U.S. Department of the Interior

U.S. Geological Survey

National Water-Quality Assessment Program 

Assessment of Nutrient Enrichment by Use of Algal-, 

Invertebrate-, and Fish-Community Attributes in Wadeable 

Streams in Ecoregions surrounding the Great Lakes 

Scientific Investigations Report 2011–5009 
Cover images (from let to right):

Tractor applying fertilizer in the Sugar Creek watershed in Hancock County, Indiana, June 7, 2007. (Photograph by John T. 

Wilson, U.S. Geological Survey (USGS))

Cows drinking in the Mad River, August 25, 2003. (Photograph by Stephanie D. Janosy, USGS)

An algal mass in a stream from an unknown location in Indiana.

USGS employees collecting an invertebrate community sample from the Popple River, Wisconsin, on September 10, 2003. 

(Photograph by Barbara C. Eikenberry, USGS)

USGS employees collecting a fish-community sample from Sugar Creek at New Palestine, Indiana, in Hancock County on 

August 15, 2008. (Photograph by David S. Nail, USGS)

Scatter plot showing breakpoint analysis of total nitrogen and Achnanthidium minutissimum in the Glacial North diatom 

ecoregion. 
Assessment of Nutrient Enrichment by 

Use of Algal-, Invertebrate-, and FishCommunity Attributes in Wadeable Streams 

in Ecoregions surrounding the Great Lakes 

By Jeffrey W. Frey, Amanda H. Bell, Julie A. Hambrook Berkman, and 

David L. Lorenz 

National Water-Quality Assessment Program 

Scientific Investigations Report 2011–5009 

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2011

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reproduce any copyrighted materials contained within this report.

Suggested citation:

Frey, J.W., Bell, A.H., Hambrook Berkman, J.A., and Lorenz, D.L., 2011, Assessment of nutrient enrichment by use of 

algal-, invertebrate-, and fish-community attributes in wadeable streams in ecoregions surrounding the Great Lakes: 

U.S. Geological Survey Scientific Investigations Report 2011–5009, 49 p. 
iii

Contents

Foreword ........................................................................................................................................................vi

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................2

Purpose and Scope .......................................................................................................................................3

Approach and Methods................................................................................................................................3

Site Selection.........................................................................................................................................3

Nutrient Sample Collection .................................................................................................................6

Biological Sample Collection..............................................................................................................6

Data Analysis.........................................................................................................................................6

Regional Differences...................................................................................................................6

Nutrient Conditions of the Sites ................................................................................................7

Biological Conditions of the Sites .............................................................................................7

Using Biological and Nutrient Conditions to Describe Differences between Sites .......12

Description of Regional Differences and the Nutrient and Biological Conditions in 

the Nutrient Categories.................................................................................................................14

Regional Biological Differences.......................................................................................................14

Regional Comparisons of the Nutrient Categories........................................................................15

Comparison of Seasonal Nutrient Concentrations by Nutrient Categories..............................19

Biological Conditions Within the Nutrient Categories..................................................................20

Algae............................................................................................................................................23

Central and Western Plains Ecoregion.........................................................................23

Glaciated North Ecoregion..............................................................................................23

Invertebrates ..............................................................................................................................28

Central and Western Plains Ecoregion.........................................................................28

Glaciated North Ecoregion..............................................................................................28

Fish ...............................................................................................................................................32

Central and Western Plains Ecoregion.........................................................................32

Glaciated North Ecoregion..............................................................................................32

Breakpoint Analysis Between Nutrients and Biological Attributes....................................................35

Algae............................................................................................................................................35

Invertebrates ..............................................................................................................................35

Fish ...............................................................................................................................................39

Management Implications..........................................................................................................................39

Study Limitations..........................................................................................................................................42

Summary and Conclusions.........................................................................................................................43

References Cited..........................................................................................................................................44 
iv

Figures

1. Map showing National Water-Quality Assessment Program Major River 

Basins (MRB).................................................................................................................................4

2. Map showing locations of the nutrient and diatom ecoregions and sampling sites ........5

3. Ordination plots showing the algae-, invertebrate-, and fish-community data ...............14

4–10. Boxplots showing:

4. Distribution of median annual total nitrogen concentrations in the low-, 

medium-, and high-nutrient categories in the Central and Western Plains 

and Glacial North diatom ecoregions ............................................................................18

5. Distribution of median annual total phosphorus concentrations in the low-, 

medium-, and high-nutrient categories in the Central and Western Plains 

and Glacial North diatom ecoregions ............................................................................18

6. Monthly concentrations of total nitrogen at the 64 sites within Central and 

Western Plains and Glacial North diatom ecoregions used to determine 

nutrient categories ............................................................................................................19

7. Seasonal concentrations of total nitrogen (TN) at the low-TN site 

Muscatatuck River, Indiana .............................................................................................20

8. Seasonal concentrations of total nitrogen (TN) at the medium-TN site 

Sugar Creek, Indiana.........................................................................................................21

9. Seasonal concentrations of total nitrogen (TN) at the high-TN site 

Little Cobb River, Minnesota ............................................................................................21

10. Monthly concentrations of total phosphorus at the 64 sites within Central 

and Western Plains and Glacial North diatom ecoregions used to 

determine nutrient categories.........................................................................................22 

Tables

1. Location, drainage area, diatom ecoregion, and period-of-record data of 

the 64 sampling sites in the Central and Western Plains and Glacial North 

diatom ecoregions........................................................................................................................8

2. Nutrient categories for total nitrogen and total phosphorus in the Central 

and Western Plains and Glaciated North diatom ecoregions ..............................................7

3. Spearman rank correlation coefficients from the analysis of similarity 

between the three diatom ecoregions....................................................................................14

4. Summary of Spearman rho correlations for the nutrients total nitrogen and 

total phosphorus and the algae, invertebrate, and fish community..................................15

5. Level III ecoregion, diatom ecoregion, and nutrient categories of the 

54 sampling sites in the Central and Western Plains and Glacial North 

diatom ecoregions......................................................................................................................16

6. Summary statistics for total nitrogen and total phosphorus in the Central 

and Western Plains and Glacial North diatom ecoregions .................................................19
v

7. Spearman rank correlation coefficients from the analysis of similarity 

between the three nutrient categories ...................................................................................22

8. Most abundant algal taxa in the total nitrogen and total phosphorus 

nutrient categories in the Central and Western Plains and Glacial North 

diatom ecoregions......................................................................................................................24

9. Most abundant invertebrate taxa in the total nitrogen and total phosphorus 

nutrient categories in the Central and Western Plains and Glacial North 

diatom ecoregions......................................................................................................................29

10. Most relative abundant fish species in the total nitrogen and total phosphorus 

nutrient categories in the Central and Western Plains and Glacial North 

diatom ecoregions......................................................................................................................33

11. Breakpoints between nutrients and the most significant biological metrics 

in the Central and Western Plains and Glacial North diatom ecoregions ........................36

12. Comparison of the number of sites in low, medium, and high nutrient categories 

based on nutrient and biological classifications of stream conditions.............................42 

Conversion Factors 

Multiply By To obtain

Length

micrometer (cm) 0.00003937 inch (in.) 

Area

square kilometer (km2

) 0.3861 square mile (mi2) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

Horizontal coordinate information is referenced to the North American Datum of 1983 (NAD 83).

Concentrations of chemical constituents in water are given in milligrams per liter (mg/L). 
vi

Foreword 

The U.S. Geological Survey (USGS) is committed to providing the Nation with reliable scientific 

information that helps to enhance and protect the overall quality of life and that facilitates 

effective management of water, biological, energy, and mineral resources (http://www.usgs.

gov/). Information on the Nation’s water resources is critical to ensuring long-term availability 

of water that is safe for drinking and recreation and is suitable for industry, irrigation, and fish 

and wildlife. Population growth and increasing demands for water make the availability of that 

water, measured in terms of quantity and quality, even more essential to the long-term sustainability of our communities and ecosystems.

The USGS implemented the National Water-Quality Assessment (NAWQA) Program in 1991 to 

support national, regional, State, and local information needs and decisions related to waterquality management and policy (http://water.usgs.gov/nawqa). The NAWQA Program is 

designed to answer: What is the quality of our Nation’s streams and ground water? How are 

conditions changing over time? How do natural features and human activities affect the quality 

of streams and ground water, and where are those effects most pronounced? By combining 

information on water chemistry, physical characteristics, stream habitat, and aquatic life, the 

NAWQA Program aims to provide science-based insights for current and emerging water issues 

and priorities. From 1991 to 2001, the NAWQA Program completed interdisciplinary assessments and established a baseline understanding of water-quality conditions in 51 of the Nation’s 

river basins and aquifers, referred to as Study Units (http://water.usgs.gov/nawqa/studies/

study_units.html).

In the second decade of the Program (2001–2012), a major focus is on regional assessments 

of water-quality conditions and trends. These regional assessments are based on major river 

basins and principal aquifers, which encompass larger regions of the country than the Study 

Units. Regional assessments extend the findings in the Study Units by filling critical gaps in 

characterizing the quality of surface water and ground water, and by determining water-quality 

status and trends at sites that have been consistently monitored for more than a decade. In 

addition, the regional assessments continue to build an understanding of how natural features 

and human activities affect water quality. Many of the regional assessments employ modeling 

and other scientific tools, developed on the basis of data collected at individual sites, to help 

extend knowledge of water quality to unmonitored, yet comparable areas within the regions. 

The models thereby enhance the value of our existing data and our understanding of the hydrologic system. In addition, the models are useful in evaluating various resource-management 

scenarios and in predicting how our actions, such as reducing or managing nonpoint and point 

sources of contamination, land conversion, and altering flow and (or) pumping regimes, are likely 

to affect water conditions within a region.
vii

Other activities planned during the second decade include continuing national syntheses of 

information on pesticides, volatile organic compounds (VOCs), nutrients, trace elements, and 

aquatic ecology; and continuing national topical studies on the fate of agricultural chemicals, 

effects of urbanization on stream ecosystems, bioaccumulation of mercury in stream ecosystems, effects of nutrient enrichment on stream ecosystems, and transport of contaminants to 

public-supply wells.

The USGS aims to disseminate credible, timely, and relevant science information to address 

practical and effective water-resource management and strategies that protect and restore 

water quality. We hope this NAWQA publication will provide you with insights and information 

to meet your needs, and will foster increased citizen awareness and involvement in the protection and restoration of our Nation’s waters. 

The USGS recognizes that a national assessment by a single program cannot address all waterresource issues of interest. External coordination at all levels is critical for cost-effective management, regulation, and conservation of our Nation’s water resources. The NAWQA Program, 

therefore, depends on advice and information from other agencies—Federal, State, regional, 

interstate, Tribal, and local—as well as nongovernmental organizations, industry, academia, and 

other stakeholder groups. Your assistance and suggestions are greatly appreciated.

 William H. Werkheiser

USGS Associate Director for Water 

Assessment of Nutrient Enrichment by Use of Algal-, 

Invertebrate-, and Fish-Community Attributes in 

Wadeable Streams in Ecoregions surrounding the 

Great Lakes 

By Jeffrey W. Frey, Amanda H. Bell, Julie A. Hambrook Berkman, and David L. Lorenz

Abstract 

The algal, invertebrate, and fish taxa and community 

attributes that best reflect the effects of nutrients along a 

gradient of low to high nutrient concentrations in wadeable, 

primarily midwestern streams were determined as part of the 

U.S. Geological Suvey’s National Water-Quality Assessment 

(NAWQA) Program. Nutrient data collected from 64 sampling 

sites that reflected reference, agricultural, and urban influences 

between 1993 and 2006 were used to represent the nutrient 

gradient within Nutrient Ecoregion VI (Cornbelt and Northern 

Great Plains), VII (Mostly Glaciated Dairy Region), and VIII 

(Nutrient Poor Largely Glaciated Upper Midwest and Northeast). Nutrient Ecoregions VII and VIII comprise the Glacial 

North diatom ecoregion (GNE) and Nutrient Ecoregion VI 

represents the Central and Western Plains diatom ecoregion 

(CWPE). The diatom-ecoregion groupings were used chiefly 

for data analysis. 

The total nitrogen (TN) and total phosphorus (TP) data 

from 64 sites, where at least 6 nutrient samples were collected within a year at each site, were used to classify the sites 

into low-, medium-, and high-nutrient categories based upon 

the 10th and 75th percentiles of for sites within each Nutrient Ecoregion. In general, TN and TP concentrations were 

3–5 times greater in Nutrient Ecoregion VI than in Nutrient 

Ecoregions VII and VIII. 

A subgroup of 54 of these 64 sites had algal-, invertebrate-, and fish-community data that were collected within 

the same year as the nutrients; these sites were used to assess 

the effects of nutrients on the biological communities. Multidimensional scaling was used to determine whether the 

entire region could be assessed together or whether there were 

regional differences between the algal, invertebrate, and fish 

communities. The biological communities were significantly 

different between the northern sites, primarily in the GNE and 

the southern sites, primarily in the CWPE. 

In the higher nutrient concentration gradient in the 

streams of the CWPE, algae exhibited greater differences than 

invertebrates and fish between all of the nutrient categories 

for both TN and TP; however, in the lower nutrient gradient 

in the streams of the GNE, invertebrates exhibited greater 

differences between the nutrient categories. Certain species of 

algae, invertebrates, and fish were more prevalent in low- and 

high-nutrient categories within each of the diatom ecoregions. 

Breakpoint analysis was used to identify the concentration at 

which the relations between the response variable (biological 

attribute) and the stressor variable (TN and TP) change. There 

were significant breakpoints for nutrients (TN and TP) and 

multiple attributes for algae, invertebrates, and fish communities within the CWPE and GNE diatom ecoregions. In general, 

more significant breakpoints, with lower concentrations, were 

found in the GNE than the more nutrient-rich CWPE. The 

breakpoints from all biological communities were generally 

about 3–5 times higher in the south (CWPE) than the north 

(GNE). In the north, breakpoints with similar lower concentrations were found for TN from all biological communities 

(around 0.60 milligram per liter) and for TP (between 0.02 

and 0.03 milligram per liter) for the algae and invertebrate 

communities. 

The findings from our study suggest that the range in 

breakpoints for TN and TP from the GNE can be used as 

oligotrophic and eutrophic boundaries derived from biological 

response based on this ecoregion having (1) a gradient with 

sufficiently low to high nutrient concentrations, (2) distinctive 

differences in the biological communities in the low- to highnutrient streams, (3) similarity of breakpoints within algal, 

invertebrate, and fish communities, (4) significant attributes 

with either direct relations to nutrients or traditional changes 

in community structure (that is, decreases in sensitive species 

or increases in tolerant species), and (5) similar breakpoints 

in other studies in this and other regions. In nutrient-rich 

areas like the CWPE, all of the breakpoints were substantially 

higher than those for the lower nutrient conditions of the GNE, 

suggesting that streams are nutrient saturated to the point that 

low-end breakpoints cannot be detected.
2 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Introduction

Nutrients—an element or compound used in animal and 

plant growth—are essential to all life, as well as to the health 

and diversity of surface waters; in excessive amounts, however, nutrients can have potentially negative human-health, 

ecological, and economic consequences. Excess amounts of 

nitrogen (N) and phosphorus (P) in particular have been shown 

to cause eutrophication in aquatic ecosystems, which sometimes has been linked to fish kills, shifts in species composition, unpleasant taste and odor in drinking water, blooms of 

harmful algae in freshwater bodies and estuaries, and hypoxic 

and anoxic zones in gulfs and estuaries (U.S. Environmental 

Protection Agency, 2000c,d; Munn and Hamilton, 2003). 

Many streams within the United States have been 

placed on the Clean Water Act (CWA) Section 303(d) list of 

impaired water bodies because of excess nutrients. The CWA, 

as amended in 1976, established a national goal of achieving water quality to provide for the protection and propagation of aquatic organisms, wildlife, and recreation in and on 

water; regulation to achieve this goal is the responsibility 

of the U.S. Environmental Protection Agency (USEPA). In 

1996, the USEPA National Water Quality Inventory identified 

excess nutrients as second only to siltation as a leading cause 

of impairment of U.S. rivers and streams (U.S. Environmental Protection Agency, 1997). In 2000, the USEPA proposed 

ecoregion-specific nutrient water-quality criteria for the causal 

variables total nitrogen (TN) and total phosphorus (TP) and 

for the response variables periphyton chlorophyll a (CHLa), 

seston CHLa, and turbidity, to improve the health in rivers 

and streams from excess nutrients. However, little or no CHLa

data existed or were available for most of the USEPA Nutrient Ecoregions, and criteria were proposed for periphyton 

CHLa for limited Nutrient Ecoregions in the United States 

(U.S. Environmental Protection Agency, 2000a,b,c,d). 

USEPA drinking-water criteria (Maximum Contaminant 

Levels) are 10 mg/L for nitrate as N and 1mg/L for nitrite as 

N (U.S. Environmental Protection Agency, 2005). In addition, 

aquatic-life criteria established to protect aquatic organisms 

have been set for ammonia as N (the ammonia as N aquaticlife criterion varies with pH, temperature, and life stage) 

(U.S. Environmental Protection Agency, 2005). Yet, these 

criteria do not address the effects of increased nutrients on 

the biological communities in rivers and streams. Typically, 

nutrient concentrations must be extremely high to be toxic to 

biological communities; such concentrations rarely are found 

in the environment (Rankin and others, 1999). Exceptions are 

concentrations of ammonia associated with accidental discharges from wastewater-treatment facilities, combined-sewer 

overflows, or concentrated-animal feeding operations (Rankin 

and others, 1999). 

Although the USEPA proposed criteria for nutrients 

as causal variables and periphyton and seston CHLa as 

response variables, direct correlations or models that predict 

CHLa on the basis of nutrients often are weak, and statistical 

significance of the correlations has varied from study to study. 

For example, previous studies of nutrient-rich agricultural 

streams in Illinois (Figueroa-Nieves and others, 2006) and 

Indiana (Lowe and others, 2008) found few significant relations among concentrations of N or P and periphyton or seston 

CHLa. In contrast, Biggs (2000) listed several studies that 

found significant relations between nutrients and periphyton 

CHLa. Dodds and others (1998) found significant relations 

between TN and TP and periphyton CHLa for temperate streams. In Wisconsin, significant relations were found 

between nitrate, TN, dissolved P (DP), and TP and periphyton 

CHLa in wadeable streams (Robertson and others, 2006) and 

seston CHLa in nonwadeable streams (Robertson and others, 

2008). Dodds and others (2002) presented regression models 

that predict periphyton CHLa as a function of N and (or) P 

and found coefficients of determination (r2) values that ranged 

from 0.1 to 0.40. A primary reason for the weak relations 

between algal biomass and nutrients in streams is the complex 

interactions of physical and biological factors. In general, the 

addition of nutrients stimulates the growth of algae; however, 

physical variables such as light limitation from canopy shading (Mosisch and others, 2001), turbidity (Munn and others, 

1989), and water temperature (Kilkus and others, 1975; Munn 

and others, 1989) can limit algal growth. Moreover, various 

physical and biological factors can decrease existing algal 

growth and confound the nutrient-algal biomass relations 

(Munn and others, 2010). For example, increased streamflow, 

elevated suspended-sediment concentrations, and scouring are physical factors that decrease algal biomass (Power, 

1992; Biggs, 1995), whereas grazing by invertebrates or fish 

biologically decreases algal biomass (Lamberti and Resh, 

1983; Power, 1992). Additionally, pesticides used to control 

unwanted plant growth also have been reported to decrease 

algal biomass in agricultural streams (Kosinski, 1984). 

