U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2007–5231

Prepared in cooperation with the Indiana Department of Environmental Management, 

Division of Water, Assessment Branch

Relations of Principal Components Analysis Site Scores to 

Algal-Biomass, Habitat, Basin-Characteristics, Nutrient, and 

Biological-Community Data in the Upper Wabash River 

Basin, Indiana, 2003

Relations of Principal Components 

Analysis Site Scores to Algal-Biomass, 

Habitat, Basin-Characteristics, Nutrient, 

and Biological-Community Data in the 

Upper Wabash River Basin, Indiana, 2003

By Donald R. Leer, Brian J. Caskey, Jeffrey W. Frey, and B. Scott Lowe

Prepared in cooperation with the Indiana Department of Environmental 

Management, Division of Water, Assessment Branch

Scientific Investigations Report 2007-5231

U.S. Department of the Interior

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

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2007

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Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Leer, D.R., Caskey, B.J., Frey, J.W., and Lowe, B. S., 2007, Relations of Principal Component Analysis site scores algalbiomass, habitat, basin-characteristics, nutrient, and biological community data in the Upper Wabash River Basin, 

Indiana, 2003: U.S. Geological Survey Scientific Investigations Report 2007-5231, 25 p.
iii

Contents

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

Introduction.....................................................................................................................................................1

Purpose and Scope ..............................................................................................................................2

Description of the Upper Wabash River Basin................................................................................4

Study Methods ...............................................................................................................................................4

Site Selection and Sampling Strategies ...........................................................................................4

Algal-Biomass, Habitat, Basin-Characteristics, Nutrient, and Biological-Community 

Data-Collection and Processing Methods .........................................................................4

Data Analysis.........................................................................................................................................7

Basin-Characteristics Data........................................................................................................8

Nutrient Data ................................................................................................................................8

Principal Components Analysis.................................................................................................8

Comparison With U.S. Environmental Protection Agency Data .........................................9

Relations of the Principal Components Analysis Site Scores to Algal-Biomass, Habitat, 

Basin-Characteristics, Nutrient, and Biological-Community Data ..........................................9

Drainage Area and Land-Use Analysis....................................................................................................11

Comparison of the Data to Ecoregion Nutrient Criteria ........................................................................15

Conclusions...................................................................................................................................................20

Acknowledgments.......................................................................................................................................21

Reference......................................................................................................................................................21

Appendix 1.....................................................................................................................................................24

Figures

1. Map showing location of the 38 algal-bilmass sampling sites in the Upper Wabash 

River Basin, Indiana, 2003. ..........................................................................................................3

2. Graphs showing the percentage of drainage area and agricultural, forest, and 

urban land use in the Upper Wabash River Basin, Indiana, 2003.......................................12

Tables

1. Location and basin characteristics of the 38 algal-biomass sampling sites in the 

Upper Wabash River Basin, Indiana, May through October 2003........................................5

2. Significant Spearman’s rho relations of the yearly periphyton Principal Components 

Analysis axis 1 site scores (PC1) to algal-biomass, habitat, basin-characteristics, 

and nutrient parameters and fish- and invertebrate-community attributes and 

metric scores, 2003.....................................................................................................................10

3. Significant Spearman’s rho relations of the yearly seston Principal Components 

Analysis axis 1 site scores (PC1) to algal-biomass, habitat, basin-characteristics, 

and nutrient parameters and fish- and invertebrate-community attributes and 

metric scores, 2003.....................................................................................................................11
iv

4. Basin characteristics of the 38 algal-biomass sampling sites in the Upper Wabash 

River Basin, Indiana, May through October 2003..................................................................13

5. Total Kjeldahl nitrogen as N values collected in 2003 from the Indiana Department 

of Environmental Management/U.S. Geological Survey study and the published 

U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions 

VI and VII and Level III Ecoregions 55 and 56........................................................................15

6. Nitrite plus nitrate as N values collected in 2003 from the Indiana Department of 

Environmental Management/U.S. Geological Survey study and the published U.S. 

Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI 

and VII and Level III Ecoregions 55 and 56.............................................................................16

7. Total nitrogen as N values collected in 2003 from the Indiana Department of Envi

ronmental Management/U.S. Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and VII 

and Level III Ecoregions 55 and 56. .........................................................................................17

8. Total phosphorus as P values collected in 2003 from the Indiana Department of 

Environmental Management/U.S. Geological Survey study and the published U.S. 

Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI 

and VII and Level III Ecoregions 55 and 56.............................................................................18

9. Mean periphyton chlorophyll a values collected in 2003 from the Indiana Depart

ment of Environmental Management/U.S. Geological Survey study and the pub

lished U.S. Environmental Protection Agency values for Aggregate Nutrient 

Ecoregions VI and VII and Level III Ecoregions 55 and 56...................................................19

10. Mean seston chlorophyll a values collected in 2003 from the Indiana Department 

of Environmental Management/U.S. Geological Survey study and the published 

U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions 

VI and VII and Level III Ecoregions 55 and 56........................................................................20
v

Multiply By To obtain

centimeter (cm) 0.3937 inch (in.)

square kilometer (km2) 0.3861 square mile (mi2)

cubic meter per second (m3/s) 35.31 cubic foot per second (ft3/s)

liter (L) 0.2642 gallon (gal)

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

°F=(1.8×°C)+32

Horizontal coordinate information used in the map is referenced to the North American Datum 

of 1927 (NAD 27). Sampling-site coordinate information used a Global Positioning System (GPS) 

referenced to North American Datum of 1983 (NAD 83), which then was converted to NAD 27.

Concentrations of chemical and algal-biomass constituents in water are given either in 

milligrams per liter (mg/L) or micrograms per liter (μg/L). Concentrations of periphyton algal 

biomass are given in milligrams per meter squared (mg/m2).

Conversion Factors and Datum
vi

Abbreviations 

Abbreviations and acronyms used in this report:

PC1 Principal components analysis first axis site score

EPT Ephemeroptera, Plecoptera, and Trichoptera

USEPA U.S. Environmental Protection Agency

N nitrogen

P phosphorus

CWA Clean Water Act

IBI Index of Biotic Integrity

TN total nitrogen

TP total phosphorus

CHLa chlorophyll a

TKN total Kjeldahl nitrogen

IDEM Indiana Department of Environmental Management

USGS U.S. Geological Survey

WMP Watershed Monitoring Program

PCA Principal components analysis

AFDM ash-free dry mass

POC particulate organic carbon

IN Indiana

NAWQA National Water-Quality Assessment Program

NWQL National Water Quality Laboratory

QHEI Qualitative Habitat Evaluation Index

NLCD National Land Cover Dataset

r

s

 Spearman’s rho statistic
Relations of Principal Components Analysis Site Scores to 

Algal-Biomass, Habitat, Basin-Characteristics, Nutrient, 

and Biological-Community Data in the Upper Wabash 

River Basin, Indiana, 2003

By Donald R. Leer, Brian J. Caskey, Jeffrey W. Frey, and B. Scott Lowe

Abstract

Data were gathered from May through October 2003 at 

38 randomly selected sites in the Upper Wabash River Basin, 

Indiana, for algal biomass, habitat, nutrients, and biological 

communities (fish and invertebrates). Basin characteristics 

(drainage area and land use) and biological-community attributes and metric scores were determined for each sampling 

site’s basin. Yearly Principal Components Analysis site scores 

were calculated for algal biomass (periphyton and seston). The 

yearly Principal Components Analysis site scores for the first 

axis (PC1) were related, using Spearman’s rho, to the seasonal 

algal-biomass, basin-characteristics, habitat, seasonal nutrient, 

biological-community attribute and metric score data.

The periphyton PC1 site score, which was most influenced by September chlorophyll a, was not related to the five 

basin-characteristics, nine habitat, or 12 nutrient variables 

examined. Of the 43 fish-community attributes and metric 

scores examined, the periphyton PC1 was negatively related 

to one attribute (tolerant percent) and positively related to 

one metric score (insectivore percent metric score). Of the 43 

invertebrate-community attributes and metric scores examined, 

the periphyton PC1 was negatively related to three attributes 

(Ephemeroptera, Plecoptera, and Trichoptera (EPT) count, 

EPT-to-total ratio, and number of taxa) and four metric scores 

(EPT count metric score, EPT to total ratio metric score, 

macroinvertebrate Index of Biotic Integrity metric score, and 

number of taxa metric score). The seston PC1 was not related 

to the five basin-characteristics, nine habitat, or 12 nutrient 

variables or to the 43 fish- and 21 invertebrate-community 

attributes or metric scores examined.

To understand how the choice of sampling sites might 

have affected the results, an analysis of the drainage area and 

land use was done. The random site selection in 2003 in the 

Upper Wabash River Basin was skewed to small streams. 

The lack of large streams in the Upper Wabash River Basin 

could account for the few relations among the seston and the 

biological-community data.

The values for nutrients (nitrate, total Kjeldahl nitrogen, 

total nitrogen, and total phosphorus) and chlorophyll a

(periphyton and seston) were compared to published 

U.S. Environmental Protection Agency (USEPA) values 

for Aggregate Nutrient Ecoregions VI and VII and USEPA 

Level III Ecoregions 55 and 56. Several nutrient values were 

greater than the 25th percentile of the published USEPA values. 

