maine 1985). This law required the State to “restore and 

maintain the chemical and biological integrity” of Maine waters. This law also established a 

classification system, and narrative biological and habitat criteria were described for each of 

these classes. Furthermore, the statute details specific numerical standards to which each class 

must adhere for dissolved oxygen and bacterial concentrations (Table 3-1). The water quality 

classes are AA and A, B, and C (Table 3-1). Water quality below Class C is given NonAttainment status. 

Once water bodies were assigned a discrete water quality classification (i.e., A, B, C, 

NA) and narrative aquatic life standards were established, MDEP began testing whether 

empirical and ecological data collected from Maine’s streams would show the gradients of 

environmental quality reflected in the narrative standards (Figure 3-1). They were able to 

conclude that the four established categories of biological condition did fit well with the State’s 

four-tiered standards for dissolved oxygen, bacteria and habitat (Davies et al. Draft). Multiple 

exploratory multivariate analyses were performed, including k-means clustering, 

multidimensional scaling, principal coordinate analysis, principal components analysis, multiple 

regression analysis, two-way indicator species analysis, log-linear modeling, logistic regression, 

detrended correspondence analysis, and variance component analysis, but MDEP ultimately 

chose discriminant analysis to determine the probability of a stream’s membership to an 

established class (Davies et al. Draft). 

3-1 
Table 3-1. Water quality classification system for rivers and streams in Maine. (M.R.S.A. 

Title 38 Article 4-A § 464-465). Class 

Management Narrative Habitat and 

Aquatic Life Standards 

Dissolved 

Oxygen 

Numeric 

Criteria 

Bacteria 

(E. coli) 

Numeric 

Criteria 

AA Highest quality water for 

recreation and ecological 

interests. Minimal human 

influence. No discharges or 

impoundments permitted. 

Habitat natural and free 

flowing. Aquatic life as 

naturally occurs. 

As 

naturally 

occurs 

As 

naturally 

occurs 

A High quality water with 

limited human interference. 

Discharges restricted to noncontact process water or 

highly treated wastewater 

equal to or better than the 

receiving water. 

Impoundments allowed. 

Habitat natural. Aquatic life 

as naturally occurs. 

7 ppm; 

75% 

saturation 

As 

naturally 

occurs 

B Good quality water. 

Discharge of well-treated 

effluent with ample dilution 

permitted. Impoundments 

allowed. 

Habitat unimpaired. 

Ambient water quality 

sufficient to support life 

stages of all indigenous 

aquatic species. Only nondetrimental changes in 

community composition 

allowed. 

7 ppm; 

75% 

saturation 

64/100 ml 

(geometric 

mean) or 

427/100 

ml 

(instantane 

ous level) 

C Acceptable water quality. 

Maintains the interim goals 

of the Federal Water Quality 

Act (Fishable/swimmable). 

Discharge of well-treated 

effluent permitted. 

Impoundments allowed. 

Habitat for fish and other 

aquatic life. Ambient water 

quality sufficient to support 

life stages of all indigenous 

aquatic species. Change in 

community composition may 

occur but structure and 

function of the community 

must be maintained. 

5 ppm; 

60% 

saturation 

142/100 

ml 

(geometric 

mean) or 

949/100 

ml 

(instantane 

ous level) 

* Classes AA and A are indistinguishable in the discriminant model because narrative criteria 

are both described “as naturally occurs”. 

3-2 
3.2 

Figure 3-1. Maine’s narrative aquatic life standards with the human disturbance and 

biological condition gradients (Taken from Courtemanch 2003). 

The MDEP must report the specific class attainment of each stream under Section 305(b) 

of the CWA. Those sites that are found in non-attainment must be listed on the state’s 303(d) 

list. For those streams placed on the 303(d) list, the MDEP is then expected to develop and 

implement a total maximum daily load (TMDL) for each stressor that is preventing the stream 

from reaching attainment status. 

Key Elements of the Biological Assessment Approach 

3.2.1 Index Period and/or Temporal Conditions 

It is important to select a sampling season that is indicative of the conditions needed to 

collect the most suitable data to answer the objectives for the intended study. Thus, the MDEP 

has chosen the index sampling period to be July 1 - September 30th, the low flow period for 

streams in Maine. All baseline data from streams were collected during this period because late 

summer represents the time of the year when organisms may be exposed to maximum stressful 

conditions (Davies and Tsomides 2002). During this period, water levels tend to drop, which 

may increase the concentration of soluble contaminants or nutrients in the stream, and water 

temperatures tend to be at a maximum level. 

3-3 
3.2.2 Monitoring Program Survey Approach/Natural Classification of Water 

Bodies 

Maine has divided the state into 5 main watersheds that are sampled every five years on a 

rotational basis. These basins are the Androscoggin River; Kennebec and Mid-Coast Basin; 

Penobscot, St. Croix, and North Coastal Rivers; Piscataqua, Saco, and Southern Coast; and the 

St. John and Presumpscot Basins (Figure 3-2). Although the water bodies have been divided into 

five main watersheds for monitoring purposes, exploratory data analysis concluded that it was 

not necessary to stratify the modeling approach climatically or geographically or to create 

separate southern Maine and northern Maine models (Davies et al. Draft). 

Approximately 50-60 sites are sampled each year within a single basin. Sites are chosen 

based on a “targeted approach” that incorporates a variety of factors that document the 

degradation or improvement of each stream. These factors include: 1) a prior knowledge of 

existing activities that may degrade the water body and impact the biological community; 2) a 

continued effort to monitor the effect a potential threat may have on a water body; 3) the 

requirement (or scientific endeavor) to monitor remediation activities or water quality 

management changes; and 4) to increase documentation of natural variability by including 

previously unmonitored sites (MDEP 2002). Furthermore, the rotation schedule provides 

assessment information for scheduled wastewater renewals. 

3.2.3 Indicator Assemblage 

Benthic macroinvertebrates were chosen as the biological endpoint because “they have 

limited mobility; as a group, they include species representing a wide range of pollution 

tolerances, including those found in extremely polluted sites; they are diverse and have relatively 

long, complex life cycles; they are a food source for fish; methods of sample collection and 

analysis are well-established for this assemblage; and they are a cost-effective group to sample” 

(MDEP 1995, Davies et al. Draft). 

3.2.4 Reference Condition (Establishing a priori Groups) 

The MDEP used linear discriminant analysis for model construction, which requires that 

a priori groups be established. For this reason, reference condition is more appropriately 

addressed as a discussion of a priori groups. The MDEP used best professional judgment to set 

these a priori groups (please see Section 3.5 Analysis of Biological Data and Appendix A for an 

in-depth description of a priori group assignment). 

Furthermore, MDEP does evaluate upstream and downstream of disturbed sites in order 

to collect information about the biological condition before and after a disturbance. This 

information is evaluated and used when determining the use attainment of a site (Davies et al. 

Draft). 

3-4 
3.3 

Figure 3-2. Map of basins sampled by MDEP (2002). 

Field and Laboratory Protocols 

3.3.1 Macroinvertebrate Protocols (from Davies et al. 1999, Davies and Tsomides 

2002) 

3.3.1.1 Field Methods 

The MDEP follows a highly standardized and quantitative “Classification Attainment 

Evaluation” protocol to collect data (see Davies et al. 1999, Davies and Tsomides 2002). 

Depending on water depth encountered, the MDEP uses rock baskets, riffle bags, or rock-filled 

cones to collect macroinvertebrates. Rock-filled mesh bags are used in small streams with 

depths of at least 5 cm, and rock-filled cones are used in non-wadeable rivers that must be 

accessed by boats for placement and retrieval. Rock baskets are used to sample wadeable 

streams deep enough to allow the baskets to be fully submerged. Rock baskets consist of a 

cylindrical wire barbecue basket filled with substrate. Each basket is constructed according to 

U.S. EPA methods and has at least 1.5 cm spaces between wires, with a hinged opening and a 

secure closure (Klemm et al. 1990). The substrate material is a clean cobble, relatively uniform 

in diameter ranging from 3.8-7.6 cm. Each basket contains approximately 7.25 ± 0.5 kg of 

3-5 
substrate. Rock baskets are placed in the riffle/run portion of a stream reach at a depth to ensure 

that they will remain fully submerged. The apparatus is positioned in a portion of the stream that 

reflects the overall characteristic of the stream sampled, located in the middle 50% of bank-tobank width or in an area that depicts the overall flow of the channel. At each site, a minimum of 

three baskets are used, and baskets are placed in the stream so that the long axis is parallel to 

stream flow. Samplers remain in the water for a period of 28 days (± 4 days) within the 

sampling season (typically late August/early September). However, baskets may be required to 

remain in the stream for 56 days (± 4 days) if the stream has a low velocity or is impounded. 

Baskets are placed so that influences including bridges, culverts, channelized areas, slack water 

areas, and eddies are avoided. 

During removal of the rock baskets, they are approached from downstream to minimize 

the kicking up of sediments and subsequent addition of organisms to the basket. Macrophytes, 

algae, and debris are carefully removed from the surface of the basket. A 600-μm mesh net is 

then placed against the substrate downstream of the basket and the basket is then quickly lifted 

into the net. All contents of the net and the basket are then processed through a 600-μm sieve. 

The basket and each rock are inspected to ensure the complete removal of all macroinvertebrates. 

All of the sieved materials from each basket are then placed into a separate sample jar and 

preserved with 95% ethanol and stream water to a final concentration of approximately 70% 

ethanol in the field. 

3.3.1.2 Laboratory Methods 

The MDEP requires that all samples be handled by qualified personnel working under the 

supervision of a professional aquatic biologist and that all taxonomy be performed by a person 

who has experience in freshwater macroinvertebrate taxonomy. Valid samples must yield at 

least 50 organisms each. The entire sample is sorted in small portions (1-2 tablespoons at a time) 

until no more organisms can be found. Sorting is considered complete when no organisms are 

found after 45 seconds of searching. While sorting, the sample is kept wet using water but does 

not remain unpreserved for more than eight hours. All sorted organisms are then placed in a vial 

containing a 70% alcohol and 5% glycerin solution. The detritus is returned to the sample jar 

and preserved with 70% alcohol. Any samples used for regulatory purposes are kept for five 

years. A qualified Biologist evaluates 10% of samples for sorting completeness. 

3.3.1.2.1 Subsampling and Identification 

If the mean number of organisms in a sampler is greater than 500, then subsampling may 

be performed to yield at least 100 organisms per sampler. The MDEP does note that 

subsampling will reduce sample richness by an amount that may affect the outcome when 

performing linear discriminant analysis. This subsampling effect is taken into account by 

biologists when making the final determination of classification attainment. If subsampling is 

required, MDEP follows the methods outlined by Wrona et al (1982). First, sorted 

macroinvertebrates are gently agitated in an Imhoff settling cone fitted with an aquarium bubbler 

stone for two to five minutes. Then 25% of the sample is removed from the settling cone in five 

aliquots using a settling cone and combined into one sample vial, insuring that the required 100 

organisms have been obtained. It is important that the individual performing the subsampling 

randomly dips from the cone and does not target specific organisms. Once the five aliquots are 

3-6 
combined, the sample vial is then labeled indicating the fraction that the subsample represents. 

An important precaution taken is assuring that large, dense organisms are not included in the 

subsample because they are too heavy to be suspended for capture in the subsampler cone. 

These organisms (e.g., crayfish, molluscs or caddisflies with stone cases) are counted separately. 

The MDEP has tested the randomness of the sample distribution to conclude that five aliquots 

can be combined into one sample (Elliott, 1977). After sorting, samples are identified by a 

qualified taxonomist. The MDEP recommends that all macroinvertebrates be identified to 

species; however, all numbers are adjusted to genus for use in metric calculations. In cases 

where taxonomic expertise is lacking or when the specimen is too small or in poor condition, the 

organism is identified to the lowest taxonomic level possible. Each taxonomist submitting data 

to the MDEP is required to submit a reference collection of identified taxa and a list of 

references used to identify samples. The reference collections are checked by a MDEP 

taxonomist using the MDEP’s master collection. 

3.3.1.2.2 Chironomidae Identification and Subsampling 

 Chironomid midges are identified using slide mounts of the cleared head capsule and 

body parts. For identification purposes, Euparol or Berlese mounting medium is preferred; 

however, for permanent slide mounting, MDEP recommends that CMCP-9 be used (Wiederholm 

1983). 

For any sample containing less than 100 midges, all midges are identified to the lowest 

possible taxonomic level. In samples containing 100-199 midges, a subsample containing 50% 

of randomly selected specimens for identification, and in samples containing 200-499 midges, 

25% of the specimens are randomly subsampled. The subsamples are identified to genus/species 

level, and then the unsampled midges are examined for unusual or rare specimens. Those rare or 

unusual specimens are also identified to genus/species level and kept separate from the 

subsample. If a sample contains 500 or more midges, midges are grouped by genus, and a 

random subsample of 100 organisms is selected from the remaining ungrouped midges. These 

midges are identified to species level. If any rare or unusual specimens are found after 

examination, they are identified to species level and kept separate from the subsample. 

3.4 Data Management/Quality 

The data generated (i.e., identified organisms) are entered into the database management 

system ORACLE. ORACLE stores the taxonomic code table and all sampling event data, 

computes analytical variables, and computes and reports the results of linear discriminant models 

(Davies et al. 1999). All data are checked and verified following rigorous data entry and data 

editing protocols. After entry into the database, all data are adjusted to the same taxonomic 

resolution (genus) for comparison (Davies et al. 1995). All site data in ORACLE are also 

georeferenced in ARCINFO so that spatial relationships can be studied. 

3.5 Analysis of Biological Data (Information compiled from: Davies et al. 1995, 

Davies et al. 1999, Davies and Tsomides 2002, MDEP 2003) 

The MDEP uses linear discriminant analysis to assess the attainment of aquatic life 

standards. A series of discriminant models based on macroinvertebrate metrics is used to divide 

3-7 
observations among two or more predetermined classes (i.e., A, B, and C in Maine WQS). The 

M.R.S.A. 38 Public Law Chapter 698: The Classification System for Maine Waters (State of 

Maine 1985) outlines the system for assuring both the attainment of aquatic life and that the 

objectives of maintaining the chemical, physical and biological integrity of waters are upheld by 

the state. Minimum standards for dissolved oxygen, bacteria and aquatic life for each class, as 

well as a detailed explanation of the classification system (Table 3-1), are described in this 

document. The aquatic life standards are narratively established for attainment of each class and 

are derived from measurable parameters. The results of the discriminant analysis are ultimately 

used to assign each site to a class and determine ALUS. 

The MDEP originally developed the linear discriminant models based on 145 rock basket 

samples collected from across the state and representing a range of water quality during 1983­

1989. They recalibrated the models in 1998 using a much larger macroinvertebrate database 

with a total of 376 sampling events (Davies et al. 1999). Quantifiable measures for each class 

(A, B, C, and NA) were determined (Figure 3-3). The final step involved assigning each of the 

376 sites in the database to one of four a priori groups using the quantifiable measures. 

Linear discriminant analysis requires that a priori groups be established. A priori groups 

consist of samples of “known” classification from which a predictive model can be developed to 

characterize streams with unknown classifications (Davies et al. Draft). Based on Maine’s Water 

Quality Laws, MDEP chose four groups to which streams could be assigned: AA/A, B, C and 

non-attainment (Figure 3-1). After testing multiple statistical modeling techniques (e.g., kmeans clustering, Two-Way Indicator Species Analysis, multivariate ordination), they decided 

that the use of best professional judgment of expert aquatic biologists would be the most ideal 

way to assign a priori groups. An explanation of the criteria biologists followed to establish the 

a priori groups can be found in Appendix A. Once these groups were determined subjectively 

and independently by three biologists, univariate and multivariate analysis of variance (ANOVA 

and MANOVA, respectively) confirmed that the assigned groups were statistically distinct. To 

determine variability in expert judgment assignments, a subset of data was assigned to a priori 

groups by two non-MDEP biologists, yielding an average concurrence with MDEP biologists’ 

assignments of 80%. Furthermore, to check the model, MDEP chose 27 minimally disturbed 

sites that were not originally used to build the model to determine the success of the model and 

to assign them to Class A conditions. These sites had no known point sources and land uses 

comprised 97% forested (3% logged), 2% crop and 1% residential/industrial/commercial. 

Next, 25 biological community variables were ultimately identified from a list of 400 

variables using stepwise discriminant analysis and iterative backward selection procedures to 

best predict membership of an unknown stream sample to one of the four water quality classes 

(A, B, C, and non-attainment). The 25 variables and the methods used to calculate them are 

found in Table 3-2. 

3-8 
Maine Tiered Uses Based on Measurable Ecological Values 

Narrative Standard Biological Value Quantifiable Measures 

Class A Taxonomic and numeric Similarity, Richness, 

natural equality; Presence of 

indicator taxa 

Abundance, Diversity, EPT, 

Indicator taxa, Biotic Index 

Class B Retention of taxa and Community loss, Richness, 

unimpaired, maintain 

indigenous taxa 

numbers; Absence of 

hyperdominance; 

Presence of sensitive 

Abundance, Diversity, 

Equitability, Evenness, EPT, 

Indicator taxa, Biotic Index 

taxa 

Class C Resistance; Richness, Diversity, 

maintain structure and 

function 

Redundancy; Resilience; 

Balanced distribution 

Equitability, Evenness 

Energy transfer; Trophic groups, Richness, 

Resource assimilation; Abundance, Community loss, 

Reproduction Fecundity, Colonization rate 

Figure 3-3. Maine tiered uses based on measurable ecological values (modified from 

Courtemanch 2003). 

Linear discriminant functions were developed from the 25 quantitative macroinvertebrate 

variables (Table 3-2). The discriminant functions determine the probability that a site belongs to 

a given water quality class. Using a linear optimization algorithm to calculate the discriminant 

function coefficients, multivariate space distance was minimized between sites within a class, 

while the distance between classes was maximized. The linear discriminant model consists of 

functions to compute an association value in the following form (MDEP 2003): 

Z = C +W1X1 +W2X2+.....WnXn. 

Z = discriminant score 

C = constant 

Wi = the coefficients or weights 

Xi = the predictor variable (metric) values 

For each site, 25 quantitative metrics are calculated with the data from the three replicate 

samples combined. Then, the discriminant function is calculated using one four-way model and 

three two-way models. First, using only nine variables and calculated coefficients (Table 3-3), 

the four-way model calculates the probability (range 0.0 - 1.0) that a site fits into each of the 

three attainment classes (AA/A, B, or C), and the non-attainment (NA) class. The resultant 

probabilities are then transformed and used as variables in the three two-way models (Table 3-4). 

3-9 
Table 3-2. Methods for the calculation of variables and measures of community structure 

used in linear discriminant models (from Davies and Tsomides, 2002). 

Variable Calculation Method 

1 Total Mean 

Abundance 

Count all individuals in all replicate samples from one site and divide by 

the number of replicates to yield the mean number of individuals per 

sample. 

2 Generic Richness Count the number of different genera found in all replicates from one site. 

Counting rules for Generic Richness: 

a) All population counts at the species level are aggregated to the generic 

level. 

b) A family level identification that includes no more than one taxon 

identified to the generic level is counted as a separate taxon in generic 

richness counts. 

c) A family level identification with more than one taxon identified to 

generic level is not counted towards generic richness. Counts are divided 

proportionately among the genera that are present. 

d) Higher level taxonomic identifications (Phylum, Class, Order) are not 

counted toward generic richness unless they are the only representative. 

e) Pupae are ignored in all calculations. 

3 Plecoptera Mean 

Abundance 

Count all individuals from the order Plecoptera in all replicate samplers 

from one site and divide by the number of replicates to yield mean number 

of Plecoptera individuals per basket. 

4 Ephemeroptera 

Mean Abundance 

Count all individuals from the order Ephemeroptera in all replicate 

samplers from one site and divide by the number of replicates to yield 

mean number of Ephemeroptera individuals per basket. 

5 Shannon-Wiener 

Generic Diversity 

(Shannon and 

Weaver, 1963) 

After adjusting all counts to genus following counting rules in Variable 2: 

N log( 10 i 10 nn i)log N 

c d = − ∑

Where: C= 3.321928 (converts base 10 log to base 2) 

 N= Total abundance of individuals 

ni= Total abundance of individuals in the ith taxon 

6 Hilsenhoff Biotic 

Index (Hilsenhoff, 

1987) N 

a n 

HBI ii = ∑

Where: HBI= Hilsenhoff Biotic Index 

ni= number of individuals in the ith taxon 

ai = tolerance value assigned to taxon i 

 N= total number of individuals in sample with tolerance values. 

7 Relative 

Chironomidae 

Abundance 

Calculate the mean number of individuals in the family Chironomidae, 

following counting rules in Variable 4, and divide by total mean 

abundance (Variable 1). 

3-10 
Variable Calculation Method 

8 Relative Diptera 

Richness 

Count the number of different genera from the order Diptera, following 

counting rules in Variable 2, and divide by generic richness (Variable 2). 

9 Hydropsyche Mean 

Abundance 

Count all individuals from the genus Hydropsyche in all replicate samplers 

from one site, and divide by the number of replicates to yield mean number 

of Hydropsyche individuals per basket. 

10 Probability (A + B + 

C) from First Stage 

Model 

Sum of probabilities for Classes A, B, and C from first stage model. 

11 Cheumatopsyche 

Mean Abundance 

Count all individuals from the genus Cheumatopsyche in all replicate 

samplers from one site and divide by the number of replicates to yield 

mean number of Cheumatopsyche individuals per basket. 

12 EPT- Diptera 

Richness Ratio 

EPT Generic Richness (Variable 19) divided by the number of genera from 

the order Diptera, following counting rules in Variable 2. If the number of 

genera of Diptera in the sample is 0, a value of 1 is assigned to the 

denominator. 

13 Relative Oligochaeta 

Abundance 

Calculate the mean abundance in the class Oligochaeta, following counting 

rules in Variable 4, and divide by total mean abundance (Variable 1). 

14 Probability (A+B) 

from First Stage 

Model 

Sum of probabilities for Classes A and B from the First Stage Model. 

15 Perlidae Mean 

Abundance (Family 

Count all individuals 

from the family 

Perlidae Functional Group 

Functional Group) Perlidae in all replicate 

samplers from one site. 

Divide by the number 

of replicates to yield 

mean number of 

Perlidae per basket. 

Acroneuria 

Agnetina 

Attaneuria 

Beloneuria 

Eccoptura 

Neoperla 

Paragnetina 

Perlesta 

Perlinella 

16 Tanypodinae Mean 

Abundance (Family 

Functional Group) 

Count all individuals 

from the subfamily 

Tanypodinae in all 

replicate samplers from 

one site and divide by 

the number of replicates 

to yield mean number 

of Tanypodinae per 

basket. 

Tanypodinae Functional Group 

Ablabesmyia 

Clinotanypus 

Coelotanypus 

Conchapelopia 

Djalmabatista 

Guttipelopia 

Husonimyia 

Labrundinia 

Larsia 

Meropelopia 

Natarsia 

Niltanypus 

Paramerina 

Pentaneura 

Procladius 

Psectrotanypus 

Rheopelopia 

Tanypus 

Telopelopia 

Thienemannimyia 

Trissopelopia 

Zavrelimyia 

3-11
Variable Calculation Method 

17 Chironomini Mean 

Abundance (Family 

Functional Group) 

Count all individuals in 

the tribe Chironomini in 

all replicate samplers 

from one site and divide 

by the number of 

replicates to yield the 

mean number of 

Chironomini per basket. 