 The lack of strong relations between nutrients and CHLa

suggests that biological communities will be important in 

understanding the true nutrient condition in streams. Multimetric indices of biotic integrity for fish and invertebrate 

communities have been successfully used throughout the 

United States (Karr and others, 1986; Ohio Environmental 

Protection Agency, 1987a,b, 1989; Bryce and Hughes, 2002) 

and worldwide to measure effects of human disturbances on 

streams. More recently, periphyton (benthic) diatom indices 

have been successfully used to measure differences in water 

quality between reference (high-quality) and impaired sites at 

scales ranging from state (Fore and Grafe, 2002) to ecoregion 

(Wang and others, 2005) and geographic region (Fore, 2003; 

Porter and others, 2008). Idaho, Kentucky (Kentucky Division of Water, 2002), Michigan, Montana (Teply and Bahls, 

2005), and New York (Passy, 2000) have developed diatom 

indexes to determine whether streams need to be included on 

the 303(d) list of the Clean Water Act. In some studies, multimetric diatom indices were better than fish or invertebrate 

multimetric indices at differentiating the effects of nutrients 

(U.S. Environmental Protection Agency, 2006a; Justus and 

others, 2009). Fish and invertebrate indices developed by 
Approach and Methods 3

many states effectively distinguish between impaired and 

unimpaired streams; however, it has been more problematic 

to discover whether the primary ecosystem stressor is nutrient 

impairment or whether the stressor or stressors are related to 

land use, hydrology, habitat, or inputs of chemicals other than 

nutrients. 

The study described herein is an attempt by the NAWQA 

Program to discover statistical relations between nutrient 

stressors and biotic response across a geographically wide 

network of water-quality sampling sites. Biological, waterquality, and ancillary data collected at certain sites as part of 

the NAWQA Program provide a description of water-quality 

conditions and allow for assessments of how natural features and human activities affect those conditions. Regional 

assessments are possible because of a consistent study design 

and uniform methods of data collection. Monitoring data are 

integrated with geographic information on hydrological characteristics, land use, and other landscape features in models 

to extend water-quality understanding to unmonitored areas. 

Local, State, Tribal, and national stakeholders use NAWQA 

information to design and implement strategies for managing, 

protecting, and monitoring water resources in many different 

hydrologic and land-use settings across the Nation.

Purpose and Scope

The purpose of this regional report is to describe which 

algal, invertebrate, and fish taxa and attributes best reflect 

the effects of nutrients at sites along a gradient of low- to 

high-nutrient conditions in the Great Lakes, Ohio, Upper 

Mississippi, and Souris-Red-Rainy River Basins; the U.S. 

Geological Survey (USGS) National Water-Quality Assessment (NAWQA) Program refers to these basins collectively as 

Major River Basin 3 (MRB3; fig. 1). These taxa and attributes 

can be used to determine more precise qualifications of nutrient impairments of streams in this area of the United States, 

particularly USEPA Nutrient Ecoregions VI, VII, and VIII.

The MRB3 region includes parts of seven USEPA 

Nutrient Ecoregions, but the sampling sites ultimately used 

in our study were all within four of them: VI, the Cornbelt 

and Northern Great Plains; VII, the Mostly Glaciated Dairy 

Region; VIII, Nutrient Poor Largely Glaciated Upper Midwest 

and Northeast; and IX, Southeastern Temperate and Forested 

Plains and Hills (U.S. Environmental Protection Agency, 

2000c,d). Potapova and Charles (2007) combined three of 

these Nutrient Ecoregions into two diatom ecoregions based 

on similarities of algal diatom communities. Specifically, 

Nutrient Ecoregion VI corresponds to the Central and Western 

Plains diatom ecoregion (CWPE), and the combined Nutrient Ecoregions VII and VIII correspond to the Glacial North 

diatom ecoregion (GNE); Potapova and Charles’ categorization was used as the regional basis for this study (fig. 2), so the 

study results will be discussed in terms of CWPE and the GNE 

hereafter in this report.

Approach and Methods

The approach used in our study was first to determine 

whether the invertebrate and fish communities within the 

CWPE and GNE are similar or whether they needed to be 

assessed separately, as was true for the algae. The analyses 

found the composition of the algae, invertebrate, and fish communities to be significantly different, so subsequent analyses 

on sites within the CWPE and the GNE were done separately. 

Nutrient variables were evaluated to determine which had the 

strongest relation with the biological communities and should 

be used in later analyses. The sampling sites were classified 

into low-, medium-, and high-nutrient categories based upon 

selected percentiles of TN and TP concentrations from a total 

of 64 NAWQA sites sampled during 1993–2006; these sites 

included those reflecting reference, agricultural, and urban 

conditions for both diatom ecoregions. The three nutrient 

categories provide the basis for comparisons of differences 

between the biological communities within each of the nutrient categories. A subgroup of 54 of the 64 NAWQA sites with 

at least 6 nutrient samples per year and with corresponding 

algae-, invertebrate-, and fish-community data were used 

to assess biological differences in nutrient categories. The 

attributes for the algae, invertebrates, and fish were analyzed 

to determine which biological attributes had the strongest 

correlations with TN and TP. Breakpoint analysis was used to 

find the concentration of TN and TP where the biological community dramatically changed with respect to the statistically 

strongest and most ecologically significant attributes. The 

differences in the biological communities and the breakpoints 

were assessed for use as measures of trophic level, management implications, and the development of nutrient criteria. 

Data for our study were collected by use of standard 

NAWQA field and analytical methods. The following sections 

describe the site-selection and sampling strategies; field and 

laboratory methods used in collecting, processing, and analyzing nutrients; and statistical analyses used.

Site Selection

Biological communities are influenced by multiple 

instream habitat and water-quality-related variables such as 

substrate, water depth, velocity, nutrients, and clarity; they 

are additionally influenced by landscape-level variables such 

as land use, climate, and geology (Wehr and Sheath, 2003). 

Typically, most studies that examine how biological communities are affected by a specific stressor or groups of stressors 

try to minimize the variability associated with these regional 

differences so that the effects of the stressor(s) can be maximized. For example, most indices of biological integrity are 

developed for specific watersheds or ecoregions (Simon 1991, 

1997; Potapova and Charles, 2007), in part to account for 

these regional differences.
EXPLANATION MRB 1 - New England and Mid-Atlantic MRB 2 - South Atlantic-Gulf and Tennessee MRB 3 - Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy MRB 4 - Missouri MRB 5 - Lower Mississippi, Arkansas-White-Red, and Texas-Gulf

MRB 6 - Rio Grande, Colorado, and Great BasinMRB 7 - Pacific NorthwestMRB 8 - California

MR

MRB 2

MRB 5

MRB 3

MRB 4

MRB 6

MRB 8

MRB 7

Figure 1. National Water-Quality Assessment Program Major River Basins (MRB). 
EXPLANATION

Major river basin, three sites

Total phosphorus categories

 0: Low 1: Medium 2: High

Total nitrogen categories

 0: Low 1: Medium 2: High

Central and Western Plains diatom ecoregion

Nutrient ecoregion V

Nutrient ecoregion XI

Nutrient ecoregion VI

Nutrient ecoregion VIINutrient ecoregion VIIINutrient ecoregion IXNutrient ecoregion X

Other nutrient ecoregions shown

Glaciated North diatom ecoregion

Sites used for nutrient analysisSites used for nutrient and biological analyses

J

I

H

G

F

E

D

C

B

A

9 8

7

6

4

3

2

1

54

53

52

51

50

49

48

47

46

45

44

43

42

41

40

39

38

37

36

35

34

33

32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

Figure 2. Locations of the nutrient and diatom ecoregions and sampling sites. 
6 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Potapova and Charles (2007) classified the United States 

into five diatom ecoregions based on the U.S. Environmental 

Protection Agency’s scheme of Nutrient Ecoregions, which 

itself is an aggregation of Omernik’s level III ecoregions 

(Omernik, 1987, 1995; fig. 2). The development of the five 

diatom ecoregions entailed use of NAWQA algal data collected from 1993 through 2001 at 1,240 sites throughout the 

United States. For our study, site selection focused primarily 

on streams in the upper Midwest, with most sites located in 

the GNE or CWPE diatom ecoregions; two sites were in the 

Eastern Highlands (EHE) diatom ecoregion (Potapova and 

Charles, 2007). Multidimensional scaling analyses (Clarke and 

Ainsworth, 1993) were done to see whether regional differences such as those found in the diatom community were also 

characteristic of the invertebrate and fish communities.

Biological communities, especially fish communities, 

are influenced by stream size (Simon 1991, 1997; Goldstein 

and Meador, 2004; Hambrook Berkman and others, 2010). 

Most Index of Biological Integrity (IBI) methods distinguish 

attributes on the basis of basin size. To minimize the effect of 

varied drainage areas that could mask the effects of nutrients 

on biological communities, only wadeable sites are discussed 

in this report. Sites were selected to maximize the number of 

sites within each diatom ecoregion. Sites were classified by 

basin size (headwater, less than 52 km2; wadeable, greater 

than 52 km2 and less than 1,000 km2; and boat, greater than 

1,000 km2) on the basis of classifications used by several states 

within the region (for example, Ohio Environmental Protection Agency, 1989; Indiana Department of Environmental 

Management, 1999). 

Nutrient Sample Collection 

All nutrient samples were collected according to standard 

NAWQA protocols as detailed below. Typically, samples were 

collected by using a depth-integrating sampler at multiple 

vertical locations in the stream cross section (Shelton, 1994). 

Samples for analysis of dissolved constituents were filtered 

in the field, within 2 hours of collection, through either a 

nitrocellulose filter or a polyether-sulfone medium with a pore 

size of 0.45 μm. Nutrient samples were chilled and maintained 

at 4°C until analyzed at the laboratory. The number of samples 

collected at a NAWQA site varied from 6 to 26 per year.

Ammonia (NH3), nitrite (NO2) plus nitrate (NO3), and 

orthophosphate (OP) were analyzed according to methods 

described by Fishman (1993). Kjeldahl nitrogen (TKN) was 

analyzed as described by Patton and Truitt (1992). During 

1992–98, TP and DP were analyzed by means of a modified Kjeldahl procedure (Patton and Truitt, 2000). In 1999, 

these analyses were changed to follow USEPA method 365.1, 

low-level persulfate digestion (U.S. Environmental Protection 

Agency, 1993).

Biological Sample Collection

A biological sample was collected one time per site per 

year at the 54 sites used in this study between May and September, generally during periods of stable low flow, according 

to NAWQA methods as detailed below. Quantitative benthic 

algal samples (hereafter, algae samples) were collected from 

a defined area of substrate (rocks in riffle areas or submerged 

woody snags) to enable estimation of abundance per unit area 

and were composited into a single algae sample (Porter and 

others, 1993; Moulton and others, 2002). Algal taxa were identified at the Academy of Natural Sciences in Philadelphia, Pa. 

(Charles and others, 2002). Semiquantitative benthic-macroinvertebrate samples (hereafter, invertebrate samples) were collected from areas where maximum taxa richness was likely to 

occur, generally in the same habitats and locations as the areas 

sampled for algae, according to NAWQA methods (Cuffney 

and others, 1993; Moulton and others, 2002). Invertebrates 

were identified at the U.S. Geological Survey National Water 

Quality Laboratory, Lakewood, Colo. (Moulton and others, 

2000). Fish were collected by field personnel using electrofishing units (backpack and towed-barge) and seines (Meador and 

others, 1993; Moulton and others, 2002). Fish were identified, 

weighed, and measured in the field and then returned to the 

stream. When species identification could not be confirmed 

in the field, several individuals of that species were vouchered, and their identification confirmed by taxonomists in the 

laboratory.

Data Analysis

This section details the statistical procedures used for 

data analysis of the nutrient and biological data. It includes 

descriptions of how nutrient and biological conditions at sites 

were determined and how breakpoints along a nutrient gradient were found for attributes of each biological community.

Regional Differences

For each of the three biological communities (algae, 

invertebrate, and fish), nonparametric statistical analyses were 

used to determine whether there was a regional difference 

between GNE and CWPE. By use of a Bray-Curtis similarity 

matrix based on squared-root-transformed community data, a 

non-metric multidimensional scaling (MDS) bi-plot configuration (Shepard, 1962; Kruskal, 1964a,b; Kruskal and Wish, 

1978; Clarke and Warwick, 2001; Clarke and Gorley, 2006b) 

was created to depict the distribution of the sites. An analysis 

of similarity was done to determine whether it was possible to 

analyze ecoregions together or separately by testing the null 

hypothesis that the ecoregions are the same.
Approach and Methods 7

Nutrient Conditions of the Sites

The water-quality data were summarized in three ways 

to assess which nutrient variables had the strongest relations 

with the biological data: the value on the date closest to the 

biological sample, the mean annual value, and mean annual 

maximum value. These three variables represent acute and 

chronic nutrient conditions that would affect the biological 

community. The water-quality data included the nitrogen and 

phosphorus nutrient variables NO2+NO3 as N, TN as N, OP as 

P, and TP as P; nutrient ratio variables that were used to assess 

nutrient limitation or the amount of dissolved nutrients (bioavailable) to total nutrients (long-term source) were NO3

 to 

TKN, NO2+NO3 to TN, OP to TP, NO3 to OP, and TN to TP. A 

measure of algal biomass (chlorophyll a or ash-free dry mass) 

would have greatly helped in assessing plant growth associated with the uptake component for the nutrient conditions 

at each sampling site. Specifically, the algal-biomass parameter could help assess whether low nutrient concentrations 

were due to oligotrophic conditions or whether algal uptake 

of nutrients accounted for the low concentrations. However, 

because few NAWQA study units collected algal biomass data 

during the 1990s, algal biomass measures were not included. 

One approach of our study was to classify sites solely on 

the basis of instream nutrient concentrations and was based on 

the assumption that different biological communities would 

be found at sites with “low,” “medium,” and “high” nutrient 

concentrations. To maximize the gradient of nutrient concentrations for each ecoregion, sites were included for this part of 

the analysis based solely upon having at least six TN and TP 

samples collected throughout the year. A total of 64 sites, 27 

sites in the GNE and 37 sites in the CWPE, had sufficient data 

to be included in this analysis (table 1). 

“Low-nutrient” sites were those with concentrations at 

or below approximately the 10th percentile for TN and TP, 

“High-nutrient” sites were those approximately at or above the 

75th percentile, and “Medium-nutrient” sites were between 

these two concentrations. The 10th percentile was used for 

“Low-nutrient” sites as a conservative target to assess reference-like conditions. Additionally, the 10th-percentile values 

for TN and TP were similar to proposed nutrient criteria for 

Nutrient Ecoregions VI, VII, and IX (U.S. Environmental 

Protection Agency, 2000d). Dodds and others (1998) used 

the 66th percentile as the eutrophic category, which was 

based solely on nutrient concentrations and not a measure of 

biological response; the 75th percentile was used in our study 

as a more conservative measure of the high-nutrient conditions. Multiple years of nutrient data were available for most 

NAWQA sampling sites. There were 54 sites where (1) at 

least 6 nutrient samples were collected per year, and (2) algal, 

invertebrate, and fish data were collected within the same year 

as the nutrient samples to describe the nutrient condition at 

these sites. Data from some of these intensively sampled sites 

spanned 13 years, including the period of widespread implementation of Best Management Practices designed to keep 

nutrients out of streams. All of the annual data for each site 

were included because changing nutrient concentrations at a 

site should be reflected by changes in species composition. To 

best represent the true nutrient condition of these streams, the 

median annual average concentration was used in the analyses, 

not the value from a low or high runoff year. Because some 

of the sites that had multiple years of nutrient data and annual 

average concentrations that spanned the 10th and 75th percentiles (low, medium, and (or) high categories), the percentile 

values were adjusted up or down slightly so that sites would 

be included only in one nutrient category. The final low, 

medium, and high concentrations for TN and TP in the CWPE 

and GNE diatom ecoregions are listed in table 2. 

Table 2. Nutrient categories for total nitrogen and total 

phosphorus in the Central and Western Plains and Glaciated 

North diatom ecoregions. 

[mg/L, milligrams per liter; <, less than; >, greater than]

Nutrient

Nutrient category

Low 

sites

Medium 

sites

High 

sites

Central and Western Plains diatom ecoregion (CWPE)

Total nitrogen (mg/L) < 1.74 1.74 – 7.80 > 7.80

Total phosphorus (mg/L) < .05 .05 – .17 > .17

Glaciated North diatom ecoregion (GNE)

Total nitrogen (mg/L) < 0.60 0.60 – 2.08 > 2.08

Total phosphorus (mg/L) < .01 .01 – .13 > .13

Biological Conditions of the Sites

All biological data were retrieved from either the USGS 

NAWQA biological database (BioTDB, for algae) or Data 

Warehouse (U.S. Geological Survey, 2007, 2008, for fish and 

invertebrates). Biological samples were checked for possible 

errors on the basis of species richness, biovolume, and individual richness. Biological communities and attributes were 

summarized and computed by using the Algal Data Analysis 

System (ADAS) for algae and the Invertebrate Data Analysis 

System (IDAS) for invertebrates. IDAS was developed to 

reduce ambiguous taxa and calculation of tolerance, feeding 

guild, water-quality preference, and other assemblage attributes (Cuffney, 2003). ADAS is a similar program, modified 

for algal communities. Fish attributes were calculated by use 

of Microsoft Excel. Statistical analysis of biological, nutrient and environmental data was done with PRIMER version 

6.1.10 (Clarke and Warwick, 2001; Clarke and Gorley, 2006a, 

b) and S-Plus version 8.1 (TIBCO, 2008).