Chlorophyll a (periphyton and seston) values either were 

greater than the 25th percentile of published USEPA values or 

extended data ranges in the Aggregate Nutrient and Level III 

Ecoregions. If the proposed values for the 25th percentile were 

adopted as nutrient water-quality criteria, many samples in the 

Upper Wabash River Basin would have exceeded the criteria.

Introduction

Excessive inputs of nutrients into streams have humanhealth, economic, and ecological consequences. Excess 

amounts of nutrients—nitrogen (N) and phosphorus (P)—have 

been shown to be a source of eutrophication in aquatic ecosystems, which sometimes has been linked to fish kills, shifts in 

species composition, taste and odor in drinking-water supplies, and blooms of harmful algae in freshwater and estuaries 

(Munn and Hamilton, 2003; U.S. Environmental Protection 

Agency, 2000a,b).

The Clean Water Act (CWA) established a national goal 

of achieving water quality that provides for the protection and 

propagation of aquatic organisms and wildlife, and recreation 

in and on the water. In 1996, the U.S. Environmental Protection Agency’s (USEPA) National Water Quality Inventory 

identified excess amounts of nutrients as the second leading 
2 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

cause of impairment in rivers and streams across the United 

States (the first cause was siltation) (U.S. Environmental 

Protection Agency, 1997a). The excess amounts of nutrients 

that have been documented in many rivers and streams have 

resulted in streams that do not meet the goal of the CWA in 

Indiana and the nation.

USEPA drinking-water criteria (maximum contaminant 

levels) are 10 mg/L for nitrate as N and 1 mg/L 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 criteria varies with pH, temperature, and life-stage) (U.S. 

Environmental Protection Agency, 2005). These criteria do 

not address the effects on the biological communities resulting from increased nutrients 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 (Ohio Environmental Protection Agency, 

1999). For example, nitrate as N concentrations below 90 

mg/L would not have direct effects on warmwater fish. Exceptions are concentrations of ammonia after accidental discharges from wastewater-treatment facilities, combined-sewer 

overflows, or confined-animal feedlots (Ohio Environmental 

Protection Agency, 1999). Previous analysis of the effects of 

nutrients on biological communities in Ohio found few relations between nutrients and fish and invertebrate-community 

data (Miltner and Rankin, 1998). Only total phosphorus was 

significantly correlated with any of the fish or invertebrate 

attributes or metrics (fish Index of Biotic Integrity [IBI] scores 

in headwater streams). 

Many streams have been placed by the USEPA on the 

CWA Section 303(d) list of impaired water bodies because of 

excess amounts of nutrients. In 2000, the USEPA proposed 

nutrient water-quality criteria for causal variables—total 

nitrogen (TN) and total phosphorus (TP)—and for response 

variables—periphyton and seston chlorophyll a (CHLa) and 

turbidity. Criteria also have been proposed for nitrate and total 

Kjeldahl nitrogen (TKN) because TN is the sum of nitrate 

and TKN. These proposed nutrient water-quality criteria are 

based on Aggregate Nutrient Ecoregions, areas with similar 

geographic features (topography, soils, geology, land use, 

and biogeography) (U.S. Environmental Protection Agency, 

2000a,b). USEPA reviewed selected data and set the proposed 

nutrient water-quality criteria for nitrate, TKN, TN, TP, CHLa 

(periphyton and seston), and turbidity at the 25th-percentile 

value of all data for each parameter.

USEPA mandated that by 2004 states either accept the 

proposed nutrient water-quality criteria or provide their own 

set of criteria that are more appropriate to the waters within 

each state (U.S. Environmental Protection Agency, 2000a,b). 

An extension was given to Indiana and other states that 

adopted plans describing the data needs and the process to 

develop nutrient water-quality criteria. Beginning in 2001, the 

Indiana Department of Environmental Management (IDEM) 

and the U.S. Geological Survey (USGS) are cooperating on 

several studies that will assist the State of Indiana in developing nutrient water-quality criteria as mandated by the USEPA. 

The multivariate approach used in this report should allow the 

results to be used in similar ecoregions in Illinois and Ohio.

Purpose and Scope

Data in this report were collected as part of an ongoing 

cooperative effort between IDEM and the USGS in which similar studies have been conducted as part of the IDEM probabilistic Watershed Monitoring Program (WMP) in the West Fork 

White River Basin (2001), Whitewater River and East Fork 

White River Basins (2002), Lower Wabash River and Kankakee River Basins (2004), and Ohio River and Great Lakes 

Basins (2005) (Indiana Department of Environmental Management, 2001). In all of these yearly WMP studies, IDEM collected habitat and biological community data and the nutrients 

from 2001 through 2003; to augment the IDEM WMP studies 

the USGS collected algal biomass during all years of the study 

and also collected nutrients in 2004 and 2005. The long-term 

goal of these studies is to provide data and analysis to aid 

IDEM in the development of nutrient water-quality criteria. 

An objective of this report was to develop a preliminary understanding of how algal biomass relates to biological community 

and environmental variables in the Upper Wabash River Basin 

in 2003. An additional purpose of this report was to compile a 

list of the most statistically significant relations between algal 

biomass, nutrients, and biological metrics that may be helpful 

in future investigations.

Two approaches were used for the preliminary analysis 

of the data sets. The first approach included ordination and 

regression analyses of the algal biomass, nutrient, and environmental data. The second approach compared the CHLa 

and nutrient values collected by IDEM and USGS personnel 

to USEPA published values. The purpose of this preliminary 

analysis was to investigate all potential relations and identify 

those relations that were the strongest and warrant further 

investigation. 

This report discusses the relations of yearly Principal 

Components Analysis (PCA) site axis scores, which represent algal biomass (periphyton and seston), to habitat, basincharacteristics, nutrient, and biological-community (fish 

and invertebrates) attributes and metric scores (appendix 1; 

Dufour, 2002). Data were collected at 38 randomly selected 

sites in the Upper Wabash River Basin in 2003 (fig. 1). A 

discussion of the basin characteristics of the 38 sites describes 

how drainage area and land use affect the analysis. This report 

also compares nutrient values (nitrite plus nitrate as N, TKN 

as N, TN as N, TP as P) and CHLa (periphyton and seston) 

collected by IDEM and USGS for the Upper Wabash River 

Basin to values published by the USEPA for Aggregate Nutrient Ecoregion VI—Corn Belt and Northern Great Plains, for 

Aggregate Nutrient Ecoregion VII—Mostly Glaciated Dairy 

Region, for Level III Ecoregion 55—Eastern Corn Belt Plains, 

and for Level III Ecoregion 56—Southern Michigan/Northern 

Indiana Drift Plains. Aggregate Nutrient Ecoregions consist of 
Purpose and Scope 3

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 EXPLANATION

U.S. ENVIRONMENTAL PROTECTION AGENCY AGGREGATE NUTRIENT ECOREGION VI,

LEVEL III ECOREGION 54

U.S. ENVIRONMENTAL PROTECTION AGENCY AGGREGATE NUTRIENT ECOREGION VI,

LEVEL III ECOREGION 55

U.S. ENVIRONMENTAL PROTECTION AGENCY AGGREGATE NUTRIENT ECOREGION VII,

LEVEL III ECOREGION 56

MAJOR STREAMS IN UPPER WABASH RIVER BASIN

UPPER WABASH RIVER DRAINAGE BASIN BOUNDARY

UPPER WABASH RIVER SAMPLING SITES WITH SITE IDENTIFIER

U.S. GEOLOGICAL SURVEY STREAMFLOW-GAGING STATION

622

Base from U. S. Geological Survey digital data,

scales ranging from 1:24,000 to 1:250,000, 1999

Albers Equal Area projection

Standard parallels 29° 30' and 45° 30', central meridian -86°

North American Datum, 1927

INDIANA

OHIO

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40 MILES 

40 KILOMETERS 

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WABASH RIVER

AT LAFAYETTE, IND

UPPER WABASH RIVER BASIN

Wabash

River

Eel

River

TippecanoeTippecanoe

River

Wildcat CreekWildcat Creek

MississinewaMississinewa

River

Figure 1. Map showing location of the 38 algal-bilmass sampling sites in the Upper Wabash River Basin, Indiana, 2003.
4 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

one or more Level III Ecoregions (U.S. Environmental Protection Agency, 2000a,b).

In the text, the Aggregate Nutrient Ecoregions are 

referred to as Ecoregion VI or VII; Level III Ecoregions are 

referred to as Ecoregion 55 or 56. The nutrients are described 

as concentrations of nitrate, TKN, TN, and TP. In this report, 

periphyton CHLa, ash-free dry mass (AFDM), seston CHLa, 

and particulate organic carbon (POC) are measures of algal 

biomass.

Description of the Upper Wabash River Basin

The Upper Wabash River Basin (fig. 1) drains more 

than 18,655 km2 of central Indiana and parts of western Ohio 

before draining into the middle branch of the Wabash River 

(Hogatt, 1975). The annual mean streamflow in the 2003 water 

year at the USGS streamflow-gaging station Wabash River at 

Lafayette, IN (03335500) (fig. 1) was 234.3 m3/s (Morlock 

and others, 2004). Streamflow in the 2003 water year was 

above average; the annual mean long-term streamflow 

(1924-2003) was 189.7 m3/s at Lafayette (Morlock and others, 

2004). The gaging station at Lafayette is 8.2 km downstream 

from the basin boundary of the Upper Wabash River and is the 

gaging station closest to the basin boundary. 