Chironomini Functional Group 

Axarus 

Chironomus 

Cryptochironomus 

Cladopelma 

Cryptotendipes 

Demicriptochirono-mus 

Dicrotendipes 

Endochironomus 

Einfeldia 

Gyptotendipes 

Harnishchia 

Kiefferulus 

Lauterborniella 

Microchironomus 

Microtendipes 

Nlothauma 

Parachironomus 

Paracladopelma 

Paralauterborniella 

Paratendipes 

Paenopsectra 

Polypedilum 

Pseudochironomus 

Pagastiella 

Robackia 

Stelochomyia 

Stenochironomus 

Stictochironomus 

Tribelos 

Xenochironomus 

18 Relative 

Ephemeroptera 

Abundance 

Variable 4 divided by Variable 1. 

19 EPT Generic 

Richness 

Count the number of different genera from the orders Ephemeroptera, 

Plecoptera, and Trichoptera in all replicate samplers, according to counting 

rules in Variable 2, generic richness. 

20 Variable Reserved* 

21 Sum of Mean 

Abundances of: 

Dicrotendipes, 

Microspectra, 

Parachironomus and 

Helobdella 

Sum the abundances of the 4 genera and divide by the number of replicates 

(as performed in Variable 4). 

22 Probability of Class 

A from First Stage 

Model 

Probability of Class A from First Stage Model 

23 Relative Plecoptera 

Richness 

Count number of genera of Order Plecoptera, following counting rules in 

variable 2, and divide by generic richness (Variable 2). 

24 Variable Reserved* 

3-12 
Variable Calculation Method 

25 Sum of Mean 

Abundances of 

Cheumatopshyche, 

Cricotopus, 

Tanytarsus and 

Ablabesmyia 

Sum of the number of individuals in each genus in all replicate baskets and 

divide by the number of replicates (as performed in Variable 4). 

26 Sum of Mean 

Abundances of 

Acroneuria and 

Stenonema. 

Sum the number of individuals in each genus in all replicate baskets and 

divide by the number of replicates (as performed in Variable 4). 

27 Variable Reserved* 

28 Ratio of EP Generic 

Richness 

Count the number of different genera from the orders Ephemeroptera (E), 

and Plecoptera (P) in all replicate baskets, following counting rules in 

Variable 2, and divide by 14 (maximum expected for Class A). 

29 Variable Reserved* 

30 Ratio of Class A 

Indicator Taxa 

Count the number 

of Class A 

indicator taxa that 

are present in the 

sample and 

divide by 7 (total 

possible number). 

Indicator Taxa: Class A 

Brachycentrus (Trichoptera Brachycentridae) 

Serratella (Ephemeroptera: Ephemerellidae) 

Leucrocuta (Ephemeroptera: Heptageniidae) 

Glossosoma (Trichoptera: Glossosomatidae) 

Paragnetina (Plecoptera: Perlidae) 

Eurylophella (Ephemeroptera: Ephemerellidae) 

Psilotreta (Trichoptera: Ondontoceridae) 

*These variable numbers are not used in discriminant models. 

3-13 
Table 3-3. Coefficients for the First Stage Model (from MDEP 2003). 

Variable # Coefficients 

Transformation Class A Class B Class C Nonattainment 

Constant -99.95508 -105.70948 -112.67581 -107.74283 

1 ln (value + 0.001) 10.77061 11.46981 11.80888 11.26793 

2 -0.38619 -0.43340 -0.50051 -0.48822 

3 ln (value + 0.001) 0.23940 0.03946 -0.60923 -0.95480 

4 ln (value + 0.001) -0.59970 -0.55500 -0.67722 -1.79032 

5 21.22732 20.91256 21.07602 19.46547 

6 8.01620 9.12163 10.31492 10.72746 

7 ln (value + 0.001) -11.70298 -11.52650 -11.49414 -11.66371 

8 70.77937 71.09637 72.46514 70.22517 

9 -0.00535 -0.00398 -0.00152 0.00007 

Table 3-4. Coefficients for the Final Classification Models (AA/A, B, and C) (MDEP 2003). 

Class C or Better Model 

Variable # Coefficients 

Transformation Class A-B-C Nonattainment 

Constant -25.70020 -8.55844 

10 Arcsin 19.98470 3.36032 

11 ln (value + 0.001) -0.26001 -0.43781 

12 Square Root 5.57672 5.92732 

13 ln (value + 0.001) -2.33229 -1.89945 

Class B or Better Model 

Variable # Coefficients 

Transformation Class A-B Nonattainment 

Constant -17.81016 -6.93836 

14 Arcsin 12.04826 3.63707 

15 ln (value + 0.001) -1.11091 -1.03934 

16 ln (value + 0.001) -0.10582 0.01978 

17 ln (value + 0.001) 0.17813 0.10825 

18 4.03202 -1.14508 

19 0.87400 0.63310 

21 ln (value + 0.001) -0.69360 -0.53194 

3-14 
Class A Model 

Variable # Coefficients 

Transformation Class A-B-C Nonattainment 

Constant -9.59254 -4.08552 

22 Arcsin 8.34341 1.52080 

23 3.78999 4.27447 

25 ln (value + 0.001) 0.53110 0.77851 

26 ln (value + 0.001) -0.55838 -0.51448 

28 12.32529 9.81592 

30 6.94828 -0.67475 

The next step is to use three two-way models to test the probability of a site belonging to 

class AA/A, B, or C. These models distinguish between a given class plus any higher classes as 

a group and any lower classes as a group (i.e., Classes AA/A + B + C vs NA; Classes AA/A + B 

vs Class C + NA; Class AA/A vs Classes B + C + NA). The three models are applied using the 

equations in Table 3-4 (MDEP 2003, Davies and Tsomides 2002). 

The discriminant scores (Z) are known as the Mahalanobis Distances (MDEP 2003) 

where: 

Mahalanobis Distance = Zt (sample x) = g1 (x,t) + g2 (t) 

And where: 

Zt = discriminant score for sample x, class t 

g1 (x,t) = (x- mt) S-1 (s-mt) 

g2 (t) = -2 loge (qt) = 0 (if prior probabilities are equal) 

x = a vector containing all the values of all the variables for a given linear discriminant 

function, for a given sample, of class t 

mt = a vector, as for x, but containing the means of all predictor variables in the given linear 

discriminant function, for the given sample, of class t 

S = pooled covariance matrix (the variance of the multivariate observation) 

qt = value of the prior probability that a given sample is Class A, B, C, or NA 

The probability (association value) of a sample x belonging to a particular class t is: 

3-15 
e[. t −0 5Z x( )] 

Px NA () = t 

[ . −0 5Z A B C NA ( x)] ] ,,, ∑ [e 

A 

Where: 

Pt(x) = the probability that sample x belongs to class t (for Classes AA/A, B, C, NA) 

e = the exponential function 

-0.5 = a standardized constant from the normal distribution 

Zt = the discriminant score or Mahalanobis Distance for class t (Classes AA/A, B, C, NA) 

Once the probability that a site belongs to a certain class is calculated, the MDEP follows 

a process to determine whether the site attains at least that particular class (Davies and Tsomides 

2002, MDEP 2003). In order to determine whether a site attains at least Class C or is in nonattainment, the association value (ZC) calculated using the ‘C or better model’ needs to be used. 

If the association value is greater than 0.6, then the sample attains to Class C or higher, but if it is 

less than 0.4 then the site is in non-attainment. If a site falls within 0.4 - 0.6, then best 

professional judgment is used to determine if the site belongs in Class C, is in non-attainment, or 

is indeterminate of Class C. For any site found to be indeterminate, additional monitoring may 

be needed in order to make a decision. 

Those samples that do attain to at least Class C are then tested for Class B attainment 

using the association values (ZB) from the ‘B or better model’. If the association value is greater 

than 0.6 then the sites are at the minimum attaining to Class B status. Those values below 0.4 

are now considered to be sites that attain to Class C. If a value falls between 0.4 - 0.6 then it is 

indeterminate of Class B and may require additional monitoring to determine to which 

attainment class the site belongs. 

When the association value for a site is 0.6 or greater using the Class B or better model, it 

is then tested using the ‘A or better model’. If the association value (ZA) is 0.6 or greater, then 

the class attains to A. If the value is 0.4 or less, then the class attains to Class B. If the value is 

between 0.4 - 0.6, the finding is indeterminate of Class A and additional sampling may be 

required. Figure 3-4 graphically shows the process for calculating model variables and 

association values using linear discriminant models and Figure 3-5 shows how the attainment 

classes are determined using association values (MDEP 2003). 

After a site is placed into an aquatic use attainment class, a provision in MDEP regulation 

Chapter 579 (MDEP 2003) allows for professional judgment to make an adjustment to the 

evaluation. Any adjustment may be made using analytical, biological, and habitat data. 

Professional judgment also may be employed when the condition of the stream does not allow 

for the accurate use of the linear discriminant models. Such factors may include habitat 

influences (e.g., lake outlets, impounded waters, substrate characteristics, tidal waters), sampling 

issues (e.g., disturbed samples, unusual taxa assemblages, human error in sampling), or 

analytical and sample processing issues (e.g., subsample vs. whole sample analysis or human 

error in processing) (Davies and Tsomides 2002). 

If a water body falls into a lower class than its assigned statutory class after a MDEP 

biologist determines that adjustment is not needed, then the site is determined to be in nonattainment of its statutory class. When a site is found to be in non-attainment of its legally 

3-16 
3.6 

defined class, then certain actions must be taken that include: 1) the notification of other 

programs (e.g., Licensing or Land Use Regulation); 2) the listing of the stream in question on the 

303(d) list of impaired water bodies; and 3) the development of a total maximum daily load 

(TDML) for pollutants. 

If a water body falls into a higher class than its assigned statutory class, then the higher 

aquatic life conditions must be maintained if the finding confirmed under critical water quality 

conditions. This finding also requires certain actions to ensure the findings were accurate and 

include: 1) the confirmation of the finding by resampling; 2) the confirmation that higher aquatic 

life quality exists at the maximal pollution loads; and 3) MDEP can propose that the water body 

be upgraded if dissolved oxygen, bacteria and habitat standards also adhere to the next higher 

class (MDEP 2002). Furthermore, data collected from two or more sampling events over 

different seasons must be evaluated to determine if a site has improved in quality and moved into 

the next higher statutory class. 

All results and classification attainment decisions made by MDEP must be reported in the 

State of Maine Water Quality Assessment Report that is required under Section 305(b) of the 

CWA. If a water body is found to be in non-attainment of its statutory classification, then it is 

placed on Maine’s list of impaired waters as required in Section 303(d) of the CWA. 

Literature Cited 

Courtemanch, D. L. 2003. RFC 101: Development of Reference Conditions for Management 

Classes. National Biological Assessment and Criteria Workshop: Advancing State and 

Tribal Programs. Coeur d’Alene, ID. March 31-April 3, 2003. 

http://www.epa.gov/waterscience/biocriteria/modules/rfc101-07-maine.pdf 

Davies, S.P., L. Tsomides, D.L. Courtemanch, and F. Drummond. 1995. Maine Biological 

Monitoring and Biocriteria Development Program, 2nd edition. Maine Department of 

Environmental Protection, Augusta, ME. 

Davies, S.P., L. Tsomides; J.L. DiFranco and D.L. Courtemanch. 1999. Biomonitoring 

Retrospective: Fifteen Year Summary for Maine Rivers and Streams. Maine Department of 

Environmental Protection, Augusta, ME. 

http://www.state.me.us/dep/blwq/docmonitoring/biomonitoring/biorep2000.htm

 Davies, S.P. and L. Tsomides. 2002. Methods for the Biological Sampling and Analysis of 

Maine’s Rivers and Streams. Maine Department of Environmental Protection, Augusta, ME. 

http://www.state.me.us/dep/blwq/docmonitoring/finlmeth1.pdf 

Davies, S.P., L. Tsomides, D.L. Courtemanch, and F. Drummond. Draft. Stream Biological 

Monitoring and Numeric Criteria Development in Maine. Maine Department of 

Environmental Protection, Augusta, ME. 

Elliot, J.M. 1977. Some Methods for the Statistical Analysis of Samples of Benthic 

Macroinvertebrates. Freshwater Biological Association, Science Publication No. 25. 

3-17 
1.1 “C or Better” Second Stage LDM

(2-way model: A, B, or C vs. NA)1. .Model calculates Discriminant Score

using Var10 (pA1+pB1+pC1) and 

Var11 – Var13.

2. Model uses Discriminant Score to

calculate the following AssociationValues:

• probability C or better (pABC) 

• probability NA (pNA)

1.2 “B or Better” Second Stage LDM(2-way model: A or B vs. C or NA)1. .Model calculates Discriminant Score

using Var14 (pA1+pB1) and Var15 –

Var21.

2. .Model uses Discriminant Score to

calculate the following AssociationValues1:

• probability B or better (pAB) 

• probability C or NA (pCNA) 

Computer calculates model variables (Var1 – Var30) 

using taxa counts from a sample event usingprocedures described in Table 3-2.

First Stage Linear Discriminant Model (LDM)(4-way model: A vs. B vs. C vs. NA)

1. Model calculates Discriminant Score using Var1 – Var9.

2. Model uses Discriminant Score to calculate the following

Association Values1:

• probability Class AA/A (pA1) 

• probability Class B (pB1) 

• probability Class C (pC1) 

• probability Nonattainment (pNA1) 

1.3 “A” Second Stage L(2-way model: A vs. B, C, 1. .Model calculates Di

using Var22 (pA1) a

2. .Model uses Discrim

calculate the followiValues1:

• probability A

• probability B

(pBCNA) 

1.1 “C or Better” Second Stage LDM(2-way model: A, B, or C vs. NA)1. .Model calculates Discriminant Score

using Var10 (pA1+pB1+pC1) and 

Var11 – Var13.

2. Model uses Discriminant Score to

calculate the following AssociationValues:

• probability C or better (pABC) 

• probability NA (pNA)

1.2 “B or Better” Second Stage LDM(2-way model: A or B vs. C or NA)1. .Model calculates Discriminant Score

using Var14 (pA1+pB1) and Var15 –

Var21.

2. .Model uses Discriminant Score to

calculate the following AssociationValues1:

• probability B or better (pAB) 

• probability C or NA (pCNA) 

Computer calculates model variables (Var1 – Var30) 

using taxa counts from a sample event usingprocedures described in Table 3-2.

First Stage Linear Discriminant Model (LDM)(4-way model: A vs. B vs. C vs. NA)

1. Model calculates Discriminant Score using Var1 – Var9.

2. Model uses Discriminant Score to calculate the following

Association Values1:

• probability Class AA/A (pA1) 

• probability Class B (pB1) 

• probability Class C (pC1) 

• probability Nonattainment (pNA1) 

1.1 ( ) 1. 

(pA1+pB1+ )

. 

2. 

• pABC) 

• pNA) 

1.2 

) 

1. 

(pA1+pB1 Var15 – 

. 

2. 

1: 

• (pAB) 

• ( ) 

Var1 – ) 

. 

(

( ) 

– . 

2. 

1: 

• ) 

• (pB1) 

• (pC1) 

• (pNA1) 

1.3 ( 1. 

(pA1 

2. 

1: 

• 

•

( ) 

“C or Better” Second Stage LDM 

2-way model: A, B, or C vs. NA

.Model calculates Discriminant Score 

using Var10 pC1 and 

Var11 – Var13

Model uses Discriminant Score to 

calculate the following Association Values: 

probability C or better (

probability NA (

“B or Better” Second Stage LDM 

(2-way model: A or B vs. C or NA

.Model calculates Discriminant Score 

using Var14 ) and 

Var21

.Model uses Discriminant Score to 

calculate the following Association Values

probability B or better

probability C or NA pCNA

Computer calculates model variables ( Var30using taxa counts from a sample event using procedures described in Table 3-2

First Stage Linear Discriminant Model LDM) 

4-way model: A vs. B vs. C vs. NA

1. Model calculates Discriminant Score using Var1 Var9

Model uses Discriminant Score to calculate the following 

Association Values

probability Class AA/A (pA1

probability Class B 

probability Class C 

probability Nonattainment 

“A” Second Stage L

2-way model: A vs. B, C, 

.Model calculates Di

using Var22 ) a

.Model uses Discrim

calculate the followiValues

probability A

probability B

pBCNA

Figure 3-4. Process of calculating model variables and association values using linear discriminant models 2003). 3-18 
YES NO 

Use BPJ 

≥ ≥0.4

At least C NA NA

e 

pAB ≥ ≥ 0.4

At least B C C

pA ≥ 0.6 ≥

A A B B

e 

Is the sample appropriate for Linear Discriminant Model? 

Is the sample Class C or better? 

pABC 0.6 0.6> pABC pABC < 0.4 

At least C Indeterminat 

Is the sample Class B or better? 

 0.6 0.6 > pAB pAB < 0.4 

At least B Indeterminate 

Is the sample Class A? 

0.6 > pA 0.4 pA < 0.4 

Indeterminat 

Figure 3-5. Process for determining attainment class using association values (modified 

from MDEP 2003). 

3-19 
Klemm, D.J., P.A. Lewis, F. Fulk, and J.M. Lazorchak. 1990. Macroinvertebrate 

and Field Methods for Evaluating the Biological Integrity of Surface Water. 

EPA/600/4-90/030. U.S. Environmental Protection Agency, Cincinnati, OH. 

(MDEP) Maine Department of Environmental Protection. 2002. Board of 

Environmental Protection Meeting, Frequently Asked Questions. Unpublished 

material. 

MDEP. 2003. Classification Attainment Evaluation Using Biological Criteria for Rivers and 

Streams. Rules of Maine State Government Agencies, 06 096 Chapter 579. 

http://www.maine.gov/sos/cec/rcn/apa/06/096/096c579.doc 

State of Maine. 1985. Maine Laws Ch. 698 § 15 (in part). An Act to Amend the 

Classification System for Maine Waters and Change the Classifications of 

Certain Waters. Augusta, Maine. 

Wiederholm, T. 1983. Chironomidae of the Holarctic Region. Entomologica 

Scandinavica, Suppl. No. 19. 

Wrona, F.J., J.M. Culp and R.W. Davies. 1982. Macroinvertebrate subsampling: 

a simplified apparatus and approach. Canadian Journal of Fisheries and 

Aquatic Science 39:1051-1054. 

3.7 Resources 

Maine Department of Environmental Protection, Biological Monitoring Web Page: 

http://www.state.me.us/dep/blwq/docmonitoring/biomonitoring/index.htm 

Maine Revised Statutes Annotate. Title 38, Chapter 3: Protection and Improvement of Waters, 

Sections 464 and 465. http://janus.state.me.us/legis/statutes/38/title38ch3sec0.html 

U.S. EPA. Biological Indicators of Watershed Health, Maine Webpage: 

http://www.epa.gov/bioindicators/html/state/me-bio.html 

U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria 

Development for States, Tribes, Territories, and Interstate Commissions: 

Streams and Wadeable Rivers. EPA-822-R-02-048. U.S. Environmental 

Protection Agency. 

3-20 
4 MASSACHUSETTS 

This document was prepared using documents written by the 

Massachusetts Department of Environmental Protection. Any 

questions concerning bioassessment methods should be directed to: 

Arthur Johnson, Environmental Monitoring Coordinator 

Massachusetts Department of Environmental Protection (MA 

DEP) 

627 Main Street 

Worcester, MA 01608 

Phone: (508) 767-2873; Fax (508) 791-4131 

Email: Arthur.Johnson@state.ma.us 

4.1 Introduction 

As required by the CWA, MA DEP submits a biennial 305(b) report that describes the 

status of the state’s water resources with respect to the classes defined by the Massachusetts 

Surface Water Quality Standards. The Massachusetts Surface Water Quality Standards assign 

support classes according to intended use, which include aquatic life, fish and shellfish 

consumption, drinking water supply, primary recreational contact, and secondary recreational 

contact (314 CMR 4.00 2000, Table 4-1). These standards were set to account for the most 

severe hydrologic conditions. In rivers, the standards must apply to waters at or above the sevenday ten-year flow statistic (7Q10). In regulated water systems, the standards must apply to the 

lowest discharge that meets or exceeds criteria 99% of the time on a yearly basis and any 

alternatives must be approved by the MA DEP Commissioner or the entity controlling flow (MA 

DEP 2003). 

The ALUS criteria of the standards require that a suitable habitat be provided, “to sustain 

a native, naturally diverse community of aquatic flora and fauna” (MA DEP 2003). For 

assessment purposes each attainment class is composed of two sub-classes, the cold water fishery 

(capable of sustaining a year-round population of cold water aquatic organisms) and the warm 

water fishery (not capable of sustaining a year-round population of cold water organisms). Each 

stream that is assessed is designated as fully supporting, partially supporting, or non-supporting 

for its ALU class. Sites that are designated as “fully supporting” for a particular attainment class 

also may be given a threatened status if the stream is in danger of becoming polluted within a 

two-year period. Sites with too little or no data are not assessed. 

The MA DEP uses biocriteria to aid in the assessment of the ALUS of surface waters. 

Macroinvertebrate, periphyton, and fish data are used by Massachusetts State Biologists to aid in 

determining ALUS attainment. Furthermore, MA DEP combines the biological data with habitat 

evaluations, toxicological and chemical data, and other environmental variables in order to make 

a final ALUS decision. 

4-1 
4.2 

Table 4-1. Massachusetts attainment classes with management strategy and narrative 

biological and habitat criteria as stated in 314 CMR 4.00 (2000). Class

Management Biological and Habitat Narrative 

Criteria 

A These waters are designated as a source of public 

water supply. To the extent compatible with this use 

they shall be an excellent habitat for fish, other 

aquatic life and wildlife, and suitable for primary 

and secondary contact recreation. These waters shall 

have excellent aesthetic value. These waters are 

designated for protection as Outstanding Resource 

Waters under 314 CMR 4.04(3). 

Excellent habitat for fish, other 

aquatic life and wildlife, supporting 

normal species diversity, successful 

migration, reproductive functions or 

growth of aquatic organisms. 

B These waters are designated as a habitat for fish, 

other aquatic life, and wildlife, and for primary and 

secondary contact recreation. Where designated they 

shall be suitable as a source of public water supply 

with appropriate treatment. They shall be suitable 

for irrigation and other agricultural uses and for 

compatible industrial cooling and process uses. 

These waters shall have consistently good aesthetic 

value. 

C These waters are designated as a habitat for fish, 

other aquatic life and wildlife, and for secondary 

contact recreation. These waters shall be suitable for 

the irrigation of crops used for consumption after 

cooking and for compatible industrial cooling and 

process uses. These waters shall have good aesthetic 

value. 

These waters are designated as a 

habitat for fish, other aquatic life 

and wildlife, protect normal species 

diversity, successful migration, 

reproductive functions or growth of 

aquatic organisms. 

Key Elements of the Biological Assessment Approach 

4.2.1 Index Period and/or Temporal Conditions 

Originally, in order to represent the “worst-case” scenario, all sampling events were 

performed during low-flow, dry-weather, high-stress conditions. However, when MA DEP 

began to consider the impact of non-point source pollution, they altered the schedule to include a 

larger range of weather conditions. Typically, the sampling season is July through September 

and this sampling schedule is consistent from year to year so that data collected from surveys can 

be compared to make historical inferences. This time frame also coincides with streams 

returning to base flow conditions and occurs after the spawning season for most fish species 

present within the state. 