A total of 566 algal species were identified at the 54 

sites; this taxa list was used to calculate the algal attributes, 

including oxygen requirements, pollution tolerance, nutrient 

trophic preference, saprobic condition, and pH preference. 
Table 1. Location, drainage area, diatom ecoregion, and period-of-record data of the 64 sampling sites in the Central and Western Plains and Glaciaecoregions.—Continued [km2, square kilometer; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; NA, no biological data] Site number Station identification Station name Latitude Longitude Drainage area (km2) Diatom ecoregion Used for the biological analyses Number of nutrient samples per site 

1 04186500 AUGLAIZE RIVER NEAR FORT JENNINGS, OH 40.94866 -84.26606 858.4 CWPE YES 92 

A 393306086585201 BIG WALNUT CREEK AT CO RD 700 W AT REELSVILLE, IN 39.55171 -86.98110 823.6 CWPE NO 31 

2 03357330 BIG WALNUT CREEK NEAR ROACHDALE, IN 39.81616 -86.75334 340.1 CWPE YES 33 3 040853145 BLACK CREEK AT CURRAN ROAD NEAR DENMARK, WI 44.33722 -87.74537 56.1 GNE YES 6 4 04159492 BLACK RIVER NEAR JEDDO, MI 43.15253 -82.62409 1,197.8 CWPE YES 29 5 04213500 CATTARAUGUS CR AT GOWANDA, NY 42.46395 -78.93503 1,128.8 GNE YES 26 6 05286290 CEDAR CREEK NEAR COOPERS CORNER, MN 45.39191 -93.21245 70.7 GNE YES 6 

B 05536995 CHICAGO SANITARY AND SHIP CANAL AT ROMEOVILLE, IL 41.64059 -88.06060 1,852.0 CWPE NO 33 C 391732085414401 CLIFTY CREEK AT CO RD 1150 E NEAR HARTSVILLE, IN 39.29227 -85.69550 227.6 CWPE NO 35 

7 04161820 CLINTON RIVER AT STERLING HEIGHTS, MI 42.61448 -83.02659 802.8 GNE YES 83 8 04208504 CUYAHOGA RIVER NEAR NEWBURGH HEIGHTS, OH 41.46255 -81.68096 2,043.6 GNE YES 33 9 05532500 DES PLAINES RIVER AT RIVERSIDE, IL 41.82225 -87.82089 1,634.2 CWPE YES 74 

10 05527800 DES PLAINES RIVER AT RUSSELL, IL 42.48919 -87.92647 317.7 CWPE YES 39 

11 04072050 DUCK CREEK AT SEMINARY ROAD NEAR ONEIDA, WI 44.46582 -88.21899 247.2 GNE YES 111 

D 04072150 DUCK CREEK NEAR HOWARD, WI 44.53583 -88.12972 247.2 GNE NO 95 

12 03246400 EAST FORK LITTLE MIAMI RIVER 

NEAR WILLIAMSBURG, OH

39.05895 -84.05132 606.5 CWPE YES 19 

E 04085108 EAST RIVER AT CT HIGHWAY ZZ 

NEAR GREENLEAF, WI

44.38667 -88.07972 122.2 GNE NO 14 
Table 1. Location, drainage area, diatom ecoregion, and period-of-record data of the 64 sampling sites in the Central and Western Plains and Glaciaecoregions.—Continued[km2, square kilometer; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; NA, no biological data] 

Site 

number

Station 

identification

Station 

name

Latitude Longitude

Drainage 

area 

(km2)

Diatom 

ecoregion

Used for 

the 

biological 

analyses

Number of 

nutrient 

samples 

per site

13 04085109 EAST RIVER AT MIDWAY ROAD 

NEAR DE PERE, WI

44.38666 -88.07982 122.2 GNE YES 26 

F 04075365 EVERGREEN RIVER BLW EVERGREEN 

FALLS NR LANGLADE, WI

45.06583 -88.67611 161.6 GNE NO 31 

G 04177810 FISH CREEK NEAR ARTIC, IN 41.46500 -84.81417 256.4 CWPE NO 9 

14 05461390 FLOOD CREEK NEAR POWERSVILLE, IA 42.90720 -92.72075 321.2 CWPE YES 35 15 04211820 GRAND RIVER AT HARPERSFIELD, OH 41.75533 -80.94843 1,431.5 GNE YES 33 16 393944084120700 HOLES CK IN HUFFMAN PARK AT 

KETTERING, OH

39.66228 -84.20188 51.9 CWPE YES 121 

17 05568800 INDIAN CREEK NEAR WYOMING, IL 41.01889 -89.83556 163.7 CWPE YES 29 18 05449500 IOWA RIVER NEAR ROWAN, IA 42.75994 -93.62185 1083.9 CWPE YES 92 19 03360895 KESSINGER DITCH NEAR 

MONROE CITY, IN

38.57060 -87.27696 145.6 CWPE YES 55 

20 05584500 LA MOINE RIVER AT COLMAR, IL 40.33032 -90.89625 1695.7 CWPE YES 73 21 05320270 LITTLE COBB RIVER NEAR 

BEAUFORD, MN

43.99663 -93.90856 336.0 CWPE YES 103 

22 03373530 LOST RIVER NEAR LEIPSIC, IN 38.63644 -86.36526 90.1 CWPE YES 31 23 03267900 MAD RIVER AT ST PARIS PIKE AT 

EAGLE CITY, OH

39.96423 -83.83160 802.9 CWPE YES 118 

24 04087000 MILWAUKEE RIVER AT MILWAUKEE, WI 43.10001 -87.90897 1805.2 GNE YES 144 25 03366500 MUSCATATUCK RIVER NEAR 

DEPUTY, IN

38.80422 -85.67386 758.9 CWPE YES 28 

26 05331833 NAMEKAGON RIVER AT LEONARDS, WI 46.17134 -91.32935 332.6 GNE YES 32 27 05330902 NINE MILE CREEK NEAR JAMES CIRCLE 

AT BLOOMINGTON, MN

44.80719 -93.30161 115.5 GNE YES 54 

28 05548105 NIPPERSINK CREEK ABOVE 

WONDER LAKE, IL

42.38530 -88.36954 219.4 CWPE YES 38 

29 040863075 NORTH BRANCH MILWAUKEE RIVER 

NEAR RANDOM LAKE, WI

43.55694 -88.05287 129.9 GNE YES 52 
Table 1. Location, drainage area, diatom ecoregion, and period-of-record data of the 64 sampling sites in the Central and Western Plains and Glaciaecoregions.—Continued [km2, square kilometer; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; NA, no biological data] Site number Station identification Station name Latitude Longitude Drainage area (km2) Diatom ecoregion Used for the biological analyses Number of nutrient samples per site 

30 05276005 NORTH FORK CROW RIVER ABOVE PAYNESVILLE, MN 45.37719 -94.78362 601.4 GNE YES 35 31 04087204 OAK CREEK AT SOUTH MILWAUKEE, WI 42.92502 -87.87008 66.8 GNE YES 33 32 05455100 OLD MANS CREEK NEAR IOWA CITY, IA 41.60641 -91.61572 521.8 CWPE YES 43 33 05030150 OTTER TAIL RIVER NEAR PERHAM, MN 46.64274 -95.60449 870.1 GNE YES 24 34 05567000 PANTHER CREEK NEAR EL PASO, IL 40.77333 -89.07694 243.4 CWPE YES 29 35 04071795 PENSAUKEE RIVER NEAR KRAKOW, WI 44.75249 -88.27649 86.8 GNE YES 34 36 04062085 PESHEKEE RIVER NEAR MARTINS LANDING, MI 46.60966 -88.02235 114.4 GNE YES 33 

H 04066500 PIKE RIVER AT AMBERG, WI 45.50000 -88.00000 639.1 GNE NO 27 

37 04087258 PIKE RIVER AT CTH A NEAR KENOSHA, WI 42.65363 -87.85035 100.3 CWPE YES 9 38 04063700 POPPLE RIVER NEAR FENCE, WI 45.76357 -88.46318 362.6 GNE YES 87 39 04085188 RIO CREEK AT PHEASANT ROAD NEAR RIO CREEK, WI 44.60333 -87.52703 55.8 GNE YES 6 40 04175600 RIVER RAISIN NEAR MANCHESTER, MI 42.16809 -84.07606 330.8 GNE YES 61 41 05531500 SALT CREEK AT WESTERN SPRINGS, IL 41.82583 -87.90028 290.6 CWPE YES 92 42 05572000 SANGAMON RIVER AT MONTICELLO, IL 40.03087 -88.58895 1426.5 CWPE YES 117 43 05288705 SHINGLE CREEK AT QUEEN AVE IN MINNEAPOLIS, MN 45.04997 -93.31023 73.0 GNE YES 120 

I 05085900 SNAKE RIVER ABOVE ALVARADO, MN 48.17415 -96.99900 564.6 CWPE NO 28 

J 05086000 SNAKE RIVER AT ALVARADO, MN 48.17417 -96.99861 800.3 CWPE NO 6 

44 05451210 SOUTH FORK IOWA RIVER NE OF 

NEW PROVIDENCE, IA

42.31498 -93.15243 580.5 CWPE YES 107 

45 04178000 ST JOSEPH RIVER NEAR NEWVILLE, IN 41.38561 -84.80163 1600.2 CWPE YES 88 46 395355084173600 STILLWATER R. ON MARTINDALE R. 

NEAR UNION, OH

39.89867 -84.29328 1673.1 CWPE YES 30 
Table 1. Location, drainage area, diatom ecoregion, and period-of-record data of the 64 sampling sites in the Central and Western Plains and Glaciaecoregions.—Continued[km2, square kilometer; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; NA, no biological data] 

Site 

number

Station 

identification

Station 

name

Latitude Longitude

Drainage 

area 

(km2)

Diatom 

ecoregion

Used for 

the 

biological 

analyses

Number of 

nutrient 

samples 

per site

47 394340085524601 SUGAR CREEK AT CO RD 400 S AT 

NEW PALESTINE, IN

39.72782 -85.87942 246.2 CWPE YES 289 

48 05525500 SUGAR CREEK AT MILFORD, IL 40.63004 -87.72392 1158.8 CWPE YES 75 49 04080798 TOMORROW RIVER NEAR 

NELSONVILLE, WI

44.52442 -89.33789 114.0 GNE YES 32 

50 05082625 TURTLE R AT TURTLE R STATE PARK 

NEAR ARVILLA, ND

47.93832 -97.50036 311.0 GNE YES 83 

51 05420680 WAPSIPINICON RIVER NEAR TRIPOLI, IA 42.83609 -92.25740 897.0 CWPE YES 76 52 03275000 WHITEWATER RIVER NEAR ALPINE, IN 39.57310 -85.15746 1370.1 CWPE YES 27 53 05062500 WILD RICE RIVER AT TWIN VALLEY, MN 47.26663 -96.24478 2407.1 CWPE YES 48 54 05464220 WOLF CREEK NEAR DYSART, IA 42.25166 -92.29880 775.2 CWPE YES 55 
12 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

The original taxa list was then reduced by removing rare taxa: 

those that occurred in three or fewer samples and at less than 

3 percent of the summed relative abundance for all samples. 

This reduction resulted in 448 species used for community 

analysis: 334 diatom species, 70 blue-green algae species, 33 

green algae species, 5 euglenoid species, 4 red algae species, 

and 2 species with an undetermined algal division. Many of 

the benthic algal attributes focused on a large and diverse 

group of single-celled algae called “diatoms” and that means 

the diatoms discussed are “benthic” diatoms. Algal attributes 

based on species attributes, such as dissolved oxygen (DO) 

requirements, nutrient preferences, nitrogen fixation, and 

motility, among others, were calculated for individual abundance, richness, and relative percentages of both (Lange-Bertalot, 1979; Bahls, 1993; Van Dam and others, 1994; Porter, 

2008).

A total of 481 invertebrate taxa were found at the 54 

sites after ambiguous taxa were divided among the children of 

the parent taxa on the basis of relative composition (Cuffney, 

2003). Functional feeding-group and regional-tolerance attributes, based on this taxa list, were derived from appendix B of 

the USEPA’s Rapid Bioassessment Protocol (RBP) included 

in the Invertebrate Data Analysis System (IDAS) program 

(Barbour and others, 1999; Cuffney, 2003). Similar to the algal 

assemblage, the original taxa list was then reduced by removing rare taxa (those that occurred in three or fewer samples 

and at less than 3 percent of the summed relative abundance 

for all samples). The reduction resulted in a final taxa list of 

260 species. 

A total of 159 fish species for all 54 sites were identified. 

Because of the relatively small species list compared to the 

algae and invertebrates, no rare taxa were removed for fishcommunity attributes, and all analyses were completed on the 

full taxa list. Fish-community attributes were based on species 

traits such as preferences for substrate, geomorphic units, 

trophic ecology, feeding guild, stream size, locomotion, and 

reproductive strategy. Additionally, commonly used attributes 

from fish Indices of Biotic Integrity (IBI) in the Midwest were 

included, such as number of individuals (abundance) and taxa 

(richness) and abundance of certain families (Karr and others, 

1986; Lyons, 1992; Barbour and others, 1999; Goldstein and 

Meador, 2004). 

Using Biological and Nutrient Conditions to 

Describe Differences between Sites

The calculation of the biological attributes resulted in a 

large number of variables for use in further data analysis; however, because of the number of samples in our dataset, the list 

of over 400 attributes needed to be reduced to a manageable 

number. The goal in reducing the variable set was to identify 

the fewest attributes that distinguish different responses to 

the same environmental stressors—in this case, nutrients. For 

example, the number of individuals or taxa may increase with 

increasing nutrients, but the number of sensitive or intolerant 

species may decrease. The first task in attribute reduction 

was grouping the variables for each community into logical 

categories such as trophic level (fish), feeding group (invertebrates), nutrient/eutrophic preference (algae), and pollution 

tolerance (all). Secondly, Spearman rank correlations between 

the attributes were examined within each biological category 

to select attributes that were not strongly correlated with each 

other. For example, there may be a strong relation of motile 

algae to nonmotile algae, but it would be redundant to retain 

both attributes because they are the inverse of each other; so, 

only one of the attributes was retained. 

The third step in biological-attribute reduction was 

examining the Spearman rank correlation between the retained 

biological attributes and the different nutrient variables 

(NO2+NO3-N, TN, OP, TP, NO3:TKN, NO3:TN, OP:TP, 

NO3:OP, and TN:TP, based on annual, maximum, and nearestbiological-sample values). Several biological attributes within 

each community showed strong linear relations to the nutrient variables. The last step in biological-attributes reduction was to examine the attributes on the basis of correlation 

values with nutrients, relations found in previous studies, and 

best professional judgment in selecting attributes that were 

believed to provide the best ecologically-based response.

An ordination, which is a nonparametric, multivariate 

analysis, was used to determine which species were most 

related to the distribution of the sites (Clarke and Ainsworth, 

1993). Basically, MDS plots were used to display the similarity or difference between samples as revealed by a stepwise 

regression to examine which species within each biological 

assemblage and ecoregion account for those similarities or 

differences by maximizing a rank correlation between the biological assemblage and the resemblance matrix. For example, 

if sites A and B are relatively close together on the MDS plot, 

they have similar species composition, whereas if site C is 

distant from sites A and B, the x species composition differs 

correspondingly. The species common between A and B and 

not for C are identified by using this technique. Once the species with the highest rank correlation to the site distribution 

is identified, the species with the next highest correlation is 

identified and combined with correlation of the first species. 

This additive, stepwise process is repeated for all species until 

either the addition of species does not enhance the correlation statistic or a list of the 10 species with the highest rank 

correlations is identified. The list of species is assumed to be 

responsible for the majority of the distribution of sites. This 

process was repeated using the biological attributes for each 

assemblage to determine which selected biological attributes 

were most related to the site distribution.

A similar analysis was done to determine which nutrient 

variables most closely represent the biological assemblages for 

each of the community assemblages for both diatom ecoregions, individually and combined. The resemblance matrices 

for the nutrient data were calculated by using Euclidean 

distance. The nutrient variable or combination of variables 

most related to the similarities of the biological data was determined by the stepwise comparison. It was assumed that these 
Approach and Methods 13

variables, attributes, and species represent the most significant 

ecological and nutrient relations.

Use of the biological communities to evaluate nutrient 

categories was a multistep process, the first step being a modification of multivariate regression tree analysis developed by 

De’ath (2002). The procedure takes abiotic variables (in this 

case the nutrient concentrations) and uses them to “describe 

how best the [biological] assemblages are split into groups 

by successive binary division” (Clarke and Gorley, 2006b). 

Secondly, to assess the differences in the biological communities between the low, medium, and high nutrient sites, a 

similarity analysis was done for the algal-, invertebrate-, and 

fish-community data. 

The goal of breakpoint or change-point analysis is to 

identify the concentration at which the relations between 

the response variable and the stressor variable (TN and TP) 

change. The form of the change in relation can be any of a 

wide variety of changes—change in mean, change in variance, change in slope, and so forth. The procedures described 

in this section were developed by using Spotfire S+® (TIBCO, 

2008) and using the resample library for the bootstrapping and 

permutation methods.

The deviance-reduction method of breakpoint analysis 

was used in this study. This method sorts observations along 

the stressor-variable gradient and identifies the concentration 

where the deviance is minimized (Qian and others, 2003). The 

method is based on tree-based modeling (Breiman and others, 

1984) and selects the first binary split as the change point. 

The deviance is the sum of the squared differences between 

the response variable and the respective mean for all observations less than or equal to the breakpoint and all observations 

greater than the breakpoint, as follows (equation 1):

k

j k

n

i

k

D i y k j y = k − − + − ∑ ∑ + = = +

2 2

1 1

( )  ( )  (1)

where 

	 Dk is the deviance computed for observation, 

	 k, yi and yj	 are response variables, 

μk- is the mean of response variables less than or 

equal to k, and 

μk+ is the mean of response variables greater 

than k. 

The method divides the observations into two groups, which 

are assumed to be relatively homogenous (Qian and others, 

2003). To reduce the effect of influential observations near the 

ends of the range of the stressor variables, a minimum group 

size of 8 was used for all analyses.

The deviance-reduction method will always find a minimum deviance and an associated breakpoint regardless of the 

real ecological change. The achieved significance level (ASL) 

of the minimum deviance is computed by using a permutation test (Efron and Tibshirani, 1993). The permutation test 

permutes the values of the response variable many times, 

999 times for this study, and computes the minimum deviance 

from each permutation. The ASL is computed as the number 

of the simulated minimum deviances less than the observed 

value plus 1 divided by the total number of simulations 

(permutation) plus 1 (equation 2) and is treated much like a 

p-value from a parametric significance test:

ASL N = + ( )1 1 / (B + ) (2)

where 

N is the number of the simulated minimum 

deviances less than the observed value and 

B is the total number of simulations. 

Parametric tests have been used in other breakpoint-analysis 

software applications (Tetra Tech, 2009), under the assumption 

that the parametric distribution is approximately correct, but 

simulations done for this study indicate that substantial differences can occur.

An estimate of the error of the breakpoint estimate can be 

obtained by bootstrapping methods (Qian and others, 2003). 

A specialized sampling method was developed to preserve 

the range of the stressor variable and the local behavior of 

the environmental-response variable. The method is called 

random block resampling. In contrast to block resampling, 

which divides the set of observations into known blocks that 

are resampled, random block resampling creates blocks of 

observations along a specific gradient that vary in size for each 

simulation. The response variable is resampled with replacement in each block for each value of the stressor variable. The 

average group size used for this study was about 4.

The following example can help describe the random 

block resampling method. Consider eight samples with values 

of 1 though 8 for the stressor variable and values of 11 though 

18 the corresponding response variable. The first step is to 

select observations along the environmental gradient that 

divides the observations into a known number of groups. 

For this example, one observation will be selected to subdivide the data into two groups. Assume that it is observation 

number 5, which creates groups of 1 through 5 and 6 through 

8. The response variables are also grouped 11 through 15 and 

16 through 18. For each stressor value in the first group, one 

value from the first group of response values is selected. For 

this example, the selections could be 12, 14, 11, 12, 13. The 

same procedure is repeated for each group. The selection for 

the second group could be 16, 17, and 16. These data would be 

analyzed to determine the breakpoint and the method repeated 

several times to create a sample of possible breakpoint values. 

The simulations can be used to estimate the uncertainty in the 

change point. Plots of the counts of simulated change points 

can be useful in indentifying other problems such as alternate 

change points indicated by bimodal density.
14 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Description of Regional Differences 

and the Nutrient and Biological 

Conditions in the Nutrient Categories

Regional Biological Differences

Potapova and Charles (2007) divided the MRB3 region 

into the GNE and CWPE because of significant differences 

in the algal community. Ordination analysis showed all three 

biological communities were significantly different, with the 

fish community showing the most dissimilarity between the 

two diatom ecoregions (fig. 3). 

As indicated by the ordination analysis and confirmed 

by an analysis of similarities (table 3), the community assemblages in the GNE and CWPE diatom ecoregions are significantly dissimilar and therefore should be analyzed separately. 

There was a significant dissimilarity between GNE and EHE 

invertebrate and fish communities but no such dissimilarity 

for the algae communities. Because only two sites were in 

the EHE and the land use, nutrient concentrations, algal and 

fish communities and general location were more like those 

in the CWPE than the GNE, those two sites were included in 

the analysis of the CWPE. MDS ordinations were recreated 

separately for the two diatom ecoregions (GNE and CWPE) 

for each biological community. The ordination scores derived 

from these diatom-ecoregion-scaled MDS were included in the 

biological attributes.

Algae Invertebrates Fish

Diatom ecoregion

Glaciated North

Central and Western Plains

Eastern Highlands

EXPLANATION

Table 3. Spearman rank correlation coefficients from the 

analysis of similarity between the three diatom ecoregions. 