The climate in the Upper Wabash River Basin is characterized as humid continental with mild to warm summers 

and cold winters. The mean monthly temperature ranges from 

-4.3oC in winter to 22.9oC in summer (Purdue Applied Meteorology Group, 2005). The mean annual precipitation ranges 

from 94.5 cm to 104 cm (Purdue Applied Meteorology Group, 

2005). 

The dominant land use of the Upper Wabash Basin is 

agriculture (92 percent), primarily corn and soybeans. Approximately 5 percent of the basin area is forested, 1 percent is 

urban, and 2 percent is other land-use types. Typical of many 

streams in the State, streams in the Upper Wabash River Basin 

have low gradients and velocities. The total population of the 

97 cities and towns in the basin is 294,680; the largest urban 

areas are Kokomo (population of 46,113) and Marion (population of 31,320) (U.S. Census Bureau, 2000) (fig. 1). 

Study Methods

This study used field and analytical methods from the 

IDEM and the USGS. The following sections describe the site 

selection and sampling strategies; field and laboratory methods 

used in collecting, processing, and analyzing algal-biomass, 

habitat, basin-characteristics, nutrients, and biological-community data; and data analysis used in this report.

Site Selection and Sampling Strategies

Sampling sites were selected randomly by the USEPA as 

part of the IDEM probabilistic Watershed Monitoring Program 

(WMP) (Indiana Department of Environmental Management, 

1999, 2001). Each selected sampling site (table 1) represents 

a specific stream order; therefore, statistically valid extrapolations can be made from the randomly sampled streams to the 

entire class of streams in a particular basin. The IDEM WMP 

works on a 5-year rotating basin schedule, focusing on 1-2 

selected basins each year, with a complete assessment of the 

state at the end of each 5-year cycle. In 2003, the focus was 

the Upper Wabash River Basin. After the sampling sites were 

selected and prior to collection of field data, IDEM personnel 

completed a visual assessment of the potential sampling sites 

and determined the area to be sampled at each site. At each of 

the USEPA randomly selected sites, the latitude and longitude 

was used as the middle point of the reach, with half of the 

reach upstream and half downstream of the middle point.

During periods of stable flow from May through October 

2003 in the Upper Wabash River Basin, 38 sites (fig. 1, 

table 1) were sampled three times for algal biomass and 

nutrients. The samples were collected three times to measure 

seasonal changes in the algal biomass and nutrients. Round 

one sampling was done in May and June (spring); round two 

sampling was done in July and August (summer); and round 

three sampling was done in September (fall). In this report, 

round one sampling will be referred to as May or spring, round 

two as July or summer, and round three as September or fall. 

The algal-biomass and nutrient samples were collected on the 

same day at about the same time. The biological community 

(fish and invertebrate) and habitat were sampled one time by 

IDEM personnel from June through October 2003.

Algal-Biomass, Habitat, Basin-Characteristics, 

Nutrient, and Biological-Community DataCollection and Processing Methods 

Algal-biomass samples were collected and processed for 

periphyton and seston, as described in USGS protocols, with 

several modifications. The National Water-Quality Assessment 

(NAWQA) Program algal protocols for periphyton (Moulton 

and others, 2002) are a reach-based sampling methodology in 

which five periphyton subsamples were collected from five 

different locations within the sampling reach. At each location, the stream depth, velocity, shading, and substrate were 

recorded. The subsamples were composited and marked as a 

single sample. At each site periphyton samples were collected 

using the same substrate type—epilithic (rocks), epidendric 

(sticks), or epipsammic (sand)—during the sampling period. 

One modification to this study from the NAWQA protocols 

was that ten periphyton subsamples were collected from the 

same substrate as close to the center of the reach as possible. 

Then five subsamples were selected that best represented the 
Study Methods 

5

Table 1. Location and basin characteristics of the 38 algal-biomass sampling sites in the Upper Wabash River Basin, Indiana, May through October 2003.[IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; ID, identification number; USEPA, U.S. Environmental Protection Agency; FT, feet; US, upstream; trib, tributary; CR, county road; R, River; SR, state route; DS, downstream; CK, Creek; FK, Fork; DCH, Ditch; NR, near; AV, Avenue; ST, Street; N, North; S,South; E, East; W, West; USHWY, U.S. Highway; IN, Indiana; OH, Ohio]

Short IDEM 

site 

number1

USGS station ID USGS station name

USEPA 

Aggregate 

Nutrient 

Ecoregion2

Level III 

Ecoregion3

Latitude

(degrees)

Longitude

(degrees)

601 403857085593501 PIPE CK 100 FT US PIPE CK AT SANTA FE, IN VI 55 40.64950 -85.99200602 401643084521501 HARSHMAN CK 0.9 MI US MISSISSINEWA R NR SALEM, IN VI 55 40.27870 -84.87020603 404805086333701 GALBREATH DITCH 0.5 MI DS CR 200N NR LK CICOTT, IN VII 56 40.80107 -86.56016604 402554085305901 MISSISSINEWA R 0.8 MI US SR 26 NR FOWLERTON, IN VI 55 40.43180 -85.51480605 405236086495201 BIG MONON CK 0.5 MI US SR 16 NR MONON, IN VI 54 40.87680 -86.83150606 405243085564701 PAW PAW CK 0.3 MI US CR 800N NR ROANN, IN VI 55 40.87859 -85.94648607 401943086385101 S FK WILDCAT CK 0.6 MI US CR 800W NR MULBERRY, IN VI 55 40.32850 -86.64740608 410301086184501 MUD CK 0.3 MI US GRUBE DITCH NR PERSHING, IN VII 56 41.04981 -86.31161609 403712086593901 MEYERS DITCH 820 FT US I-65 NR BADGER GROVE, IN VI 54 40.61974 -86.99414610 410543085373101 CLEAR CK 980 FT US SPRING CK AT SOUTH WHITLEY, IN VII 56 41.09500 -85.62430611 404156086024501 LITTLE PIPE CK 900 FT DS CR 150E NR SOUTH PERU, IN VI 55 40.69930 -86.04680612 403633085115401 ROCK CK 770 FT US CR 900S NR PETROLEUM, IN VI 55 40.60936 -85.19817613 403542086305901 DEER CK 0.7 MI US MERIDIAN LINE RD NR CAMDEN, IN VI 55 40.59506 -86.51631614 405208085093701 EIGHTMILE CK 0.4 MI US SR 1 AT OSSIAN, IN VI 55 40.86910 -85.16019615 403603086325101 DEER CK 0.6 MI DS SR 75 AT CAMDEN, IN VI 55 40.60070 -86.54740616 411707085593601 EASTERDAY DITCH 0.3 MI DS CR 400N NR ATWOOD, IN VII 56 41.28654 -85.99596617 402612086094301 E FK LITTLE WILDCAT CK 880 FT US CR200W AT ALTO, IN VI 55 40.43653 -86.16117618 405346085595201 WILSON RHODES DITCH 0.6 MI US CR850N NR ROANN, IN VII 56 40.89640 -85.99760619 404912086080401 UNNAMED TRIB 1.2 MI US EEL R AT MEXICO, IN VI 55 40.82060 -86.13430621 405856086223001 MILL CK 600 FT US HINES DITCH NR MARSHTOWN, IN VII 56 40.98213 -86.37508
6 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003 Table 1. Location and basin characteristics of the 38 algal-biomass sampling sites in the Upper Wabash River Basin, Indiana, May through October 2003.—Continued [IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; ID, identification number; USEPA, U.S. Environmental Protection Agency; FT, feet; US, upstream; trib, tribu- tary; CR, county road; R, River; SR, state route; DS, downstream; CK, Creek; FK, Fork; DCH, Ditch; NR, near; AV, Avenue; ST, Street; N, North; S,South; E, East; W, West; USHWY, U.S. Highway; IN, Indiana; OH, Ohio] Short IDEM site number1 USGS station ID USGS station name USEPA Aggregate Nutrient Ecoregion2 Level III Ecoregion3 Latitude (degrees) Longitude (degrees) 622 410526085291501 STONY CK 720 FT US MERIDIAN RD AT PEABODY, IN VII 56 41.09090 -85.48870 623 402423086423501 MID FK WILDCAT CK 0.4 MI DS HOG RUN AT PETTIT, IN VI 55 40.40630 -86.71070 624 411753085493801 TIPPECANOE R 880 FT DS CR 100E NR ISLAND PARK, IN VII 56 41.29765 -85.82738 625 403639086402301 WABASH R 1.9 MI US SR 39 NR DELPHI, IN VI 55 40.61088 -86.67318 626 404739085210101 ROCK CK 0.4 MI US SR 3 NR MARKLE, IN VI 55 40.79580 -85.35150 627 402135086211701 KILMORE CK 500 FT US CR 800E NR FOREST, IN VI 55 40.35993 -86.35473 628 410048086350101 MILL CK 0.6 MI US TIPPECANOE R NR WINAMAC, IN VI 55 41.01320 -86.58340 629 404248086341901 UNNAMED TRIB 0.8 MI US BURNETTS CK AT LOCKPORT, IN VI 55 40.71320 -86.57180 630 404203085274901 DETAMORE DITCH 700 FT DS I-69 NR WARREN, IN VI 55 40.70118 -85.46385 631 401517084495201 LITTLE MISSISSINEWA R 2.8 MI DS SR28 NR COSMOS, OH VI 55 40.25505 -84.83130 632 411625085372301 UNNAMED TRIB 450 FT US GAFF DITCH NR ETNA, IN VII 56 41.27390 -85.62304 633 402739085413701 DEER CK 0.25 MI US CR 100W NR WEAVER, IN VI 55 40.46050 -85.69430