4.2.2 Monitoring Program Survey Approach 

The protocol of the MA DEP biomonitoring program divides the state into 27 major 

watersheds and coastal drainage areas that are sampled using a five-year basin rotation strategy 

4-2 
(MA DEP 2003, Figure 4-1). Within each basin, the first year of the program involves 

reconnaissance of basins and research to identify available data and/or information gaps. During 

the second year, streams are sampled and data are collected according to a MA DEP Quality 

Assurance Project Plan (QAPP). These sites are targeted based on known or suspected water 

quality degradation. In the third year, the stream assessments are made and published in State 

documents and/or 305(b) reports. Over the next two years, other watershed management issues 

are targeted (e.g., TMDL calculations, permit issuance, targeting of problem watersheds, 

outreach program implementation). In order to determine ALUS, MA DEP samples 

approximately 75 streams per year. 

4.2.3 Natural Classification of Water Bodies 

Massachusetts contains three Level III Ecoregions: the Northeastern Highlands, the 

Northeastern Coastal Zone, and the Atlantic Coastal Pine Barrens (Griffith et al. 1994). The 

Northeastern Highlands contains seven Level IV ecoregions (Taconic Mountains, Western New 

England Marble Valleys, Green Mountain/Berkshire Highlands, Lower Berkshire Hills, 

Berkshire Transition, Vermont Piedmont, and Worcester/Monadnock Plateau). The Northeastern 

Coastal Zone contains five Level IV ecoregions (Connecticut Valley, Lower Worcester 

Plateau/Eastern Connecticut Upland, Southern New England Coastal Plains and Hills, Boston 

Basin, and Narraganset/Bristol Lowland). The Atlantic Coastal Pine Barrens contains one Level 

IV ecoregion (Cape Cod/Long Island). For sampling purposes, the state is assessed on a basinlevel scale (Figure 4-1), which may incorporate more than one Level IV ecoregion (Figure 4-2). 

Cold water and warm water streams are assessed as separate categories. 

4.2.4 Indicator Assemblages 

Biological, toxicological and chemical data are collected and used to make ALUS 

decisions based on a “weight of evidence” approach. Biological indicators include assessments 

of the macroinvertebrate, fish, and algal communities. An index based on the RBP II and III 

(Plafkin et al. 1989) is used to assess the macroinvertebrate assemblage. The overall structure 

and condition of the fish assemblage is assessed using some measurements from the RBP V 

(Plafkin et al. 1989). Water quality condition is also assessed using algal measurements (i.e., 

Chlorophyll a concentration, percent cover of green algae, and biomass). 

4.2.5 Reference Condition (Arthur Johnson, Personal Communication) 

Reference sites are identified by examining criteria and by using the best professional judgment 

of MA DEP Biologists. Sites that are described as “least impacted” have minimal or no potential 

to receive point or non-point source pollution and lack land use patterns that would degrade 

water or habitat quality. Maps and field reconnaissance are both used to locate unique reference 

sites when RPBs are used to assess a site. Multiple reference sites often exist for a study and 

occasionally reference sites of adjoining watersheds of the study site may be used. Sites are only 

considered reference for streams of similar elevation and drainage area. 

4-3 
4.3 

Figure 4-1. Massachusetts 5-Year Basin Rotation Strategy (taken from the Massachusetts 

Department of Environmental Protection website 

www.mass.gov/dep/brp/wm/files/cyclemap6.jpg). 

Field and Laboratory Protocols 

4.3.1 Macroinvertebrate Protocols (Taken from Nuzzo 2003) 

4.3.1.1 Field Methods 

The MA DEP uses one of three different methods to collect macroinvertebrates, 

depending on the depth and substrate of the stream. Kick sampling is the most commonly used 

method and is designed for use in wadeable streams that have coarse substrates, rock baskets are 

used in streams that are not wadeable or have fine substrates and for studies that require 

quantitative measurements, and Hester-Dendy multi-plate samplers are used in deep rivers. Each 

of the methods requires a habitat assessment to accompany the macroinvertebrate data to identify 

problematic areas and habitat destruction or loss. The MA DEP uses a visual-based rapid habitat 

assessment method (as described in Barbour et al. 1999) that includes ten habitat categories that 

are rated from 0 (lowest) to 20 (optimal). 

4-4 
Figure 4-2. Level III and Level IV Ecoregions of Massachusetts (taken from Griffith et al. 1994, 

http://www.epa.gov/wed/pages/ecoregions/mactri_eco.htm). 
4.3.1.1.1 Kick Sampling 

Kick sampling is used for routine biomonitoring in wadeable streams with coarse 

substrates and riffle habitats. A 100-meter reach is chosen to represent the best habitat for the 

particular sample area. Then, a 0.46-m x 0.46-m, 500-μm mesh kick net is placed into the riffle 

and pressed firmly against the bottom so that the net is facing upstream. A biologist then kicks 

upstream in an area approximately the same size as the kick net, allowing material to be caught 

in the net. Following a kick (minimally 30 seconds), the net is examined, any 

macroinvertebrates residing on large debris are rinsed off into the net, and the excess debris is 

then placed back into the stream. In streams where the riffles within the reach are inadequate to 

allow for a 2-m2

 composite, other productive habitats are sampled by jabs into snags and/or by 

rubbing substrates. The contents of the net are then emptied into a 2-L wide-mouth leak-proof 

Nalgene bottle. Ten kick samples (approximately equaling 2-m2

) are collected at a site and 

composited into one bottle. The contents are preserved with enough denatured 100% reagent 

alcohol (5% methanol, 5% isopropanol, 90% ethanol) to cover the residue. If preservative is not 

added to the sample, it must be placed on ice and processed within 48 hours of collection. 

4.3.1.1.2 Rock Basket Sampling 

When kick sampling is deemed inappropriate for stream biomonitoring, rock baskets are 

used to collect benthic macroinvertebrates. Situations where rock baskets may be employed 

include planned statistical treatments of benthos data requiring quantitative collection methods, 

water depth too great for kick sampling, and substrate which is too fine for kick sampling at one 

or more sites used for comparison studies. The MA DEP uses rock baskets that are filled with 

roofing stone. Each basket is approximately the same weight and uses 2.5 -7.5 cm sized rocks. 

Three baskets are placed separately at the bottom of the stream in a riffle for six to eight weeks 

(in a current velocity of 15 - 76 cm/s). Upon retrieval, a kick-net is pressed tightly against the 

stream bed along the edge of the basket’s downstream edge. The basket is then lifted onto the 

net. If the removal is difficult due to water depth, a cover is draped over the basket 

(Courtemanch 1984). After removal, each basket is placed into a separate large bucket or tub of 

water and then opened. All of the contents are emptied into the bucket and each rock is rinsed 

and set aside. After all of the rocks have been rinsed, the contents of each bucket are run through 

a #30 mesh (600 μm) and then placed into a sample container and preserved with denatured 

100% reagent alcohol. If the sample is to be processed within 48 hours, the sample is put on ice 

without preservative. 

4.3.1.1.3 Hester-Dendy Multi-plate Sampling 

Hester-Dendy multi-plate samplers are used in deep rivers or where kick sampling is 

inappropriate and rock baskets are impractical. The MA DEP uses round-plate samplers as 

specified by U.S. EPA (Klemm et al. 1990). The samplers are placed in deep waters and 

tethered to an anchored float so that the sampler is suspended 1 m below the surface. Samplers 

placed in shallow water are mounted to a patio block or 4-in cinder blocks. In order for 

comparable data to be collected, each sampler is placed in a riffle of a similar velocity (within 

the range of 15 and 76 cm/s). Three Hester-Dendy multi-plate samplers are independently 

deployed in a stream at the same time for six to eight weeks. When they are retrieved, samplers 

4-6 
are completely surrounded with a 500-μm mesh net, a plastic bag, or a 2-L wide-mouth jar and 

pulled from the water. Enough water is placed in the 2-L wide-mouth jar to completely cover 

each sampler and then it is tightly capped and placed on ice and labeled. All multi-plate 

samplers are removed, placed in separate bottles in a refrigerator, and processed within 48 hours. 

After processing, samples are preserved with 100% reagent ethanol. 

4.3.1.2 Laboratory Methods (Nuzzo 2003) 

4.3.1.2.1 Processing of Kick Net and Rock Basket Samples 

The MA DEP uses RBPs to process the macroinvertebrate data collected using kick nets 

or rock baskets (RBP II and RBP III; Plafkin et al. 1989) to obtain 100 organisms in the 

subsample. Each sample to be sorted is first run through a 600-μm mesh sieve and held over a 

waste collection vessel to collect the decanted alcohol preservative, then rinsed three times with 

enough water so that the sample is washed free of any preservative. Following rinsing, the 

sample is left to drain in the sieve for one minute and then the sample is spread evenly across a 

gridded pan that contains 25 squares 6-cm x 6-cm. Multiple pans are used to distribute the 

material evenly and to reduce the density of organisms per grid to a workable number (e.g., pan 

A contains grids 1-25, and pan B contains grids 26 - 50). Sufficient water is then added to 

distribute the material evenly across the pans. Next, the sorter chooses random numbers within 

the range of the sum of total squares used to hold the sample. Once a random grid square is 

chosen, only the material from that square is removed and it is placed into a petri dish that has 

been divided into quarters. One of the quadrants inside the petri dish is randomly selected and 

all organisms are sorted from that portion. The remaining debris in the petri dish is discarded 

into the waste collection vessel. The process is then repeated, so that complete quadrants are 

sorted until at least 100 organisms are obtained in the sample or until sorting yields no fewer 

than 90 organisms and the spread between the highest and lowest count among subsamples being 

compared is not greater than ±10%. If a problem arises from the original sample because the 

primary data are questioned, an additional 100-200 organisms are sorted, stored separately and 

labeled as “subsample extras”. Furthermore, if any specimens in good condition are 

encountered, they may be kept separately from the subsample and stored in separate vials as 

voucher specimens. 

4.3.1.2.2 Processing of Hester-Dendy Multi-plate Samples 

Hester-Dendy multi-plate sampling of the macroinvertebrate assemblage uses a different 

protocol because all processing must take place within 48 hours of collection. After removal 

from the refrigerator, the entire multi-plate sampler and the water in which it is stored are 

transferred into a large pan. The sample container is then rinsed into the pan three times with 

small volumes of water to ensure that no organisms remain attached to the inside of the 

container. The multi-plate is then disassembled and all pieces are rinsed. The sample is then 

poured through a #30 mesh sieve, and the resultant debris is placed into a jar and preserved with 

denatured 70% ethanol or sorted immediately as required by the study design. Normally all 

macroinvertebrates recovered are identified; however, if the macroinvertebrate density is high, 

only a subsample is identified. 

4-7 
4.3.1.2.3 Taxonomic Identification 

A taxonomist identifies macroinvertebrates to genus or species level if the RBP III 

protocol is used. Identification may be to the family or order level if the RBP II protocol is being 

followed, or if the organism is in poor condition, immature, or not identified in an available key. 

MA DEP uses a number of keys to identify macroinvertebrates (Klemm 1985; Kathman and 

Brinkhurst 1998; Jokinen 1983; Smith 1986; 1987; 1988; Merritt and Cummins 1996; and 

Peckarsky et al. 1990). After identification, specimens are placed in labeled glass vials and 

preserved with denatured 70% ethanol. 

4.3.1.2.4 Oligochaeta and Chironomidae Identification 

Chironomid midges and oligochaetes are identified using slide mounts under a compound 

microscope using permanent or semi-permanent mounting medium. A 3-in x 1-in microscope 

slide is situated with the label end facing left. Three worms or midges are then placed side-byside with heads towards the top. A sufficient amount of CMC-10 is then added to the specimens 

to cover them and surround them. Worms are uncoiled and placed on their sides using forceps. 

The heads of the midges are separated from the body using an insect pin and forceps and then 

they are oriented so that their bodies are on their side and the heads are placed with the ventral 

side up. A #1 cover slip is then added and gentle pressure is applied to ensure that all air bubbles 

have been removed and so that the mandibles of midges are opened. CMC-10 is added to the 

edge of the cover slips and the slides are dried for at least one week horizontally before 

placement into a storage box. Identification by taxonomists are made using the keys by Kathman 

and Brinkhurst (1998), Bode (1990), Coffman and Ferrington (1996), and Wiederholm (1983, 

1986). 

4.3.2 Periphyton Protocols (Beskenis 2002, Draft 2003; Barbour et al. 1999) 

4.3.2.1 Field Methods 

MA DEP biologists evaluate stream condition using assessments of the periphyton 

assemblage above and below point and non-point sources or compared to a reference stream to 

look for toxicity issues, nutrient impacts and habitat alterations. The data from the periphyton 

sampling is used to evaluate if either the aesthetics or ALU is affected. MA DEP identifies both 

diatoms and soft algae to the genus level, and they incorporate other algal measurements: 

biomass (AFDM), chlorophyll a determination, and percent coverage. 

4.3.2.1.1 Algal Abundance and Identification 

Within the same 100-m reach used to collect macroinvertebrate samples (see section 

4.3.1), benthic algal samples are collected using the RBP Single Habitat Approach from riffles 

with a current of 10-50 cm/s. All samples are scraped from either natural or artificial substrates 

following U.S. EPA’s RBPs (Barbour et al. 1999). Substrate (preferably cobble) within the 

stream reach is scraped with a knife and the detached algae are then washed into a labeled glass 

vial using stream water. All vials collected from a site are tightly closed and placed into a large 

plastic jar containing stream water to ensure a regular temperature is maintained. Then the 

4-8 
samples are transported back to the laboratory to be stored in the refrigerator until identified and 

counted. If a sample is held for an extended time before analysis the sample is preserved with 

M3 fixative. 

4.3.2.1.2 Biomass 

MA DEP uses artificial substrates to collect algae data that determine periphyton biomass 

and chlorophyll a measurements (APHA 1992). The artificial substrate apparatus is constructed 

from a wooden tray attached to a cinder block with nylon twine. Slides are placed vertically in 

the wooden tray and the apparatus is placed into the water at a depth of at least 0.25 m and less 

than 1 m for a period of approximately three weeks. Any duplicate apparatuses are placed at 

comparable light and flow regimes. 

4.3.2.1.3 Chlorophyll a 

Chlorophyll a samples are typically measured using two slides from trays that have been 

deployed into a stream for three weeks to measure biomass. The two slides removed from the 

tray are then placed on ice, brought back to the lab and either filtered and ground or frozen until 

analysis (Beskenis Draft 2003). An alternative method to collect periphyton chlorophyll a is to 

scrape a known area of rocks clean. Then, the material is rinsed into a bottle with water and a 

subsample is removed for chlorophyll a analysis. Samples are typically processed immediately 

upon arrival and must be analyzed within 21 days of first filtering or freezing. 

4.3.2.1.4 Percent coverage calculation (Beskenis 2002) 

Percent algal cover is determined in the riffle zones and is usually determined visually by 

the biologist conducting the on-site survey. The surveyor will also note whether the algal 

composition is microalgae or macroalgae. If a stream consists of greater than 50% macroalgae, 

it may be considered to be threatened based upon review of other data including, if available, DO 

measurements and impacts on the macroinvertebrate assemblage. 

MA DEP also uses the point-intercept method to determine percent algal cover. For this 

method, a bottom viewer constructed by cutting a hole in the bottom of a bucket greater than 0.5 

m wide) and attaching a piece of clear acrylic material to the bottom with silicon caulk is used to 

assess the algal cover. The viewing bucket has 50 evenly spaced dots forming a grid on the clear 

acrylic bottom. Three transects are then laid out across areas where benthic algae are observed, 

insuring that the left and right banks and the middle are covered. The viewer is then placed into 

the stream and the number of dots covering the macroalgae are counted and recorded. A 

subsample of the macroalgae is collected for identification. Then, the process is repeated to 

count the number of rocks that contain substrata with microalgae on them. The microalgal 

composition is assessed to determine if it is composed of diatomaceous, green algae, or bluegreen algae and a subsample is collected for identification. If an algal mat exists, the thickness is 

determined in the field using a ruler and a score is given to describe the mat as a function of 

thickness: 

4-9 
Score 

0 Substrate is rough with no visible evidence of microalgae 

0.5 Substrate is slimy and no visible accumulation of microalgae is present 

1 A thin layer of microalgae is present 

2 Accumulation of a microalgae mat from 0.5 - 1 mm thick is present 

3 The microalgae mat is 1- 5 mm thick 

4 The mat is 5 mm - 2 cm thick 

5 The mat is > 2 cm 

Abundance is calculated by counting the number of organisms of each genus in each field 

view. If less than one cell per field is counted, the genus is considered rare (R). If at least one 

cell of a genus (but fewer than five cells) is counted per field, the genus is common (C). If 

between five and 25 cells are counted per field then the genus is very common (VC). A genus is 

abundant (A) if greater than 25 cells per field are present and very abundant (VA) if the cells are 

too abundant to count. 

4.3.2.1.5 Biomass Determination 

When the artificial substrate sampling device is retrieved, the wooden trays and slides are 

removed from the cinder blocks and placed in plastic bags on ice. The two outside slides are 

discarded and a random number generator is used to choose two slides to be analyzed for 

chlorophyll a (See chlorophyll a determination). The remaining four slides are used to measure 

biomass as AFDM. After removal from the tray the slides are first air-dried. China crucibles are 

cleaned and dried at 105oC and then cooled in a desiccator and weighed. Crucibles are returned 

to the oven for one hour, cooled and re-weighed until there is less than a 1% change in weight for 

each of the crucibles. After the slides are removed from the muffle furnace, one slide is retained 

for the archive and three of the slides are used as replicates and scraped into the pre-cleaned and 

pre-weighed china crucibles. A few drops of water are added to the crucibles and then they are 

placed into the combustion oven at 500oC for one hour (Thermolyne Sybron furnatrol 11, type 

13300). After the oven cools to 200oC, the samples are removed and placed in a dessicator 

before water is added for re-hydration. After the sample is re-hydrated, it is dried to a constant 

weight at 105oC and then the final weight is recorded. To calculate productivity, the following 

equation is used: 

P = ( slide per AFDM of mg )

t ∗A

P= net productivity (mg ash-free weight/m2

/day)

t= exposure time (days) 

A= area of slide (m2

) 

The mean weight from the slides is calculated and reported as dry weight and ash-free dry 

weight/m2

 using the formula: 

Mean g in AFDM g / m

2 = 00375.0 m

2 

4-10 
To calculate the Autotrophic Index (Biggs 1996): 

2 ( AI = Biomass organic of weight free ash mg in matter / m )

Chlorophyl mg in a l / m2

AI values range from 50 - 200. Larger values indicate a greater number of heterotrophic (fungi, 

bacteria) than autotrophic (algae) organisms. 

4.3.2.1.6 Chlorophyll a Determination (Beskenis Draft 2003) 

MA DEP has modified the U.S. EPA Fluorometric Method 445.0 to measure chlorophyll 

a samples using a Turner Design Fluorometer TD-700. The modified method used does not 

require acidification of the sample since background compounds are eliminated with the use of a 

filter and blue (mercury) lamp for analysis. Before any samples are run on the fluorometer, it is 

calibrated using chlorophyll a standards, and samples are analyzed according to Massachusetts 

Division of Watershed Management (MA DWM) methods (Beskenis 2003 Draft). All 

chlorophyll a concentrations are reported as mg/m3

 and used in conjunction with abundance, 

biomass, and percent coverage data to make water quality condition inferences. 

4.3.3 Fish Protocols (from Maietta and Decesare 2001) 

MA DEP evaluates fish populations using a method based on U.S. EPA RBP V (Plafkin 

et al. 1989) to aid in the determination of ALUS classifications in streams. While monitoring 

streams for fish, biologists also assess the habitat and characterize the physical and chemical 

water quality within the sample reach. A representative 100-m reach is selected and measured 

such that the primary physical habitat characteristics of the stream are included within the reach 

(i.e., riffle, run and pool habitats, when available). MA DEP selects sample reaches away from 

the influences of major tributaries and major bridge and road crossings. Fish are collected using 

a pulsed direct current (DC) backpack electrofisher. A crew of at least 3 people begins at a 

shallow riffle or other physical barrier at the downstream limit of the stream reach and moves 

upstream sweeping from bank to bank until an upstream barrier is reached. If a natural barrier is 

not present, block nets are set prior to sampling. All fish caught are added to a bucket or live 

well. All wadeable habitats within the reach are sampled with one pass. However a second pass 

is required at sites where estimated capture efficiency of observed fish is less than 75%. Fish are 

identified to species or subspecies on-site by a qualified/trained fish taxonomist, familiar with 

Massachusetts ichthyofauna. Fish are also examined for any deformities, eroded fins, lesions, 

and tumors. Any young-of-the-year fishes less than 20 mm in length are not identified or 

counted and are released back to the water. A subsample (maximum of 25 specimens) of each 

species is measured to the nearest mm in length and weighed to the nearest g. Any unknown 

specimens are preserved in 10% formalin solution and transported to the lab for identification by 

a second qualified fish taxonomist. 

4-11 
4.4 Data Management/Quality 

All assessment data collected by the MA DEP are entered into the U.S. EPA’s Water 

Body System (WBS) electronic database. This system aids in the determination of use support 

and compiles the data collected throughout the state. The MA DEP also uses a database called 

MABEN to analyze macroinvertebrate samples and to determine the condition of the community 

at a site using a multimetric approach (Nuzzo 2002). Within the database, taxa richness, HBI (or 

FBI), EPT index, ratio of EPT abundances to Chironomidae abundance, ratio of scrapers to 

filtering collectors, % contribution of dominant family, and similarity are calculated (Table 4-2). 

4.5 Analysis of Biological Data 

4.5.1 Macroinvertebrate Data (Nuzzo 2003) 

MA DEP uses modifications of the RBPs (II and III) to calculate metrics for the benthic 

macroinvertebrate assemblage (Plafkin et al. 1989). The RBP II is designed for family level 

taxonomic identification of macroinvertebrates, while the RBP III protocol separates streams 

based on generic- and species-level identifications. The metrics are nearly identical between the 

two with the exception that the FBI is used for the RBP II (Table 4-2), and the HBI is used for 

the RBP III analysis (Table 4-3). Each metric is calculated and then assigned a metric score (6, 

3, or 0 for the RBP II; 6, 4, 2, or 0 for the RBP III) based on a comparison with values from 

reference sites. Details of metric calculation and scoring are provided in Tables 4-2 and 4-3. 

The scores given to each of the individual metrics are then summed to yield a single index value. 

Based on the overall index value, the RBP II has the capability to discern between three different 

impact categories: Not Impacted, Moderately Impacted, and Severely Impacted. The RBP III 

discerns between four different impact categories: Not Impacted, Slightly Impacted, Moderately 

Impacted, and Severely Impacted. The overall impact category is determined by comparing the 

index score of the test stream to that of the reference site(s), and then determining if the score 

lies within the threshold value assigned to the expected level of impairment (Tables 4-2 and 4-3). 

Table 4-2. Methods for the calculations of metrics and scoring ranges used in RBP II 

determinations of level of biological impact (Plafkin 1989; Nuzzo 2003). 