[Bold indicates significant difference between the diatom ecoregions 

(p value = 0.05)]

Community

Diatom ecoregion

Glaciated North 

to Central and 

Western Plains

Glaciated North 

to Eastern 

Highlands

Central and 

Western Plains 

to Eastern 

Highlands

Algae 0.231 0.123 0.273

Invertebrates .324 .124 .467

Fish .399 .481 .269

Figure 3. Ordination plots of the algae-, invertebrate-, and fish-community data. 
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 15

Regional Comparisons of the Nutrient 

Categories

The sampling strategies and the number of samples 

collected for nutrients that are used to assess the nutrient 

condition within streams vary by State and study, depending upon the objectives of the study and available funding. In 

many previous studies relating stressors such as nutrients to a 

biological response, the nutrient data used have been a measure of nutrient concentrations nearest the biological sample 

(U.S. Environmental Protection Agency, 2006b; Caskey and 

Frey, 2009) or some measure of nutrient loading affecting 

the biota throughout the year; for example, the annual average or annual maximum concentrations (Indiana Department 

of Environmental Management, 1999). Typically, within the 

GNE and CWPE ecoregions, most biological community data 

are collected during stable low flow conditions between July 

and October, when nutrients are at the lowest concentrations 

for the year. 

A nonparametric multivariate correlation analysis was 

done to compare which measure of nutrient condition (nutrient 

concentration or nutrient ratio) had the strongest relations with 

algal-, invertebrate-, and fish-community structure for each 

ecoregion separately and both ecoregions combined. For this 

analysis, the nutrient measures were the (1) nutrient concentration or nutrient ratio on the day of the biological sampling or 

as close to the day of the sampling as possible, (2) the annual 

average, or (3) the annual maximum concentration. For all 

biological communities and all nutrient categories (nearest 

biological sample, annual mean, and annual maximum concentrations), the ratio of nitrate to total nitrogen (NO3

:TN) had the 

strongest single relation with all the biological communities; 

however, TN and TP had the most significant relations with 

the different biological attributes. The nutrient sample collected nearest the biological sample had the weakest relation 

with algae, invertebrate, and fish communities for the GNE, 

CWPE, and all samples combined (table 4). For all samples 

combined, the nutrient sample with the strongest correlation 

to the biotic community assemblage was the annual maximum 

concentration. However, for the two ecoregions, the nutrient 

sample with the highest correlation to the biotic community 

assemblage was usually the annual average concentration. On 

the basis of these results and the use of TN and TP in proposed 

nutrient criteria, the breakpoint analyses used the annual average for TN and TP.

Using the criterion for sites to have at least 6 nutrient 

samples and algal-, invertebrate-, and fish-community data 

collected within the same year, we identified 23 and 31 sites 

in the GNE and CWPE, respectively. The number of sites 

within each nutrient category ranged from 4 (TN and TP, low 

category) to 18 (TN, medium category) in the CWPE and from 

3 (TN and TP, low category) to 15 (TN, medium category) in 

the GNE (table 5). The TN concentrations ranged from 0.74 to 

16.1 mg/L with a median of 5.48 mg/L in the CWPE and from 

0.05 to 5.52 mg/L with a median of 1.45 mg/L in the GNE. 

The TP concentrations ranged from 0.02 to 2.10 mg/L with 

a median of 0.11mg/L in the CWPE and from 0.01 to 0.47 

mg/L with a median of 0.08 mg/L in the GNE (table 6). The 

main difference between the two diatom ecoregions was that 

TN and TP concentrations were about 3–5 times higher in the 

more agricultural and nutrient-rich CWPE than in the GNE 

(figs. 4 and 5). 

Table 4. Summary of Spearman rho correlations for the nutrients total nitrogen and total phosphorus and the algae, invertebrate, and 

fish community. 

[NS, not significant; bold, strongest correlation of the three nutrient variables]

Nutrient

Algae Invertebrates Fish

All 

samples

Glaciated 

North 

diatom 

ecoregion

Central and 

Western 

Plains 

diatom 

ecoregion

All 

samples

Glaciated 

North 

diatom 

ecoregion

Central and 

Western 

Plains 

diatom 

ecoregion

All 

samples

Glaciated 

North 

diatom 

ecoregion

Central and 

Western 

Plains 

diatom 

ecoregion

Bio-nutrient1 0.248 0.261 0.243 0.152 0.168 0.196 0.16 0.323 NS

Annual 

average2

.309 .271 .252 .283 .183 .230 .346 .524 0.253

Annual 

maximum3

.337 .259 .306 .289 NS .214 .355 .395 .323

1

 Nutrient concentration closest to the biological sample.

2

 Average nutrient concentration for the year.

3

 Maximum nutrient concentration for the year.
Table 5. Level III ecoregion, diatom ecoregion, and nutrient categories of the 54 sampling sites in the Central and Western Plains and Glacial North ecoregions.—Continued [TN, total nitrogen; TP, total phosphorus; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; Bold shows sites that chawhen biological response was used to assign low, medium, and high categories] Site number Site identification Site name Diatom ecoregion Nutrient category 

TN TP 

1 04186500 AUGLAIZE RIVER NEAR FORT JENNINGS, OH CWPE Med High 2 03357330 BIG WALNUT CREEK NEAR ROACHDALE, IN CWPE Med Low 3 040853145 BLACK CREEK AT CURRAN ROAD NEAR DENMARK, WI GNE Med High 4 04159492 BLACK RIVER NEAR JEDDO, MI CWPE Med Med 5 04213500 CATTARAUGUS CR AT GOWANDA, NY GNE Med Low 6 05286290 CEDAR CREEK NEAR COOPERS CORNER, MN GNE Med Med 7 04161820 CLINTON RIVER AT STERLING HEIGHTS, MI GNE Med Med 8 04208504 CUYAHOGA RIVER NEAR NEWBURGH HEIGHTS, OH GNE High High 9 05532500 DES PLAINES RIVER AT RIVERSIDE, IL CWPE Med High 

10 05527800 DES PLAINES RIVER AT RUSSELL, IL CWPE Med Med 

11 04072050 DUCK CREEK AT SEMINARY ROAD NEAR ONEIDA, WI GNE High High 

12 03246400 EAST FORK LITTLE MIAMI RIVER NEAR WILLIAMSBURG, OH CWPE Low Med 13 04085109 EAST RIVER AT MIDWAY ROAD NEAR DE PERE, WI GNE Med High 14 05461390 FLOOD CREEK NEAR POWERSVILLE, IA CWPE High Med 15 04211820 GRAND RIVER AT HARPERSFIELD, OH GNE Med Med 16 393944084120700 HOLES CK IN HUFFMAN PARK AT KETTERING, OH CWPE Low Low 17 05568800 INDIAN CREEK NEAR WYOMING, IL CWPE High Med 18 05449500 IOWA RIVER NEAR ROWAN, IA CWPE Med High 19 03360895 KESSINGER DITCH NEAR MONROE CITY, IN CWPE Med Med 20 05584500 LA MOINE RIVER AT COLMAR, IL CWPE Med High 21 05320270 LITTLE COBB RIVER NEAR BEAUFORD, MN CWPE High High 22 03373530 LOST RIVER NEAR LEIPSIC, IN CWPE Med Med 23 03267900 MAD RIVER AT ST PARIS PIKE AT EAGLE CITY, OH CWPE Med Med 24 04087000 MILWAUKEE RIVER AT MILWAUKEE, WI GNE Med Med 25 03366500 MUSCATATUCK RIVER NEAR DEPUTY, IN CWPE Low Med 26 05331833 NAMEKAGON RIVER AT LEONARDS, WI GNE Low Low 27 05330902 NINE MILE CREEK NEAR JAMES CIRCLE AT BLOOMINGTON, MN GNE Med Med 28 05548105 NIPPERSINK CREEK ABOVE WONDER LAKE, IL CWPE Med Med 29 040863075 NORTH BRANCH MILWAUKEE RIVER NEAR RANDOM LAKE, WI GNE High High 
Table 5. Level III ecoregion, diatom ecoregion, and nutrient categories of the 54 sampling sites in the Central and Western Plains and Glacial North ecoregions.—Continued[TN, total nitrogen; TP, total phosphorus; CWPE, Central and Western Plains diatom ecoregion; GNE, Glacial North diatom ecoregion; Med, medium; Bold shows sites that chawhen biological response was used to assign low, medium, and high categories] 

Site 

number

Site 

identification Site name Diatom ecoregion

Nutrient 

category

TN TP 

30 05276005 NORTH FORK CROW RIVER ABOVE PAYNESVILLE, MN GNE Med Med 31 04087204 OAK CREEK AT SOUTH MILWAUKEE, WI GNE Med Med 32 05455100 OLD MANS CREEK NEAR IOWA CITY, IA CWPE High High 33 05030150 OTTER TAIL RIVER NEAR PERHAM, MN GNE Med Med 34 05567000 PANTHER CREEK NEAR EL PASO, IL CWPE High Med 35 04071795 PENSAUKEE RIVER NEAR KRAKOW, WI GNE Med High 36 04062085 PESHEKEE RIVER NEAR MARTINS LANDING, MI GNE Low Low 37 04087258 PIKE RIVER AT CTH A NEAR KENOSHA, WI CWPE Med Med 38 04063700 POPPLE RIVER NEAR FENCE, WI GNE Low Med 39 04085188 RIO CREEK AT PHEASANT ROAD NEAR RIO CREEK, WI GNE High High 40 04175600 RIVER RAISIN NEAR MANCHESTER, MI GNE Med Med 41 05531500 SALT CREEK AT WESTERN SPRINGS, IL CWPE Med High 42 05572000 SANGAMON RIVER AT MONTICELLO, IL CWPE High Med 43 05288705 SHINGLE CREEK AT QUEEN AVE IN MINNEAPOLIS, MN GNE Med Med 44 05451210 SOUTH FORK IOWA RIVER NE OF NEW PROVIDENCE, IA CWPE High Med 45 04178000 ST JOSEPH RIVER NEAR NEWVILLE, IN CWPE Med Med 46 395355084173600 STILLWATER R. ON MARTINDALE R. NEAR UNION, OH CWPE Med Med 47 394340085524601 SUGAR CREEK AT CO RD 400 S AT NEW PALESTINE, IN CWPE Med Med 48 05525500 SUGAR CREEK AT MILFORD, IL CWPE High Med 49 04080798 TOMORROW RIVER NEAR NELSONVILLE, WI GNE High Med 50 05082625 TURTLE R AT TURTLE R STATE PARK NEAR ARVILLA, ND GNE Med Med 51 05420680 WAPSIPINICON RIVER NEAR TRIPOLI, IA CWPE Med Med 52 03275000 WHITEWATER RIVER NEAR ALPINE, IN CWPE Med Low 53 05062500 WILD RICE RIVER AT TWIN VALLEY, MN CWPE Low Low 54 05464220 WOLF CREEK NEAR DYSART, IA CWPE High Med 
18 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes TOTAL NITROGEN, IN MILLIGRAMS PER LITER

x

x o

x

TN2L

TN2M

TN2H

TN3L

TN3M

TN3H

10

15

20 17 93 44 13 62 22

30 Number of values 

EXPLANATION 

 schematic boxplot

x

x

o

o

Upper adjacent

75th percentile

Median

25th percentile

Lower adjacent

Lower outside

Lower detached

Upper detached

Upper outside

LowLow Med High High Low Med

Central and Western Plains Glacial North

Low Med High Low Med

5

0

TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER

xx

o

x

x

o

TP2L

TP2M

TP2H

TP3L

TP3M

TP3H

15 91 48

Central and Western Plains Glacial North

Low Med High Low Med High

2.5

2.0

1.5

1.0

0.5

0.0

7 63 26

30 Number of values 

EXPLANATION 

 schematic boxplot

x

x

o

o

Upper adjacent

75th percentile

Median

25th percentile

Lower adjacent

Lower outside

Lower detached

Upper detached

Upper outside

Figure 4. Distribution of median annual total nitrogen concentrations in the low-, medium-, and high-nutrient 

categories in the Central and Western Plains and Glacial North diatom ecoregions. 

Figure 5. Distribution of median annual total phosphorus concentrations in the low-, medium-, and high-nutrient 

categories in the Central and Western Plains and Glacial North diatom ecoregions. 
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 19

Comparison of Seasonal Nutrient 

Concentrations by Nutrient Categories 

Seasonally, the highest TN concentrations within the 

CWPE and GNE were found in the spring, after the application of fertilizer to fields; concentrations decreased steadily 

from June through October because of (1) nutrient uptake 

from crops and terrestrial plants, (2) nutrient uptake by algae 

and macrophytes in streams, and (3) decreased nutrient runoff 

to streams from drier soils, which are better able to absorb precipitation (fig. 6). Concentrations of TN increased in the fall 

and winter because of decreased plant uptake and application 

of manure and fertilizer. In general, the seasonal patterns of 

high and low concentrations in the low-, medium-, and highnutrient categories were similar for TN; however, the main 

difference between the sites in these categories was the magnitude of nutrient concentrations. For example, one can compare 

the TN concentrations in three similarly sized agricultural 

sites in the CWPE, all within USEPA Nutrient Ecoregion VI, 

where the proposed criterion is 2.18 mg/L. At the low-TN 

site, Muscatatuck River in Indiana (site 25), TN ranged from 

1.02 to 3.30 mg/L, and concentrations in 75 percent of the 

samples were below the USEPA proposed criterion (fig. 7). 

Table 6. Summary statistics for total nitrogen and total 

phosphorus in the Central and Western Plains and Glacial North 

diatom ecoregions. 

[mg/L, milligrams per liter; concentrations in bold slightly differ from the 

values used to classify nutrient categories]

Statistic Total nitrogen 

(mg/L)

Total phosphorus 

(mg/L)

Central and Western Plains diatom ecoregion

Minimum 0.74 0.02

10th percentile 1.55 .05

25th percentile 2.99 .07

Median 5.48 .11

75th percentile 7.77 .16

90th percentile 10.8 .71

Maximum 16.1 2.10

Glacial North diatom ecoregion

Minimum 0.05 0.01

10th percentile .52 .02

25th percentile .87 .03

Median 1.45 .08

75th percentile 2.00 .13

90th percentile 2.58 .20

Maximum 5.52 .47

x

x

x

x

x

x

x

x xx

x

x

x

x

x

x

x

x

x

o

x

x

x x

x

x

x

x

x

x

January

February

March

April

May

June

July

August

September

October

November

December

0.1

0.5

1.0

5.0

50.0

118 126 140 150 149 168 221 302 293 312 297 310 255 290 223 214 135 170 132 150 125 142 137 136

TOTAL NITROGEN, IN MILLIGRAMS PER LITER

Central and Western Plains 

 diatom ecoregion

Glacial North diatom 

 ecoregion 

10.0

30 Number of values 

EXPLANATION 

 schematic boxplot

x

x

o

o

Upper adjacent

75th percentile

Median

25th percentile

Lower adjacent

Lower outside

Lower detached

Upper detached

Upper outside

Figure 6. Monthly concentrations of total nitrogen at the 64 sites within Central and Western Plains and Glacial North 

diatom ecoregions used to determine nutrient categories. 
20 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

At the medium-TN site, Sugar Creek in Indiana (site 47), TN 

ranged from 0.57 to 7.70 mg/L, and concentrations in about 

63 percent of the samples were below the USEPA proposed 

criterion for TN (fig. 8). At the high-TN site, Little Cobb River 

in Minnesota (site 21), TN ranged from 3.03 to 14.4 mg/L, and 

concentrations in 100 percent of the samples were above the 

USEPA proposed criterion for TN (fig. 9). Similar trends for 

TN were found in the low, medium, and high categories within 

the CWPE and GNE ecoregions.

Seasonally, the highest TP concentrations in streams 

within the CWPE and GNE ecoregions were found in June and 

July, after application of fertilizer and manure (fig. 10). Unlike 

the pattern of concentrations of TN, however, concentrations 

of TP in streams were low in the winter and increased to the 

highest concentrations during May and June and then steadily 

decreased through the fall and winter. Variability was greater 

for seasonal concentrations of TP than for TN. Likewise, the 

differences in the magnitude and seasonal trends of nutrient 

concentrations between the low, medium, and high categories 

for TP was less consistent than the patterns found for TN. 

Biological Conditions Within the Nutrient 

Categories 

The next step was to determine whether the designated 

nutrient categories within the ecoregions were significantly 

different on the basis of species composition of the samples. 

Comparable to the analysis to determine differences in nutrients between the ecoregions, a similarity analysis based on the 

community composition was used to distinguish significant 

differences in the biological communities among the nutrient 

categories (table 7). All biological communities were significantly different between at least one nutrient category for TN 

and TP in each ecoregion but were not consistently different 

from one ecoregion to the other. In the GNE, the algal community was most dissimilar between the low and high and low 

and medium sites for both TN and TP. In the more nutrientrich CWPE, however, fish were more different between the 

low and high and low and medium sites for both TN and TP. 

0.0

1.0

2.0

3.0

4.0

Jan−93 Mar−93 May−93 Jun−93 Aug−93 Oct−93 Nov−93

TOTAL NITROGEN, IN MILLIGRAMS PER LITER

Biological sample

collected

Drainage area − 759 square kilometers

Proposed U.S. Environmental Protection Agency 

total nitrogen value, nutrient ecoregion VI − 2.18 milligrams per liter

Jan−94 Mar−94 Apr−94

Figure 7. Seasonal concentrations of total nitrogen (TN) at the low-TN site Muscatatuck River, Indiana. (The 

blue line on the graph indicates a Lowess smooth.) 
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 21

TOTAL NITROGEN, IN MILLIGRAMS PER LITER

0.0

2.0

4.0

6.0

8.0

7.0

5.0

3.0

1.0

Mar−93 May−93 Jun−93 Aug−93 Oct−93 Nov−93 Jan−94 Mar−94 Apr−94

Drainage area − 246 square kilometers

Biological sample

collected

Proposed U.S. Environmental Protection Agency 

total nitrogen value, nutrient ecoregion VI − 2.18 milligrams per liter

0

2

4

6

8

10

12

14

16

TOTAL NITROGEN, IN MILLIGRAMS PER LITER

Drainage area − 336 square kilometers

Biological sample

collected

Proposed U.S. Environmental 

Protection Agency 

total nitrogen value, 

nutrient ecoregion VI − 

2.18 milligrams per liter

Jan−97 Mar−97 Apr−97 Jun−97 Aug−97 Sep−97 Nov−97 Jan−98 Feb−98 Apr−98

Figure 8. Seasonal concentrations of total nitrogen (TN) at the medium-TN site Sugar Creek, Indiana. (The 

blue line on the graph indicates a Lowess smooth.) 

Figure 9. Seasonal concentrations of total nitrogen (TN) at the high-TN site Little Cobb River, Minnesota. 

(The blue line on the graph indicates a Lowess smooth.) 
22 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

x

x

x

xx

x

x

x

x x x

xx

x

xx

x

x

x

x

x

x

x

x

x

x

x

xx

x

x

x

x

x

x

x

x

x

x

xx

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

x

January

February

March

April

May

June

July

August

September

October

November

December

0.010

0.100

1.000

10.000 117 63 140 75 149 85 217 151 292 156 297 155 256 144 223 106 135 85 132 75 125 71 137 69

TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER

Central and Western Plains 

 diatom ecoregion

Glacial North diatom 

 ecoregion 

30 Number of values 

EXPLANATION 

 schematic boxplot

x

x

o

o

Upper adjacent

75th percentile

Median

25th percentile

Lower adjacent

Lower outside

Lower detached

Upper detached

Upper outside

Figure 10. Monthly concentrations of total phosphorus at the 64 sites within Central and Western Plains and Glacial 

North diatom ecoregions used to determine nutrient categories. 