634 401401085413701 ELKHORN CK 0.25 MI US CR 500N NR FARMLAND, IN VI 55 40.23280 -85.16620635 405501086490201 BIG MONON CK 0.2 MI US BIG MONON DITCH NR MONON, IN VI 54 40.91790 -86.81700636 402425084574101 LITTLE SALAMONIE R 0.4 MI US CR 120S AT LIBER, IN VI 55 40.40696 -84.96156637 405617086260401 TRAVERS DITCH 560 FT DS CR1000W NR GRASS CREEK, IN VII 56 40.93800 -86.43380638 410106085191601 ABOITE CK 0.3 MI US USHWY 24 AT TIMBERCREST, IN VI 55 41.01850 -85.32080639 402710086510501 WILDCAT CK 150 FT US SR 25 AT LAFAYETTE, IN VI 55 40.45283 -86.85122

1The full IDEM site number has a prefix of INRB, for example INRB03-601.2U.S. Environmental Agency, 2000a,b.3U.S. Environmental Protection Agency, 1997b.
Study Methods 7

average algal cover at the sampled reach and these subsamples 

were composited into a single sample (Charles and others, 

2000). 

Seston samples were collected in the center of the sampling reach along a line that extended from the left edge to the 

right edge of the streambank (transect). The wetted channel 

width and water depths (one-quarter, one-half, and three-quarters points) along the chosen transect were recorded. The seston samples were collected with a 3-L bottle and a 0.476-cm 

nozzle; either a grab sample or a multiple vertical method was 

used (Shelton, 1994). 

Algal-biomass samples were collected, homogenized, and 

filtered onto glass-fiber filters in the field by USGS personnel. 

All filters collected by the USGS were placed on dry ice and 

transported to the USGS Indiana Water Science Center laboratory for analysis. The CHLa and AFDM filters were analyzed 

at the USGS Indiana Water Science Center laboratory; all the 

POC filters were analyzed at the USGS National Water Quality Laboratory (NWQL) in Lakewood, Colorado. 

Concentrations of CHLa were determined, following 

USEPA method 445, with a Turner Designs TD-700 fluorometer outfitted for CHLa analysis (Arar and Collins, 1997). There 

were two exceptions to method 445; filters were ground in 

Nalgene centrifuge tubes instead of glass to counter the problem of tube breakage, and samples were centrifuged at 1,500 

revolutions per minute (approximately 320 g) for 15 minutes. 

At the modified centrifuge rate, the filter residue and acetone 

solution separated well. If samples did not separate well, they 

were placed in the centrifuge a second time or care was taken 

not to decant the solute. For consistency, all samples were 

allowed to steep for 2.5 hours. Concentrations of AFDM were 

determined, following USGS method B–3520–85 (Britton and 

Greeson, 1988) with two exceptions: the samples were filtered 

in the field, and the filters were not baked and weighed in the 

crucibles before use. 

Quality-assurance methods for algal biomass samples 

included triplicate filters from the same sample to measure 

variability and a blank filter collected at each sampling reach 

to measure bias. Additionally, a fifth filter was collected at 

each site and 10 percent of these fifth CHLa filters were analyzed at the NWQL to measure laboratory variability.

Habitat assessments were collected from June through 

October 2003 at each site by IDEM personnel following standard IDEM methods (Indiana Department of Environmental 

Management, 1992, 2002). Habitat assessments were made 

at the same time fish communities were sampled. Habitat 

assessments include in-stream and riparian measurements that 

are incorporated into the Qualitative Habitat Evaluation Index 

(QHEI). 

Nutrient samples (ammonia, nitrate, TKN, TN, and TP) 

were collected by IDEM personnel following approved IDEM 

methods (Beckman, 2000). Nutrient quality-assurance methods followed approved IDEM methods (Bowren and Ghiasuddin, 1999). The nutrient samples were preserved by IDEM 

personnel, placed on wet ice, and taken to an independent 

laboratory (Test America, Indianapolis) for analysis.

Biological-community (fish and invertebrates) samples 

were collected one time, June through October 2003, at each 

site by IDEM personnel following standard IDEM methods 

(Indiana Department of Environmental Management, 1992, 

2002; Barbour and others, 1999). IDEM personnel calculated 

the biological-community attributes and metric scores for 

this study. Attributes are the raw data and metric scores are 

rankings of the data from poor (a score of one), fair to good (a 

score of three), and excellent (a score of 5). The metric scores 

are ranked using a large data set that includes unimpaired 

reference sites to impaired sites. Each attribute has a corresponding metric score (Dufour, 2002). A list of the fish and 

invertebrate attributes and metrics are listed in appendix 1.

Data sets for sampling dates, laboratory-analysis dates, 

and algal-biomass (periphyton and seston) are available at: 

http://in.water.usgs.gov/NAWQAWHMI/neet.php

Data Analysis

In large environmental datasets, natural variability often 

masks the relations among variables. An objective of this 

report was to develop a preliminary understanding of how 

algal biomass relates to biological community and environmental variables collected in the Upper Wabash Basin in 2003. 

In this report, the environmental variables included nutrients, 

habitat, and basin characteristics. Two approaches were used 

for the preliminary analysis of the data sets. The first approach 

included ordination and regression analyses of the algal biomass, nutrient, and environmental data. The second approach 

compared the CHLa and nutrient values collected by IDEM 

and USGS personnel to USEPA published values.

The purpose of this preliminary analysis was to investigate all potential relations and identify those relations that 

were the strongest and warrant further investigation. In this 

report, for a relation to be considered statistically significant 

using PCA, the Spearman’s rho statistic (rS) was required to be 

greater than the absolute value of 0.45 and have at least a 

95 percent significance level based on the sample size. 

Although an rS of 0.45 is considered significant, it has a 

possibility of introducing a Type I error. The methods used 

provided an exploratory analysis to identify which biological 

and environmental variables were significantly related to algal 

biomass. This technique also was used as a data censoring tool 

and allowed researchers to determine the relations of interest 

to use as a starting point in future studies.

The ordination approach consisted of determining yearly 

site scores for the periphyton and seston data using PCA. The 

ordination approach consisted of determining site scores for 

the periphyton and seston data using PCA. In each PCA two 

measures of algal biomass for periphyton (CHLa and AFDM) 

and seston (CHLa and POC) were used. The site scores are 

considered yearly because all of the seasonal algal biomass 

data are included in the PCA site score determination. The 

regression approach related the periphyton and seston PCA 

site scores from the first axis to five basin characteristics, nine 
8 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

habitat, 12 nutrient variables, and 43 fish and 21 invertebrate 

attribute and metric scores. All data were normalized to a 

z-score prior to use in the data analyses, allowing for comparison of variables that were recorded in different units.

Basin-Characteristics Data

The basin characteristics used in this analysis were drainage area and land use (percentage of agriculture, forest, other, 

and urban) and these were determined by the USGS for this 

study. Drainage area was derived from the basin boundaries. 

Basin boundaries for each site were generated following the 

method outlined by Ries, III, and others (2004). This method 

combines the National Elevation Dataset, Digital Elevation 

Model data, and the National Hydrography Dataset, which is a 

comprehensive set of digital surface-water features. The basin 

boundaries were used to extract land-use and soil information 

from the National Land Cover Dataset (NLCD) (U.S. Geological Survey, 2000). This conversion allowed the land-use data 

to be compared among and between basins. Each sampling site 

was assessed to determine in which Aggregate Nutrient Ecoregion (U.S. Environmental Protection Agency, 2000a,b) and 

Level III Ecoregion (U.S. Environmental Protection Agency, 

1997b) it was located.

Nutrient Data

The nutrients used in this analysis were dissolved ammonia as N, dissolved nitrate plus nitrite as N, total Kjeldahl 

nitrogen as N, total nitrogen as N, and total phosphorus as 

P. Because concentrations of nitrate typically are two orders 

of magnitude greater than nitrite and because nitrite usually does not exceed 0.5 mg/L in surface water (National 

Research Council, 1978), concentrations of nitrite plus nitrate 

are referred to as nitrate in this report. Concentrations of 

total nitrogen were calculated as the sum of nitrate and TKN. 