Metric Method Scoring Ranges 

6 3 0 

Taxa Richness (a) The total number of distinct taxa in a sample >80% 40­

80% 

<40% 

EPT(a) The number of taxa within the orders of 

Ephemeroptera, Plecoptera, and Trichoptera 

>90% 70­

90% 

<70% 

EPT/Chironomidae 

(abundance ratio) (a) 

(Abundance of EPT organisms)/(Abundance of 

EPT + Chironomidae) 

>75% 25­

75% 

<25% 

FBI (modified)(b) (Number of individuals in family i) x (Family i 

tolerance value)/(Total number organisms in 

sample) 

>85% 50­

85% 

<50% 

4-12 
Metric Method Scoring Ranges 

6 3 0 

Scraper/Filtering 

Collector Ratio(a) 

(Number of scrapers)/(Number of filtering 

organisms) 

>50% 25­

50% 

<25% 

% Contribution of 

Dominant Taxon (c) 

(Number of individuals in most common taxon)/ 

(Total number of organisms) x 100 

<30% 30­

50% 

>50% 

Community Similarity Index 

Community 

Loss (d) 

A measure of the dissimilarity between a test 

site and a reference site (Plafkin et al. 1989). 

Metric values increase as biological impairment 

increase. Values have no limits. CLI = a - c / b 

where: a = number of families in reference 

sample, b = number of families in test sample, 

c = number of families common to both samples 

<0.5 0.5-

4.0 

>4.0 

% Similarity 

(d) = ∑

i 

Similarity ba ii % ),min(

Where ai is the percentage of taxon i in sample a 

and bi is the percentage of taxon i in sample b. 

Higher values indicate a healthier site. 

>70% 30­

70% 

<30%

% Reference 

Affinity(d) 

A test site compared to the reference site for 

seven faunal groups (Ephemeroptera, 

Trichoptera, Plecoptera, Chironomidae, 

Oligochaetes, Coleoptera, and Other. 

Calculated similarly to % Similarity but only 

percentages for the aggregate groups are used 

(Novak and Bode 1992). 

>64% 35­

64% 

<35% 

% Compared to Reference and Impact Category 

> 79% Not Impaired 

29-72% Moderate Impaired 

21% Severe Impaired 

a) Value is converted to ratio of test to reference site *100 

b) Value is converted to ratio of reference to test site *100 

c) Actual percent contribution used in scoring, not ratio to reference 

d) Uses range of values actually obtained 

4-13
Table 4-3. Methods for the calculations of metrics and scoring ranges used in RBP III 

determinations of level of biological impact (Plafkin 1989; Nuzzo 2003). 

Metric Method Scoring Ranges 

6 4 2 0 

Taxa Richness(a) The total number of distinct taxa in a sample >80% 60­

80% 

40­

59% 

<40% 

EPT(a) The number of taxa within the orders of >90% 80­ 70- <70% 

Ephemeroptera, Plecoptera, and Trichoptera 90% 79% 

EPT/ Chironomidae 

(abundance ratio)(a) 

(Abundance of EPT organisms)/(Abundance 

of EPT + Chironomidae) 

>75% 50­

75% 

25­

49% 

<25% 

HBI (modified)(b) (Number of individuals in taxon i)x(tolerance >85% 70­ 50- <50% 

value of taxon i)/(Total number of organisms 85% 69% 

in sample) 

Scraper/Filtering 

Collector Ratio(a) 

Number of scrapers/filtering organisms. >50% 35­

50% 

20­

34% 

<20% 

% Contribution of 

Dominant Taxon(c) 

Number of individuals in most common 

taxon/ total number of organisms x 100. 

<20% 20­

29% 

30­

40% 

>40% 

Community 

Loss(d) 

A measure of the dissimilarity between a test 

site and a reference site (Plafkin et al. 1989). 

Metric values increase as biological 

impairment increase. Values have no limits. 

<0.5 0.5-

1.5 

1.6-

4.0 

>4.0 

CLI = a - c / b 

where: a = number of genera in reference 

sample, b = number of genera in test sample, c 

= number of genera common to both samples 

% Similarity 

(d) = ∑

i 

Similarity ba ii % ),min( >70% 50­

70% 

30­

49% 

<30%

Community Similarity Index

Where ai is the percentage of taxon i in 

sample a and bi is the percentage of taxon i in 

sample b. 

% Reference 

Affinity(d) 

A test site compared to the reference site for 

seven faunal groups (Ephemeroptera, 

Trichoptera, Plecoptera, Chironomidae, 

>64% 50­

64% 

35­

49% 

<35% 

Oligochaeta, Coleoptera, and Other). 

Calculated similarly to % Similarity but only 

percentages for the aggregate groups are used 

(Novak and Bode 1992). 

% Compared to Reference Sites 

> 83% Not Impaired 

54-79% Slight Impaired 

21-50% Moderate Impaired 

<17% Severe Impaired 

4-14 
Metric Method Scoring Ranges 

6 4 2 0 

a) 

b) 

c) 

d) 

Value is converted to ratio of test to reference site *100 

Value is converted to ratio of reference to test site *100 

Actual percent contribution used in scoring, not ratio to reference 

Uses range of values actually obtained 

4.5.2 Algal Data (Arthur Johnson, Personal Communication) 

Algal data are used in aquatic use attainment as an indicator of nutrient enrichment of a 

water body. MA DEP has not developed metrics for algae but does use chlorophyll a, percent 

cover of green algae, and biomass to determine if the levels of algae are high enough to disrupt 

the biological value of the stream. Aquatic life support is considered fully supporting if no algal 

blooms are detected, while persistent algal blooms may represent impairment of ALU support. 

4.5.3 Fish Data 

MA DEP uses a modification of the RBP V (Plafkin et al. 1989) to assess some aspects of 

the fish assemblage in streams. A suite of measurements is used to assess the overall structure 

and condition of the fish assemblage. These calculations require the taxonomic identification of 

fish to the species level and include: number of fish species, number of fluvial 

specialists/dependents, number of intolerant species and number of salmonid species. Although 

MA DEP does not use an IBI, they do use the fish assemblage data with macroinvertebrate 

metrics qualitatively to help determine biological attainment. Furthermore, a detailed habitat 

assessment (Barbour et al. 1999) is made to accompany the data. 

4.5.4 Summary: Determining ALU Support 

The MA DEP uses a “weight of evidence” approach to assign ALU designations and 

attainment status for monitored streams. Data are collected from the biological (biotic and 

habitat), toxicological and chemical components and referenced against criteria (narrative or 

numerical) to make a final decision on the ALUS (Table 4-4). 

The biological component used to make the decision takes into consideration the 

outcome of the macroinvertebrate index (i.e., impairment designation: Tables 4-2, 4-3 and 4-4), 

the structure and condition of the fish assemblage, the habitat and flow regime of the stream, and 

algal presence as measured by chlorophyll a, biomass, and percent coverage. Once the available 

evidence is analyzed and weighed, a site is placed into an attainment category (e.g., Supported or 

Impaired) for the water quality class to which it was originally assigned (e.g., A, B or C). These 

assignments are then outlined in the 305(b) report and any streams not in attainment are placed 

on the Massachusetts 303(d) list. 

4-15 
Table 4-4. Biological, toxicological, and chemical parameters that are used collectively to 

determine ALUS. Attainment is assigned based on a “weight of evidence” evaluation. (MA 

DEP 2003) (Numerical criteria for dissolved oxygen, pH, and temperature can be found in 

314 CMR 4.00 (MA DEP 2000). MA DEP uses the recommended limits published by EPA 

pursuant to Section 304(a) of the Federal Act for Toxic Pollutant Criteria). 

Variable Support – Data available clearly 

indicates support or minor 

modification of the biological 

community. Excursions from 

chemical criteria not frequent or 

prolonged and may be tolerated if the 

biosurvey results demonstrate 

support. 

Impaired – There are frequent or severe 

violations of chemical criteria, presence 

of acute toxicity, or a moderate or severe 

modification of the biological 

community. 

BIOLOGY 

Macroinvertebrates: 

Rapid Bioassessment 

Protocol (RBP) III* 

Non/Slightly impacted Moderately or Severely Impacted 

Fish Assemblage Best Professional Judgment (BPJ) BPJ 

Habitat and Flow BPJ Dewatered streambed due to artificial 

regulation or channel alteration, BPJ 

Eelgrass Bed Habitat No/minimal loss, BPJ Moderate/severe loss, BPJ 

Macrophytes BPJ Exotic species present, BPJ 

Plankton/ 

Periphyton 

No/infrequent algal blooms Frequent and/or prolonged algal blooms 

TOXICITY TESTS** 

Water 

Column/Ambient 

>75% survival either 48 hr or 7-day 

exposure 

<75% survival either 48 hr or 7-day 

exposure 

Sediment >75% survival <75% survival 

CHEMISTRY-WATER** 

Dissolved 

oxygen/percent 

saturation (MA DEP 

1996, U.S. EPA 1997) 

Infrequent excursion from criteria, 

BPJ (minimum of three samples 

representing critical period) 

Frequent and/or prolonged excursion 

from criteria [river and shallow lakes: 

exceedances >10% of measurements; 

deep lakes (with hypolimnion): 

exceedances in the hypolimnetic area 

>10% of the surface area]. 

pH (MA DEP 1996, 

U.S. EPA 1999) 

Infrequent excursion from criteria Criteria exceeded >10% of 

measurements. 

Temperature (MA 

DEP 1996, U.S. EPA 

1997) 

Infrequent excursion from criteria1 Criteria exceeded >10% of 

measurements. 

Toxic Pollutants (MA 

DEP 1996, U.S. EPA 

1999) 

Ammonia-N (MA 

DEP 1996, U.S. EPA 

1999) 

Chlorine (MA DEP 

1996, U.S. EPA 1999) 

Infrequent excursion from criteria 

Ammonia is pH and temperature 

dependent2 

0.011 mg/L (freshwater) or 0.0075 

mg/L (saltwater) total residual 

chlorine (TRC)3 

Frequent and/or prolonged excursion 

from criteria (exceeded >10% of 

measurements). 

4-16 
4.6 

 Variable Support – Data available clearly 

indicates support or minor 

modification of the biological 

community. Excursions from 

chemical criteria not frequent or 

prolonged and may be tolerated if the 

biosurvey results demonstrate 

support. 

Impaired – There are frequent or severe 

violations of chemical criteria, presence 

of acute toxicity, or a moderate or severe 

modification of the biological 

community. 

CHEMISTRY-SEDIMENT** 

Toxic Pollutants 

(Persaud et al. 1993) 

Concentrations < Low Effect Level 

(L-EL), BPJ 

Concentrations ≥ Severe Effect Level (SEL)4

, BPJ 

CHEMISTRY-TISSUE 

PCB – whole fish 

(Coles 1998) 

<500 μg/kg wet weight BPJ 

DDT (Environment 

Canada 1999) 

<14.0 μg/kg wet weight BPJ 

PCB in aquatic tissue <0.79 ng TEQ/kg wet weight BPJ 

(Environment Canada 

1999) 

*RBP II analysis may be considered for assessment decision on a case-by-case basis. 

**For identification of impairment, one or more of the following variables may be used to 

identify possible causes/sources of impairment: NPDES facility compliance with whole effluent 

toxicity test and other limits, turbidity and suspended solids data, nutrient (nitrogen and 

phosphorus) data for water column/sediments. 1

Maximum daily mean T in a month (minimum 

six measurements evenly distributed over 24-hours) less than criterion. 2 Saltwater is temperature 

dependent only. 3 The minimum quantification level for TRC is 0.05 mg/L. 4

For the purpose of 

this report, the S-EL for total polychlorinated biphenyl compounds (PCB) in sediment (which 

varies with Total Organic Carbon (TOC) content) with 1% TOC is 5.3 ppm while a sediment 

sample with 10% TOC is 53 ppm. 

Literature Cited 

(APHA) American Public Health Association. 1992. Standard Methods for the Examination of 

Water and Wastewater, 18th edition. American Public Health Association, Washington, 

D.C. 

Barbour, M, 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. 

http://www.epa.gov/owow/monitoring/rbp/wp61pdf/rbp.pdf 

Beskenis, J. 2002. Standard Operating Procedures, Benthic Algae: Micro and Macro 

Identifications and Biomass Determinations. CN: 060.0. Department of Environmental 

Protection, Division of Watershed Management, Worcester, MA. 

4-17
Beskenis, J. 2003 (Draft). Standard Operating Procedures. Extracted Chlorophyll a (SM – 

10200H) (USEPA Flourometric Method 445 and 445 with the Welschmeyer 

modification). CN 0003.0. Department of Environmental Protection, Division of 

Watershed Management, Worcester, MA. 

Biggs, B.J.F. 1996. Patterns of benthic algae in streams. Pages 31-35 in R.J. Stevenson, M. 

Bothwell, and R.L. Lowe (editors). Algal Ecology: Freshwater Benthic Ecosystems. 

Academic Press, San Diego, CA. 

Bode, R.W. 1990. Chironomidae. Pages 225-267 in B.L.Peckarsky, P.R. Fraissinet, M.A. 

Penton, and D.J. Conklin, Jr. (editors). Freshwater Macroinvertebrates of Northeastern 

North America. Comstock Publishing Association, Ithaca, NY. 

Coffman, W.P. and L.C. Ferrington. 1996. Chironomidae. Pages 635-754 in R.W. Merritt and 

K.W. Cummins (editors). An Introduction to the Aquatic Insects of North America. 

Kendall/Hunt Publishing Company, Dubuque, IA. 

Coles, J.C. 1998. Organochlorine Compounds in Fish Tissue from Connecticut, Housatonic and 

Thames River Basins Study Unit, 1992-1994. National Water-Quality Assessment 

Program, U.S. Department of the Interior, U.S. Geological Survey, Marlbourough, MA. 

Courtemanch, D.L. 1984. A closing artificial substrate device for sampling benthic 

macroinvertebrates in deep rivers. Freshwater Invertebrate Biology 3(3):143-146. 

Environment Canada. 1999. Canadian Environmenatal Quality Guidelines. Environment 

Canada. 

Griffith, G.E., J.M. Omernik, S.M. Pierson, and C.W. Kiilsgaard. 1994. Massachusetts 

Ecological Regions Project. EPA/600/A-94/111. U.S. Environmental Protection 

Agency, Corvallis, OR. 

Jokinen, E.H. 1983. The Freshwater Snails of Connecticut. State Geological and Natural 

History Survey of Connecticut, Bulletin 109. 

Kathman, R.D. and R.O. Brinkhurst. 1998. Guide to the Freshwater Oligochaetes of North 

America. Aquatic Resources Center, College Grove, TN. 

Klemm, D.J. (editor). 1985. A Guide to the Freshwater Annelida (Polychaeta, naidid and 

tubificid Oligochaeta, and Hirudinea) of North America. Kendall/Hunt Publishing 

Company, Dubuque, IA. 

Klemm, D.J., P.A. Lewis, F. Fulk, and J.M. Lazorchak. 1990. Macroinvertebrate Field and 

Laboratory Methods for Evaluating the Biological Integrity of Surface Waters. 

EPA/600/4-90/030. U.S. Environmental Protection Agency, Cincinnati, OH. 

4-18 
Maietta, B. and G. Decesare. 2001. Standard Operating Procedures: Fish Collection Procedures 

for Evaluation of Resident Fish Populations. CN: 75.0. Department of Environmental 

Protection, Division of Watershed Management, Worcester, MA. 

(MA DEP) Massachusetts Department of Environmental Protection. 1996. Massachusetts 

Surface Water Quality Standards. Massachusetts Department of Environmental 

Protection, Division of Pollution Control, Technical Services Branch, Westbourough, 

MA. 

MA DEP. 2000. 314 CMR 4.00, Massachusetts Surface Water Quality Standards. 

Massachusetts Department of Environmental Protection, Division of Water Pollution 

Control. http://www.mass.gov/dep/bwp/iww/files/314cmr4.htm

 MA DEP. 2003. Massachusetts Year 2002 Integrated List of Waters: Part 1, Context and 

Rational for Assessing and Reporting the Quality of Massachusetts Surface Waters. 

CN:125.1. Massachusetts Department of Environmental Protection, Division of 

Watershed Management. http://www.mass.gov/dep/brp/wm/files/2002-il2.pdf 

Merritt, R.W., and K.W. Cummins (editors). 1996. An Introduction to the Aquatic Insects of 

North America. Kendall/Hunt Publishing Company, Dubuque, IA. 

Novak, M. A., and R. W. Bode. 1992. Percent model affinity: a new measure of 

macroinvertebrate community composition. Journal of the North American 

Benthological Society 11:80-85. 

Nuzzo, R.M. (editor). 2002. Benthic macroinvertebrate database descriptions and procedures 

(rev. 1.1). Massachusetts Department of Environmental Protection, Division of 

Environmental Management, Worcester, MA. 

Nuzzo, R.M. 2003. Standard Operating Procedures. Water Quality Monitoring in Streams 

Using Aquatic Macroinvertebrates. Massachusetts Department of Environmental 

Protection, Division of Watershed Management, Worcester, MA. 

Peckarsky, B.L., P.R. Fraissinet, M.A. Penton, and D.J. Conklin, Jr. 1990. Freshwater 

Macroinvertebrates of Northeastern North America. Comstock Publishing Association, 

Ithaca, NY. 

Persaud, D., R. Jaaguamagi, and A. Hayton. 1993. Guidelines for the Protection and 

Management of Aquatic Sediment Quality in Ontario. Water Resources Branch, Ontario 

Ministry of the Environment, Queen’s Printer for Ontario, Canada. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA/444/4-89-001. U.S. EPA, Washington D.C. 

4-19 
Smith, D.G. 1986. Keys to the Freshwater Macroinvertebrates of Massachusetts (No. 1): 

Mollusca Pelecypoda (clams, mussels). Massachusetts Division of Water Pollution 

Control, Westbourough, MA. 

Smith, D.G. 1987. Keys to the Freshwater Macroinvertebrates of Massachusetts (No. 2): 

Mollusca Mesogastropoda (operculate snails). Massachusetts Division of Water 

Pollution Control, Westborough, MA. 

Smith, D.G. 1988. Keys to the Freshwater Macroinvertebrates of Massachusetts (No. 3): 

Crustacea Malacostraca (crayfish, isopods, amphipods). Massachusetts Division of Water 

Pollution Control, Wesborough, MA. 

U.S. EPA. 1997. Guidelines for Preparation of the Comprehensive State Water Quality 

Assessments (305(b) Reports) and Electronic Updates: Supplement. U.S. Environmental 

Protection Agency, Office of Water, Office of Wetlands, Oceans and Watersheds, 

Assessment and Watershed Protection Division, Washington, D.C. 

U.S. EPA. 19 November 1999. Federal Register Document. [Online]. Environmental Protection 

Agency. http://www.epa.gov/fedrgstr/EPA-WATER/1998/December/Day10/w30272.htm 

Wiederholm, T. (editor). 1983. Chironomidae of the Holarctic Region: Keys and Diagnoses 

(Part 1. Larvae). Entomologica Scandinavica, Supplement No. 19. 

Wiederholm, T. (editor). 1986. Chironomidae of the Holarctic Region: Keys and Diagnoses 

(Part 2. Pupae). Entomologica Scandinavica, Supplement No. 28. 

4.6.1 Resources 

Massachusetts Department of Environmental Protection, Bureau of Resource Protection Web 

Page: http://www.state.ma.us/dep/brp/ 

U.S. EPA. Biological Indicators of Watershed Health, Massachusetts Webpage: 

http://www.epa.gov/bioindicators/html/state/ma-bio.html 

U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria Development or 

States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

4-20 
5 NEW HAMPSHIRE 

This document was prepared using documents written by the State of 

New Hampshire. Any questions concerning bioassessment methods 

should be directed to: 

Dave Neils, Biomonitoring Program Coordinator 

New Hampshire Department of Environmental Services (NH DES) 

Concord, NH 03302-0095 

Phone: (603) 271-8865; Fax (603) 271-7894 

Email: dneils@des.state.nh.us 

5.1 Introduction 

The State of New Hampshire Water Quality Standards (NH WQS), as defined by the NH 

DES, recognizes six designated uses in surface freshwaters: aquatic life, drinking water supply, 

fish consumption, primary contact recreation, secondary contact recreation, and wildlife. The 

narrative standards for water use classifications are stated as: “(a) State surface waters shall be 

divided into class A and class B, pursuant to RSA 485-A:8, I, II and III. Each class shall identify 

the most sensitive use which it is intended to protect; (b) All surface waters shall be restored to 

meet the water quality criteria for their designated classification including existing and 

designated uses, and to maintain the chemical, physical, and biological integrity of surface 

waters; (c) All surface waters shall provide, wherever attainable, for the protection and 

propagation of fish, shellfish and wildlife, and for recreation in and on the surface waters; (d) 

Unless the flows are caused by naturally occurring conditions, surface water quantity shall be 

maintained at levels adequate to protect existing and designated uses” (NH DES 1999). 

Furthermore, NH DES standards define biological and aquatic community integrity such 

that “a) The surface waters shall support and maintain a balanced, integrated, and adaptive 

community of organisms having a species composition, diversity, and functional organization 

comparable to that of similar natural habitats of a region” and where “b) Differences from 

naturally occurring conditions shall be limited to non-detrimental differences in community 

structure and function” (NH DES 1999). For rivers and streams and associated impoundments 

(4th order or less), ALUS is determined based on ten indicators, with the macroinvertebrate 

assemblage as the core indicator of biological condition (NH DES 2004a). The other indicators 

are: dissolved oxygen, pH, habitat, water quality criteria for toxic substances, toxicity tests of 

ambient water, sediment quality, exotic macrophytes, flow, and benthic deposits. In all other 

surface waters, ALUS is determined based on biological assemblage data or a minimum of DO 

and pH. If these constituents are within allowable standards or if there is documentation from a 

trained biologist that there is no other obvious impairment to the biological community, then the 

water body can be listed as fully supporting. 

5-1 
Table 5-1. NH DES water quality classes and the defined designated uses for each class. 

Dissolved oxygen exceedance values for aquatic life criteria are also listed (NH DES 1999 ). 

Classification Designated Uses Exceedances of the water quality criteria 

(aquatic life) for dissolved oxygen 

(Applies to any depth in free flowing rivers) 

Class A Generally of the 

highest quality and 

considered 

potentially usable 

for water supply 

after adequate 

treatment. 

Discharge of 

sewage or wastes 

prohibited to waters 

of this 

classification. 

Daily Average Measurement: < 75% saturation; 

Instantaneous Measurement: <6 mg/l 

In cold water naturally producing fisheries 

Daily Average Measurement: From October 1 to 

May 14, a 7 day mean DO based on the daily 

average of < 9.5 mg/L; 

Instantaneous Measurement: From October 1 to 

May 14 DO < 8 mg/L 

Class B Of the second 

highest quality, 

considered 

acceptable for 

fishing, swimming 

and other 

recreational 

purposes, and, after 

adequate treatment, 

for use as water 

supplies. 

Daily Average Measurement:<75% saturation; 

Instantaneous Measurement: < 5mg/l 

In cold water naturally producing fisheries 

Daily Average Measurement: From October 1 to 

May 14, a 7 day mean DO based on the daily 

average of < 9.5 mg/L; 

Instantaneous Measurement: From October 1 to 

May 14 DO < 8 mg/L 

The NH DES uses a weight of evidence approach when taking in consideration 

biological, RBP habitat, in situ chemistry, physical (e.g., landuse coverages and point sources) 

and toxicological data to support narrative criteria that determine ALUS (NH DES 2004a). 