Table 7. Spearman rank correlation coefficients from the analysis of similarity between the three nutrient categories. 

[Bold indicates significant difference between the nutrient categories (p value = 0.05)]

Community

Diatom ecoregion

Central and Western Plains Glaciated North

Low to 

medium Low to high Medium to high

Low to 

medium Low to high Medium to high

Annual average total nitrogen

Algae 0.257 0.485 0.070 0.493 0.665 −0.025

Invertebrates .003 .529 .071 .262 .411 .160

Fish .297 .562 .115 .301 .099 .079

Annual average total phosphorus

Algae 0.180 0.347 0.160 0.408 0.721 0.095

Invertebrates .055 .234 −.004 .182 .552 .154

Fish .079 .652 .037 .308 .602 −.028
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 23

Certain species of algae, invertebrates, and fish were 

more prevalent in and characteristic of either the low- or the 

high-nutrient condition within each of the ecoregions. For 

the most part, the species in the medium-nutrient categories 

consisted of a mix of species in the low or high categories 

and reflected a transition from the low to high nutrient levels. 

Some species were found in similar relative abundance regardless of nutrient category. In particular, the species that increase 

or decrease in abundance with corresponding changes in 

nutrient concentrations could be useful indicators of nutrient 

conditions within streams and may be useful for development of nutrient biotic indices for states within these regions. 

For this analysis, a given algae, invertebrate, or fish taxa was 

considered indicative of low nutrient (TN or TP) concentrations if the relative abundance consistently decreased in value 

from low- to medium- to high-nutrient categories or was found 

only in the low-nutrient category. To be considered an indicator of high nutrient concentrations, the taxa had to consistently 

decrease in value from high to medium to low categories or 

be found only in the high-nutrient category. Taxa that occur in 

all nutrient categories without a consistent upward or downward trend or those only occurring in the medium category are 

considered common and ubiquitous.

Algae

To describe nutrient conditions in streams on the basis 

of algal-community data, identification to genus of algae is 

not specific enough to distinguish low- and high-nutrient 

taxa (Hill and others, 2000). For example, the freshwater 

diatom Nitzschia, a genus with more than 600 species (Wehr 

and Sheath, 2003), includes species that are abundant in 

low- or high-nutrient conditions, as well as species that are 

ubiquitous in all nutrient conditions. Many Nitzschia species 

are recognized as indicators of organic enrichment (Lowe, 

1974) and are pollution tolerant. Cholnoky (1938) suggested 

that Nitzschia are nutrient tolerant because many species are 

nitrogen heterotrophs and use organic nitrogen in the water 

column. One strength of incorporating the algal community 

structure into nutrient stressor studies is that algae directly 

use nutrients (unlike invertebrates or fish), so the abundance, 

diversity, and evenness of the algal species are limited by the 

available nutrients (Pringle, 1990; Biggs and Smith, 2002). 

When nutrients are abundant they can stimulate algal blooms, 

whereas with lower concentrations of N or P, the algal community will grow only to the level that can be supported by 

these nutrients.

Central and Western Plains Diatom Ecoregion 

In the high-nutrient conditions in streams of the CWPE, 

12 species in 3 genera dominated the algal community 

(table 8). The most abundant taxa indicative of high TP conditions were five Navicula species (recens, minima, subminuscula, erifuga, and trivalis), three Nitzschia species (amphibia, 

palea, and palea debilis), and a single Cyclotella species

(meneghiniana). The most abundant taxa indicative of high 

TN conditions were four Navicula species (cryptotonella, subminiscula, palea, and symmetrica). Three species indicative of 

conditions in the low TP category (Gomphonema kobayasii, 

Nitzschia inconspicua, and Amphora pediculus) accounted 

for nearly one-third of the relative abundance, unlike the high 

TP category, where 10 Navicula and Nitzschia species were 

relatively evenly represented. Rhoicosphenia abbreviata, 

Reimeria sinuata f. antiqua, Achnanthidium minutissimum,

and Cocconeis pediculus were indicative of conditions in the 

low TN category. Gomphonema minutum was ubiquitous in all 

nutrient categories, along with Navicula cryptotonella in all 

TP categories and Nitzschia inconspicua, Amphora pediculus,

and Nitzschia dissipata in all TN categories.

Glaciated North Diatom Ecoregion 

Several of the same algal species that indicated highnutrient conditions in the CWPE also were indicators of 

high-nutrient conditions in streams in the GNE. Amphora

pediculus in particular dominates in the high TN and TP 

categories, with about 20 and 22 percent relative abundance, 

respectively. Three Navicula species (tripunctata, cryptotonella, and secreta) were common in streams with high TN or 

TP conditions, as was Navicula minima in high TP conditions. 

Nitzschia inconspicua was the only ubiquitous species found 

in all of the GNE TN nutrient categories. 

In low TN and TP categories, Achnanthidium minutissimum and Achnanthidium deflexum dominated the algal 

community with about 25 and 30 percent of the relative abundance, respectively (table 8). Many other studies have found 

Achnanthidium minutissimum to be indicative of low TN and 

TP conditions (Van Dam and others, 1994; Fore and Grafe, 

2002). Additionally, Pseudostaurosira brevistriata preferred 

the low category for TN and TP. Six species from the family 

Fragilariaceae were found exclusively in the low TN category 

(table 8). Although these six species prefer varying trophic 

conditions ranging from mesoeutrophic to eutrophic, four of 

the six species are in the Bahls’ pollution class that are considered moderately tolerant to intolerant of nutrient and organic 

enrichment, and the other two are only somewhat tolerant of 

nutrient and organic enrichment (Bahls, 1993; Van Dam and 

others, 1994; Porter, 2008), which may explain the occurrence 

of these species at the lower TN sites.
Table 8. Most abundant algal taxa in the total nitrogen and total phosphorus nutrient categories in the Central and Western Plains and Glacial North ecoregions.—Continued [mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category] Central and Western Plains diatom ecoregion – Total phosphorus Low sites (<0.05 mg/L) Medium sites (>0.05 – <0.17 mg/L) High sites (>

Taxa Mean Taxa Mean Taxa 

Gomphonema kobayasii 11.06 Amphora pediculus 8.18 Amphora pediculus Nitzschia inconspicua 10.33 Navicula cryptotenella 5.88 Navicula recens Amphora pediculus 10.30 Nitzschia inconspicua 5.10 Navicula cryptotenella Rhoicosphenia abbreviata 7.28 Navicula minima 4.29 Navicula minima Cymbella affinis 5.39 Rhoicosphenia abbreviata 4.15 Cyclotella meneghiniana Navicula cryptotenella 4.30 Nitzschia dissipata 3.29 Nitzschia inconspicua Achnanthidium minutissimum 3.73 Cocconeis placentula var. lineata 2.91 Rhoicosphenia abbreviata Nitzschia dissipata 3.51 Cyclotella meneghiniana 2.41 Nitzschia amphibia Cocconeis pediculus 3.00 Cymbella atomus 2.12 Cocconeis placentula var. lineata Unknown Cyanophyte Oscillatoriales 3.00 Gomphonema minutum 2.03 Nitzschia palea Gomphonema minutum 2.43 Navicula subminuscula 1.93 Navicula subminuscula Navicula capitatoradiata 2.17 Cocconeis placentula var. euglypta 1.69 Navicula erifuga Amphora inariensis 2.08 Nitzschia palea 1.57 Cymbella atomus Encyonema silesiacum 1.29 Gomphonema kobayasii 1.55 Navicula trivialis Unknown Cyanophyte Oscillatoriales (no sheath) 1.22 Nitzschia amphibia 1.53 Nitzschia palea var. debilis 

Navicula tripunctata 1.34 Gomphonema kobayasii Navicula germainii 1.28 Gomphonema minutum Navicula reichardtiana 1.25 Cocconeis placentula var. euglypta Nitzschia frustulum 1.16 Gomphonema parvulum Simonsenia delognei 1.15 Sellaphora seminulum Navicula symmetrica 1.10

Total top 5 species 44.37 27.59 
Table 8. Most abundant algal taxa in the total nitrogen and total phosphorus nutrient categories in the Central and Western Plains and Glacial North ecoregions.—Continued[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Glacial North diatom ecoregion – Total phosphorus

Low sites (<0.01 mg/L) Medium sites (>0.01 – <0.13 mg/L) High sites (>

Taxa Mean Taxa Mean Taxa 

Achnanthidium

minutissimum

28.34 Amphora pediculus 6.57 Amphora pediculus 

Oscillatoria limnetica 4.56 Achnanthidium

minutissimum

5.85 Navicula tripunctata 

Cocconeis placentula 2.34 Nitzschia inconspicua 5.80 Navicula minima Cymbella delicatula 2.30 Navicula cryptotenella 3.68 Navicula cryptotenella Achnanthidium deflexum 2.17 Rhoicosphenia abbreviata 3.53 Rhoicosphenia abbreviata Navicula cryptotenella 2.02 Navicula tripunctata 2.47 Navicula secreta

var. apiculata

Pseudostaurosira

brevistriata

2.00 Cyclotella meneghiniana 2.00 Nitzschia inconspicua 

Staurosirella pinnata 1.34 Nitzschia dissipata 1.93 Gomphonema olivaceum Staurosira construens

var. venter

1.14 Staurosira construens

var. venter

1.92 Nitzschia dissipata 

Reimeria sinuata 1.13 Cocconeis placentula

var. euglypta

1.86 Platessa conspicua 

Navicula minima 1.76 Melosira varians Staurosirella pinnata 1.58 Unknown Rhodophyte 

Florideophycidae (chantransia)

Nitzschia amphibia 1.43 Planothidium rostratum Gomphonema parvulum 1.40 Homoeothrix janthina Cocconeis placentula 1.33 Gomphonema parvulum Fragilaria vaucheriae 1.17 Navicula capitatoradiata Cocconeis placentula

var. lineata

1.12

Navicula capitatoradiata 1.00

Total top 5 species 39.71 25.44 
Table 8. Most abundant algal taxa in the total nitrogen and total phosphorus nutrient categories in the Central and Western Plains and Glacial North ecoregions.—Continued [mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category] Central and Western Plains diatom ecoregion – Total nitrogen Low sites (<1.74 mg/L) Medium sites (>1.74 – <7.80 mg/L) High sites (>

Taxa Mean Taxa Mean Taxa 

Nitzschia inconspicua 9.49 Amphora pediculus 9.43 Navicula cryptotenella Amphora pediculus 8.99 Rhoicosphenia abbreviata 5.14 Nitzschia inconspicua Rhoicosphenia abbreviata 6.33 Navicula cryptotenella 5.04 Amphora pediculus Reimeria sinuata f. antiqua 5.00 Navicula minima 4.57 Navicula minima Achnanthidium minutissimum 3.73 Nitzschia inconspicua 4.55 Nitzschia amphibia Cocconeis pediculus 3.71 Nitzschia dissipata 3.30 Gomphonema minutum Cocconeis placentula var. euglypta 3.64 Cyclotella meneghiniana 3.18 Navicula subminuscula Unknown Cyanophyte Oscillatoriales (sheath) 3.19 Cocconeis placentula var. lineata 3.16 Cocconeis placentula var. euglypta Cymbella affinis 2.62 Navicula recens 3.13 Rhoicosphenia abbreviata Navicula cryptotenella 2.26 Amphora inariensis 1.86 Nitzschia palea Gomphonema minutum 2.21 Cymbella atomus 1.82 Cyclotella meneghiniana Amphora inariensis 1.81 Navicula subminuscula 1.49 Cocconeis placentula var. lineata Unknown Cyanophyte Oscillatoriales (no sheath) 1.56 Gomphonema kobayasii 1.43 Nitzschia dissipata Navicula capitatoradiata 1.34 Nitzschia palea 1.37 Gomphonema kobayasii Nitzschia dissipata 1.20 Gomphonema minutum 1.22 Navicula symmetrica Navicula tripunctata 1.11 Nitzschia amphibia 1.13 Mayamaea atomus 

Navicula tripunctata 1.11 Simonsenia delognei Melosira varians 1.04 Navicula erifuga Navicula gregaria 1.04 Nitzschia palea var. debilis 

Navicula germainii Nitzschia frustulum Craticula molestiformis Navicula tripunctata 

Total top 5 species 33.54 28.72 
Table 8. Most abundant algal taxa in the total nitrogen and total phosphorus nutrient categories in the Central and Western Plains and Glacial North ecoregions.—Continued[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Glacial North diatom ecoregion – Total nitrogen

Low sites (<0.60 mg/L) Medium sites (>0.60 – <2.08 mg/L) High sites (>

Taxa Mean Taxa Mean Taxa 

Achnanthidium

minutissimum

21.71 Amphora pediculus 9.65 Amphora pediculus 

Staurosira construens

var. venter

5.15 Nitzschia inconspicua 6.52 Navicula tripunctata 

Staurosirella pinnata 3.92 Rhoicosphenia abbreviata 4.64 Navicula cryptotenella Achnanthidium deflexum 3.63 Navicula cryptotenella 4.01 Rhoicosphenia abbreviata Gomphonema pumilum 2.94 Navicula minima 3.73 Achnanthidium

minutissimum

Pseudostaurosira

brevistriata

2.88 Navicula tripunctata 2.72 Gomphonema olivaceum 

Fragilaria vaucheriae 2.62 Cyclotella meneghiniana 2.18 Nitzschia inconspicua Cymbella affinis 2.57 Nitzschia dissipata 2.17 Navicula minima Staurosira construens

var. venter

1.82 Achnanthidium

minutissimum

1.89 Nitzschia dissipata 

Homoeothrix janthina 1.69 Cocconeis placentula

var. euglypta

1.85 Navicula secreta

var. apiculata

Gomphonema parvulum 1.35 Gomphonema parvulum 1.66 Homoeothrix janthina Synedra ulna 1.10 Nitzschia amphibia 1.57 Unknown Rhodophyte

Florideophycidae(chantransia)

Cocconeis placentula 1.41 Planothidium rostratum Navicula capitatoradiata 1.32 Cocconeis placentula

var. euglypta

Cocconeis placentula

var. lineata

1.13

Total top 5 species 37.35 28.54 

High-nutrient taxa.Low-nutrient taxa.Taxa common at all nutrient levels.
28 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Invertebrates

As with the algae, identification to family level for 

invertebrates is not specific enough to distinguish differences 

between low- and high-nutrient conditions. For example, the 

Chironimidae and Hydropsychidae include genera that are 

abundant in low- or high-nutrient conditions, as well as genera 

that are common in all nutrient conditions (Resh and Unzicker, 

1975). For this section, the invertebrate taxa are identified to 

the lowest taxonomic level possible, with the order name first, 

followed by the family and genus (and species if available) 

italicized.

With increased disturbance in the aquatic environment, 

invertebrate communities may decline in abundance, diversity, and evenness of distribution; however, in some situations, there seems to be an intermediate disturbance effect 

whereby a small increase in disturbance increases abundance, 

diversity, and evenness but, past a certain point, community 

structure begins to diminish (Biggs, 1995; Death and Winterbourn, 1995). For invertebrates, this “intermediate disturbance 

hypothesis” has been explored for stream hydrology and 

habitat characteristics such as flooding, substrate size, and 

refugia (Death and Winterbourn, 1995; Townsend and others, 

1997; Lake, 2000) but has not been examined as it relates to 

nutrients.

Central and Western Plains Diatom Ecoregion 

Three taxa were indicators of conditions of the lownutrient category for both TN and TP in CWPE: Trichoptera

Hydropsychidae Ceratopsyche sp., Ephemeroptera Baetidae 

Baetis flavistriga, and Coleoptera Psephenidae Psephenus

herricki (table 9). Apart from the common taxa, two Diptera

Chironomidae genera (Cricotopus sp. and Rheotanytarsus sp.) 

and Trichoptera Hydroptilidae Hydroptila sp. among the TP 

sites and the Trichoptera Hydropsychidae Cheumatopsyche

sp., Ephemeroptera Caenidae Caenis sp., Coleoptera Elmidae 

Stenelmis sp. and Diptera Chironomidae Microtendipes sp. 

among the TN sites were indicative of low-nutrient conditions.

Four taxa were indicative of the high-nutrient category 

for TN and TP in the CWPE: two Ephemeroptera families 

(Heptageniidae and Leptohyphidae Tricorythodes sp.), Diptera 

Chironomidae Stenochironomus sp., and Trichoptera Hydropsychidae Hydropsyche sp. High-TP conditions were also 

favored by Trichoptera Hydropsychidae Hydropsyche bidens, 

whereas high-TN conditions included higher relative abundances of six other taxa: two Diptera genera (Chironomidae 

Tanytarsus sp. and Empididae Hemerodromia sp.), two Coleoptera Elmidae species (Stenelmis grossa and Macronychus

glabratus), a Trichoptera Hydroptilidae Hydroptila sp. and a 

Tubificida Naididae. 

In the environment of higher overall nutrient concentrations of the CWPE, there were five and six common invertebrate taxa found in all nutrient categories in the TP and TN 

sites, respectively, compared to only one and two in the GNE. 

This finding suggests that as nutrient concentrations increase, 

the increase fuels the ability of multiple taxa to become 

abundant. This increased evenness of taxa could be used as an 

indicator of eutrophic conditions.

Glaciated North Diatom Ecoregion 

Four taxa were indicative of low-nutrient conditions in the TN and TP categories: two Trichoptera genera

(Philopotamidae Chimarra sp. and Hydropsychidae Ceratopsyche sp.) and two Diptera genera (Chironomidae Tvetenia

sp. and Empididae Hemerodromia sp.) (table 9). Along with 

these taxa were seven additional taxa commonly abundant 

at sites in the low-TN category: four Ephemeroptera genera 

(Leptophlebiidae Paraleptophlebia sp., Heptageniidae Maccaffertium sp., Heptageniidae Leucrocuta sp., and Isonychiidae Isonychia sp.), Diptera Chironomidae Micropsectra sp., 

Plecoptera Perlidae Acroneuria sp., and a Trichoptera Hydroptilidae Hydroptila sp. In low-TP conditions, six additional taxa 

were found: four Diptera Chironomidae taxa (Thienemanniella

sp., Lopescladius sp., Cricotopus sp., and Constempellina

brevicosta), Ephemeroptera Heptageniidae, and Coleopteran 

Elmidae.