Because almost ninety percent of the ammonia concentrations were below the reporting level, it was not included in 

the analysis. About thirteen percent of the TP concentrations 

were below the reporting level, 0.03 mg/L, and one-half of the 

reporting level was used in the analysis. For nutrient analyses, the scope of this report is narrowed to nitrate, TKN, TN, 

and TP. Nutrient data was analyzed separately for the spring 

(May), summer (July), and the fall (September) samples; the 

seasonal nutrient and the seasonal algal-biomass data were 

then compared to the PC1 sites scores using Spearman’s rho to 

determine the most significant seasonal relations.

Principal Components Analysis

Principal Components Analysis, an ordination technique, 

was used to calculate individual yearly algal-biomass site 

scores for the periphyton and seston samples. PCA site scores 

(Gauch, 1982; Jongman and others, 1995; McCune and Grace, 

2002) are theoretical variables that minimize the total residual 

sum of squares after fitting straight lines to the algal-biomass 

data. Mean CHLa (periphyton and seston) and AFDM values 

were calculated from the three filters for each sample. Mean 

algal-biomass (periphyton and seston) and other data were 

normalized prior to running the ordination analysis. To calculate the yearly periphyton PCA site scores, the mean periphyton CHLa and mean AFDM data from the May and June, July 

and August, and the September samplings were used; for the 

yearly seston PCA site scores the mean seston CHLa and POC 

data from the May and June, July and August, and the September samplings were used.

In this report, a positive site score indicates an increase in 

algal-biomass along the axis and a negative site score indicates 

a decrease in algal-biomass along the axis. Only the principal 

components (PC) site scores from the first axis (PC1) are presented in this preliminary analysis, because the PC1 axis best 

explains the algal-biomass data (McCune and Grace, 2002). 

In theory, the PC1 site scores should be related to the variables that were used in the calculations, and the variable with 

the highest loading accounts for the majority of the variation, 

which should also have the highest correlation coefficient. 

As a validation step, the yearly periphyton and yearly seston 

PC1 site scores were related to the algal biomass-values, using 

Spearman’s rho. The purpose of this preliminary analysis was 

to investigate all potential relations and identify those relations that were the strongest and warrant further investigation. In this report, for a relation to be considered statistically 

significant using PCA, the Spearman’s rho statistic (rS) was 

required to be greater than the absolute value of 0.45 and have 

at least a 95 percent significance level based on the sample 

size. Although an rS of 0.45 is considered significant, it has a 

possibility of introducing a Type I error.

Several procedures – such as the Bonferroni correction – 

are available for adjusting the significance level when performing a large number (or “family”) of tests simultaneously 

(Van Sickle, 2003). This adjustment reduces the chances of a 

Type I error (the relation is declared present when the relation is not present) at a specific alpha level. Although useful 

in reducing Type I error, this technique increases the chance 

of producing a Type II error (no relation declared when a 

relation is present). In this study, the goal was to investigate 

all potential relations and identify which relations were the 

strongest. Because this was a preliminary analysis and there 

were a limited number of significant relations, no corrections 

were applied. 

The yearly PC1 site scores spatially represent the 

(periphyton and seston) algal-biomass and were related, 

using Primer V.6.1.5 (Primer-E Ltd, 2006), to the habitat, 

basin-characteristics (drainage area and land use), seasonal 

nutrient (spring (May), summer (July), and fall (September) 

nitrate, TKN, TN, and TP), seasonal algal biomass (spring 

(May), summer (July), and fall (September) CHLa, AFDM, 

and POC), and biological-community (fish and invertebrates) 

attributes and metric score data collected in 2003. Spearman’s 

rank order (rho) correlations are the preferred method when 

determining relations among environmental data because (1) 
Relations of the Principal Component Analysis Site Scores to Data 9

the initial sample size was greater than 30 for most variables 

and (2) environmental data typically are not normally distributed. Although the data from this study were normalized prior 

to analysis, all variables were not normally distributed, requiring a non-parametric statistic. PC site scores were related in 

a correlation matrix to determine the strongest relations. The 

variables with the strongest relations could help in the development of nutrient criteria. 

Comparison With U.S. Environmental Protection 

Agency Data 

The USEPA proposed criteria in 2000 for nitrate, TKN, 

TN, TP, CHLa (periphyton and seston), and turbidity at the 

25th-percentile value of all data for each variable for each 

Aggregate Nutrient and Level III Ecoregions. Consequently, 

the second analytical approach was to determine how the 

data collected by IDEM and USGS for CHLa and nutrients 

compared to USEPA published values (U.S. Environmental 

Protection Agency, 2000a,b). For the USEPA method, the 

median value for each variable for each stream was calculated. Then, percentiles were determined for each of the 

four seasons in each ecoregion and the 25th-percentile of the 

combined four seasons was used as the proposed criteria. In 

this report, median nutrient (nitrate, TKN, TN, and TP) and 

CHLa values were calculated for all streams sampled within 

the same Aggregate Nutrient and Level III Ecoregions. However, seasonal statistics were not calculated because IDEM and 

USGS did not collect winter samples and spring samples were 

limited—USEPA considers May as part of spring—which left 

a small number of samples in the USEPA “spring” season. 

In this report, the median values for all the streams within a 

specific ecoregion were used to calculate descriptive statistics 

(ranges—minimum and maximum—and percentiles—10th, 

25th, 50th, 75th, and 90th). For CHLa (periphyton and seston), a 

mean of the three filters collected at each site was calculated 

and then a median of the means for all streams within each 

ecoregion was used to calculate the descriptive statistics. The 

descriptive statistics then were compared to published USEPA 

values for Ecoregions VI, VII, 55, and 56 (U.S. Environmental 

Protection Agency, 2000a,b).

Relations of the Principal Components 

Analysis Site Scores to Algal-Biomass, 

Habitat, Basin-Characteristics, 

Nutrient, and Biological-Community 

Data

PCA was used to calculate PC1 site scores from the 

algal-biomass (periphyton and seston) data for each site (all 

seasons). In the preliminary analysis of the 2003 IDEM/USGS 

data, the periphyton PC1 site score, which represents yearly 

algal biomass was constrained by CHLa (September); the PC2 

site score was constrained by AFDM (July). The combination of PC1 (38.2 percent) and PC2 (18.8 percent) site scores 

accounted for 57.0 percent of the total variation in the data set. 

The seston PC1 site score was constrained by CHLa (May); 

the PC2 site score was constrained by POC (September). The 

combination of PC1 (42.3 percent) and PC2 (26.2 percent) 

site scores accounted for 68.5 percent of the variation in the 

data set. Because the PC1 accounted for a large amount of the 

total variation in both of the algal-biomass data sets, only PC1 

(periphyton and seston) site scores were related to the seasonal 

algal-biomass, basin-characteristics (drainage area and land 

use), habitat, seasonal nutrient, and biological-community 

(fish and invertebrates) attributes and metric score data.

The periphyton PC1 site score (table 2) was positively 

related to two algal-biomass variables in three seasons: mean 

AFDM (May and September) and mean periphyton CHLa 

(May, July, and September). Although these relations do not 

offer inferences to the relations of the periphyton PC1 site 

score to the environmental or biological-community (fish and 

invertebrate attributes and metric scores) data, these relations 

demonstrate that the periphyton PC1 site score represents an 

algal-biomass gradient that is largely defined by AFDM and 

CHLa. 

There were no statistically significant relations between 

the periphyton PC1 site score and the five basin-characteristics, nine habitat, and 12 nutrient variables. The lack of 

relations between nutrients and periphyton algal biomass 

is surprising. Biggs (2000) listed several studies that found 

significant relations between periphyton chlorophyll and 

nutrients. However, in similar studies conducted by IDEM 

and USGS in 2001 in the West Fork White River Basin (Frey 

and others, 2007) and in 2002 in the East Fork White River 

and Whitewater River Basins (Caskey and others, 2007) no 

significant relations were found between the nutrients and the 

periphyton algal biomass. Several factors could account for 

this lack of relations between the nutrients and the periphyton 

algal biomass. Scouring of algal growth by increased streamflow that causes algal growth to restart in the stream (Biggs 

and others, 1999) and annual and seasonal differences in storm 

events can allow for “wet” and “dry” years with high levels of 

algal biomass corresponding to high and low nutrients depending upon the season. Other factors include shading from 

canopy cover and turbidity (Wehr, 2003) and grazing by snails, 

invertebrates, and fish (Lamberti and others, 1987).

The periphyton PC1 site score was most influenced by 

September CHLa. Of the 43 fish-community attributes and 

metric scores examined, the periphyton PC1 was negatively 

related to one attribute (tolerant percent) and positively related 

to one metric score (insectivore percent metric score). These 

relations suggest that as periphyton algal biomass increases, 

the amount of tolerant species decreases and the number of 

insectivores increases. However, an opposite relation was seen 

in the 2001 data analysis for the West Fork White River Basin, 
10 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

as periphyton algal-biomass increased, tolerant species also 

increased (Frey and others, 2007).