Biological and physical habitat data are given the highest weight as “they are a direct 

measurement of the aquatic life and detect the cumulative impact on the aquatic community 

including new or previously undetected stressors over time” (NH DES 2004a). NH DES collects 

fish and macroinvertebrate data in their biomonitoring program to assess the condition of streams 

for 305(b) reporting and 303(d) listing. A benthic macroinvertebrate multimetric index has been 

developed recently, and a fish index is currently under development through the modification of 

the Vermont Cold Water Index of Biotic Integrity (CWIBI) and Mixed Water Index of Biotic 

Integrity (MWIBI) (see chapter 7 in this document, as well as VT DEC 2004). All other data 

collected (chemical, physical habitat and toxicological) provide complementary information and 

assist the biologists in determining ALUS. 

5-2 
5.2 Key Elements of the Biological Assessment Approach 

5.2.1 Index Period and/or Temporal Conditions 

The NH DES biomonitoring program collects both macroinvertebrates and fish during 

the mid-summer to early fall index period. This period represents the lowest flows and most 

stressful hydrologic conditions for instream biota. During this period, the larvae of the 

macroinvertebrate assemblages are easily identifiable because the late instars are larger. 

Furthermore, the fish assemblage is more stable and resident species are more likely to remain in 

a localized area when flow conditions are not fluctuating dramatically. The sampling index 

period is consistent from year to year in order to accurately compare monitoring data through 

time. 

5.2.2 Monitoring Program Survey Approach 

When developing the biocriteria program, an effort was made by NH DES to initially 

sample least impacted sites from a variety of habitats. Since then the program has collected 

samples from moderate and high impact sites in order to develop a multimetric index that best 

represents a response of the biological community to human disturbance. Sites have also been 

chosen for macroinvertebrate and fish monitoring using a targeted approach for 305(b) reporting 

and for identifying sites that cover geographical data gaps. Approximately 25 – 30 sites are 

sampled statewide per year. 

5.2.3 Natural Classification of Water Bodies 

New Hampshire consists of five major river basins: Androscoggin, Connecticut, Saco, 

Merrimack, and Piscataqua (Figure 5-1). For macroinvertebrate biomonitoring purposes, the 

state is separated into bioregions defined by distinct biological community types. The 

boundaries for the “Northern” and “Southern” bioregions represent similar Ecological Drainage 

Units as defined by The Nature Conservancy (Figure 5-1). For fish, streams are divided into two 

categories: cold and warm water. 

5.2.4 Indicator Assemblages 

Currently (for the 2004 305 (b) report), NH DES is using the macroinvertebrate 

assemblage to determine ALUS. A Benthic Index of Biotic Integrity (B-IBI) is used to assess 

the macroinvertebrate assemblages (with separate biocriteria for northern and southern 

bioregions). A fish Index of Biotic Integrity (IBI) is currently being developed to assess the fish 

assemblages of both cold and mixed waters. NH DES is basing the fish IBI on Vermont’s 

CWIBI and MWIBI because of the presumed similarity in fish assemblage composition, 

structure, and function. 

5.2.5 Reference Condition 

NH DES defines reference condition as the least disturbed sites. Reference sites were 

chosen based on a state-wide scoring system that takes into account both local and watershed 

5-3 
scale variables using GIS coverages of land use and point sources, as well as an immediate instream measure of human influence using the U.S. EPA’s RBP habitat assessment (Barbour et al. 

1999). On a watershed scale, land use was portioned into percentages developed, undeveloped 

and agricultural lands categories using the New Hampshire Land Cover (NHLC) dataset. 

Figure 5-1. Major New Hampshire basins and the northern and southern bioregion 

boundaries used for macroinvertebrate sampling (indicated by the red line). 

Percent water impounded was also calculated at the watershed scale. Local scale variables were 

estimated for a 300-ft buffer on either side of all hydrologic features within the watershed and 

within a 1-mile radius of the site. Densities of Ground Water Hazard Inventory (GWHI) sites, 

Resource Conservation and Recovery Act (RCRA) sites, junkyards, dams, water withdrawals, 

National Pollutant Discharge Elimination System (NPDES) sites, and roads were calculated at 

this local scale for each site. Each variable was scored on a 0-3 scale, with 0 representing 

5-4 
5.3 

minimal disturbance for that variable and 3 representing maximum disturbance, based upon 

overall distributions across all sites sampled between 1997 and 2001. Scores were summed for 

both the local and watershed scale disturbance scores, as well as across all variables to yield a 

total disturbance score (the human disturbance gradient (HDG) score). The total score was 

divided into four disturbance levels with equal scoring ranges: 0-4 (Best), 5-9 (Good), 10-14 

(Fair), and >14 (Worst). The entire range observed across all sites was 0 to 19. Those sites 

scoring in the best range were identified as the least disturbed sites. This HDG resulted in the 

identification of 46 reference condition sites within New Hampshire, 26 in the northern bioregion 

and 20 in the southern region. 

Field and Laboratory Protocols 

5.3.1 Macroinvertebrates Protocols 

5.3.1.1 Field Methods (NH DES 2004b) 

NH DES uses rock baskets to collect macroinvertebrates for B-IBI calculations. Rock 

baskets consist of an 11-in cylindrical plastic coated wire barbecue basket with 1-in2

 mesh 

openings. The bottom of the basket has a hinged opening. The basket is filled with regionally 

indigenous bank run gravel ranging in diameter from 1.5-3.0 in. The hinge is then secured with a 

plastic tie wrap. Baskets are placed in groups of three replicates attached to a 1⁄2-in steel rod 

anchor. Baskets are submerged to depths of greater than 5 in. Each of the attached baskets is 

arranged so that the bottom is facing downstream. 

The baskets are left in the stream for eight weeks to allow for colonization. At removal, 

the baskets are approached from downstream and a 3-gal bucket containing a 600-μm sieve 

bottom is placed downstream of the rock baskets. The plastic cable ties that secure the three 

baskets to the steel rod are cut and the baskets are quickly placed into the bucket. Any extra 

debris or algae clinging to the outside of the rock baskets are removed and discarded. The 

bucket containing the baskets is then transported to the stream side for sample retrieval. Using a 

knife, the plastic tie wraps are cut and the rocks are emptied into the sieve bucket. Then, the 

empty rock basket is added to a 5-gal bucket containing 3-4 gal of water. The basket is scraped 

free of organisms using a soft bristle brush. The 5-gal bucket is then emptied into the sieve 

bucket. Next, 2-3 gal of water are added to the 5-gal bucket and the sieve bucket is nested 

inside. Using a soft bristle brush, the rocks inside the sieve bucket are scrubbed free of attached 

organisms, and each scrubbed rock is then returned to the rock basket. After all organisms and 

detritus have been removed from the rocks, the sieve bucket is lifted from the 5-gallon bucket, 

capturing the targeted sample in the sieve. The contents of the sieve bucket are then placed into 

a one-quart, wide-mouthed jar and preserved with a solution of 1/3 water and 2/3 ethanol. This 

process is repeated for each of the replicate baskets. The scraped rocks from the baskets are 

allowed to dry before baskets are reused. 

5.3.1.2 Laboratory Methods (NH DES 2004b) 

NH DES processes macroinvertebrate samples collected in rock baskets using the U.S. 

EPA Caton Method (Caton 1991). A tray is fitted with a gridded screen with 16 uniform 

squares. Enough water is added to the tray so that the entire sample can be dispersed evenly over 

5-5 
the screen. The screen is then removed so that the organisms are settled on the screen. Random 

numbers are then selected to choose the grids from which to sort organisms. A metal square is 

used to delineate a grid square and the organisms are removed using a scoop. Those organisms 

that occupy more than one grid are counted in the grid that contains the head, unless the head is 

missing. Then, the grid with the largest portion of the organism is counted. At least 25% of the 

squares are counted yielding a minimum sample of 100 organisms. In the event that the 100­

organism target is not achieved, the entire sample is sorted. After sorting, benthic organisms are 

identified to the lowest possible taxonomic level. For data analysis, however, all Chironomidae 

were aggregated to the family level. 

5.3.2 Fish Protocol (NH DES 2004b) 

NH DES follows U.S. EPA’s RBP V (Barbour et al. 1999) to collect fish from wadeable 

streams using backpack electrofishing units. For wadeable streams, a minimum reach length of 

150 m is selected in close proximity to the location where the macroinvertebrate rock baskets 

were placed. NH DES has established a minimum 150-m reach as “the reasonable limit to 

prevent oversampling, while optimizing efficiency and representation of the resident species” 

(NH DES 2004b). A field sampling crew comprised of one shocker, at least two netters, and one 

person to carry an aerated bucket, begins at the most downstream portion of the reach and moves 

upstream. The crew collects a representative fish sample along the available instream habitat 

using a single-pass method. All fish identifications are made streamside. The identities, number 

of fish and external anomalies are recorded, and the fish are then released. For important cold 

water game fish, young-of-the-year (YOY) are recorded in order to document natural spawning 

activity. Fish that cannot be easily identified are taken back to the laboratory for identification 

by a trained biologist or using the fishes of New Hampshire key (Scarola 1973). 

5.4 Data Management/Quality 

NH DES has adapted the Ecological Data Assessment System (EDAS) to manage the 

chemical, physical and biological data collected in New Hampshire (EDAS 1999). EDAS is a 

Microsoft Access-based data storage and retrieval warehouse that contains station information, 

basic water chemistry data, fish data, habitat assessments, macroinvertebrate data and flow data. 

Updated GIS coverages of sample locations and upstream watersheds are also maintained using 

ARCVIEW software. The GIS coverages include data pertaining to watershed size, station 

elevation, latitude, longitude, stream order, Human Disturbance Gradient (HDG) status, 

bioregion, and hydrologic unit code (HUC). The NH DES uses the Assessment Database (ADB) 

developed by the U.S. EPA to submit electronic reports. Since 2002, the new Oracle-based 

version has been used by NH DES to submit surface water assessments. 

5.5 Analysis of Biological Data 

5.5.1 Macroinvertebrate Data (Neils and Blocksom 2004) 

The following seven metrics comprise the New Hampshire Benthic Index of Biotic 

Integrity (B-IBI): Total number of taxa, number of Plecoptera taxa, % Chironomidae, % noninsects, % clingers, number of tolerant taxa, and % individuals in intolerant taxa. NH DES used 

5-6 
the 95th percentile across all sites as a threshold for metrics that decrease in response to 

disturbance (i.e., positive metrics) and the 5th percentile across all sites for metrics that increase 

in response to disturbance (i.e., negative metrics) (Table 5-2). Scoring for each metric is on a 

continuous scale ranging from 0 to 100. Positive metrics are scored using the following 

equation: 

observed SCORE = 100 * 

threshold 

Negative metric scores are calculated using the following equation: 

(max− observed ) SCORE = 100 * 

(max− threshold ) 

The maximum is either the maximum possible (for percentage metrics) or the maximum 

observed in the calibration data set (for all other metrics). The metric scores for each site are 

calculated and averaged across all metrics, and the score is compared to the bioregional threshold 

value. 

Within each bioregion, the distribution of reference sites was used to set the biocriterion 

for the New Hampshire B-IBI. NH DES does not regard the reference sites used as truly 

unimpaired conditions for the state and has set the thresholds for attainment of ALU standards to 

take into consideration the possibility of incomplete information about sites. The 25th percentile 

of the reference distribution within each bioregion was used as the biocriterion. In the Northern 

Region, the threshold score is 77, and in the Southern Region, the threshold score is 66.4. Sites 

with B-IBI scores at or above the threshold in each bioregion are considered to be in attainment 

of ALUS. 

5.5.2 Fish Data (personal communication, David Neils, NH DES; VT DEC 2004) 

Currently, NH DES is modifying the Vermont Cold Water Index of Biotic Integrity 

(CWIBI) and the Mixed Water Index of Biotic Integrity (MWIBI) for use in New Hampshire 

streams. Detail of the Vermont indices can be found in Chapter 7. Vermont uses the CWIBI in 

streams that contain two to four species, and the MWIBI in streams that contain greater than four 

species (VT DEC 2004). NH DES used native fish species richness to adapt the method to use 

the MWIBI for any stream containing five or more taxa and used the CWIBI for other streams 

containing less than five taxa. Best professional judgment was used to ensure a stream was 

placed into the correct category. After this initial categorization, sites identified as belonging in 

MWIBI category were further refined as cold or warm water fish communities utilizing Eastern 

Brook Trout and Slimy Sculpin as indicator species. Site elevations were also used to classify 

MWIBI sites into cold and warm fish communities. Within the MWIBI, the differentiation 

between cold and warm water fish assemblages was applied in scoring individual metrics. 

Although the use of the CWIBI and MWIBI is currently under development, NH DES found the 

indices to be promising because the method showed significant differences between reference 

and non-reference sites. Further refinement of the CWIBI and MWIBI will be completed in the 

near future to calibrate final index scores and establish attainment cutoffs specific to New 

5-7 
Hampshire fish communities. Although the CWIBI or MWIBI appeared to work well for many 

sites, as in VT, neither index is applicable in slow, winding sites. 

Table 5-2. Metrics and scoring for the New Hampshire B-IBI. 

Metric Definition Expected 

Response to 

Disturbance 

Scoring Equation 

Total taxa 

Number of distinct 

macroinvertebrate 

taxa. 

Decrease Total taxa/21.5*100 

Plecoptera taxa 

Number of taxa in 

the order Plecoptera 

(genus or species 

level). 

Decrease Plecoptera taxa/4.4*100 

% 

Chironomidae 

% individuals in the 

family 

Chironomidae. 

Increase (100 - % Chironomidae)/100-

0)*100 

% Non-insects 

% individuals in a 

sample that are not 

in the class Insecta. 

Increase (100- % Non-insects)/100-

0)*100 

% Clingers 

% insects having 

fixed retreats or 

adaptations for 

attachment to 

surfaces in flowing 

water. 

Decrease % Clingers/94.6*100 

% Intolerant 

% of individuals 

considered to be 

sensitive to various 

types of pollution. 

Decrease % Intolerant/76.1*100 

Tolerant taxa 

Taxa richness of 

those organisms 

considered to be 

tolerant to 

increased 

disturbance. 

Increase (6.2-Tolerant taxa)/(6.2-0)*100 

Summary: Determining ALU Support 

NH DES follows the guidelines listed in the Comprehensive Assessment and Listing 

Methodology (CALM) to assess streams for ALUS (NH DES 2004a). This document lists the 

assessment criteria necessary for making the decision for specific segments (Assessment Units, 

AUs) of wadeable streams. Non-support in an ALU segment can be determined based on the BIBI score or the failure to meet chemical criteria (e.g., DO or pH). However, Full Support status 

cannot be given to a segment without a B-IBI evaluation within that segment. This assessment 

must also include data from the most recent calendar year. Other data, such as RBP habitat 

5-8 

5.6 
5.7 

assessments, fish assessments, benthic deposits, flow, macrophyte composition, sediment and 

ambient water toxicity tests are also used to aid in the decision-making process. 

Literature Cited 

Barbour, M, 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. 

http://www.epa.gov/owow/monitoring/rbp/wp61pdf/rbp.pdf 

Caton, L.W. 1991. Improving subsampling methods for the EPA "Rapid Bioassessment" 

benthic protocols. Bulletin of the North American Benthological Society 8(3):317-319. 

Ecological Data Assessment System (EDAS). 1999. Tetra Tech, Inc. 

Neils, D., and K.A. Blocksom. 2004. Development of the New Hampshire Benthic Index of 

Biotic Integrity. New Hampshire Department of Environmental Services, Concord, New 

Hampshire. 

(NH DES) New Hampshire Department of Environmental Services. 1999. State of New 

Hampshire Surface Water Quality Regulations, Chapter 1700. 

http://www.des.state.nh.us/wmb/env-ws1700.pdf 

NHDES. 2004a. 2004 Section 305(b) and 303(d) Consolidated Assessment and Listing 

Methodology. New Hampshire Department of Environmental Services, Concord, NH. 

http://www.des.state.nh.us/wmb/swqa/2004/pdf/CALM.pdf 

NH DES. 2004b. Biomonitoring Program Protocols. New Hampshire Department of 

Environmental Services, Concord, NH. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA 440-4-89-001. U.S. Environmental Protection Agency, Office of Water 

Regulations and Standards, Washington, D.C. 

Scarola, J.F. 1973. Freshwater Fishes of New Hampshire. New Hampshire Fish and Game 

Department, Division of Inland and Marine Fisheries. 

(VT DEC) Vermont Department of Environmental Conservation. 2004. Biocriteria for Fish and 

Macroinvertebrate Assemblages in Vermont Wadeable Streams and Rivers – 

Implementation Phase. Vermont Department of Environmental Conservation, 

Waterbury, VT. http://www.vtwaterquality.org/bass/htm/bs_biomon.htm 

5-9 
5.8 Resources 

New Hampshire Department of Environmental Services, Watershed Management Bureau, 

Biomonitoring Program Web Page: http://www.des.state.nh.us/wmb/biomonitoring/ 

U.S. EPA. Biological Indicators of Watershed Health, New Hampshire Webpage: 

http://www.epa.gov/bioindicators/html/state/nh-bio.html 

U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

http://www.epa.gov/bioindicators/html/program_summary.html 

5-10 
6 RHODE ISLAND 

This document was prepared using documents written by the State 

of Rhode Island. Any questions concerning bioassessment 

methods should be directed to: 

Connie Carey, Principal Environmental Scientist 

Rhode Island Department of Environmental Management (RI 

DEM) 

Office of Water Resources 

235 Promenade Street 

Providence, Rhode Island 02908 

Phone: (401) 222-3961 ext. 7239; Fax: (401) 222-3564 

Email: ccarey@dem.state.ri.us 

6.1 Introduction 

Rhode Island WQS define water quality goals for the state’s waters by designating uses 

and setting criteria necessary to protect those uses. Rhode Island WQS provide for the 

protection of the waters from pollutants so that the waters shall, where attainable, be available 

for all designated uses (i.e., drinking water supply, shell fish consumption, fish consumption, 

swimming, aquatic life) and thus assure protection for the public health, welfare, and the 

environment (RI DEM 2000). Rhode Island WQS define ALU as “providing suitable habitat 

and water quality for the protection, maintenance, and propagation of a viable community of 

aquatic life”. In accordance with Section 305(b) of the CWA, states are required to evaluate 

water quality of all water body types (i.e., rivers/streams, lakes/ponds, estuarine waters) for 

attainment of their designated uses. The water quality criteria and assessment methodology used 

for the evaluation is detailed in the Integrated Water Quality Monitoring and Assessment Report. 

Any water bodies that are not attaining their designated uses are placed on the 303(d) List of 

Impaired Waters and a TMDL must be developed for each exceedance that results in nonattainment status. 

The Rhode Island Department of Environmental Management (RI DEM) has used 

biological assessments to supplement physical and chemical water quality monitoring data for 

evaluation of ALU attainment in rivers and streams. The Rhode Island WQS currently only 

contain narrative biological criteria to utilize in evaluating biological assessment data. RI DEM 

implements a reference site approach for evaluation of the macroinvertebrate assemblage, as 

described below. The data collected from the bioassessment program is compared to 

corresponding reference sites and scored relative to conditions observed at the reference station. 

Scoring information from multiple years is used to determine a site’s ALU attainment status for 

305(b) reporting and 303(d) listing purposes. 

6-1 
6.2 Key Elements of the Biological Assessment Approach 

6.2.1 Index Period and/or Temporal Conditions 

The RI DEM conducts its biological monitoring program during the summer and fall 

seasons. Sites are sampled annually during this index period and any data collected and 

evaluated during drought conditions are noted. A long-term database allows for current and 

historical comparisons of data using these seasons. 

6.2.2 Monitoring Program Survey Approach 

Currently, RI DEM samples 45 fixed-river stations located throughout the state. The 

sampling sites are located within one of two subecoregions in Rhode Island: the New England 

Coastal Plains and Hills, or the Narragansett/Bristol Lowland. The stations were originally 

located on rivers/streams that were considered unassessed for ALU. They generally consist of 

smaller, wadeable rivers that do not have point-source discharges located on them, and range 

from first order to fifth order in size. 

6.2.3 Natural Classification of Water Bodies 

Rhode Island has two Level IV Omernik subecoregions represented within the 

Northeastern Coastal Zone ecoregion: the New England Coastal Plains and Hills subecoregion 

and the Narragansett/ Bristol Lowland subecoregion. Each subecoregion is represented by a 

reference site that is used to compare to test sites within the subecoregion. 

6.2.4 Indicator Assemblages 

RI DEM currently uses only the macroinvertebrate assemblage as a biological indicator. 

Macroinvertebrates are typically identified to the lowest practical taxonomic level. Rhode Island 

follows the U.S. EPA’s RBP for evaluation of benthic macroinvertebrate communities (Plafkin et 

al. 1989). 

6.2.5 Reference Condition 

The RI DEM currently uses two reference sites in their bioassessment program, one 

representing each of the two Level IV Omernik subecoregions present in the state (Figure 6-1). 

One reference site, the Wood River, is located in the New England Coastal Plains and Hills 

subecoregion, while Adamsville Brook serves as the reference site for the Narragansett/Bristol 

Lowland subecoregion. Currently, no specific reference criteria exist, but each reference site 

represents minimally disturbed, high quality, historically natural sites within its subecoregion. 

The Wood River site is located on a fourth order, minimally disturbed portion of the river within 

the Pawcatuck River Basin. This reference site is located almost completely within the 

boundaries of a state park, and, therefore, is not expected to undergo the degradation that would 

remove it from the reference category. The Adamsville Brook reference station is located on a 

second order portion of the brook in the Cape Cod Basin. This area consists of very low density 

residential and 82% rural land use. 

6-2 
6.3 

Figure 6-1. Level IV Omernik subecoregions and reference streams used in RI DEM’s 

biological monitoring program. 

Field and Laboratory Protocols 

6.3.1 Macroinvertebrate Protocols 

6.3.1.1 Field Methods (RI DEM 2002a, RI DEM 2003) 

RI DEM uses the U.S. EPA’s RBP III Single Habitat Approach (Barbour et al. 1999) to 

assess the macroinvertebrate assemblage of wadeable streams. This method has been in use 

since 1991 at the 45 fixed-station riffle stream sites. A physical evaluation is conducted at each 

of the sites that includes: surrounding land use; subsystem classification; documentation of dams, 

erosion and non-point source pollution; width, depth and flow measurements; inorganic and 

organic substrate types; and presence of odors, oils and deposits. Along with a physical 

evaluation, water quality measurements are taken (DO, pH, specific conductance, turbidity, and 

water temperature), and a habitat assessment is conducted. The habitat assessment and 

biological data are scored and compared against the reference station’s data. The information is 

6-3 
6.4 

then integrated with water quality, physical, rainfall, and historical trends data, to determine the 

ALU attainment status relative to the reference site from the same subecoregion. 

RI DEM uses the RBP III Single Habitat Approach to collect representative samples of 

the stream reach. A 100-m reach is chosen for sampling, assuring that the reach is located at 

least 50 m upstream of bridges or road crossings to minimize their effect on stream velocity, 

depth and overall habitat quality. An effort is also made to not sample immediately downstream 

of stream confluences. The reach is approached from the downstream to upstream direction 

collecting a composite sample of macroinvertebrate kick samples from riffles (multiple if 

present) with a dominant cobble substrate. A 0.3-m wide D-Frame net with 500-μm mesh is 

placed into the bottom of the riffle, and the substrate is disturbed for three minutes. Larger 

substrate particles and debris are rubbed to remove attached organisms. Collected material is 

rinsed using stream water before being transferred to a sample container. If multiple riffles are 

sampled, all contents of the net from each riffle are placed into a single jar. The sample is 

preserved with 70% ethanol. 