Two Diptera Chironomidae genera (Polypedilum sp. 

and Stictochironomus sp.) and Trichoptera Hydropsychidae 

Cheumatopsyche sp. were indicators of the high-nutrient category for both TP and TN. Along with those three genera, two 

Diptera Chironomidae taxa (Orthocladiinae and Paratanytarsus sp.), two Coleoptera Elmidae genera (Optioservus sp. and 

Stenelmis sp.), and the Haplotaxida Naididae were indicative 

of the high-TP category, and two Diptera Chironomidae genera (Microtendipes sp. and Cladotanytarsus sp.), Trichoptera 

Glossosomatidae Protoptila sp., and Ephemeroptera Baetidae 

Acerpenna sp. were indicative of the high-TN category. Only 

one genus was commonly found in all three nutrient categories: Diptera Chironomidae Pentaneurini sp.
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 29

Table 9. Most abundant invertebrate taxa in the total nitrogen and total phosphorus nutrient categories in the Central and 

Western Plains and Glacial North diatom ecoregions.—Continued

[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Central and Western Plains diatom ecoregion – Total phosphorus

Low sites (<0.05 mg/L) Medium sites (>0.05 – <0.17 mg/L) High sites (>0.17 mg/L)

(n=13) (n=61) (n=30)

Taxa Mean Taxa Mean Taxa Mean

Ceratopsyche sp. 14.01 Tricorythodes sp. 8.93 Hydropsyche sp. 12.32

Cheumatopsyche sp. 12.21 Hydropsyche sp. 6.24 Tricorythodes sp. 11.68

Stenelmis sp. 5.94 Cheumatopsyche sp. 5.81 Cheumatopsyche sp. 9.82

Cricotopus sp. 5.64 Polypedilum sp. 5.46 Baetis intercalaris 9.04

Polypedilum sp. 5.51 Macronychus glabratus 5.36 Polypedilum sp. 7.85

Baetis intercalaris 4.98 Baetis intercalaris 3.94 Stenelmis sp. 4.00

Rheotanytarsus sp. 3.93 Rheotanytarsus sp. 3.11 Heptageniidae 2.92

Tricorythodes sp. 3.82 Ceratopsyche sp. 3.00 Ceratopsyche sp. 2.75

Hydropsyche sp. 3.45 Caenis sp. 2.92 Macronychus glabratus 2.22

Hydroptila sp. 2.80 Stenelmis sp. 2.82 Hydropsyche bidens 2.15

Baetis flavistriga 2.37 Cricotopus sp. 2.51 Pentaneurini 1.45

Pentaneurini 2.04 Heptageniidae 2.39 Arachnida 1.43

Arachnida 1.95 Pentaneurini 2.00 Rheotanytarsus sp. 1.42

Psephenus herricki 1.47 Arachnida 1.84 Stenochironomus sp. 1.19

Naididae 1.71

Total top 5 species 43.31 31.79 50.71

Glacial North diatom ecoregion – Total phosphorus

Low sites (<0.01 mg/L) Medium sites (>0.01 - <0.13 mg/L) High sites (>0.13 mg/L)

(n=8) (n=47) (n=20)

Taxa Mean Taxa Mean Taxa Mean

Heptageniidae 8.17 Tubificida 5.66 Orthocladiinae 11.58

Simulium sp. 7.57 Polypedilum sp. 5.66 Microtendipes sp. 9.25

Tvetenia sp. 5.94 Cheumatopsyche sp. 5.60 Cheumatopsyche sp. 7.10

Chimarra sp. 5.80 Rheotanytarsus sp. 4.55 Polypedilum sp. 6.83

Elmidae 2.94 Orthocladiinae 4.06 Tubificida 5.78

Polypedilum sp. 2.82 Hydropsyche sp. 3.78 Stictochironomus sp. 4.98

Hemerodromia sp. 2.69 Cricotopus sp. 3.67 Baetis flavistriga 4.70

Ceratopsyche sp. 2.31 Naididae 3.58 Naididae 4.63

Nematoda 2.24 Baetis intercalaris 2.42 Optioservus sp. 3.46

Thienemanniella sp. 2.01 Arachnida 2.29 Paratanytarsus sp. 3.45

Eukiefferiella sp. 1.89 Pentaneurini 2.13 Simulium sp. 2.79

Lopescladius sp. 1.65 Ceratopsyche sp. 2.02 Eukiefferiella sp. 2.55

Hydroptila sp. 1.49 Caenis sp. 1.95 Stenelmis sp. 1.65

Baetis flavistriga 1.46 Stenelmis sp. 1.62 Nematoda 1.61

Pentaneurini 1.41 Tanytarsus sp. 1.29 Pentaneurini 1.30

Rheotanytarsus sp. 1.29

Arachnida 1.22

Microtendipes sp. 1.19

Constempellina brevicosta 1.16

Cricotopus sp. 1.08

Total top 5 species 30.42 25.53 40.54
30 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Table 9. Most abundant invertebrate taxa in the total nitrogen and total phosphorus nutrient categories in the Central and 

Western Plains and Glacial North diatom ecoregions.—Continued

[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Central and Western Plains diatom ecoregion – Total nitrogen

Low sites (<1.74 mg/L) Medium sites (>1.74 – <7.80 mg/L) High sites (>7.80 mg/L)

(n=11) (n=57) (n=36)

Taxa Mean Taxa Mean Taxa Mean

Cheumatopsyche sp. 12.74 Cheumatopsyche sp. 9.19 Tricorythodes sp. 14.61

Ceratopsyche sp. 10.92 Tricorythodes sp. 6.88 Hydropsyche sp. 10.31

Stenelmis sp. 7.99 Polypedilum sp. 6.55 Macronychus glabratus 7.07

Caenis sp. 6.66 Hydropsyche sp. 6.52 Baetis intercalaris 5.84

Polypedilum sp. 6.29 Baetis intercalaris 5.63 Polypedilum sp. 5.48

Hydropsyche sp. 4.77 Ceratopsyche sp. 3.82 Cheumatopsyche sp. 4.00

Baetis intercalaris 4.11 Cricotopus sp. 3.45 Heptageniidae 3.51

Pentaneurini 3.03 Stenelmis sp. 3.30 Ceratopsyche sp. 3.05

Baetis flavistriga 2.77 Rheotanytarsus sp. 2.96 Stenelmis sp. 2.59

Baetis sp. 2.65 Macronychus glabratus 2.40 Rheotanytarsus sp. 2.40

Rheotanytarsus sp. 2.58 Tubificida 2.39 Pentaneurini 2.04

Cricotopus sp. 2.43 Caenis sp. 2.29 Naididae 1.88

Tricorythodes sp. 2.39 Arachnida 1.69 Tanytarsus sp. 1.83

Arachnida 2.30 Pentaneurini 1.49 Stenochironomus sp. 1.76

Tubificida 2.03 Naididae 1.32 Arachnida 1.64

Psephenus herricki 1.74 Leucrocuta sp. 1.12 Stenelmis grossa 1.40

Microtendipes sp. 1.14 Cricotopus sp. 1.27

Hydroptila sp. 1.22

Hemerodromia sp. 1.07

Total top 5 species 44.60 34.77 43.32
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 31

Table 9. Most abundant invertebrate taxa in the total nitrogen and total phosphorus nutrient categories in the Central and 

Western Plains and Glacial North diatom ecoregions.—Continued

[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Glacial North diatom ecoregion – Total nitrogen

Low sites (<0.60 mg/L) Medium sites (>0.60 – <2.08 mg/L) High sites (>2.08 mg/L)

(n=13) (n=43) (n=16)

Taxa Mean Taxa Mean Taxa Mean

Orthocladiinae 7.87 Tubificida 7.89 Orthocladiinae 13.16

Chimarra sp. 6.37 Cheumatopsyche sp. 5.73 Cheumatopsyche sp. 8.63

Polypedilum sp. 4.40 Polypedilum sp. 5.58 Polypedilum sp. 7.47

Paraleptophlebia sp. 3.35 Naididae 4.93 Microtendipes sp. 6.76

Simulium sp. 3.25 Rheotanytarsus sp. 4.74 Baetis flavistriga 5.77

Stenelmis sp. 2.92 Hydropsyche sp. 4.14 Optioservus sp. 5.08

Tvetenia sp. 2.83 Cricotopus sp. 3.64 Simulium sp. 3.47

Rheotanytarsus sp. 2.61 Hyalella azteca 3.20 Eukiefferiella sp. 2.95

Maccaffertium sp. 2.45 Baetis intercalaris 2.63 Stictochironomus sp. 2.71

Ceratopsyche sp. 2.38 Microtendipes sp. 2.12 Naididae 2.70

Baetis flavistriga 2.36 Pentaneurini 2.02 Tubificida 2.43

Optioservus sp. 2.07 Caecidotea sp. 1.86 Arachnida 2.42

Micropsectra sp. 2.07 Paratanytarsus sp. 1.82 Stenelmis sp. 2.07

Cricotopus sp. 2.05 Caenis sp. 1.79 Protoptila sp. 2.01

Acroneuria sp. 2.05 Arachnida 1.68 Nematoda 1.77

Nematoda 1.98 Tricorythodes sp. 1.67 Plecoptera 1.75

Leucrocuta sp. 1.90 Ceratopsyche sp. 1.58 Pentaneurini 1.55

Pentaneurini 1.66 Acerpenna sp. 1.51

Cheumatopsyche sp. 1.63 Caecidotea sp. 1.43

Arachnida 1.56 Cladotanytarsus sp. 1.12

Hydroptila sp. 1.46

Hemerodromia sp. 1.20

Isonychia sp. 1.02

Eukiefferiella sp. 1.01

Total top 5 species 25.23 28.87 41.80

High-nutrient taxa.

Low-nutrient taxa.

Taxa common at all nutrient levels.
32 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Fish

Fish are larger than invertebrates and algae, which makes 

identification to species easier in the field and limits the need 

for laboratory verification. Although identification below family level is relatively easy (unlike algae and invertebrates), it 

is possible to find whole families that have relatively similar 

tolerances to disturbances; for example, trout (in the family Salmonidae) are considered intolerant of disturbance. 

For some families, however, it is necessary to look at lower 

taxonomic levels; for example, the Cyprinidae, which range 

from the very tolerant Cyprinus carpio (common carp) to the 

sensitive Exoglossum laurae (tonguetied minnow).

Although fish do not directly utilize nutrients, nutrients 

are needed to support the base of their food web. The nutrients 

support the algae, which feed the invertebrates and smaller fish 

which, in turn serve as food for the larger fish. In oligotrophic 

conditions, there generally is not enough food at the base of 

the food web. Excessive nutrients in eutrophic streams may 

cause an algal bloom that can consume DO when the algae 

die, cause toxic blooms, and cover nesting areas.

Central and Western Plains Diatom Ecoregion 

Three species, Campostoma anomalum (central 

stoneroller), Semotilus atromaculatus (creek chub), and 

Rhinichthys atratulus (blacknose dace), were indicators of low 

nutrients for the TN and TP categories in the CWPE (table 10). 

Additionally, Luxilus chrysocephalus (striped shiner) and 

Moxostoma duquesnii (black redhorse) were indicator species in the low-TP category, and Luxilus cornutus (common 

shiner) and Rhinichthys cataractae (longnose dace) were 

indicator species in the low-TN category. Despite our classifying the study area into two regions to account for regional 

biotic differences, there appears to still be a north-south or 

coldwater-warmwater artifact within the CWPE fish community. The longnose dace is typically found in northern or 

montane regions (Barnes and others, 1985) and the common 

shiner generally prefers cooler waters and is often replaced 

by the striped shiner as streams become warmer (eNature.

com, 2009). Within the low-nutrient category, three of the sites 

were in Indiana or Ohio, and one was in Minnesota. The common shiner and longnose dace dominated the Minnesota site, 

whereas the striped shiner was dominant in the Indiana and 

Ohio sites. 

Within the CWPE, Cyprinella spiloptera (spotfin shiner) 

and Notropis stramineus (sand shiner) were indicator species 

in the high-TN and high-TP categories. Additionally, Lepomis

cyanellus (green sunfish), Lepomis macrochirus (bluegill), 

Pimephales promelas (fathead minnow), common carp, and 

Ameiurus natalis (yellow bullhead) were indicator species in 

the high-TP category and the Hybopsis dorsalis (bigmouth 

shiner), Moxostoma erythrurum (golden redhorse), Cyprinella

lutrensis (red shiner), and Moxostoma macrolepidotum (shorthead redhorse) were indicator species in the high-TN category. 

Like the longnose dace and common shiners in low-TP conditions, the bigmouth shiner (McCulloch, 2003) and red shiner 

(Messaad and others, 2000) prefer cooler water temperatures. 

Three indicator species in the high-TP category are species 

that can withstand low DO concentrations (green sunfish, 

fathead minnow, and common carp). The Catostomus commersonii (white sucker) and Pimephales notatus (bluntnose 

minnow) were commonly found within all nutrient categories 

for TN and TP within the CWPE.

Glaciated North Diatom Ecoregion 

Within the GNE, Salmo trutta (brown trout) and Cottus

sp. (freshwater sculpins) were indicator species in the low-TP 

and low-TN categories along with Cottus bairdii (mottled 

sculpin), longnose dace, and Lota lota (burbot) in the low-TP 

category and common shiner, Nocomis biguttatus (hornyhead 

chub), and mottled sculpin in the low-TN category (table 10). 

All of the low-nutrient indicator fish species are commonly 

found in cool or cold water. Etheostoma nigrum (johnny 

darter), Umbra limi (central mudminnow), Etheostoma duryi

(blackside darter), and common carp were indicator species 

in the high-TP and high-TN categories, along with creek chub 

and Culaea inconstans (brook stickleback) in the high-TP 

category and green sunfish in the high-TN category. Three 

species in the GNE—brook stickleback, central mudminnow 

(Klinger and others, 1982), and common carp—are able to 

withstand low-DO concentrations, similar to the species found 

within the high-TP conditions in the CWPE. The high number 

of indicator species for low TP within the CWPE and GNE 

capable of surviving in low-DO conditions suggests that the 

sites with low phosphorus concentrations may be influenced 

by increased algal growth and subsequent senescence, which 

leads to low-DO conditions. Within the GNE, white sucker 

was commonly found within all nutrient conditions for both 

TN and TP, along with blacknose dace and common shiner for 

TP and creek chub for TN. 
Description of Regional Differences and the Nutrient and Biological Conditions in the Nutrient Categories 33

Table 10. Most relative abundant fish species in the total nitrogen and total phosphorus nutrient categories in the Central and 

Western Plains and Glacial North diatom ecoregions.—Continued

[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Central and Western Plains diatom ecoregion – Total phosphorus

Low sites (<0.05 mg/L) Medium sites (>0.05 – <0.17 mg/L) High sites (>0.17 mg/L)

(n=13) (n=61) (n=30)

Species Mean Species Mean Species Mean

central stoneroller 21.25 spotfin shiner 8.76 spotfin shiner 15.00

bluntnose minnow 12.14 bluntnose minnow 8.03 bluntnose minnow 13.10

creek chub 11.83 sand shiner 5.92 sand shiner 10.80

common shiner 6.30 green sunfish 5.76 green sunfish 9.82

sand shiner 4.76 white sucker 5.36 bluegill 6.26

blacknose dace 4.04 central stoneroller 5.09 fathead minnow 5.63

hornyhead chub 3.10 creek chub 4.26 common carp 4.87

striped shiner 2.82 golden redhorse 3.90 white sucker 2.64

black redhorse 2.49 bluegill 2.78 yellow bullhead 2.35

northern hog sucker 2.33 northern hog sucker 2.68 creek chub 2.06

white sucker 2.26

golden redhorse 2.06

Total top 5 species 56.29 33.83 54.98

Glacial North diatom ecoregion – Total phosphorus

Low sites (<0.01 mg/L) Medium sites (>0.01 – <0.13 mg/L) High sites (>0.13 mg/L)

(n=8) (n=47) (n=20)

Species Mean Species Mean Species Mean

mottled sculpin 19.42 common shiner 11.23 johnny darter 27.66

longnose dace 12.93 white sucker 10.76 creek chub 11.58

brown trout 10.56 creek chub 6.65 central mudminnow 6.28

freshwater sculpins 10.72 hornyhead chub 5.48 white sucker 6.20

blacknose dace 7.94 bigmouth shiner 5.41 brook stickleback 5.76

creek chub 5.02 fathead minnow 5.29 common shiner 5.71

white sucker 2.89 bluntnose minnow 4.35 blacknose dace 3.78

burbot 2.28 rock bass 4.00 blackside darter 3.42

johnny darter 1.04 northern hog sucker 3.93 hornyhead chub 2.83

johnny darter 2.27 common carp 2.79

blacknose dace 2.17

bluegill 2.01

Total top 5 species 61.56 39.52 57.48
34 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

Table 10. Most relative abundant fish species in the total nitrogen and total phosphorus nutrient categories in the Central and 

Western Plains and Glacial North diatom ecoregions.—Continued

[mg/L, milligrams per liter; Mean, mean relative abundance for each taxa or species within each nutrient category]

Central and Western Plains diatom ecoregion – Total nitrogen

Low sites (<1.74 mg/L) Medium sites (>1.74 – <7.80 mg/L) High sites (>7.80 mg/L)

Species Mean Species Mean Species Mean

central stoneroller 24.36 bluntnose minnow 10.64 spotfin shiner 16.69

creek chub 15.21 spotfin shiner 8.33 sand shiner 12.02

bluntnose minnow 13.11 green sunfish 7.46 bluntnose minnow 8.01

common shiner 8.19 sand shiner 5.53 green sunfish 6.69

blacknose dace 5.25 bluegill 5.04 bigmouth shiner 5.99

hornyhead chub 4.03 central stoneroller 4.94 golden redhorse 4.80

sand shiner 3.23 common carp 4.11 red shiner 3.51

longnose dace 3.05 longear sunfish 3.99 common carp 2.21

fantail darter 2.59 creek chub 3.92 creek chub 1.89

white sucker 2.05 golden redhorse 2.86 northern hog sucker 1.75

bigmouth shiner 1.17 northern hog sucker 2.37 shorthead redhorse 1.51

johnny darter 1.12 striped shiner 2.23 hornyhead chub 1.47

northern hog sucker .91

Total top 5 species 45.79 31.31 37.50

Glacial North diatom ecoregion – Total nitrogen

Low sites (<0.60 mg/L) Medium sites (>0.60 – <2.08 mg/L) High sites (>2.08 mg/L)

Species Mean Species Mean Species Mean

common shiner 22.44 white sucker 10.11 johnny darter 15.37

hornyhead chub 17.19 johnny darter 8.56 creek chub 11.15

mottled sculpin 10.65 common shiner 6.89 white sucker 6.85

creek chub 10.19 creek chub 6.19 central mudminnow 6.35

longnose dace 7.03 fathead minnow 5.62 common shiner 4.79

white sucker 6.87 bigmouth shiner 5.55 hornyhead chub 3.90

brown trout 5.59 bluntnose minnow 4.80 blackside darter 3.51

freshwater sculpins 5.36 northern hog sucker 4.55 common carp 3.44

rock bass 4.11 green sunfish 2.01

blacknose dace 3.15 blacknose dace 1.77

largemouth bass 2.08

smallmouth bass 2.05

bluegill 2.04

green sunfish 1.78

Total top 5 species 40.34 29.05 25.40

High-nutrient taxa.

Low-nutrient taxa.

Taxa common at all nutrient levels.
Breakpoint Analysis Between Nutrients and Biological Attributes 35

Breakpoint Analysis Between 

Nutrients and Biological Attributes

In all biological communities, there are species that 

thrive in either low- or high-nutrient conditions. In this study, 

within each of the biological communities, certain attributes 

increased with increasing nutrient concentrations (positive 

relation), whereas others decreased with increasing nutrient 

concentrations (negative relation). There tended to be a wide 

range of breakpoints in most of the biological communities. In 

previous studies in Wisconsin (Robertson and others, 2008), 

Indiana (Caskey and others, 2010), and Kentucky (Crain and 

Caskey, 2010) the mean or median of breakpoints from several 

relations were used to determine the appropriate breakpoint for 

a specific State or ecoregion. In our study, instead of averaging the significant breakpoints for attributes of each biological 

community, the breakpoints for each biological community 

were associated with either low nutrient concentrations indicative of lower trophic levels (oligotrophic) or high nutrient 

concentrations indicative of higher trophic (eutrophic) levels. 

These breakpoints for specific attributes can be viewed as a 

gradient that can help environmental managers and researchers 

to assess the nutrient conditions within a stream.

Significant breakpoints were found for nutrients (TN 

and TP) and multiple attributes for algae, invertebrates, and 

fish communities within the CWPE and GNE ecoregions. In 

general, there were more significant breakpoints, with lower 

concentrations, for the GNE than for the more nutrient-rich 

CWPE (table 11). The significant algal attributes resulted in a 

wider range of breakpoints than the fish or invertebrate attributes; the invertebrate community did not have any low-TN 

or low-TP breakpoints in the CWPE. In general, TP correlated 

more strongly with all of the biological attributes than did TN. 