Of the 21 invertebrate-community attributes examined, 

the periphyton PC1 site score was negatively related to three 

attributes (EPT count, EPT-to-total ratio, and number of taxa) 

and four metric scores (EPT count metric score, EPT-to-total 

ratio metric score, macroinvertebrate Index of Biotic Integrity 

(MIBI) metric score, and number of taxa metric score). These 

relations suggest that as periphyton algal biomass increases, 

the diversity and quality of the invertebrate community 

decreases. This decrease in the invertebrate community also 

could affect the community of insectivore fish previously mentioned. This is the first time in the IDEM/USGS 2001 through 

2003 studies that significant relations between invertebrates 

and algal biomass were more numerous than those between 

fish and algal biomass; there were in total four significant 

relations with invertebrates compared to nine for fish in the 

2001 study in the West Fork White River Basin (Frey and others, 2007) and in 2002 in the Whitewater River and East Fork 

White River Basins (Caskey and others, 2007). 

The seston PC1 site score, which was most influenced 

by July CHLa (table 3), was positively related to two algalbiomass variables in three seasons: mean seston CHLa (May, 

July, and September) and POC (May, July, and September). 

Although these relations do not offer inferences to the relations of the seston PC1 site score to the environmental or 

biological-community (fish and invertebrate attributes and 

metric scores) data, these relations demonstrate that the seston 

PC1 site score represents an algal-biomass gradient that is 

largely defined by CHLa and POC. There were no statistically 

significant relations between the seston PC1 score and the 

nine habitat, five basin characteristics, or 12 nutrient variables 

examined or the 43 fish- and 21 invertebrate-community attributes and metric scores examined. 

The lack of a significant relation between the seston PC1 

site score and drainage area was surprising. Vannote demonstrated in the River Continuum Concept, that as drainage area 

(surrogate for stream order) increases, the amount of seston 

CHLa and POC should increase (Vannote and others, 1980). 

Because of this concept, it was expected that seston CHLa 

Table 2. Significant Spearman’s rho relations of the yearly periphyton Principal Components Analysis axis 1 site scores (PC1) to algalbiomass, habitat, basin-characteristics, and nutrient parameters and fish- and invertebrate-community attributes and metric scores, 

2003.

[rs, Spearman’s rho statistic (probability <0.05); n, number of samples; ns, no statistically significant relations; EPT, ephemeroptera, plecoptera, trichoptera; 

MIBI, macroinvertebrate Index of Biotic Integrity]

Category Parameter and attribute/metric r

s

Parameter

Periphyton algal biomass (n=35) Mean ash-free dry mass, May-June 0.7952

Mean ash-free dry mass, September .5940

Mean periphyton chlorophyll a, May-June .7689

Mean periphyton chlorophyll a, July-August .7022

Mean periphyton chlorophyll a, September .8644

Habitat (n=35) ns ns

Basin characteristics (n=35) ns ns

Nutrients (n=35) ns ns

Attribute/metric

Fish (n=35) Tolerant percent - .4840

Insectivore percent metric score .4503

Invertebrates (n=21) EPT count - .5271

EPT count metric score - .5755

EPT to total ratio - .5377

EPT to total ratio metric score - .4747

MIBI metric score - .4865

Number of taxa - .6062

Number of taxa metric score - .4963
Drainage Area and Land-Use Analysis 11

would become more significant in this study in comparison 

to a similar study conducted by IDEM and USGS in 2001 

because basins with large drainage areas (boat sites) were 

sampled in 2003 and not in 2001 (Frey and others, 2007). 

There were also no significant relations between seston PC1 

and drainage area in 2001 (Frey and others, 2007) or 2002 

(Caskey and others, 2007).

The USEPA proposed nutrient criteria in 2000 based on 

previous studies that had found significant relations between 

nutrients and seston and periphyton CHLa. The lack of relations in this study between nutrients and seston and periphyton 

algal biomass suggests that biological communities may be 

useful in measuring nutrient enrichment in streams. In a previous study in Ohio streams, the only significant relation found 

between nutrients and biological communities was the fish 

community (Miltner and Rankin, 1998). No relations between 

nutrients and the invertebrate community were found. In the 

2001 (Frey and others, 2007) and 2002 (Caskey and others, 

2007) IDEM and USGS studies, fish and invertebrate community variables had statistically significant relations to algal 

biomass, indicating both communities may be a useful measure of nutrient enrichment in streams. Additionally, in the 

IDEM and USGS studies conducted in 2001, 2002, and 2003 

it appears that periphyton algae may be more important than 

seston algae in relating algal biomass with biological communities. The periphyton algae (17) had more significant relations 

with fish and invertebrate attributes and metric scores than 

seston algae (5).

Drainage Area and Land-Use Analysis

Basin characteristics such as drainage area and land use 

can affect the relations between nutrients, algal-biomass, and 

biological-community data. To understand how the sampling 

sites used in this analysis might have affected the results, an 

analysis of the drainage area and land use was done.

The random site selection in 2003 in the Upper Wabash 

River Basin was skewed to small streams. Forty-seven percent 

of the basins were less than 50 km2 (headwater streams); 45 

percent ranged between 501 and 1,000 km2 (wading streams); 

and 8 percent ranged from greater than 1,000 km2 to 11,000 

km2 (boat streams) (fig. 2A). Of the three boat sites, the two 

largest sites were about 2,000 km2 and 11,000 km2 (table 4). 

The lack of large streams in the Upper Wabash River Basin 

could account for the few relations among the seston and the 

biological-community data. The River Continuum Concept 

(Vannote and others, 1980) suggests that in smaller streams, 

periphyton would dominate primary production with moresuitable substrate (rock, sticks, sand), shallower water, and 

less turbidity than in larger streams. As streams get larger and 

deeper, seston would dominate the primary production. 

Although there were few large streams sampled in the 

Upper Wabash, there was still a gradient from headwater to 

boat sites (fig. 2A). The large range of basin drainage areas 

that was sampled could account for or influence the significant 

relations documented by the PC1 (periphyton and seston) and 

Table 3. Significant Spearman’s rho relations of the yearly seston Principal Components Analysis axis 1 site scores (PC1) to algalbiomass, habitat, basin-characteristics, and nutrient parameters and fish- and invertebrate-community attributes and metric scores, 

2003.

[rs, Spearman’s rho statistic (probability <0.05); n, number of samples; ns, no statistically significant relations]

Category Parameter and attribute/metric r

s

Parameter

Seston algal biomass (n=38) Mean seston chlorophyll a, May-June 0.5465

Mean seston chlorophyll a, July-August .7387

Mean seston chlorophyll a, September .5779

Particulate organic carbon, May-June .6266

Particulate organic carbon, July-August .6759

Particulate organic carbon, September .4619

Habitat (n=38) ns ns

Basin characteristics (n=38) ns ns

Nutrients (n=38) ns ns

Attribute/metric

Fish (n=38) ns ns

Invertebrates (n=21) ns ns
12 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

the environmental and biological-community data. Previous 

studies suggest that drainage-basin size (stream order) could 

mask relations of environmental variables to biological communities. Stream size was significant in explaining relations 

between algal-community assemblages (Carpenter and Waite, 

2000) and fish-community assemblages (Caskey, 2003) and 

environmental variables. Different fish and algal communities 

were found in small streams than in large streams. Additionally, stronger relations were found between nutrients and fishand invertebrate-community attributes and metric scores when 

data was analyzed by basin size (Frey and Caskey, 2007). 

Miltner and Rankin (1998) found the only significant relations between nutrients (TP and total inorganic nitrogen) and 

biological communities (fish) were in headwater streams. For 

this report, to keep the sample size large, analysis was done on 

all sites combined. 

Every site except one in the Upper Wabash River Basin 

had basins that consisted of more than 75 percent agricultural 

land use (fig. 2B). The agricultural land use of the Upper 

Wabash River Basin sites ranged from 73.5 to 97.9 percent 

(table 4). In a study of Midwest agriculturally-dominated landscapes, Wang and others (1997) noted when land use within a 

drainage basin is less than 50 percent agriculture, biologicalcommunity relations are complex and difficult to identify. As 

the percentage of agricultural land use increases to more than 

50 percent, a significant negative relation can be seen with 

IBI scores. Because nearly all basins in this study had more 

than 80 percent agricultural land use, an agricultural land-use 

gradient was not found. This lack of an agricultural-land use 

gradient potentially could mask relations between the PC1 

(periphyton and seston) and the environmental variables and 

biological-community (fish and invertebrates) attributes and 

metric scores.

Every site except one in the Upper Wabash River Basin 

had basins that consisted of less than 25 percent forest land 

use (fig. 2C). The forested land use of the Upper Wabash River 

Basin sites ranged from 0.3 to 25.2 percent (table 4). Studies 

have shown that forested landscapes are less likely to have 

elevated nutrients, in part, because an established riparian zone 

acts as a buffer and filters surface-water runoff (Jordan and 

others, 1993).

All of the sites in the Upper Wabash River Basin had 

basins that consisted of less than 25 percent urban land use 

(fig. 2D). The urban land use of the Upper Wabash River 

Basin sites ranged from 0 to 9.4 percent (table 4). Studies 

from across the U.S. have shown that agricultural and urban 

landscapes can have elevated levels of nutrients (Mueller and 

Helsel, 1996). If nutrient concentrations were elevated at all 

the sites and a nutrient gradient was not detected, it potentially 

could mask relations between the PC1 (periphyton and seston) 

and the environmental variables and biological-community 

(fish and invertebrates) attributes and metric scores.