6.3.1.2 Laboratory Methods (RI DEM 2002a) 

Samples preserved in 70% ethanol are rinsed thoroughly with tap water through a 500-μm 

mesh sieve to remove fine sediment and preservative. Any large organic material is discarded 

and the sample is then distributed evenly in an 18-in x 13-in x 1-in gridded tray with eight 

equally-sized squares. The entire sample is scanned, and any rare or large organisms are 

removed, identified and used only to report as supplemental data. Then, one section is randomly 

selected and all of the material in that section is removed using a 6-cm flat scoop and transferred 

to a separate container. Any overhanging debris is cut using scissors. Macroinvertebrates are 

sorted under a dissecting microscope on a clean Petri dish and placed into one of the three 

following groups in glass vials containing 70% ethanol for identification: 1) Oligochaetes and 

Chironomids, 2) Crustaceans and Mollusks, and 3) other organisms. Additional, randomly 

selected sections are completely sorted until 100 organisms are sorted or until the entire sample 

has been inspected for macroinvertebrates. Random quality checks are performed to assure that 

there is less than a 10% discrepancy between the sorter and the quality assurance check. 

 After sorting, macroinvertebrates are identified to the lowest practical taxonomic level. 

Vials containing chironomids and oligochaetes are sent to a sub-contractor, ARC, where they are 

mounted on labeled slides using an appropriate medium (e.g., Eupcral, CMC-9) and identified. 

All organisms are identified using a compound microscope or a dissecting microscope (up to 

45X magnification), a fiber optic lamp, standard dissecting tools, and taxonomic keys. 

Data Management/Quality 

RI DEM has recently developed a Microsoft Access database to maintain the data 

collected by the biological monitoring program. Portions of the historic biological data have 

been entered into the database. Because the Rhode Island Office of Water Resources, where the 

biological monitoring program resides, has been without staff for over a year, the more recent 

data are maintained only in hard copy format. The Access database has been developed to 

calculate RBP metrics for macroinvertebrate data and will assist in the evaluation of developing 

a biological condition matrix for the state. 

6-4 
6.5 Analysis of Biological Data 

6.5.1 Macroinvertebrate Data (RI DEM 2002a, RI DEM 2002b) 

RI DEM uses the RBP III (Plafkin et al. 1989) metric-based method to give a 

bioassessment score relative to the reference site. Eight RBP metrics and three supplemental 

metrics (i.e., EPT abundance, Shannon Weaver Diversity Index, and % Hydropsychidae of total 

Trichoptera) are calculated (Table 6-1). Each metric is given a score (6, 4, 2, or 0), primarily 

based on the percentage of the reference site value observed at a site. The overall score is the 

sum of metric scores. Then, the overall score is compared to the reference site score by 

calculating the percentage of the reference score achieved. Thresholds of percentages relative to 

the reference site score places a stream into one of four bioassessment categories: Non-impaired, 

Slightly Impaired, Moderately Impaired, or Severely Impaired (Table 6-2). Although Table 6-2 

lists the threshold percentages for assignment to a particular bioassessment category, the actual 

threshold may be adjusted slightly based on best professional judgment of the assessor, as well as 

analysis and comparison to historical data and trends. 

Table 6-1. Metrics used by the Rhode Island Biomonitoring program and the methods for 

the calculation of metrics and their scoring ranges based on the RBP III (Plafkin et al. 

1989, RI DEM 2002a, RI DEM 2002b). 

Metric Method Scoring Ranges 

6 4 2 0 

Total Taxa Richness 

(a) 

The total number of distinct taxa 

in the sample 

>80% 60­

80% 

40­

60% 

<40% 

EPT Taxa Richness The number of taxa within the >90% 80­ 70- <70% 

(a) orders of Ephemeroptera, 90% 80% 

Plecoptera, and Trichoptera. 

EPT Abundance All Ephemeroptera, Plecoptera, 

and Trichoptera individuals are 

added together. 

Only used as supplementary 

data, not used in the RBP 

approach. 

Hilsenhoff Biotic 

Index (b) 

(Number of individuals in taxon 

i)x(tolerance value of taxon 

i)/(Sum of individuals with 

tolerance values in sample) 

>85% 70­

85% 

50­

70% 

<50% 

Shannon Weaver Number in each species is counted Used as a measure of aquatic 

Diversity Index and index is calculated as: 

Σ (pi log2 pi) 

Where pi = the proportion of 

individuals in the ith species. 

environmental health, but not 

used for RBP III. 

% Contribution of 

Dominant Taxon (d) 

((a/b) x 100) 

Where: a = the number of 

<20% 20­

30% 

30­

40% 

>40% 

individuals in the dominant taxon, 

b = the total number of individuals 

recorded at the stream segment. 

EPT/Chironomidae 

(abundance ratio)(a) 

(No. of EPT individuals)/(No. 

Chironomidae individuals) 

>75% 50­

75% 

25­

50% 

<25% 

6-5 
6.6 

Metric Method Scoring Ranges 

6 4 2 0 

Percent 

Hydropsychidae of 

Total Trichoptera 

(No. Hydropsychidae 

individuals)/(No. all other 

Trichoptera) x 100 

Used only as additional potential 

tolerance/intolerance measure 

metric (RI DEM 2003; modified 

from Barbour et al. 1999) 

Ratio of Shredders to 

Total Number of 

Macroinvertebrates (a) 

(No. shredder individuals)/(Total 

no. of other individuals) (RI DEM 

2003) 

>50 

% 

35­

50% 

20­

35% 

<20% 

Ratio of Scrapers to 

Filterers(a) 

(No. of Scraper individuals)/(No. 

of filterer individuals) 

>50 

% 

35­

50% 

20­

35% 

<20% 

Community Loss 

Index(d) 

A measure of the dissimilarity 

between a test site and a reference 

site (Plafkin et al. 1989). Metric 

values increase as biological 

impairment increase. Values have 

no limits. 

CLI = (a – b) / c 

where: a = no. genera in reference 

sample, b = no. genera in test 

sample, c = no. genera common to 

both samples 

<0.5 0.5-1.5 1.5-4.0 >4.0 

a) Value is converted to ratio of test to reference site *100 

b) Value is converted to ratio of reference to test site *100 

c) Actual percent contribution used in scoring, not ratio to reference 

d) Uses range of values actually obtained 

Summary: Determining ALU Support 

RI DEM uses a combination of biological, habitat, chemical, and physical data to assign 

ALUS (Table 6-3). RI WQS contain some numeric criteria for dissolved oxygen, pH, 

temperature and priority pollutants, but other criteria are assessed based on a narrative 

description of water quality condition. 

Taking into consideration all of the above factors, RI DEM determines the ALU 

attainment status of the streams. ALU is fully supported if there are no exceedances of the water 

quality criteria and the biological data indicate a fully supporting community. RI DEM gives the 

biological component more weight than the water chemistry data for the ALU assessment. 

Therefore, a river/stream may be considered fully supporting ALU if the biological community 

demonstrates non-impairment or slight impairment, even if minor exceedances of water quality 

criteria exist. A stream is considered partially supporting ALU if the macroinvertebrate 

assemblage indicates a bioassessment category of “slightly impaired” or “moderately impaired” 

and/or if there is an exceedance of any chemical water quality criterion (acute or chronic) more 

than once in a three-year period but in fewer than 10% of the samples. A site is determined to be 

not supporting ALU if the biological community is “severely impaired” and/or if there are severe 

6-6 
Table 6-2. Percent comparability evaluation for macroinvertebrate bioassessment scores 

used by the State of Rhode Island. 

Bioassessment 

Categories 

Definition Percent 

Comparability 

to Reference 

Non-impaired Comparable to the best condition expected within an 

ecoregion. Trophic structure is balanced and community 

structure is optimal for the stream size and habitat 

quality. 

> 83 % 

Slightly 

impaired 

Community structure is less than expected. Species 

composition is lower as evident by the loss of some 

intolerant species. The percent contribution of tolerant 

species increases. 

54 – 79 % 

Moderately 

impaired 

Fewer species are present and most intolerant species 

disappear. 

21 -50 % 

Severely 

impaired 

Few species are present and the stream is often 

dominated by one or two species. 

< 17 % 

or frequent (>10% of samples) violations of chemical water quality criteria. States are required 

by the CWA to describe the water quality of their state’s waters in the 305(b) report and any 

water bodies that are found to be not in attainment of their designated uses must be listed on the 

303(d) list. In Rhode Island, any water bodies that are considered partially or not supporting any 

designated uses are listed on the State’s 303(d) List of Impaired Waters. 

Table 6-3. Biological, physical and chemical criteria used to determine aquatic life use 

(modified from RI DEM 2000). 

Component Description of Criteria 

Biological • Macroinvertebrate Index (Plafkin et al. 1989) 

• Non-impaired, slightly impaired, moderately impaired, and 

severely impaired determined for the stream based on the 

macroinvertebrate assemblage 

Physical • Land use evaluation 

• Subsystem classification 

• Documentation of dams, erosion, and non point source 

pollution 

• Width and depth measurement 

• Flow measurement 

• Inorganic and organic substrate types 

• Presence of odors, oils and deposits 

• RBP Habitat Assessment (Barbour et al. 1999) 

Chemical 

6-7 
Component Description of Criteria 

Dissolved 

Oxygen 

• Cold water fish habitat: dissolved oxygen should be above 

75% saturation based on a daily average and than the 

instantaneous minimum concentration shall be of at least 5 

mg/l. 

o To protect early life stages that are intergravel during 

the fish spawning period of October 1st to May 14th, the 

7-day mean water column dissolved oxygen 

concentration shall not be less than 9.5 mg/l, while the 

instantaneous minimum dissolved oxygen shall not be 

less than 8 mg/l. 

o To protect those early life stages that are exposed 

directly to the water column, the 7-day mean water 

column dissolved oxygen concentration shall not be 

less than 6.5 mg/l and the instantaneous minimum 

dissolved oxygen concentration shall not be less than 

5.0 mg/l. 

• Warm water fish habitat: daily average dissolved oxygen shall 

be above 60% saturation and the instantaneous minimum 

dissolved oxygen concentration shall be at least 5.0 mg/l. The 

7-day mean water column dissolved oxygen concentration 

shall not fall below 6 mg/l. 

pH The pH should be as naturally occurs (6.5-9.0) 

Temperature Increase should not rise above the recommended limit that would 

cause the growth of nuisance species nor rise above 83oF. In cold 

water habitats, heated discharges must not raise the temperature above 

68oF and in not case shall the receiving water be raised more than 4oF. 

Secchi depth Secchi depth and chlorophyll a are used to measure the impact of 

and chlorophyll nuisance algal blooms that may degrade the quality of life for fish and 

a wildlife. 

Priority Shall not be found in concentrations or combinations that would be 

Pollutants harmful to humans or fish and wildlife for the most sensitive and 

Please refer to governing water class use, or unfavorably alter the biota, or which 

Appendix B of would make the water unsafe or unsuitable for fish and wildlife or 

the Water their propagation, impair the palatability of same, or impair waters for 

Quality any other existing or designated use. 

Regulations for • Aquatic Life Criteria: The acute and chronic aquatic life 

a list of criteria for freshwaters shall not be exceeded at or above the 

pollutant 

criteria (RI 

DEM 2000). 

lowest average 7 consecutive day low flow with an average 

recurrence frequency of once in 10 years (7Q10). 

• Human Health Criteria: The freshwater human health criteria 

for non-carcinogens are applicable at or in excess of the lowest 

average 30 consecutive day low flow with an average 

recurrence frequency of once in 5 years (30Q5). The 

freshwater human health criteria for carcinogens are applicable 

at or in excess of the harmonic mean flow, which is a long6-8 
Component Description of Criteria 

term mean flow value calculated by dividing the number of 

daily flows analyzed by the sum of the reciprocals of those 

daily flows. 

6.7 Literature Cited 

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. 

http://www.epa.gov/owow/monitoring/rbp/wp61pdf/rbp.pdf 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA 440-4-89-001. U.S. Environmental Protection Agency, Office of Water 

Regulations and Standards, Washington, D.C. 

(RI DEM) Rhode Island Department of Environmental Management. 2000. Water Quality 

Regulations. Regulation EVM 112-88.97-1. Rhode Island Department of Environmental 

Management, Office of Water Resources. 

http://www.state.ri.us/dem/pubs/regs/REGS/WATER/h20qlty.pdf 

RI DEM. 2002a. Quality Assurance Project Plan for Taxonomic Identification of Benthic 

Macroinvertebrates, Rhode Island. Rhode Island Department of Environmental 

Management, Office of Water Resources. 

RI DEM. 2002b. State of Rhode Island and Providence Plantations 2002 Section 305(b) State of 

the State’s Waters Report. Rhode Island Department of Environmental Management, 

Office of Water Resources. http://www.state.ri.us/dem/pubs/305b/index.htm 

RI DEM. 2003. Biomonitoring and Habitat Assessment Rhode Island Wadeable Streams 2002 

Data Report. Rhode Island Department of Environmental Management, Office of Water 

Resources. 

6.8 Resources 

Rhode Island Department of Environmental Management, Office of Water Resources. Web 

Page: http://www.state.ri.us/dem/programs/benviron/water/index.htm 

U.S. EPA. Biological Indicators of Watershed Health, Rhode Island Webpage: 

http://www.epa.gov/bioindicators/html/state/ri-bio.html 

U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria Development or 

States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

6-9 
7 VERMONT 

This document was prepared using documents written by the State of 

Vermont. Any questions concerning bioassessment methods should 

be directed to: 

Doug Burnham, Biomonitoring and Aquatic Studies Section 

Chief 

Vermont Department of Environmental Conservation (VT DEC) 

103 South Main Street, 10N 

Waterbury, VT 05671 

Phone: (802) 241-3784; Fax (802) 241-3008 

Email: Doug.Burnham@anr.state.vt.us 

7.1 Introduction 

The Vermont Department of Environmental Conservation (VT DEC) has developed a 

biocriteria program for wadeable streams that uses both fish and macroinvertebrate assemblages 

for the calculation of indices indicative of stream conditions and biological integrity as it pertains 

to ALU classes for 303 (d) listed streams. The foundation of this program was set in 1982 with 

the creation of the Ambient Biomonitoring Network (ABN) in the Biomonitoring and Aquatic 

Studies Section (BASS) comprised of five biologists focusing on river and stream biological 

monitoring. The objectives of the ABN within BASS are to monitor long term trends over time, 

evaluate potential impacts from non-point and point sources, establish a reference database 

specific to Vermont’s classification and attainment determinations, support VT DEC permitting 

programs that require data, and conduct special studies to assess emerging water quality and 

environmental management issues. 

In 1985, VT DEC began using standardized methods to sample fish and 

macroinvertebrate communities. These methods included guidelines on evaluating physical 

habitat, processing samples, and analyzing and evaluating the data. Vermont now uses an IBI to 

measure the condition of the fish community, and multiple metrics applied individually to assess 

the integrity of the macroinvertebrate community. These data are then used together to 

determine the level of ALUS and attainment for wadeable streams and rivers in Vermont. 

Section 3-01 of the VT WQS (Vermont Water Resources Board 2000) states the 

provisions to “establish and apply numeric biological indices to determine whether there is full 

support of aquatic biota and aquatic habitat uses” and to “establish procedures that employ 

standard sampling and analytical methods to characteristics of the biological integrity of the 

appropriate reference conditions.” VT WQS also lists the water quality management classes and 

the biological standards that define the classes. Sections 3-02, 3-03, and 3-04 identify the 

management strategies and narrative standards for these water quality management classes. 

These classes range from the highest ALUS class A1 (ecological or natural conditions) to Class 

B Water Management Classes Type 1, 2, and 3, and Class A2 (public water supplies) (Table 7­

1). 

7-1 
Table 7-1. Biological ALUS management classes and associated narrative biological 

criteria for rivers and streams in Vermont. 

Class Management Biological Standard 

A1 Ecological. Managed to achieve 

and maintain waters in a natural 

condition. Highest quality 

Reference condition, minimal impacts from 

human activity. Highest quality water that 

have significant ecological value. 

waters. 

A2 Managed for public water 

supplies and to achieve and 

maintain waters with uniformly 

excellent character and level of 

water quality. 

High quality aquatic biota and habitat 

necessary to support their life cycle and 

reproductive requirements. 

Moderate change from the reference condition 

in the relative proportions of tolerant, 

intolerant, taxonomic, and functional 

components. 

Class B, Water 

Management Type 1 

Managed so that no change from 

the reference condition would 

prevent the full support of 

aquatic biota, wildlife or aquatic 

habitat uses. 

Minor change from reference condition, minor 

changes in relative proportions of taxonomic 

and functional components, relative 

proportions of tolerant and intolerant 

components within range of reference 

conditions. Changes in the aquatic habitat 

shall be limited to minimal differences from 

the reference condition consistent with the full 

support of all aquatic biota and wildlife uses. 

Class B, Water 

Managements Type 2 

Managed so that no change from 

the reference condition would 

prevent the full support of 

aquatic biota, wildlife or aquatic 

habitat uses. 

Moderate change in the relative proportions of 

tolerant, intolerant, taxonomic, and functional 

components. Changes in the aquatic habitats 

shall be limited to minor differences from the 

reference condition consistent with the full 

support of all aquatic biota and wildlife uses. 

Class B, Water 

Managements Type 3 

Managed so that no change from 

the reference condition would 

prevent the full support of 

aquatic biota, wildlife or aquatic 

habitat uses. 

Moderate change in the relative proportions of 

tolerant, intolerant, taxonomic, and functional 

components. Changes in the aquatic habitats 

shall be limited to moderate differences form 

the reference condition consistent with the full 

support of all aquatic biota and wildlife uses. 

When such habitat changes are a result of 

hydrological modification or water level 

fluctuation, compliance may be determined on 

the basis of aquatic habitat studies. 

Other Class B Waters Managed so that no change from 

the reference condition would 

No change from the reference condition that 

would have an undue adverse effect on the 

prevent the full support of 

aquatic biota, wildlife or aquatic 

habitat uses. 

composition of the aquatic biota, the physical 

or chemical nature of the substrate or the 

species composition or propagation of fishes. 

7-2 
7.2 Key Elements of the Biological Assessment Approach 

7.2.1 Index Period and/or Temporal Conditions 

The VT DEC has chosen late summer and fall (i.e., September to mid-October) for its 

index period to conduct both fish and macroinvertebrate reference and test site sampling. 

Samples must be collected during this index period to maintain consistency between the samples 

taken from year to year and for accurate community comparisons. This index period was 

selected because of the presence of stable, low-flow conditions and because the 

macroinvertebrate larvae and fish YOY are larger and easier to identify. Each site is visited one 

time during a sampling season. 

7.2.2 Monitoring Program Survey Approach 

The majority of the streams that are selected for sampling are targeted sites. These sites 

encompass specific sampling stations that have been monitored for the purpose of discharge 

permitting and TMDLs and can include sites that have been chosen for specific projects. 

Additionally sites are selected based on human land use and interest of watershed groups or the 

VT DEC planning section’s need for data to determine the current biological condition of a 

stream reach. Vermont recognizes 17 watershed planning units that are a combination of smaller 

sub-basins. Seasonally monitored sites are sampled using a five-year rotating watershed 

strategy. This strategy covers three to four basins each year and approximately 125 sites are 

sampled per year within these basins (VT DEC 2004). 

7.2.3 Natural Classification of Water Bodies 

The 17 watershed planning units used by VT DEC were chosen nearly 30 years ago using 

the three major watersheds in the state. The three watersheds were then separated into 17 

identifiable units. These classifications into 17 units were made based on local habitat features 

including elevation, drainage order, stream gradient and substrate composition. 

7.2.4 Indicator Assemblages 

VT DEC monitors both benthic macroinvertebrates and fish. Benthic macroinvertebrates 

are identified to genus or species in the laboratory (Class Oligochaeta is identified to family). A 

set of metrics to assess the macroinvertebrate assemblage has been developed using multivariate 

statistics. The fish assemblage is identified to species in the field and a multimetric fish index 

has been developed for both cold water and mixed water categories. 

7.2.5 Reference condition 

Reference conditions have been derived from macroinvertebrate and fish assemblage data 

independently and take into consideration a range of physical and chemical parameters a 

particular community would naturally encounter. The reference sites have been chosen and 

defined using the best professional judgment of VT DEC biologists, based on human activity and 

the potential that it may affect the stream. Specific reference sites and conditions can be found in 

7-3 
VT DEC (2004). There are three reference condition stream types for macroinvertebrates and 

two reference condition stream types for fish, totaling 150 reference sites. If a stream has unique 

properties and cannot be compared to one of the reference condition stream types, then a 

localized reference stream(s), historic data and best professional judgment are used to determine 

the expected natural range of metrics. 

Macroinvertebrates reference sites are categorized into one of the following stream types 

(VT DEC 2004): 

1. Small High Gradient Streams (SHG) are small, first-to-third order headwater streams with 

drainage areas averaging 10 km2

. These streams are located typically over 1500 ft in elevation 

and are highly canopied (83% average canopy cover). These streams have a high gradient and 

substrate dominated by gravel/cobble/boulder and approximately 3% fine sediments. Water is 

soft and alkalinity would typically measure less than 20 mg/l. 

2. Medium Sized High Gradient Streams (MHG) are medium, third to fourth order streams with 

a drainage area averaging 88 km2

. These streams are found at elevations averaging 814 ft, and 

are covered by about 50% canopy. These streams have a high gradient and substrate dominated 

by gravel/cobble/boulder and approximately 6% fine sediments. Water has a moderate 

alkalinity, typically averaging 48 mg/l. 

3. Warm Water Medium Gradient Streams and Rivers (WWMG) are larger streams, fourth to 

sixth order, or small streams within the Champlain Valley. Because this category contains larger 

streams than those in the Champlain Valley, the drainage areas vary, but the average size is 480 

km2

. All streams are found at lower elevations averaging 369 ft, are less shaded with an average 

30% canopy, and are warmer. These streams have a moderate gradient and substrates dominated 

by gravel/cobble/boulder and approximately 7% fine sediments. Water has a high alkalinity, 

typically averaging 70 mg/l. 

VT DEC categorizes a site by comparing chemical and physical data from a test stream to 

those conditions found in a reference stream type. Factors that are taken into consideration when 

categorizing a stream are elevation, drainage area, stream order/size, stream gradient, substrate 

composition, pH, alkalinity, specific conductance, and other unique characteristics. If these 

characteristics at a site are outside the range of those found in the reference sites, then the VT 

DEC uses an alternative analysis to describe an appropriate reference condition for site 

comparison. This prescription may include using historical monitoring data from the same or 

adjacent or similar water body; using a regional reference site; using a site-specific reference site 

(e.g., from upstream sites or adjacent sites); using paleo-ecological data collected from the 

sediments; or using quantitative models developed from field, historical, and experimental 

laboratory data. Any deviation from methods is fully documented by VT DEC and 

determination is based on the establishment of a compelling weight-of-evidence argument 

derived from monitoring data and best professional judgment. 