Algae

In the CWPE, the breakpoints for TN ranged from 1.67 

to 4.57 mg/L and for TP from 0.070 to 0.134 mg/L (table 11). 

The correlations with algal attributes were stronger for TP than 

for TN. The lowest breakpoint for TN was with the percent

diatoms that are beta mesosaprobic1 attribute and for TP was 

with the abundance of diatoms preferring meso-eutrophic

conditions attribute. The highest breakpoint for TN was the 

percent diatoms indicative of elevated concentrations of

organically bound nitrogen and breakpoints for TP were the 

richness of pollution sensitive diatoms (Bahls) and percent

beta-mesosaprobic diatoms attributes. Three of the four algal 

attributes had positive correlations with TN (increased as TN 

concentrations increased), whereas four of five algal attributes 

had negative correlations with TP (decreased as TP concentrations increased).

1

Italic type is used simply to aid readability of the rather lengthy attribute 

names. No special emphasis is intended. 

In the GNE, the breakpoints for TN ranged from 0.63 to 

1.32 mg/L and for TP from 0.020 to 0.100 mg/L (table 11). 

The correlations with algal attributes with TN and TP were 

equally strong. The lowest breakpoints for TN and TP were 

with percent abundance of Achnanthidium minutissimum and 

percent richness diatoms preferring eutrophic conditions. 

The highest breakpoints for TN and TP were with richness of

pollution sensitive diatoms (Bahls) and taxa richness. All of 

the algal attributes had negative correlations with TN and TP 

except for percent richness diatoms preferring eutrophic conditions, which increased when TN or TP increased, and taxa

richness, which decreased when TN increased but decreased 

when TP increased.

Invertebrates

In the CWPE, the breakpoints for TN ranged from 3.28 to 

8.45 mg/L and for TP from 0.092 to 0.711 mg/L (table 11). Of 

the three biological communities, the invertebrate breakpoints 

were about twice as high as the breakpoints for the algae and 

fish communities. The lowest breakpoint for TN was with 

ratio of Tanytarsini richness to midge richness and for TP with 

abundance of Orthocladinae midges. The highest breakpoint 

for TN is with percentage of total richness composed of bivalvia and for TP with number of taxa in the intolerant class. 

Significant breakpoints could only be found for invertebrates 

at higher nutrient concentrations that reflect the higher nutrient 

conditions in the streams of the CWPE; no breakpoints were 

associated with the low nutrient concentrations such as were 

found in the GNE.

In the GNE, the breakpoints for TN ranged from 0.52 to 

1.12 mg/L and for TP from 0.020 to 0.064 mg/L (table 11). 

The lowest breakpoint for TN was with richness composed of

predators and for TP was with richness composed of midges; 

however, concentrations for six of the significant breakpoints 

for TP were relatively low (between 0.020 and 0.025 mg/L). 

The highest breakpoints for TN were for richness composed

of filtering-collectors and richness composed of Tanytarsini

midges; for TP, the highest breakpoint was with richness composed of Tanytarsini midges. All of the invertebrate attributes 

had negative correlations with TN and TP except for percentage of taxa richness in the moderately tolerant class, which 

increased when TN or TP increased. 
Table 11. Breakpoints between nutrients and the most significant biological metrics in the Central and Western Plains and Glacial North diatom eco[mg/L, milligrams per liter; %, percent; NS, not significant; Neg, negative; Pos, positive; DO, dissolved oxygen; Bold indicates a p value <0.05] Attribute (Note: All algae attributes are for diatoms) Number of samples Total nitrogen Total phosp

Breakpoint concentration (mg/L) p-value Lower 5% confidence interval (mg/L) Upper 5% confidence interval (mg/L) Correlation type Breakpoint concentration (mg/L) p-value Lower 5

confiden

interva

(mg/L) 

Central and Western Plains diatom ecoregion Algae Percent pollution sensitive diatoms (Bahls) 104 NS 0.216 NS NS NS 0.133 0.007 0.064 

Percent diatoms indicative of el- evated concentrations of organically bound nitrogen1 104 4.57 .008 3.26 10.13 Pos .075 .005 .052 Percent diatoms requiring >30% DO saturation 104 3.28 .021 1.67 5.49 Pos .082 .003 .052 Percent beta-mesosaprobic diatoms2 104 1.67 .041 1.46 4.87 Pos .133 .073 .052 Abundance of diatoms preferring meso-eutrophic conditions3 104 4.35 .086 4.07 4.73 Neg .070 .016 .053 

Invertebrates Percentage of total richness composed of bivalvia 104 8.45 0.032 3.45 8.80 Neg 0.189 0.004 0.153 

Ratio of Tanytarsanii richness to midge richness 104 3.28 .021 1.67 10.13 Neg .141 .001 .075 Number of taxa in the intolerant class 104 NS .170 NS NS NS .711 .001 .090 Abundance of Orthocladinae midges 104 4.90 .035 4.68 5.91 Neg .092 .001 .044 

Fish Number of fish 105 4.73 0.070 1.24 6.95 Neg 0.070 0.001 0.040 

Percent abundance of stonerollers 105 1.79 .001 1.24 4.87 Neg .070 .001 .039 Number of darters 105 3.51 .007 1.32 4.55 Neg NS .210 NS 
Table 11. Breakpoints between nutrients and the most significant biological metrics in the Central and Western Plains and Glacial North diatom eco[mg/L, milligrams per liter; %, percent; NS, not significant; Neg, negative; Pos, positive; DO, dissolved oxygen; Bold indicates a p value <0.05] 

Attribute 

(Note: All algae 

attributes are for 

diatoms)

Number 

of 

samples

Total nitrogen Total phosp

Breakpoint 

concentration 

(mg/L)

p-value

Lower 5% 

confidence 

interval 

(mg/L)

Upper 5% 

confidence 

interval 

(mg/L)

Correlation 

type

Breakpoint 

concentration 

(mg/L)

p-value

Lower 5

confiden

interva

(mg/L)

Glaciated North diatom ecoregion 

Algae

Percent abundance of 

Achnanthidium minutissimum

71 0.63 0.002 0.53 0.98 Neg 0.020 0.001 0.015 

Richness pollution sensitive 

diatoms (Bahls)

71 1.32 .001 .56 1.54 Neg .090 .001 .030 

Taxa richness 71 1.32 .001 .53 1.58 Neg .099 .001 .082 Percent diatom taxa generally 

intolerant to organically bound nitrogen4

71 1.20 .008 .43 1.48 Neg .030 .001 .014 

Percent diatoms requiring nearly 

100% DO saturation

71 1.11 .001 .63 1.32 Neg .030 .001 .014 

Percent richness that are 

oligosaprobic5

71 .98 .001 .63 1.32 Neg .030 .001 .020 

Percent richness diatoms preferring 

eutrophic conditions

71 .63 .001 .53 1.36 Pos .025 .003 .018 

Invertebrates

Richness composed of midges 72 0.66 0.002 0.53 1.84 Neg 0.020 0.003 0.017 Number of clinger taxa 72 .63 .001 .56 1.04 Neg .025 .001 .024 Richness composed of filteringcollecters 72 1.12 .003 .52 1.28 Neg .025 .001 .018 Percentage of abundance in the 

intolerant class

72 .56 .002 .56 1.20 Neg .044 .001 .017 

Number of taxa in the intolerant 

class

72 .63 .001 .56 1.00 Neg .024 .001 .020 

Percentage of taxa richness in the 

moderately tolerant class

72 .56 .001 .56 .98 Pos .024 .001 .020 

Richness composed of odonates 72 .98 .001 .52 1.58 Neg .046 .001 .017 Richness composed of stoneflies 72 .63 .001 .53 .93 Neg .024 .001 .014 Richness composed of predators 72 .53 .001 .50 1.58 Neg .046 .001 .018 
Table 11. Breakpoints between nutrients and the most significant biological metrics in the Central and Western Plains and Glacial North diatom eco[mg/L, milligrams per liter; %, percent; NS, not significant; Neg, negative; Pos, positive; DO, dissolved oxygen; Bold indicates a p value <0.05] Attribute (Note: All algae attributes are for diatoms) Number of samples Total nitrogen Total phosp

Breakpoint concentration (mg/L) p-value Lower 5% confidence interval (mg/L) Upper 5% confidence interval (mg/L) Correlation type Breakpoint concentration (mg/L) p-value Lower 5

confiden

interva

(mg/L) 

Glaciated North diatom ecoregion—continued Fish Richness composed of Tanytarsanii midges 72 1.12 0.002 0.63 1.68 Neg 0.064 0.001 0.020 

Percent abundance of darters 64 2.46 .013 1.28 2.46 Pos .137 .001 .098 Percent abundance of piscivorous fish 64 NS .169 NS NS NS .070 .081 .020 Percent abundance of sensitive fish 64 .63 .004 .53 1.10 Neg .046 .001 .020 Percent abundance of tolerant fish 64 NS .510 NS NS NS .060 .002 .035 

1 Obligate heterotrophs. 2 Saprobic class: II - (02 saturation: 70–80%; BOD5: 2–4 mg/L). 3 Beta-mesosaprobic: somewhat degraded conditions, mesotrophic-eutrophic. 4 May be “oligotrophic” or “mesotrophic species (autotroph). 5 Saprobic classes: I, I–II - (O2 saturation: >85%; BOD5: <2 mg/L).
Management Implications 39

Fish

In the CWPE, the breakpoints for TN ranged from 

1.79 to 4.73 mg/L; for TP, both significant breakpoints were 

0.070 mg/L (table 11). The lowest breakpoint for TN was 

with percent abundance of stonerollers; for TP, the lowest 

breakpoints were with percent abundance of stonerollers

and number of fish. The highest breakpoint for TN was with 

number of fish. Fewer significant attributes were found for fish 

than for algae or invertebrates. All of the fish attributes had 

negative correlations with TN and TP. The number of fish attribute indicated that the numbers of fish decrease as concentrations of TN and TP increase; however, the breakpoint for TN 

was relatively high (4.73 mg/L), whereas the breakpoint for 

TP was moderate (0.070 mg/L) compared to the breakpoints 

found for fish in the GNE.

In the GNE, the two significant breakpoints for TN were 

0.63 and 2.46 mg/L, and the four significant breakpoints for 

TP ranged from 0.046 to 0.137 mg/L (table 11). The percent

abundance of sensitive fish attribute had the lowest breakpoints for TN and TP. The highest breakpoints for both TN and 

TP were with percent abundance of darters. 

The darter attributes were significant in both the CWPE 

and GNE ecoregions. Because darters were significant in both 

ecoregions, and if the entire region is considered together, the 

darter attribute appears to show a threshold from positive to 

negative (bell-shaped) response. In the lower nutrient conditions of the GNE, darter abundance increases with increasing 

TN, with a breakpoint of 2.46 mg/L. In the higher nutrient 

conditions of the CWPE, darter abundance decreases with 

increasing nutrients, with a breakpoint of 3.51 mg/L. 

Management Implications 

Although the geographic area of our study generally 

encompasses regional divisions used by the USEPA (U.S. 

Environmental Protection Agency, 2010) and the USGS 

(U.S. Geological Survey, 2010), there are distinct differences 

between the northern and southern parts. The southern States 

in the study area (Illinois, Indiana, Iowa, and Ohio) are dominated by row-crop agriculture, primarily corn and soybeans, 

which requires the additions of large amounts of fertilizer 

and manure to maximize crop yields. These cornbelt States 

have the greatest inputs of N and P in the country (Mueller 

and Spahr, 2006). Consequently, some of the highest nutrient 

concentrations are found in streams in these southern States 

(Mueller and Spahr, 2006; Lorenz and others, 2009), and these 

streams ultimately are the source of greatest nutrient loadings 

to the Gulf of Mexico (Goolsby and others, 2001; Alexander 

and others, 2008). In the northern States in the study area 

(Minnesota, Wisconsin, and Michigan), agriculture also is 

extensive; however, these States tend to have more forested 

areas mixed in with the row crops and less corn production overall. The higher concentrations found in our study in 

streams in the CWPE compared to the GNE (figs. 4 and 5) 

support findings from the studies listed above. The biological communities also were significantly different between the 

northern and southern parts of the study area. Some of the differences in the biological communities between the northern 

and southern states is due to stream temperature: streams are 

typically warmwater in the south and coolwater or coldwater 

in the north. In this study, the diatom ecoregions developed 

by Potapova and Charles (2007) were used to account for the 

regional differences. This distinction seemed to work well 

for the invertebrate and fish communities; however, some 

coldwater fish species were abundant in the northern parts of 

the southern States. For these reasons, it is important when 

conducting stressor-response studies that regional differences 

in water chemistry, habitat, and biological communities be 

considered.

Previous studies have set boundaries of trophic levels in 

streams. Dodds and others (1998) used the frequency distributions of TP, TN, and CHLa to determine trophic boundaries, but the boundaries were not linked to responses by the 

biological communities to nutrient concentrations. Dodds and 

others (1998) set oligotrophic-mesotrophic boundaries for TP 

at 0.025 mg/L and for TN at 0.7 mg/L and mesotrophic-eutrophic boundary for TP at 0.075 mg/L and for TN at 1.5 mg/L. 

In New York, Smith and others (2007) used invertebratecommunity response to nutrient concentrations to establish 

trophic boundaries and found comparable boundaries for TP 

(0.0175 mg/L for oligotrophic-mesotrophic and 0.065 mg/L 

for mesotrophic-eutrophic boundaries) and slightly lower 

boundaries for nitrogen (0.24 mg/L of NO3 for oligotrophicmesotrophic and 0.98 mg/L of NO3 for mesotrophic-eutrophic 

boundaries). The boundaries in these two studies cannot be 

directly related because of the differences in forms of nitrogen, TN and NO3

; however, an estimate of TN can be made 

by using the ratio of NO3 to TN as a conversion factor. In this 

study, the median NO3 toTN ratio value was about 0.70, and 

if the NO3 values from Smith and others (2007) were thus 

converted to TN, the values would become 0.34 mg/L and 

1.4 mg/L, respectively. The converted values are close to the 

Dodds and others (1998) mesotrophic-eutrophic boundary 

and about half of the value for the oligotrophic-mesotrophic 

boundary. Our dataset allowed for comparisons of differences 

in the breakpoints between low and high nutrient concentrations within and across diatom ecoregions. The breakpoints 

show the changes in the biological-community attributes 

along the nutrient gradient and therefore reflect the changes in 

trophic status better than simple trophic levels based solely on 

nutrient concentrations. 

In general, the lower nutrient concentrations of the 

northern region, the GNE, compared to the southern region, 

the CWPE, allowed for a nutrient gradient in which species 

indicative of oligotrophic conditions could be distinguished. In 

contrast, the CWPE is nutrient saturated, and examination of 

the biological communities does not reveal indicator species 

or attributes of low nutrient concentrations. The low and high 

breakpoints from all biological communities were generally 
40 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

about 3–5 times higher in the south than the north (table 11). 

In the north (GNE), similar low breakpoints were found for 

TN with all biological communities (around 0.63 mg/L) and 

for TP (between 0.02 and 0.03 mg/L) with the algae and 

invertebrate communities. In the lower nutrient conditions in 

the streams of the GNE, the breakpoints between the fish-community attributes and TP concentrations were higher compared 

to those for the algae or invertebrate communities. However, 

Miltner and Rankin (1998) found fish IBI scores in wadeable streams of Ohio significantly decreased at concentrations 

greater than 0.61 mg/L of TN, and Wang and others (2007) 

found a similar breakpoint for TN of around 0.60 mg/L. Crain 

and Caskey (2010) found multiple significant breakpoints 

between TP and invertebrate attributes between 0.032 and 

0.035 mg/L in Kentucky.

Besides their relation to significant breakpoints with low 

nutrient concentrations in the north, the significant biological 

attributes were reflective of nutrient or ecological conditions 

(specifically, increasing percentage of eutrophic diatoms in 

algae, decreasing number of taxa in the intolerant class for 

invertebrates, and decreasing number of sensitive species 

in fish). There were also more attributes with significantly 

detectable breakpoints in the north, where the nutrient gradient 

was lower than that in the south. This finding suggests that as 

nutrients increase, the biological communities become more 

uniform, with fewer sensitive taxa, so significant nonlinear 

breakpoints cannot be determined. Additionally, the breakpoints tend to be higher and are more reflective of breakpoints 

between mesotrophic and eutrophic conditions. 

Several studies have found that among algae, invertebrates, and fish, algal communities have the strongest relations 

to nutrient condition (U.S. Environmental Protection Agency, 

2003; Justus and others, 2009). Yet, few States have adopted 

the collection of algal communities as part of their strategy 

to determine impaired streams. Most States use invertebrateand fish-community assessments as part of their regulatory 

designs; although these invertebrate- and fish-based assessments of biotic integrity are able to effectively determine 

whether a stream is impaired, they lack the sensitivity to determine whether nutrients cause the impairment (Smith and others, 2007). Our study supports the finding that algal-community assessments provide important information to link stream 

impairment to nutrients on the basis of multiple attributes 

with significant relations with and breakpoints for TN and TP. 

Invertebrate- and fish-community attributes do not necessarily 

have a direct link to nutrients in the same way algae do, but 

they appear to have the ability to distinguish breakpoints for 

both low and high concentrations, especially in low-nutrient 

regions. From our study, it appears that the invertebrate community would be less useful than the algal or fish communities 

in assessing low-level breakpoints in nutrient-rich areas. However, the consistently negative correlations at low breakpoint 

concentrations and the diverse nature of many of the TP attributes support the finding that for low-nutrient conditions, the 

invertebrate community is able to detect changes in nutrient 

levels. In the low-nutrient conditions of the GNE, the similar 

breakpoints from several algal and invertebrate attributes 

provide multiple lines of evidence and strengthen the case for 

using these breakpoints for nutrient criteria development in 

this region. In nutrient-rich areas like the CWPE, all of the 

breakpoints were substantially higher than for the lower nutrient conditions of the GNE and suggest that stream conditions 

are nutrient saturated to the point that low-end breakpoints 

cannot be detected. Invertebrates in particular seem to be less 

able to distinguish low-end breakpoints in high-nutrient conditions. Miltner and Rankin (1998) found significant relations 

between nutrients and invertebrates at high nutrient concentrations, around the 75th percentile for total inorganic nitrogen 

(TIN) (3.63 mg/L) and TP (0.32 mg/L) in Ohio. Our study 

found the lowest breakpoint for invertebrates in the nutrientrich CWPE to be 3.28 mg/L for TN (table 11). Results from 

our study also suggest that the median annual nutrient concentrations need to be around the initial low-nutrient category 

classification of 0.01mg/L for TP and 0.60 mg/L for TN to be 

able to distinguish the low-end breakpoints (figs 4 and 5). This 

finding was further supported by the large number of significant algae, invertebrate, and fish breakpoints near the 0.60-

mg/L concentration for TN (table 10) in the GNE. 

Among the frequently observed responses to increased 

nutrient concentrations in streams are changes in abundance, 

density, or richness within biological communities; however, 

the relations have been inconsistent. Numbers of diatom taxa 

have been found to increase as TP concentrations increase 

(Stevenson and others, 2008). Increased nutrient concentrations have been associated with increases in total invertebrate 

taxon density and richness (Clenaghan and others, 1998; 

Heino and others, 2003) but also with decreases in richness 

(Miltner and Rankin, 1998; Wang and others, 2007). Indices 

of Biological Integrity (IBI) for fish communities have been 

developed regionally and typically have attributes for number 

of fish and number of fish taxa. In most streams, but especially 

in warmwater or coolwater streams, the more fish and fish 

taxa, the better the water quality—but these increases are not 

linked directly to increases in nutrient concentrations. However, increased nutrient concentrations have been specifically 

associated with increases (Lenat and Crawford, 1994) and 

decreases (Gammon and others, 1999) in fish abundance and 

fish biomass.