100

PERCENTAGE OF SITES WITHIN BASIN PERCENTAGE OF SITES WITHIN BASIN

100

PERCENTAGE OF SITES WITHIN BASIN PERCENTAGE OF SITES WITHIN BASIN

B.

C.

D.

A.

PERCENTAGE OF BASIN, AGRICULTURE

DRAINAGE AREA (SQUARE KILOMETERS)

PERCENTAGE OF BASIN, FOREST

PERCENTAGE OF BASIN, URBAN

0

20

40

60

80

0

20

40

60

80

100

0-50 51-1,000 >1,000-15,000

0-25 26-50 51-75 76-100

0

20

40

60

80

0-25 26-50 51-75 76-100

0-25 26-50 51-75 76-100

0

20

40

60

80

100

Headwater Wadeable

Boat

Figure 2. Graphs showing the percentage of drainage area and 

agricultural, forest, and urban land use in the Upper Wabash River 

Basin, Indiana, 2003.
Drainage Area and Land-Use Analysis 13

Table 4. Basin characteristics of the 38 algal-biomass sampling sites in the Upper Wabash River Basin, Indiana, May through October 2003.—ContinuedIDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; ID, identification number; USEPA, U.S. Environmental Protection Agency; FT, feet; US, upstream; trib, tributary; CR, county road; R, River; SR, state route; DS, downstream; CK, Creek; FK, Fork; DCH, Ditch; NR, near; AV, Avenue; ST, Street; N, North; S,South; E, East; W, West; USHWY, U.S. Highway; IN, Indiana; OH, Ohio]

Short IDEM 

site number1 USGS station name Drainage area (km2) Land use (percentage)2 Ag Forest Urban Other

601 UNNAMED TRIB 100 FT US PIPE CK AT SANTA FE, IN 359.7 96.2 2.3 1.1 0.4602 HARSHMAN CK 0.9 MI US MISSISSINEWA R NR SALEM, IN 33.6 95.7 2.8 .5 1.0603 GALBREATH DITCH 0.5 MI DS CR 200N NR LK CICOTT, IN 6.1 73.5 25.2 .1 1.2604 MISSISSINEWA R 0.8 MI US SR 26 NR FOWLERTON, IN 1,251.2 91.2 5.8 1.5 1.5605 BIG MONON CK 0.5 MI US SR 16 NR MONON, IN 87.4 91.4 6.3 0 2.3606 PAW PAW CK 0.3 MI US CR 800N NR ROANN, IN 93.8 94.7 4.2 .4 .7607 S FK WILDCAT CK 0.6 MI US CR 800W NR MULBERRY, IN 460.1 94.8 1.6 2.5 1.1608 MUD CK 0.3 MI US GRUBE DITCH NR PERSHING, IN 131.2 94.6 3.1 .6 1.7609 MEYERS DITCH 820 FT US I-65 NR BADGER GROVE, IN 1.4 96.5 .4 3.0 .1610 CLEAR CK 980 FT US SPRING CK AT SOUTH WHITLEY, IN 24.9 82.0 14.7 0 3.3611 LITTLE PIPE CK 900 FT DS CR 150E NR SOUTH PERU, IN 28.4 93.9 5.2 0 .9612 ROCK CK 770 FT US CR 900S NR PETROLEUM, IN 7.6 96.0 3.8 0 .2613 DEER CK 0.7 MI US MERIDIAN LINE RD NR CAMDEN, IN 524.6 95.8 2.6 .7 .9614 EIGHTMILE CK 0.4 MI US SR 1 AT OSSIAN, IN 64.2 96.1 3.2 .1 .6615 DEER CK 0.6 MI DS SR 75 AT CAMDEN, IN 583.0 95.9 2.5 .7 .9616 EASTERDAY DITCH 0.3 MI DS CR 400N NR ATWOOD, IN 14.7 88.4 8.7 .1 2.8617 E FK LITTLE WILDCAT CK 880 FT US CR200W AT ALTO, IN 24.0 86.0 2.7 9.4 1.9618 WILSON RHODES DITCH 0.6 MI US CR850N NR ROANN, IN 12.1 91.3 8.7 0 0619 UNNAMED TRIB 1.2 MI US EEL R AT MEXICO, IN 4.2 92.2 6.8 0 1.0621 MILL CK 600 FT US HINES DITCH NR MARSHTOWN, IN 77.8 95.5 2.5 .5 1.5622 STONY CK 720 FT US MERIDIAN RD AT PEABODY, IN 24.9 94.4 3.9 .6 1.2623 MID FK WILDCAT CK 0.4 MI DS HOG RUN AT PETTIT, IN 289.1 97.1 1.7 .5 .7624 TIPPECANOE R 880 FT DS CR 100E NR ISLAND PARK, IN 317.3 76.2 11.3 1.6 10.9625 WABASH R 1.9 MI US SR 39 NR DELPHI, IN 10,652.1 88.2 7.7 1.9 2.2626 ROCK CK 0.4 MI US SR 3 NR MARKLE, IN 240.8 96.3 3.1 .1 .5
14 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003 Table 4. Basin characteristics of the 38 algal-biomass sampling sites in the Upper Wabash River Basin, Indiana, May through October 2003.—Continued IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; ID, identification number; USEPA, U.S. Environmental Protection Agency; FT, feet; US, upstream; trib, tributary; CR, county road; R, River; SR, state route; DS, downstream; CK, Creek; FK, Fork; DCH, Ditch; NR, near; AV, Avenue; ST, Street; N, North; S,South; E, East; W, West; USHWY, U.S. Highway; IN, Indiana; OH, Ohio] Short IDEM site number1 USGS station name Land use (percentage)2 Drainage area (km2

) Ag Forest Urban Other

627 KILMORE CK 500 FT US CR 800E NR FOREST, IN 120.2 97.9 0.7 0.6 0.8628 MILL CK 0.6 MI US TIPPECANOE R NR WINAMAC, IN 233.2 96.0 2.4 .4 1.2629 UNNAMED TRIB 0.8 MI US BURNETTS CK AT LOCKPORT, IN 8.0 87.7 10.6 0 1.7630 DETAMORE DITCH 700 FT DS I-69 NR WARREN, IN 12.9 96.2 2.3 1.0 .5631 LITTLE MISSISSINEWA R 2.8 MI DS SR28 NR COSMOS, OH 44.0 92.7 2.3 4.2 .8632 UNNAMED TRIB 450 FT US GAFF DITCH NR ETNA, IN 3.2 96.2 1.0 0 2.8633 DEER CK 0.25 MI US CR 100W NR WEAVER, IN 76.0 97.6 1.1 .2 1.1634 ELKHORN CK 0.25 MI US CR 500N NR FARMLAND, IN 9.1 95.8 3.1 .2 .9635 BIG MONON CK 0.2 MI US BIG MONON DITCH NR MONON, IN 8.7 96.0 3.4 0 .6636 LITTLE SALAMONIE R 0.4 MI US CR 120S AT LIBER, IN 50.6 88.2 9.8 .1 1.9637 TRAVERS DITCH 560 FT DS CR1000W NR GRASS CREEK, IN 3.5 88.5 8.6 0 2.9638 ABOITE CK 0.3 MI US USHWY 24 AT TIMBERCREST, IN 132.6 86.1 8.9 2.1 2.9639 WILDCAT CK 150 FT US SR 25 AT LAFAYETTE, IN 2,064.8 91.9 3.2 3.4 1.5

Minimum 1.4 73.5 .3 0 .1Maximum 10,652.1 97.9 25.2 9.4 10.9Mean 475.8 92.2 5.2 1.0 1.6Median 57.4 94.7 3.2 .4 1.1Standard deviation 1,740.1 5.7 4.8 1.8 1.8

1The full IDEM site number has a prefix of INRB, for example INRB03-601.2Land-use percentages were determined by the U.S. Geological Survey using the National Land Cover Dataset (U.S. Geological Survey, 2000).
Comparison of the Data to Ecoregion Nutrient Criteria 15

Comparison of the Data to Ecoregion 

Nutrient Criteria

The values for nutrients (TKN, nitrate, TN, and TP) and 

CHLa (periphyton and seston) were compared to published 

USEPA values for the respective ecoregions. A comparison of 

the values from the IDEM/USGS and USEPA data sets was 

done to (1) determine whether USEPA data, which were used 

to set the nutrient water-quality criteria, and the IDEM/USGS 

data had similar ranges of values and (2) determine if the 

streams in Indiana would exceed the proposed USEPA 

25th-percentile nutrient water-quality criteria for the ecoregions. Of the 38 sites sampled, 28 sites on 24 streams were in 

Ecoregion VI; 10 sites on 10 streams were in Ecoregion VII 

(fig. 1, table 1). Of the 24 streams in Ecoregion VI, 22 were in 

Level III Ecoregion 55; the 3 remaining streams were in Level 

III Ecoregion 54. Because only three streams were in Ecoregion 54, no comparison was made with published USEPA 

values. The 10 streams sampled in Ecoregion VII were in 

Level III Ecoregion 56. 