Fish reference conditions fall into two stream types for which metrics are calculated 

(VT DEC 2004): 

1. Small cold water streams: A site will be categorized for assessment by the Coldwater Index of 

Biotic Integrity (CWIBI) only if the stream can naturally support two to four native, naturally 

reproducing species of fish. Any wadeable stream site that is located at an elevation greater than 

500 ft or within the Connecticut River drainage is classified as a coldwater stream for these 

7-4 
7.3 

purposes. For other streams that are below 500 ft in the Champlain Valley, fish composition will 

determine which IBI will be used. Furthermore, the site is classified as cold water if one of the 

following is present: one salmonid species, slimy sculpin, or longnose sucker. Those sites that 

would naturally meet the cold water criteria but are populated with warm water species as a 

result of human influence areas are considered cold water sites for attainment status purposes. 

2. Warm water and cold water streams: A site will be placed in this category only if the stream 

can naturally support five or more native, naturally reproducing species of fish. Stream sites in 

this group are evaluated using the Mixed Water Index of Biotic Integrity (MWIBI). Both warm 

and coldwater streams fall into this category. 

Field and Laboratory Protocols 

7.3.1 Macroinvertebrate Protocols (taken from VT DEC 2004) 

7.3.1.1 Field Methods 

VT DEC uses a method to collect macroinvertebrates similar to that described in the 

Single Habitat RBP III (Plafkin et al. 1989). A riffle habitat is chosen within the stream 

sampling reach for macroinvertebrate sampling. Then, an 18-in wide x 12-in high D-frame net 

(500 μm mesh) is placed in the riffle and an area immediately upstream of the net (approximately 

1.5-ft x 1.5-ft) is thoroughly disturbed by hand, ensuring that all pieces of substrate are moved 

and rubbed clean of attached organisms. Moving up-stream, this is repeated at four to five 

different locations within the riffle, representing a range of velocity and substrate type 

characteristics of that riffle. Each specific location is actively sampled until all the substrate in 

approximately an 18-in x 18-in square in front of the net has been disturbed. This generally 

takes about 30 seconds of active sampling per location, and active sampling is terminated at the 

end of approximately two minutes. The contents of the net are allowed to drain of excess water, 

placed into a quart mason jar and preserved with 75% ethanol. Then, to obtain a replicate 

sample for the site, this entire process is repeated, being careful to avoid areas previously 

disturbed. This “composite” sampling methodology effectively collects samples representative 

of the macroinvertebrate assemblage of that riffle. The VT DEC then measures and records the 

physical condition of the riffle (i.e., stream wetted and bankfull widths, depth, water velocity, 

water temperature, weather conditions, substrate composition, substrate embeddedness, canopy 

cover, stream bank condition, and immediate upstream land use) and takes a water sample for 

specific conductance, pH and alkalinity determination. On a site-specific basis, other water 

chemistry parameters are determined. The site and sampling event codes are recorded on the 

field sheet. 

7.3.1.2 Laboratory Methods (VT DEC 2004) 

Sample processing takes place in the laboratory where macroinvertebrate samples are 

washed of preservative through a #30 sieve. The rinsed sample is then spread evenly over a 

white gridded tray (minimum 24 squares) by adding a small amount of water to allow the sample 

to be evenly spread, but not so much as to cause the macroinvertebrates to float freely around the 

tray. Six squares are randomly selected by first choosing one random number and then isolating 

7-5 
five surrounding squares from the rest of the sample. This method ensures that one quarter of the 

sample collected will be picked. After all macroinvertebrates from one quarter of the tray have 

been sorted, additional squares are then sorted until a minimum of 300 organisms has been 

reached. The total number of squares sorted is recorded so that sample density or relative 

abundance can be calculated. Macroinvertebrates are then identified to genus or species except 

for the Class Oligochaeta which is identified to the family level (VT DEC 2004). All 

macroinvertebrates are separated into major groups and preserved with 75% ethanol. If the 

entire sample is not sorted, it is qualitatively examined for all EPT organisms and other larger 

organisms, such as crayfish or mussels, not detected in the subsample to determine species 

distributions only. The additional EPT sample is preserved in a separate jar. A reference 

collection of all identified taxa is kept to assure consistent identifications. 

7.3.2 Fish Protocols (VT DEC 2004) 

7.3.2.1 Field Methods 

The VT DEC collects fish to determine the community condition of wadeable streams. 

Wadeable is defined as a stream or river that at some time during the year can be sampled by an 

individual wading in the thalweg of the channel. In streams less than 6 m wide, a single 

backpack electrofishing unit is used. In wider streams, the VT DEC uses multiple backpack 

units. Reach length is chosen by the overall width of the stream with a minimal reach length of 

75 m for a 3-m wide stream, 100 m for a 4- to 5-m wide stream, 120 m for a 6- to 8-m wide 

stream, 140 m for a 9- to 11-m wide stream, 160 m for a 12- to 14-m wide stream, and 180 m for 

a stream with a width of 15 m. The reach should represent a subsample of the overall stream and 

may also be dependent on the expected density of fishes. For example, if an unproductive cold 

water stream is sampled, a longer reach is sampled to compensate for the low density of fish 

expected. Those streams that are productive cool and warm water sites may have smaller sample 

reaches. Furthermore, the stream section that is sampled would represent the overall habitat of 

the surrounding stream reach. All habitat types are sampled within the reach to maximize 

species richness. 

When electrofishing, the crew begins in the most downstream portion of the reach and 

moves upstream. If a stream is being screened, only one pass is required. Two or three passes 

are made in those streams where the density is being evaluated as a result of a specific impact. If 

multiple passes are made, the VT DEC calculates a removal population estimate to ensure the 

accurate calculation of fish density. All fish that are stunned are collected using a net and placed 

into buckets of water for on-site identification to species and then released. All fish are examined 

for anomalies and salmonids are measured for length. Generally all identifications occur in the 

field. If a positive identification cannot be made, the specimen is preserved and is positively 

identified by a VT DEC biologist using keys (Smith 1985, Langdon et al. in preparation). 

Data Management/Quality 

Data are stored and managed in a Microsoft Access database, and metrics are calculated 

in this program. Data stored in the Access database can be moved to other spreadsheet programs 

such as Excel and to graphical and statistical analysis programs such as SigmaPlot, SigmaStat, 

7-6 

7.4 
7.5 

and PC-ORD. VT DEC uses SigmaStat for the statistical analysis calculations of parametric 

ANOVAs and nonparametric comparisons, and uses PC-ORD for multivariate analyses. 

Analysis of Biological Data 

7.5.1 Macroinvertebrate Data (VT DEC 2004) 

VT DEC uses multivariate statistics and multiple metrics to evaluate the 

macroinvertebrate assemblage. Eight metrics have been developed to measure the 

macroinvertebrate community integrity (Table 7-2). These metrics measure specific ecological 

attributes of the community and include: Density, Richness, EPT index, Percent Model Affinity 

Orders, HBI, Percent Oligochaeta, EPT/(EPT + Chironomidae), and the Pinkham-Pearson 

Coefficient of Similarity-Functional Groups (Table 7-2). Using macroinvertebrate data, each 

metric is scored and an overall condition rating is determined for each site sampled. 

After metrics are calculated, the values are compared to a table of threshold values for each of 

the water quality classes (Table 7-3). These values represent a single point in a continuum of 

values reflecting changes from the reference condition. Along with best professional judgment 

and a weight-of-evidence decision process, VT DEC assigns a site to an attainment class. The 

biologist assigns a pass, fail, or indeterminate value for each of the metrics depending on the 

score. If five or more metrics score a pass and no metrics are below the threshold value, then the 

ALU is supported. However, if one or more of the metrics fail the ALU is not supported. If 

neither of these conditions is met for the Water Management Class of the stream being assessed 

(i.e., Class A1 or B), then the site is assigned an indeterminate finding and may require 

additional data and/or sampling to make a support or non-support decision. 

Table 7-2. VT DEC macroinvertebrate metrics and methods used to calculate each of the 

metrics. 

Variable 

Number 

Indices/ Measures of 

Biological Integrity 

Method 

1 Density The relative abundance of animals in a sample. (Number of animals 

in subsample)/(Proportion of sample processed) 

2 Richness The number of species in a sample unit. Calculated as the total 

number of distinct taxa identified in a sample and averaged across 

replicate samples. Note: immature larva identified to family or 

genus is not considered new taxon if genus or species identification 

is determined within its group. 

3 EPT Index The number of distinct taxa identified in a sample from the orders 

Ephemeroptera, Plecoptera, and Trichoptera. Calculated as for 

Variable 2. Note: immature larva identified to family or genus is not 

considered new taxa if genus or species identification is determined 

within its group. 

7-7 
Variable Indices/ Measures of Method 

Number Biological Integrity 

4 Percent Model Affinity 

Orders (PMA-O) 

A measure of order-level similarity to a model based on the reference 

streams. PMA-O is calculated by determining the percent 

composition for each major group (Coleoptera, Diptera, 

Ephemeroptera, Plecoptera, Trichoptera, and Oligochaeta) at the 

assessment site. Then those values are compared to the mean 

percent composition of each corresponding order from the reference 

condition (model). The sum of the minimum of the two values for 

each order is the PMA-O. 

PMA-O = Σ min (Xa or Xr) 

Where: Xa= the percent composition of order X from the assessment 

site 

Xr = the percent composition of order X from the appropriate 

reference conditions (Novak and Body 1992). 

5 Hilsenhoff Biotic Index 

(HBI) 

N 

a n 

HBI i 

∑ ii

=

Where: 

ni = number of individuals in taxon i 

ai = tolerance value for taxon i (as assigned by VT DEC 2004, after 

Hilsenhoff 1987, Bode et al. 1996) 

N = number of individuals in the sample with a tolerance value 

6 Percent Oligochaeta (Abundance of Oligochaeta)/(Total number of individuals) x 100 

7 EPT/EPT + (Abundance of Ephemeroptera, Plecoptera and Trichoptera) / 

Chironomidae (Abundance of Ephemeroptera, Plecoptera, Trichoptera + 

Chironomidae) 

8 Pinkham-Pearson 

Coefficient of SimilarityFunctional Groups 

(PPCS-F) 

A measure of functional feeding groups calculated by determining 

the percent composition of the six major functional groups 

(collector-gatherer, collector-filterer, predator, shredder-detritus, 

shredder-herbivore, scraper) at the site as assigned by VT DEC 

(2004) (based on Merrit and Cummins 1996, Bode et al. 1996). 

⎟

⎟

⎠

⎞ ⎜

⎜

⎝

⎛ =− ∑=

K 

i ibia 

ibia 

xx 

xx 

K

FPPCS 

1 ),max( 

1 ),min(

Where: 

K = the number of comparisons between stations (6) 

xia = the number of individuals in functional group i in sample a 

(reference site) 

xib = number of individuals in functional group i in sample b 

The final decision is based on a number of considerations in accordance with the VT 

WQS. These include the use of chemical and physical data from the sample site to determine 

which of the three wadeable stream macroinvertebrate categories are used to determine 

7-8 
attainment, as well as the evaluation of data quality. Macroinvertebrate data must be collected 

using standard methods and trained qualified personnel. Furthermore, any diversions from 

average conditions must be documented by VT DEC. These diversions could include any 

hydrological, meteorological or other extreme events that occurred before sampling, and any 

errors committed during sampling. Finally, variability of the samples from a site is analyzed and 

evaluated on a case-by-case basis. If variability exceeds 40%, but is less than 75% the data will 

be handled with caution. If the percent standard error of the mean of abundance is greater than 

75%, the data are rejected. If this is the case, any other data collected from the site (e.g., 

physical, chemical, and/or fish data) can be used to submit a temporary ALUS classification. 

VT DEC then sets threshold index values to define how the biological indices relate to 

the narrative class standards established in the VT WQS. The threshold values were derived 

from the distribution of metrics within both the reference and the impacted data sets. Thresholds 

are also minimally adjusted at each site on a case-by-case basis by a VT DEC biologist using 

BPJ. 

Class A includes only those highest quality sites that exhibit minimal change from natural 

conditions. For those metrics that decrease in value with impairment from stressors (i.e., 

Richness, EPT, PMA-O, EPT/ EPT +C, PPCS-FG, and Density), it is reasonable to expect that 

the upper 75 percent of the reference sites best meet the true natural condition (threshold set at 

25th percentile). For the metrics that increase in value with impairment from a stressor (i.e., HBI 

and percent oligochaetes) the threshold was set at the 75th percentile. 

Streams in Class B, Water Management Type 1 exhibit minor changes from “natural 

condition”. The reference streams are likely include a small percentage of lower quality 

reference sites; therefore, the threshold values for Class B1 were set to the 5th and 95th percentiles 

of the reference streams to ensure against the influence of outlier reference values. Since a 

minor change was expected for these sites, the threshold was set at the 5th percentile (to include 

the upper 95 percent of the reference sites) for the metrics that decrease in response to stress 

(e.g., Richness). For metrics that increase in response to stress, the threshold was set at the 95th 

percentile of reference sites, such that most reference sites fell below the threshold. 

Class B Water Management Types 2 and 3 and Class A2 allow a moderate change from 

“natural condition” as a management goal. Thresholds for these classes were set based on BPJ 

using the range of reference values and the median, and 10th/90th percentile values of the 

distribution from sites known to be impacted. 

7.5.2 Fish Data (VT DEC 2004) 

The fish assemblage at sampling sites is assessed using either the Cold Water Index of 

Biotic Integrity (CWIBI) or the Mixed Water Index of Biotic Integrity (MWIBI). VT DEC 

derived index values consistent with narrative biological criteria in VT WQS using data from 

reference and impaired sites. The CWIBI consists of six metrics and is applied to small cold 

water stream fish communities, while the MWIBI consists of nine metrics and is applied to cold 

or warm water communities in Vermont. Tables 7-4 and 7-5 list the metrics, calculations, and 

scoring criteria for the CWIBI and MWIBI, respectively. The scoring criteria were derived for 

the CWIBI (7.5, 4.5, and 1.5) and the MWIBI (5, 3, and 1) to compare the cold water and mixed 

water sites with each other. For example, there are six CWIBI metrics and the highest overall 

score possible is 45 (7.5 x 6 = 45), whereas there are nine MWIBI metrics and the highest overall 

score possible is also 45 (9 x 5 = 45). Each metric is scored based on definitions and habitat or 

7-9 
7.6 

basin conditions. A final summation of each of the scores is made to place a fish assemblage 

(i.e., site) into a condition category (i.e., Excellent, Very Good, Good, Fair, Poor). “Excellent” is 

assigned to those streams that achieve a score of at least 41, “Very Good” to streams with scores 

of at least 36, “Good” for scores of at least 33, “Fair” for scores of at least 27, and anything 

below 27 is assigned a condition of “Poor”. The CWIBI or MWIBI score is then compared to 

the range of scores that correspond to each of the VT WQS Classes to determine which class 

could potentially best describe the fish assemblage (Table 7-6). For example, if a cold water fish 

assemblage scores a 36 on the CWIBI, the stream could be classified B Water Management Type 

1, defined as exhibiting a minor change from the reference condition. 

As is the case with macroinvertebrate data, fish data must undergo a stringent evaluation. 

Qualified personnel must adhere to the SOPs when collecting and analyzing the data. If a 

sample is deemed unacceptable due to error or a unique event other than what occurs in an 

average year, VT DEC can sample the site again. This event is to be scheduled at least three 

weeks from the initial sampling date. Until new data are collected, the VT DEC can assign a 

temporary attainment value based on the valid biological, chemical, and physical data already 

collected. 

Summary: Determining ALU Support 

VT DEC calculates metrics for both macroinvertebrate and fish data collected from probabilistic 

and monitoring stations for use in determining ALU attainment. Although each of the metrics 

and indices were derived independently, they each provide a quantitative way to assess the 

biological condition in the diverse types of streams in Vermont. VT WQS provide a 

classification system describing the condition of these streams (Table 7-1) that include Class A1, 

A2 and Class B1, B2, and B3 streams. Once these classes were established with management 

provisions in place, indices were developed that describe the stream condition in relation to 

reference condition site values (e.g., CWIBI, MWIBI and macroinvertebrate metrics). The VT 

DEC uses the CWIBI, MWIBI and individual macroinvertebrate metrics to determine the ALUS 

based on the thresholds described in the tables above (Tables 7-3, 7-4, 7-5, and 7-6). The 

resultant product is a narrative description for each monitored site for the ALU attainment part of 

the 305(b) report on the condition of streams in Vermont. In addition, this information is used to 

list non-supporting sites on the 303(d) list for Vermont. 

7-10 
Table 7-3. Macroinvertebrate assemblage biocriteria thresholds for the macroinvertebrate community str

associated WQ classes of Vermont (VT DEC 2004). 

Small-size High Gradient Streams Medium-size High Gradient Streams Warm Water M

(SHG) (MHG) and R

WQ Class A1 B-WMT1 B A1 B-WMT1 B A1 B-

Ecological B-WMT 2-3 

A2 (water 

supply) 

Ecological B-WMT 2-3 

A2 (water 

supply) 

Ecological 

Metric Direction of Reference Minimal Moderate Reference Minimal Moderate Reference M

metric as water 

Condition Change 

from 

Change from 

Reference 

Condition Change 

from 

Change from 

Reference 

Condition C

quality improves 

Reference 

Condition 

Condition 

(undue adverse 

effect) 

Reference 

Condition 

Condition 

(undue adverse 

effect) 

Re

Co

Richness Positive >35 >31 >27 >43 >39 >30 >40 EPT Positive >21 >19 >16 >24 >22 >18 >21 PMA-O Positive >65 >55 >45 >65 >55 >45 >65 HBI Negative <3.00 <3.50 <4.50 <3.50 <4.00 <5.00 <4.25 <% Oligo Negative <2 <5 <12 <2 <5 <12 <2 EPT/EPT +Chiron. 

Positive >0.65 >0.55 >0.45 >0.65 >0.55 >0.45 >0.65 >

PPCS-FG Positive >0.50 >0.45 >0.40 >0.50 >0.45 >0.40 >0.50 >Density Positive >500 >400 >300 >500 >400 >300 >500 

7-11 
Table 7-4. The six metrics used in scoring the fish assemblage for the CWIBI. These 

streams must naturally support two to four native species (VT DEC 2004). 

Metrics and qualifications/calculations 

Scoring Criteria 

7.5 4.5 1.5 

1. Number of intolerant species 

(One exotic trout species may be substituted for brook trout) 2 1 0 

2. Proportion of individuals as cold water stenotherms > 75% 50-75% < 50% 

3. Proportion of individuals as generalist feeders < 5% 5-9% > 9% 

4. Proportion of individuals as top carnivores > 35% 25-35% < 25% 

5. Brook trout density (number/100 m2 - 1pass) > 4.0 2.0-4.0 < 2.0 

6. Brook trout age class structure 

Young-of-the-year (YOY)  100 mm, adult  100mm) 

YOY and 

adults present YOY only YOY absent 

Index Scores Conditions for use 

Excellent 

Very Good 

Good 

Fair 

Poor 

42-45 

36 

33 

27 

< 27 

1. Only fishes over 25 mm in length should be considered. 

2. Only naturally reproducing salmonids are to be considered; no stocked fish are to be 

included. No Atlantic salmon are to be included. 

3. Only species represented by more than a single individual are entered into metrics 1 

and 6. 

4. No non-resident species shall be entered into metric calculations. 

Table 7-5. The nine metrics used in scoring cold and warm water sites for the MWIBI. 

These streams must naturally support more than four native fish species (VT DEC 2004). 

Metric and Qualification Stream descriptor Scoring Criteria 

Species Richness and Composition 5 3 1 

1. Total number of native fish species Follows maximum species 

richness lines 

2. Number and identity of native, 

intolerant species 

(A non-native trout may be substituted for 

brook trout when absent) 

Site Elevation > 400 feet > 1 1 0 

Site Elevation < 400 feet 

> 0 - 0 

3. Number and identity of native benthic 

insectivores

Site Elevation < 400 feet 

with site drainage < 25 

km2 

> 0 - 0 

All other sites > 1 1 0 

4. Proportion of individuals as white 

suckers and creek chubs. < 11% 11-30% > 30% 

Trophic Composition 

7-12 
Metric and Qualification Stream descriptor Scoring Criteria 

5. Proportion of individuals as generalist Site Elevation > 500 feet < 20% 20-45% > 45% 

feeders. Site Elevation < 500 feet < 30% 30-60% > 60% 

6. Proportion of individuals as water Site Elevation > 500 feet > 65% 30-65% < 30% 

column and benthic insectivores (score a 

“1" if blacknose dace is >60% of total 

assemblage or 100% of insectivores) 

Site Elevation < 500 feet 

> 55% 20-55% < 20% 

7. Proportion of individuals as top Cold water assemblage > 15% 5-15% < 5% 

carnivores Warm water assemblage 

(Nonnative trout included) with site drainage > 25 

km2 

> 10% 3-10% < 3% 

Warm water assemblage 

with site drainage < 25 

km2

 > 0 - -

Fish Abundance and Condition 

8. Proportion of individuals with 

deformities, fin erosion, lesions, or < 1% 1-4% >4% 

tumors. 

9. Abundance in sample 

(One pass - # 100 m2

) 

(Nonnative species included) 

Site Elevation < 500 feet 

Site Elevation > 500 feet 

Alkalinity > 9 mg/l 

> 20 

> 10 

10-20 

7-10 

< 10* 

< 7* 

* Site automatically scores Poor Site Elevation < 500 feet 

Alkalinity < 9 mg/l > 6 3-6 <3* 

Sum of Metric Scores Conditions for Use 

Excellent 41-45 1. For wadeable streams only. 

Very Good 37 2. Site should naturally support at least five native 

Good 33 species. 

Fair 27 3. Only individuals more than 25 mm TL are to be 

Poor <27 entered in the determination. 

4. Only species with more than one individual captured 

are entered into metrics 2 and 3. 

5. Stocked fish are not considered in determinations. 

6. All sites within the Connecticut River drainage are to 

be scored as > 500 m elevation. 

7-13 
7.7 

Table 7-6. All possible scores for the CWIBI and MWIBI that correspond to the VT WQS 

classification scheme (VT DEC 2004). 

WQS Classification Range 

Possible Scores 

CWIBI MWIBI 

A-1 41-45 42, 45 41, 43, 45 

A-1 or B-1 based on BPJ 39 39 39 

B-1 36-37 36 37 

B-1, A-2, or B-2, B-3 based on BPJ 35 35 

A-2, B-2, B-3 33 33 33 

B-2, B-3 or Non-Support based on BPJ 29-31 30 31, 29 

Non-Support < 29 27, 24, 21, 18, 

15, 12, 9 

27, 25, 23, 21, 

19, 17, 15, 13, 

11, 9 

Literature Cited 

Bode, R.W., M.A. Novak, and L.E. Abele. 1996. Quality Assurance Work Plan for Biological 

Stream Monitoring in New York State. New York State Department of Environmental 

Conservation, Albany, NY. 

Hilsenhoff, W.L. 1987. An improved biotic index of organic stream pollution. Great Lakes 

Entomologist 20:31-39. 

Langdon, R.W., M. Ferguson, and K. Cox. In preparation. The Fishes of Vermont. 

Merrit, R.W., and K.W. Cummins (editors). 1996. An Introduction to Aquatic Insects of North 

America, 3rd ed. Kendall/Hunt Publishing Company, Dubuque, IA. 

Novak, M.A., and R.W. Bode. 1992. Percent model affinity, a new measure of 

macroinvertebrate community composition. Journal of the North American 

Benthological Society 11(1):80-85. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA 440-4-89-001. U.S. Environmental Protection Agency, Office of Water 

Regulations and Standards, Washington, D.C. 