Although previous local or regional studies have found 

inconsistent responses in the biological community to 

increases in nutrient concentrations, results from some studies 

indicate a possible threshold response if there is a sufficient 

nutrient gradient. In streams in the low-nutrient Mid-Atlantic 

Highlands, for example, the number of diatom taxa increased 

as agricultural land use and nutrients increased (Stevenson 

and others, 2008). In the forested regions of Ireland (Clenaghan and others, 1998) and Finland (Heino and others, 2003), 

the invertebrate density or richness increased as nutrients 

increased. In streams in the Piedmont region of North Carolina with low-nutrient concentrations, comparison of forested 

to agricultural basins showed that fish abundance and number of fish taxa increased as nutrients increased (Lenat and 
Management Implications 41

Crawford, 1994). When nutrients are added to oligotrophic 

water bodies, the nutrients provide the fuel for increased 

production in all biological communities. However, as the 

nutrient concentrations increase in streams, the response 

of abundance or richness eventually becomes negative. In 

contrast to results from studies in low-nutrient areas, studies in 

Ohio and Wisconsin (where nutrient concentrations generally 

are higher) showed decreased invertebrate and fish abundance 

with increasing nutrients (Miltner and Rankin, 1998; Wang 

and others, 2007). In our study, algal taxa richness decreased 

with increasing nutrients in the lower nutrient GNE, and fish 

abundance decreased in the CWPE. The algal taxa richness 

breakpoints (1.32 mg/L for TN and 0.100 mg/L for TP) and 

fish abundance breakpoints (4.73 mg/L for TN and 0.070 mg/L 

for TP) suggest that these are the concentrations where algae 

and fish abundance or richness begin to decrease. Interestingly, 

the 1.32-mg/L breakpoint for TN and 0.07-mg/L breakpoint 

for TP are similar to the mesotrophic-eutrophic boundaries of 

Dodds and others (1998) and Smith and others ( 2007). Given 

the findings above, the “intermediate disturbance hypothesis” 

seems to apply to nutrients as well as hydrological and habitat 

characteristics such as flooding, substrate size, and refugia 

(Death and Winterbourn, 1995; Townsend and others, 1997; 

Lake, 2000). Miltner and Rankin (1998) found the greatest abundance and diversity for fish and invertebrates at the 

intermediate nutrient concentrations. This finding suggests that 

determining slope of abundance or richness attributes could be 

useful in determining the trophic level of streams. If the slope 

is positive and the nutrient gradient is at the low end, then 

oligotrophic-mesotrophic conditions are suggested; but if the 

slope is negative and the nutrient conditions are on the high 

end, then nutrient saturation and mesotrophic-eutrophic conditions are suggested. An abundance or total-richness attribute 

alone may not be sufficient to determine the nutrient condition or trophic level of the stream, but it could be a useful tool 

when multiple lines of evidence are used.

Although invertebrate and fish communities do not have 

direct attributes specifically linked to nutrients as algae do, 

excessive nutrient concentrations have been linked to shifts in 

species composition from functional assemblages of intolerant species, benthic insectivores and top carnivores typical 

of high-quality warmwater streams towards less desirable 

assemblages of tolerant species, niche generalists, omnivores, 

and detritivores typical of degraded warmwater streams (Ohio 

Environmental Protection Agency, 1999). Miltner and Rankin 

(1998) found decreases in the number of pollution sensitive

species and percent as top carnivore fish in coldwater streams 

as nutrient concentrations increased. In our study in the 

GNE, the most significant attributes in the algal, invertebrate, 

and fish community attributes consisted of fewer intolerant, insectivore, and carnivore species as nutrients increased. 

However, the biological community composition in the CWPE 

did not demonstrate these same changes in functional groups 

because these streams were nutrient saturated. In the GNE, 

attributes were associated with oligotrophic boundaries for TN 

(0.6 mg/L) and TP (0.025 mg/L) for algae (percent abundance

of Achnanthidium minutissimum and the percent richness diatoms preferring eutrophic conditions), invertebrates (number

of taxa in the intolerant class), and fish (percent abundance

of sensitive fish). This finding suggests that for low-nutrient 

conditions, multiple lines of evidence from all communities can effectively result in breakpoints with low nutrient 

concentrations.

Some biological attributes work well for assessing low or 

high breakpoints for both TN and TP. Other attributes appear 

to be associated with either TN or TP. Two GNE fish attributes 

(percent abundance of tolerant fish and percent abundance of

piscivorous fish), one CWPE invertebrate attribute (number

of taxa in the intolerant class) and one CWPE algae attribute 

(percent pollution sensitive diatoms (Bahls)) were significantly 

related only to TP; one CWPE fish attribute (number of darters) was significantly related only to TN. Darters appear to 

be a good species or attribute to show eutrophication within 

wadeable stream ecosystems, especially for TN. In the lower 

nutrient conditions of the GNE, darters increased with increasing nutrients, with a TN breakpoint of 2.46 mg/L. In the higher 

nutrient conditions of the CWPE, darters decreased with 

increasing nutrients, with a TN breakpoint of 3.28 mg/L.

The use of the 10th and 75th percentiles to classify 

sites into low, medium, and high TN and TP sites effectively 

showed different species composition within the categories. 

Because the breakpoints clustered around similar lower and 

higher concentrations from each biological community within 

each diatom ecoregion it suggests another possible way to 

categorize sites along trophic levels based on biological 

categories. Specifically, the mean concentration of the lower 

and higher breakpoint values can be used as new biologicallybased thresholds to determine nutrient categories. For TN, the 

new low and high breakpoints were 1.7 and 3.3 mg/L in the 

CWPE and 0.6 and 1.1 mg/L in the GNE. For TP, the new low 

and high breakpoints were 0.07 and 0.13 mg/L in the CWPE 

and 0.03 and 0.09 mg/L in the GNE. On the basis of these biological breakpoints, the number of low sites did not change for 

TN in the CWPE or the GNE, but many of the medium sites 

were recategorized as high sites in both the CWPE and GNE 

(table 12). For TP, the new biological breakpoints increased 

the number of low and high sites.

The initial nutrient categories were based solely on 

instream nutrient concentrations; but because no measure of 

nutrient uptake, such as algal biomass, could be used with this 

dataset, a critical intermediate variable in the nutrient-biological community interactions was missing. For this reason, the 

responses of the biological communities—which integrate 

all of the effects of nutrients and subsequent algal growth— 

probably yield a better assessment of the true nutrient condition of the streams than does a strictly concentration-based 

categorization. 
42 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

By being able to contrast the biotic responses to a nutrient gradient in a region that still has streams with low nutrient 

concentrations (GNE) to one that is more nutrient saturated 

(CWPE), our study showed that biological-community 

attributes respond as the stream trophic state changes. Some 

attributes respond at lower nutrient concentrations (oligotrophic), whereas others respond only as the stream becomes 

eutrophic. Some of these attributes have a positive response 

(increase with increases in nutrients) and others a negative 

response (decrease with increases in nutrients). Identifying the 

significant breakpoints and incorporating the type of biological response helps environmental managers to determine the 

statistically and ecologically significant attributes that can be 

used to set water-quality criteria in a specific region.

The findings from our study suggest that the breakpoints 

clustered around the lower and higher concentration from the 

GNE could be used as oligotrophic and eutrophic boundaries 

derived from biological response and not just nutrient concentrations. These oligotrophic and eutrophic boundaries are 

based on (1) a gradient with sufficiently low to high nutrient 

concentrations, (2) distinctive differences in the biological 

communities from the low-nutrient to high nutrient streams, 

(3) similarity of breakpoints within algal, invertebrate, and fish 

communities, (4) the significant attributes with either direct 

relations to nutrients or traditional changes in community 

structure (that is, decreases in sensitive species or increases in 

tolerant species), and (5) similar breakpoints in other studies 

in this and other regions. 

Study Limitations

One of the strengths of the NAWQA Program is that 

multiple lines of evidence (water chemistry, habitat, and biological community assessments) are used to assess occurrence, 

distribution, and trends of specific water quality parameters. 

The costs associated with this intensive sampling, limit the 

number of sites that can be sampled; within the MRB3 region 

a total of 54 sites had sufficient nutrient and biological community data to be assessed. The relatively small number of 

sites with sufficient nutrient and biological-community data in 

our study somewhat limits the confidence in the classification 

of the nutrient categories and the breakpoint analysis. However, the similar breakpoint values found in other studies suggests the sample size in this study may have been sufficient. 

Although algal-community data were available for our study, 

algal-biomass data—which could have helped determine the 

true nutrient condition within streams—were not. The algal 

attributes with nutrient or trophic ties in particular were helpful in understanding low- and high-nutrient conditions. The 

fact that the central stoneroller—an algivore—was so dominant in several of the low-nutrient CWPE streams suggests 

that the low stream nutrients may have been from algal uptake 

rather than oligotrophic conditions. Although these streams 

were considered less enriched on the basis of measured stream 

nutrient concentrations, they may have been considered 

more enriched if algal biomass information had been available. Future studies using data from states within this Major 

River Basin region that include (1) nutrients, algal biomass, 

and all biological communities (algal, invertebrate, and fish) 

and (2) that encompass a sufficiently low nutrient gradient as 

found in the GNE would be helpful to corroborate the findings 

presented in this report. 

[mg/L, milligrams per liter; <, less than; >, greater than]

Nutrient

Nutrient category

Low 

sites

Medium 

sites

High 

sites

Central and Western Plains diatom ecoregion (CWPE)

Total nitrogen (mg/L) < 1.74 1.74 – 7.80 > 7.80

Total phosphorus (mg/L) < .05 .05 – .17 > .17

Glaciated North diatom ecoregion (GNE)

Total nitrogen (mg/L) < 0.60 0.60 – 2.08 > 2.08

Total phosphorus (mg/L) < .01 .01 – .13 > .13

Table 12. Comparison of the number of sites in low, medium, 

and high nutrient categories based on nutrient and biological 

classifications of stream conditions. 

[TN, total nitrogen; TP; total phosphorus; bold, number of sites within a 

category changed from nutrient (percentiles) to biological (breakpoints) basis 

for categories]

Nutrient 

category

Nutrient Biological

TN1 TP2 TN3 TP4

Central and Western Plains diatom ecoregion (CWPE) 

(n=31 sites)

Low 4 4 4 7

Medium 18 20 3 11

High 9 7 24 13

Glacial North diatom ecoregion (GNE) 

(n=23 sites)

Low 3 3 3 6

Medium 15 13 4 9

High 5 7 16 8

1

 Low TN nutrient boundary is 1.74 mg/L in the CWPE and 0.60 mg/L 

in the GNE. High TN nutrient boundary is 7.80 mg/L in the CWPE and 

2.08 mg/L in the GNE.

2

 Low TP nutrient boundary is 0.05 mg/L in the CWPE and 0.01 mg/L 

in the GNE. High TP nutrient boundary is 0.17 mg/L in the CWPE and 

0.13 mg/L in the GNE.

3

 Low TN biological boundary is 1.70 mg/L in the CWPE and 0.60 mg/L 

in the GNE. High TN biological boundary is 3.30 mg/L in the CWPE and 

1.10 mg/L in the GNE.

4

 Low TP biological boundary is 0.07 mg/L in the CWPE and 0.03 mg/L 

in the GNE. High TP biological boundary is 0.13 mg/L in the CWPE and 

0.09 mg/L in the GNE.
Summary and Conclusions 43

Summary and Conclusions 

Algal, invertebrate, and fish taxa and attributes that best 

reflect the effects of nutrients in streams along a gradient of 

low to high concentrations were determined for this primarily midwestern study. Nutrient data from 64 U.S. Geological Survey sampling sites from the National Water-Quality 

Assessment (NAWQA) Program—most of which were within 

the U.S. Environmental Protection Agency (USEPA) Nutrient 

Ecoregions VI, VII, and VIII—were used to categorize the 

sites into low-, medium-, and high-nutrient conditions for total 

nitrogen (TN) and total phosphorus (TP). Nutrient categories 

were based on approximately the 10th and 75th percentiles of 

TN and TP concentrations for sites within each of two diatom 

ecoregions used for subsequent analysis: the Glacial North 

diatom ecoregion (GNE) and the Central and Western Plains 

diatom ecoregion (CWPE). “Low-nutrient sites” were those 

with concentrations at or below the 10th percentile for TN and 

TP, and “high-nutrient sites” were those at or above the 75th 

percentile; “medium-nutrient sites” were in between these two 

concentrations. Annual median TN and TP concentrations by 

nutrient category and diatom ecoregion were as follows: 

A total of 54 of these sites with at least 6 nutrient samples 

and with algal-, invertebrate-, and fish-community data collected within a year were included for further study. Nonparametric statistical analyses were used to determine whether 

there were regional differences between the algal, invertebrate, 

and fish communities. The biological communities were 

significantly different between northern sites (primarily in 

the GNE) and the southern sites (primarily in the CWPE). In 

general, TN and TP concentrations were 3–5 times greater in 

the nutrient-rich CWPE than in the GNE. 

In the nutrient-rich CWPE, algae were the best indicators of differences between all of the nutrient categories for 

both TN and TP; however, in the less nutrient-rich GNE, 

invertebrates were more indicative of differences between the 

nutrient categories. Seasonally, the greatest nutrient concentrations in the low-, medium-, and high-nutrient categories were 

found in the spring, after the application of fertilizer to fields; 

concentrations decreased steadily from June through October 

because of (1) nutrient uptake from crops and terrestrial plants, 

(2) nutrient uptake by algae and macrophytes in streams, and 

(3) decreased nutrient runoff to streams from drier soils, which 

are better able to absorb precipitation. The difference between 

sites in the nutrient categories was the magnitude and amount 

of time the elevated nutrient concentrations were found in the 

stream. An analysis of the nutrient variables annual average, annual maximum, and value closest to the timing of the 

biological sample found that annual average concentration had 

the highest correlation to the biotic-community assemblage in 

the two diatom ecoregions. 

Certain species of algae, invertebrates, and fish were 

more prevalent in low- and high-nutrient conditions within 

each of the diatom ecoregions. In high-nutrient conditions of 

the CWPE, 12 species in 3 genera dominated the algal community. For high-TP conditions, the most abundant species 

were five Navicula species (recens, minima, subminuscula,

erifuga, and trivalis), three Nitzschia species (amphibia, palea, 

and palea debilis), and one Cyclotella species (meneghiniana). 

For high-TN conditions, the most abundant were four Navicula species (cryptotonella, subminiscula, palea, and symmetrica). Additionally, Pseudostaurosira brevistriata preferred 

low-TN and low-TP conditions. Six species from the family 

Fragilariaceae were found exclusively in low-TN conditions. 

In the GNE, for low-TN and low-TP conditions, Achnanthidium minutissimum and Achnanthidium deflexum dominated the 

algal community with about 25 and 30 percent of the relative 

abundance, respectively. For invertebrates, three taxa indicated low-nutrient conditions for both TN and TP in CWPE: 

Trichoptera Hydropsychidae Ceratopsyche sp., Ephemeroptera Baetidae Baetis flavistriga, and Coleoptera Psephenidae 

Psephenus herricki. Four taxa were indicative of high-nutrient 

(TN and TP) conditions in the CWPE: two Ephemeroptera 

families (Heptageniidae and Leptohyphidae Tricorythodes

sp.), Diptera Chironomidae Stenochironomus sp., and Trichoptera Hydropsychidae Hydropsyche sp. High-TP conditions 

were also favored by Trichoptera Hydropsychidae Hydropsyche bidens, whereas high-TN conditions were indicated by 

higher relative abundances of six other taxa: two Diptera genera (Chironomidae Tanytarsus sp. and Empididae Hemerodromia sp.), two Coleoptera Elmidea species (Stenelmis grossa

and Macronychus glabratus), a Trichoptera Hydroptilidae 

Hydroptila sp., and a Tubificida Naididae. For fish communities, Campostoma anomalum (central stoneroller), Semotilus atromaculatus (creek chub), and Rhinichthys atratulus

(blacknose dace) were indicative of both low-TN and low-TP 

conditions in the CWPE; additionally, Luxilus chrysocephalus

(striped shiner) and Moxostoma duquesnii (black redhorse) 

were indicative of low-TP conditions, and Luxilus cornutus

(common shiner) and Rhinichthys cataractae (longnose dace) 

were indicative of low-TN conditions. Within the GNE, Salmo

trutta (brown trout) and Cottus sp. (freshwater sculpins) were 

indicative of both low-TP and low-TN conditions; Cottus bairdii (mottled sculpin), longnose dace, and Lota lota (burbot) 

were indicative of low-TP conditions, and common shiner, 

Nocomis biguttatus (hornyhead chub), and mottled sculpin 

were indicative of low TN.

There were significant breakpoints for nutrients (TN 

and TP) and multiple attributes for algae, invertebrates, and 

fish communities within the CWPE and GNE ecoregions. In 

general, there were more significant breakpoints, with lower 

concentrations, in the GNE than the more nutrient-rich CWPE. 
44 Assessment of Nutrient Enrichment in Wadeable Streams in Ecoregions surrounding the Great Lakes

The low and high breakpoints from all biological communities were generally about 3–5 times higher in the south 

than the north. In the north (GNE), similar low breakpoints 

were found for TN with all biological communities (around 

0.63 mg/L) and for TP with the algae and invertebrate communities (between 0.02 and 0.03 mg/L). The significant algal 

attributes provided a better range of breakpoints than did the 

attributes for fish or invertebrates; in the CWPE, for example, 

the invertebrate community did not have any low-TN or lowTP breakpoints. In general, TP had stronger correlations with 

all of the biological attributes than did TN. In the low-nutrient 

GNE, the significant attributes in the algae, invertebrate, and 

fish communities supported the changes in functional groups 

with increasing nutrient concentration as evidenced by fewer 

intolerant species, insectivores, and carnivores. In the nutrientrich CWPE, however, similar changes in functional groups 

were not evident, owing to the nutrient-saturated conditions of 

these streams. In the low-nutrient GNE, attributes associated 

with oligotrophic boundaries for TN (0.6 mg/L) and TP (0.025 

mg/L) included two attributes for algae (percent abundance

of Achnanthidium minutissimum and percent richness diatoms preferring eutrophic conditions), one for invertebrates 

(number of taxa in the intolerant class), and one for fish 

(percent abundance of sensitive fish). This result suggests that 

in low-nutrient conditions, multiple lines of evidence from all 

biological communities can effectively be used to find lownutrient breakpoints. 

The findings from our study suggest that the low and high 

breakpoints for TN and TP from the GNE could be used as 

oligotrophic and eutrophic boundaries derived from biological response based on this diatom ecoregion’s having (1) a 

gradient with sufficiently low to high nutrient concentrations, 

(2) distinctive differences in the biological communities in the 

low-nutrient to high-nutrient streams, (3) similarity of breakpoints within algal, invertebrate, and fish communities, (4) the 

significant attributes with either direct relations to nutrients or 

traditional changes in community structure (that is, decreases 

in sensitive species or increases in tolerant species), and 

(5) similar breakpoints in other studies in the GNE and other 

regions. In nutrient-rich areas like the CWPE, all of the breakpoints were substantially higher than those for the lower-nutrient conditions of the GNE; this finding suggests that streams 

in the CWPE are nutrient saturated to a level at which low-end 

breakpoints cannot be detected.

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