Smith, C.L. 1985. The Inland Fishes of New York State. New York State Department of 

Environmental Conservation, Albany, NY. 

7-14 
U.S. EPA. 2002. Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

State of Vermont. 2000. Vermont Water Quality Standards. State of Vermont, Water 

Resources Board, Montpelier, VT. http://www.state.vt.us/wtrboard/july2000wqs.htm 

(VT DEC) Vermont Department of Environmental Conservation. 2004. Biocriteria for Fish and 

Macroinvertebrate Assemblages in Vermont Wadeable Streams and Rivers – 

Implementation Phase. Vermont Department of Environmental Conservation, 

Waterbury, VT. http://www.vtwaterquality.org/bass/htm/bs_biomon.htm 

7.8 Resources 

U.S. EPA. Biological Indicators of Watershed Health, Vermont Webpage: 

http://www.epa.gov/bioindicators/html/state/vt-bio.html 

U.S. EPA. (2002). Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA-822-R-02-048. U.S. Environmental Protection Agency. 

http://www.epa.gov/bioindicators/html/program_summary.html 

VT DEC, Water Quality Division, Biomonitoring and Aquatic Studies Web page: 

http://www.vtwaterquality.org/bass.htm 

7-15 
8 SUMMARY 

8.1 Comparison Across States 

The U.S. EPA allows for each state to implement its own bioassessment program. This 

document reviews the methods used by U.S. EPA Region 1 states to monitor and assess streams 

for ALU attainment for 305(b) reporting and 303(d) listing as required by the CWA. Although 

Region 1 states share many commonalities such as the Connecticut River, which traverses four 

of the six states (New Hampshire, Vermont, Massachusetts, and Connecticut) and has a 

watershed that covers 11,000 mi2

, the entire region has diverse habitats ranging from northern 

broadleaf forests to southern New England forests, coastal systems, northern conifer forests and 

alpine regions (Alden and Cassie 2000). The diverse topography causes distinct differences in 

biota and, therefore, requires states to make adjustments to their bioassessment programs to 

account for these differences. For example, although 41 entities use the fish assemblage for 

bioassessment, Maine does not assess fish (U.S. EPA 2002b). One reason Maine has chosen to 

assess benthic macroinvertebrates and not the fish assemblage as well, is due to the great 

diversity of this assemblage in state waters compared to the fish assemblage (Personal 

Communication, Susan Davies). Meanwhile, Vermont has diverse benthic macroinvertebrate 

and fish fauna and they have adapted their bioassessment program to include a benthic metrics 

and indices to assess cold water and warm water fish assemblages. The rationale for program 

development can stem from a biological point of view but may be influenced by historical 

sampling methods, feasibility of sampling protocols, and by funding available to the program. 

Due to regional similarities, some states share many of the same protocols and have borrowed 

ideas for their own index development. Summary Table 8-1 offers a comparison and contrast of 

the key components of the state bioassessment programs and Table 8-2 lists all of the metrics 

used by the states. 

States are responsible for setting narrative criteria that will protect waters and define 

ALU. Bioassessment programs are then designed to aid in the determination of ALU using 

biological, physical, chemical and habitat data. Biological data provide the most accurate 

information about the resident aquatic organisms (U.S. EPA 2002a); therefore, it is essential that 

appropriate indicator assemblages be chosen to properly assess the natural system. All U.S. EPA 

Region 1 states minimally have chosen to assess the benthic macroinvertebrate assemblage, but 

some attempt is being made by most of the states to at least include information about fish 

assemblages (i.e., Connecticut, Massachusetts, New Hampshire, and Vermont) and/or algae (i.e., 

Connecticut and Massachusetts). It is then the responsibility of the states to decide the best way 

to analyze the biological data in order to show biotic gradients as water quality changes. 

As apparent in Table 8-1, these programs do share some commonalities such as using 

modified methods from the U.S. EPA RBP document, although the details of the methods may 

differ considerably. These differences are evident at all levels, including the strategy used for 

site selection, the decision making process used to select reference sites, field collection 

methods, laboratory sorting and taxonomic methods, and finally the selection of metrics and data 

analysis. These differences could be due to the necessity to compare to historical data by using 

similar methods, topographical or regional differences, available equipment, logistical 

constraints, and funding provided to the states. 

8-1 
Table 8-1. Comparisons of the key components of state bioassessment programs. 

Component CONNECTICUT MAINE MASSACHUSETTS NEW RHOD

HAMPSHIRE ISLAN

Index Period Late fall July-September July-September mid summerearly fall Summer a

fall 

Indicator Benthic Benthic Benthic Benthic Benthic assemblages used Fish Fish Fish 

Periphyton Periphyton 

Survey approach 5-yr rotating basin; 

Targeted. CT in the process of 

5-yr rotating basin; Targeted 

5-yr rotating basin; Targeted 

Targeted within Northern and Southern regions 

Fixed statiwithin two Level IV 

developing a probabilistic 

Omernick ecoregion

component to monitoring program 

(AdamsvilBrook and Wood Riv

Total stream miles 5,830 miles 31,672 miles 8,229 miles 10,881 miles 1,498 mile# sites sampled/year Benthic-50 sites 

Fish- 24 sites 

50-60 sites 75 sites 25-30 sites 45 sites 

Reference condition Least disturbed sites 

comparable to test sites based on natural features such as gradient 

BPJ for a priori placement into four pre-defined classes for linear discriminant analysis 

Least impacted sites; no potential to receive point or non-point source pollution and lack land use patterns that would degrade water quality 

Least impacted sites based on Human Disturbance Gradient, reference sites within each bioregion used to set attainment 

2 least disturbed reference one in eacthe ecoreg

thresholds 

Macroinvertebrates 

Sampling approach RBP III Single 

Habitat (Plafkin et al. 1989) 

Classification Attainment Evaluation (Davies et al. 1999, Davies 

RBP II and III Single Habitat Approach (Plafkin et al. 1989) 

NH DES methods (NH DES 2004) 

RBP III SiHabitat (Plafkin et 1989) 

and Tsomides 

8-2 
Component CONNECTICUT MAINE MASSACHUSETTS NEW 

HAMPSHIRE 

RHOD

ISLAN

2002) 

Field method(s) Kick net 9-in x 18-in 

rectangle, 800 x 900 

Rock baskets, riffle bags, rock 

Kick net 0.46 m x 0.46 m, 500 μm mesh. 

Rock baskets deployed for 8 

0.3 m widframe net, 

μm mesh filled cones. 

Deployed for 28-56 days. 600 

Rock baskets deployed for 6 to 8 weeks. 

weeks, 600 μm 

mesh 

μm mesh 

μm mesh sieve 

Lab sorting method Entire sample over a Entire sample RBP II and III (Plafkin Caton method 18-in x 13

56-square grid in small 

quantities 

et al. 1989) 25 squares 6-cm x 6-cm 

(Barbour et al. 1999) using 16 

1-in griddtray with 8 

square grids. equal squa

Organisms 200 Entire sample, 100 minimum of 100 100 subsample count unless >500 

organisms in sample 

Taxonomic resolution 

genus/species or lowest practical 

genus/species or lowest 

genus/species or lowest practical taxonomic 

genus/species or lowest practical 

genus/speor lowest 

taxonomic level practical 

taxonomic level 

level taxonomic level practical 

taxonomic level 

Analysis method RBPIII metrics and Linear RBP II and III metrics B-IBI for RBP III 

index (Plafkin et al. 1989) 

discriminant analysis 

and index (Plafkin et al. 1989) 

northern and southern regions (Neils and 

metrics anindex (Plaet al. 1989

Blocksom 2004) 

# of metrics (See 7 25 9 7 11 Table 8-2 for metrics) Periphyton Current Use Developing an algal Not Used Used for the detection Not Used Not Used 

indicator using probabilistic 

of algal blooms and as indicator of water 

monitoring; currently used only for 

quality to identify toxicity issues, nutrient 
Component CONNECTICUT MAINE MASSACHUSETTS NEW 

HAMPSHIRE 

RHOD

ISLAN

supplementary impacts, and habitat information. alterations. 

Field method(s) Modified RBP RBP Single Habitat 

Single Habitat method, field-based Rapid 

method, artificial substrates (biomass and chl a), and viewing 

Periphyton Survey (viewing bucket) 

bucket (% coverage) (Barbour et al. 1999) 

(Barbour et al. 1999) 

Measurements taken Chlorophyll a, Chlorophyll a, biomass, 

biomass, species composition and abundance 

and percent coverage 

Use for attainment Literature metrics to Use to evaluate if the 

derive conclusions aesthetics or Aquatic 

Life Use are affected 

Fish Field method RBP V protocols 

(Plafkin et al. 1989): electrofish 150 m reach, fish identified to species in field 

Not Used RBP V protocols 

(Plafkin et al. 1989): electrofish 100 m reach, fish identified to species in field 

RBP V protocols (Plafkin et al. 1989): electrofish 150 m reach, fish identified in field 

Not Used 

Analysis method Currently developing 

an index modeled after Vermont; data collected currently used as supplementary information for ALU 

Modification of RBP V metrics (Plafkin et al. 1989) 

IBI under development and modeled after Vermont. In the process of refining CWIBI and MWIBI for 

determination NH stream fish 

communities 

8-4 
Component CONNECTICUT MAINE MASSACHUSETTS NEW 

HAMPSHIRE 

RHOD

ISLAN

Use of fish data Fish Species Structure and function Currently 

composition, trophic structure and age class distribution 

of the fish assemblage and BPJ used to determine support or 

supplements macroinvertebrat e and other data 

along with BPJ used to make assessment 

impairment of aquatic life use 

for attainment decisions 

ALUS determination: data used 

Biological (macroinvertebrate quantitative index, supplemental fish data) physical, chemical, toxicological and habitat data also used 

Heaviest weight on biological data (outcome of the benthic linear discriminant analysis), physical, chemical, bacterial, and habitat 

Weight of Evidence approach using biological, habitat, chemical, and toxicological data 

Heavy weight placed on biological (BIBI) data, with fish assessments, benthic deposits, flow, habitat (RBP), macrophyte composition, sediment and ambient water 

Biological (macroinvbrate communitscore), physical, chemical (pH, temperatuand habita(RBP) dat

toxicity tests, pH and DO 
Table 8-2. Comparison of the macroinvertebrate metrics used by states in the New England Region. Colo

equivalent metrics across states. 

CONNECTICUT MAINE MASSACHUSETTS NEW RHOD

HAMPSHIRE ISLAN

Community Loss Cheumatopsyche 

Mean Abundance 

Ratio of Class A Indicator Taxa 

Community Loss Percent 

Chironomidae 

Community 

EPT Index Chironomini Mean Ratio of EP EPT Index Percent EPT Abund

Abundance (Family Generic Richness Clingers Functional Group) 

EPT/Chironomidae Ephemeroptera Relative EPT/Chironomidae Percent EPT Taxa (abundance ratio) Mean Abundance Chironomidae (abundance ratio) Intolerant Richness 

Abundance 

HBI EPT Generic Relative Diptera HBI/FBI Percent Non- EPT/Chiron

Richness Richness insects ae (abundan

ratio) 

Percent EPT-Diptera Relative Percent Contribution Percent HBI Contribution of Richness Ratio Ephemeroptera of Dominant Taxon Tolerant taxa Dominant Taxon Abundance Scraper/Filtering Generic Richness Relative Percent Reference Plecoptera Percent Ratio Oligochaeta Affinity Taxa Contributio

Abundance Dominant T

Taxa Richness HBI Relative Plecoptera 

Richness 

Percent Similarity Total Taxa Percent 

Hydropsychof Total 

Trichoptera 

Hydropsyche Mean Shannon-Weiner Scraper/Filtering Ratio of Abundance Generic Diversity Ratio Shredders t

Total NumbIndividuals 

8-6 
CONNECTICUT MAINE MASSACHUSETTS NEW 

HAMPSHIRE 

RHOD

ISLAN

Perlidae Mean Sum of Mean Taxa Richness Scraper/Fil

Abundance (Family Abundances of: Ratio Functional Group) Cheumatopsyche, 

Cricoptopus, Tanytarsus and Ablabesmyia 

Plecoptera Mean 

Abundance 

Sum of Mean Abundances of: Dicrotendipes, Microspectra, Parachironomus 

Shannon WDiversity In

and Helobdella 

Probability (A + B Sum of Mean Total Taxa 

+ C) from First Abundances of: Richness State Model Acroneuria and 

Stenonema 

Probability (A + B) Tanypodinae Mean 

from First Stage Abundance (Family Model Functional Group) 

Probability of Class Total Mean 

A from First Stage Abundance Model 
8.2 

Currently, U.S. EPA Region 1 is conducting the New England Wadeable Streams 

(NEWS) Project. The primary purpose of this study is to apply a random probability-based site 

selection strategy across New England states. As part of this study, fish, macroinvertebrate, 

water chemistry, physical chemistry, and habitat data are being collected at 50 sites in each 

participating state, either by the state itself or by Region 1. In some cases, individual states are 

collecting samples using both the standardized method for this study and their own method, 

allowing for a possible comparison of field sampling methods. Sampling was to be completed 

by the end of 2003, and a final report presenting the findings of this study is anticipated by late 

2004 (Personal Communication, Hillary Snook, U.S. EPA Region 1). 

Literature Cited 

Alden, P., and B. Cassie. 1998. The National Audubon Society Field Guide to New England. 

Alfred A. Knopf, New York. 

Barbour, M., 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. 

http://www.epa.gov/owow/monitoring/rbp/wp61pdf/rbp.pdf 

Davies, S.P., L. Tsomides, J.L. DiFranco, and D.L. Courtemanch. 1999. Biomonitoring 

Retrospective: Fifteen Year Summary for Maine Rivers and Streams. Maine Department 

of Environmental Protection. 

http://www.state.me.us/dep/blwq/docmonitoring/biomonitoring/biorep2000.htm 

Davies, S.P., and L. Tsomides. 2002. Methods for the Biological Sampling and Analysis of 

Maine’s Rivers and Streams. Maine Department of Environmental Protection, Augusta, 

ME. http://www.state.me.us/dep/blwq/docmonitoring/finlmeth1.pdf 

Neils, D., and K. Blocksom. 2004. Development of the New Hampshire Benthic Index of Biotic 

Integrity. New Hampshire Department of Environmental Services, Concord, NH. 

New Hampshire Department of Environmental Services. 2004. Biomonitoring Program 

Protocols. New Hampshire Department of Environmental Services, Concord, NH. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M. Hughes. 1989. Rapid 

Bioassessment Protocols for Use in Streams and Rivers: Benthic Macroinvertebrates and 

Fish. EPA 440-4-89-001. U.S. Environmental Protection Agency, Office of Water 

Regulations and Standards, Washington, D.C. 

(U.S. EPA) U.S. Environmental Protection Agency. 2002a. Consolidated Assessment and 

Listing Methodology, Toward a Compendium of Best Practices, First Edition. U.S. 

Environmental Protection Agency, Office of Water, Washington, D.C. 

http://www.epa.gov/owow/monitoring/calm.html 

8-8 
U.S. EPA. 2002b. Summary of Biological Assessment Programs and Biocriteria Development 

for States, Tribes, Territories, and Interstate Commissions: Streams and Wadeable Rivers. 

EPA 822-R-02-048. U.S. Environmental Protection Agency. 

Vermont Department of Environmental Conservation. 2004. Biocriteria for Fish and 

Macroinvertebrate Assemblages in Vermont Wadeable Streams and Rivers – 

Implementation Phase. Vermont Department of Environmental Conservation, 

Waterbury, VT. http://www.vtwaterquality.org/bass/htm/bs_biomon.htm 

8-9 
APPENDIX A: PROCESS AND CRITERIA FOR THE ASSIGNMENT OF BIOLOGIST'S 

CLASSIFICATION 

DRAFT 

From: Appendix H in Stream Biological Monitoring and Numeric Criteria Development in 

Maine 

by: 

Susan P. Davies, M.S.1 

Leonidas Tsomides, M.S.1 

David L. Courtemanch, Ph.D.1 

Francis Drummond, Ph.D. 2 

1 Maine Department of Environmental Protection, Augusta, Maine

2 Department of Biological Sciences, University of Maine, Orono, Maine 

Raters 

David Courtemanch 

MS in aquatic entomology; PhD in environmental science; employed as a Biologist in the 

Division of Environmental Evaluation and Lake Studies (DEELS) in the Water Bureau for 16 

years; currently Director, Division of Environmental Assessment. 

Susan Davies 

MS aquatic entomology; employed as a Biologist in the River and Stream section of 

DEELS for 9 years, coordinating the Instream Biological Monitoring Program. 

Leon Tsomides 

MS aquatic entomology; employed as a Biologist in the River and Stream Section of 

DEELS for 3 years, working with the Instream Biological Monitoring Program. 

Ranking Process 

Each biologist independently reviewed biological information for each sampling event, as 

listed below, including identities and abundances of taxa occurring in the biological sample and 

computed index values for the biological data (e.g. diversity, richness, EPT, etc). Physical habitat 

information was also reviewed including water depth, velocity, substrate composition, canopy 

cover, etc., in order to evaluate the effects of various habitat conditions on the structure of the 

macroinvertebrate assemblage. Sample information was reviewed for the values of the given 

measures, relative to values for other samples in the data set. The actual classification 

assignment was determined by how closely the biological information conformed to the aquatic 

life classification standards, correcting for habitat effects. Numerical ranges, per se, were not 

established, a priori, for each measure. Instead, the information was reviewed for it's 

compatibility with the mosaic of findings expected for each Class, listed in the Relative Findings 

Chart in this Appendix (H-1). The biologists did not have any knowledge of the actual location 

of the sampled sites, nor did they have knowledge of any pollution influences. Following the 

A-1 
independent assignment of classes the biologists established a consensus classification, following 

an open exchange of justifications for each biologist's assignment. 

Biologist's Classification Criteria 

Each biologist reviewed the sample data for the values of a list of measures of 

community structure and function. Criteria used by biologists to evaluate each measure are listed 

in the Relative Findings Chart, Appendix A-1. 

TOTAL ABUNDANCE OF INDIVIDUALS 

TOTAL ABUNDANCE OF EPHEMEROPTERA 

TOTAL ABUNDANCE OF PLECOPTERA 

ABUNDANCE OF EPHEMEROPTERA/TOTAL ABUNDANCE 

ABUNDANCE OF PLECOPTERA/TOTAL ABUNDANCE 

ABUNDANCE OF HYDROPSYCHIDAE/TOTAL ABUNDANCE 

ABUNDANCE OF EPHEMEROPTERA+PLECOPTERA/TOTAL ABUNDANCE 

ABUNDANCE OF GLOSSOSOMA/TOTAL ABUNDANCE 

ABUNDANCE OF BRACHYCENTRUS/TOTAL ABUNDANCE 

ABUNDANCE OF OLIGOCHAETES/TOTAL ABUNDANCE 

ABUNDANCE OF HIRUDINEA/TOTAL ABUNDANCE 

ABUNDANCE OF GASTROPODA/TOTAL ABUNDANCE 

ABUNDANCE OF CHIRONOMIDAE/TOTAL ABUNDANCE 

ABUNDANCE CONCHAPELOPIA+THIENNEMANNYMIA/TOTAL ABUNDANCE 

ABUNDANCE OF TRIBELOS/TOTAL ABUNDANCE 

ABUNDANCE OF CHIRONOMUS/TOTAL ABUNDANCE 

GENERIC RICHNESS 

EPHEMEROPTERA RICHNESS 

PLECOPTERA RICHNESS 

EPT RICHNESS 

EPHEMEROPTERA RICHNESS/GENERIC RICHNESS 

PLECOPTERA RICHNESS/GENERIC RICHNESS 

DIPTERA RICHNESS/GENERIC RICHNESS 

EPHEMEROPTERA+PLECOPTERA RICHNESS/GENERIC RICHNESS 

EPT RICHNESS/DIPTERA RICHNESS 

NON-EPT OR CHIRONOMIDAE RICHNESS/GENERIC RICHNESS 

PERCENT PREDATORS 

% COLLECTOR FILTERERS+GATHERERS/%PREDATORS+SHREDDERS 

NUMBER OF FUNCTIONAL FEEDING GROUPS REPRESENTED 

SHANNON-WEINER GENERIC DIVERSITY 

HILSENHOFF BIOTIC INDEX 

In addition, in cases where a valid, clean-water, upstream reference station existed, the 

following comparative index data was also reviewed: 

JACCARD TAXONOMIC SIMILARITY 

TAXONOMIC SIMILARITY OF DOMINANT TAXA 

COEFFICIENT OF COMMUNITY LOSS 

A-2 
PERCENT SIMILARITY 

Results 

In 64% of the cases there was unanimous agreement among the independent raters, and in 

an additional 34% of the samples two of the raters were in agreement and one had assigned a 

different classification. In 3 of the rated samples there was disagreement among all three raters 

(2%). 

Table A-1. RELATIVE FINDINGS CHART 

Measure of Relative Findings 

Community 

Structure A B C NA 

Total Abundance 

of Individuals 

often low often high variable variable: often 

very low or high 

Abundance of high high low low to absent 

Ephemeroptera 

Abundance of 

Plecoptera 

highest some present low to absent absent 

Proportion of highest variable, low zero 

Ephemeroptera depending on 

dominance by 

other groups 

Proportion of highest variable, low zero 

Pleocoptera depending on 

dominance by 

other groups 

Proportion of intermediate highest variable low to high 

Hydropsychidae 

Proportion of highest variable Low absent 

Ephemeroptera & 

Plecoptera 

Proportion of highest low to very low to absent 

Glossoma intermediate absent 

Proportion of highest low to very low to absent 

Brachycentrus intermediate absent 

Proportion of low low low to moderate highest 

Oligochaetes 

Proportion of low variable variable variable to 

Hirudinea highest 

Proportion of low low variable variable to 

Gastropoda highest 

Proportion of lowest variable, highest variable 

Chironomidae depending on the 

dominance of 

other groups 

A-3 
Measure of 

Community 

Structure 

Relative Findings 

A B C NA 

Proportion of 

Conchapelopia & 

Thienemannimyia 

lowest low to variable variable variable to 

highest 

Proportion of 

Tribelos 

low to absent low to absent low to variable variable to 

highest 

Proportion of 

Chironomus 

low to absent low to absent low to variable variable to 

highest 

Generic Richness variable highest variable lowest 

Ephemeroptera 

Richness 

highest high low very low to 

absent 

Plecoptera 

Richness 

highest variable low to absent absent 

EPT Richness high highest variable low 

Proportion 

Ephemeroptera 

Richness 

highest variable low zero 

Proportion 

Plecoptera 

Richness 

highest high low low to zero 

Proportion 

Diptera Richness 

low to variable variable highest variable to high 

Proportion 

Ephemeroptera & 

Plecoptera 

Richness 

highest high low to variable low to absent 

EPT Richness 

dvided by Diptera 

Richness 

high highest low to variable lowest to zero 

Proportion NonEPT or 

Chronomid 

Richness 

high high low lowest 

Percent Predators low low high to variable highest 

Percent Collector, 

Filterers & 

Gatherers divided 

by Percent 

Predators & 

Shredders 

high highest low lowest 

Number of 

Functional 

Feeding Groups 

variable highest variable lowest 

A-4
Measure of Relative Findings 

Community 

Structure A B C NA 

Represented 

Shannon-Weiner low to highest variable to lowest 

Generic Diversity intermediate intermediate 

Hilsenhoff Biotic lowest low intermediate highest 

Index 

A-5