The IDEM/USGS values for TKN fell within or were 

close to the range of the published values for Ecoregions VI, 

VII, 55, and 56 (table 5). The IDEM/USGS 25th-percentile 

TKN values were greater than the published values for Ecoregions VI, VII, 55, and 56. New maximum concentrations were 

measured for Ecoregions VII and 56 and were about 5 and 10 

times the published USEPA values, respectively. If the proposed USEPA TKN water-quality criteria for Ecoregions VI 

and VII were enacted, 69.6 percent of the individual samples 

collected in Ecoregion VI and 100 percent in Ecoregion VII 

would have exceeded those criteria.

The IDEM/USGS values for nitrate fell within, or were 

close to, the range of published values for Ecoregions VI, VII, 

55, and 56 (table 6). The IDEM/USGS 25th-percentile nitrate 

values were greater than the published values for Ecoregions 

VI, VII, 55, and 56. A new maximum concentration was 

measured for Ecoregion 56. If the proposed USEPA nitrate 

water-quality criteria for Ecoregions VI and VII were enacted, 

93.9 percent of the individual samples collected in Ecoregion 

VI and 93.1 percent in Ecoregion VII would have exceeded 

those criteria.

Table 5. Total Kjeldahl nitrogen as N values collected in 2003 from the Indiana Department of Environmental Management/U.S. 

Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and VII 

and Level III Ecoregions 55 and 56.

[All data except number of streams are total Kjeldahl nitrogen as nitrogen values in milligrams per liter; N, nitrogen; IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; bold text indicates value exceeds published U.S. Environmental Protection Agency value for Level III Ecoregions 55 and 56 reference conditions, all seasons; shading indicates value exceeds published U.S. Environmental Protection Agency value for Aggregate Nutrient Ecoregions VI and VII, all seasons; nc, not calculated in U.S. Environmental Protection Agency Nutrient 

Criteria documents]

Statistic

IDEM/

USGS 

values

USEPA 

values, 

all seasons1

IDEM/

USGS 

values

USEPA 

values, 

all seasons

IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 563

Number of streams 25 628 23 199 10 705 50

Minimum value .330 .025 .330 .050 .420 .000 .110

10th percentile .380 nc .480 nc .600 nc nc

25th percentile .600 .591 .600 .400 .830 .240 .580

50th percentile .690 nc .690 nc .925 nc nc

75th percentile .895 nc .920 nc 1.80 nc nc

90th percentile 1.10 nc 1.10 nc 12.7 nc nc

Maximum value 1.60 4.50 1.60 3.50 23.0 4.70 2.06

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.
16 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

The IDEM/USGS values for TN fell within, or were close 

to, the range of published values for Ecoregions VI, VII, 55, 

and 56 (table 7). The IDEM/USGS 25th-percentile TN values 

were greater than the published values for Ecoregions VI, VII, 

and 56. New maximum concentrations were measured for 

Ecoregions VII, 55, and 56 and new minimum values were 

measured for Ecoregions 55 and 56. If the proposed USEPA 

TN water-quality criteria for Ecoregions VI and VII were 

enacted, 82.3 percent of the individual samples collected in 

Ecoregion VI and 100 percent in Ecoregion VII would have 

exceeded those criteria.

The IDEM/USGS values for TP fell within, or were close 

to, the range of published values for Ecoregions VI, VII, 55, 

and 56 (table 8). The IDEM/USGS 25th-percentile TP value 

was greater than the published values for Ecoregion 55. New 

maximum concentrations were measured for Ecoregions VII 

and 56. If the proposed USEPA TP water-quality criteria for 

Ecoregions VI and VII were enacted, 52.4 percent of the individual samples collected in Ecoregion VI and 72.4 percent in 

Ecoregion VII would have exceeded those criteria.

The IDEM/USGS values for mean periphyton CHLa provide a new range for Ecoregions VI, VII, 55, and 56 (table 9). 

No comparison was made for Ecoregions VI or VII because 

of the lack of published values in the USEPA nutrient-criteria 

document (Environmental Protection Agency, 2000a,b).

The IDEM/USGS values for mean seston CHLa fell 

within the range of the published values for Ecoregions VI, 

VII, and 56 (table 10). The IDEM/USGS median seston CHLa 

values provide a new range of values for Ecoregion 55. The 

IDEM/USGS 25th-percentile median seston CHLa values were 

less than published values for Ecoregions VI, VII, and 56. If 

the proposed USEPA mean seston CHLa water-quality criteria 

for Ecoregions VI and VII were enacted, 37.8 percent of the 

individual samples collected in Ecoregion VI and 51.7 percent 

in Ecoregion VII would have exceeded those criteria.

Table 6. Nitrite plus nitrate as N values collected in 2003 from the Indiana Department of Environmental Management/

U.S. Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and 

VII and Level III Ecoregions 55 and 56.

[All data except number of streams are nitrite plus nitrate as nitrogen values in milligrams per liter; N, nitrogen; IDEM, Indiana Department of Environmental 

Management; USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; shading indicates value exceeds or equaled published U.S. 

Environmental Protection Agency value for Aggregate Nutrient Ecoregions VI and VII, all seasons; bold text indicates value exceeds or equals published U.S. 

Environmental Protection Agency value for Level III Ecoregions 55 and 56 reference conditions, all seasons; nc, not calculated in U.S. Environmental Protection 

Agency Nutrient Criteria documents]

Statistic

IDEM/

USGS 

values

USEPA 

values, 

all seasons1

IDEM/

USGS 

values

USEPA 

values, 

all seasons

IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 563

Number of streams 25 717 23 219 10 435 73

Minimum value .920 .010 .920 .025 .050 .003 .050

10th percentile 1.20 nc 1.20 nc .625 nc nc

25th percentile 2.00 .633 1.90 1.60 1.50 .300 .410

50th percentile 3.10 nc 3.10 nc 2.25 nc nc

75th percentile 4.10 nc 4.30 nc 3.60 nc nc

90th percentile 6.00 nc 6.00 nc 5.55 nc nc

Maximum value 8.00 10.7 8.00 8.13 6.60 9.54 6.45 

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.
Comparison of the Data to Ecoregion Nutrient Criteria 17

Table 7. Total nitrogen as N values collected in 2003 from the Indiana Department of Environmental Management/

U.S. Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and 

VII and Level III Ecoregions 55 and 56.

[All data except number of streams are total nitrogen as nitrogen values in milligrams per liter; N, nitrogen; IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; bold text indicates a new minimum value or exceeds the published 

U.S. Environmental Protection Agency value for Level III Ecoregions 55 and 56 reference conditions, all seasons; shading indicates a new minimum value or 

exceeds the published U.S. Environmental Protection Agency value for Aggregate Nutrient Ecoregions VI and VII, all seasons; nc, not calculated in U.S. Environmental Protection Agency Nutrient Criteria documents]

Statistic

IDEM/

USGS 

values

USEPA 

values, 

all seasons1 IDEM/

USGS 

values

USEPA 

values, 

all seasons IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 563

Number of streams 25 77 23 2 10 125 5

Minimum value 1.70 .885 1.70 3.63 .828 .100 .900

10th percentile 2.09 nc 2.09 nc 1.43 nc nc

25th percentile 2.64 2.18 2.48 3.63 2.18 .540 1.10

50th percentile 3.74 nc 3.71 nc 2.92 nc nc

75th percentile 5.27 nc 5.44 nc 5.80 nc nc

90th percentile 6.64 nc 6.64 nc 17.9 nc nc

Maximum value 7.60 10.1 7.60 3.78 26.7 7.94 2.55 

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.
18 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

Table 8. Total phosphorus as P values collected in 2003 from the Indiana Department of Environmental Management/

U.S. Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and 

VII and Level III Ecoregions 55 and 56.

[All data except number of streams are total phosphorus as phosphorus values in milligrams per liter; P, phosphorus; IDEM, Indiana Department of Environmental Management; USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; bold text indicates value exceeds published U.S. Environmental Protection Agency value for Level III Ecoregions 55 and 56 reference conditions, all seasons; shading indicates value exceeds published U.S. Environmental Protection Agency value for Aggregate Nutrient Ecoregions VI and VII, all seasons; nc, not calculated in U.S. Environmental Protection Agency Nutrient 

Criteria documents]

Statistic

IDEM/

USGS 

values

USEPA values, 

all seasons1

IDEM/

USGS 

values

USEPA values, 

all seasons IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 563

Number of streams 25 815 23 225 10 910 64

Minimum value .015 .005 .015 .010 .015 .001 .004

10th percentile .041 nc .043 nc .015 nc nc

25th percentile .069 .076 .069 .063 .015 .033 .031

50th percentile .082 nc .085 nc .070 nc nc

75th percentile .110 nc .130 nc .082 nc nc

90th percentile .275 nc .275 nc 1.55 nc nc

Maximum value .390 2.23 .390 1.82 2.00 1.72 .300

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.
Comparison of the Data to Ecoregion Nutrient Criteria 19

Table 9. Mean periphyton chlorophyll a values collected in 2003 from the Indiana Department of Environmental Management/U.S. 

Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and VII 

and Level III Ecoregions 55 and 56.

[All data except number of streams are mean periphyton chlorophyll a values in milligrams per square meter; IDEM, Indiana Department of Environmental 

Management; USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; nd, no data collected or published in U.S. Environmental Protection Agency Nutrient Criteria documents] 

Statistic

IDEM/

USGS 

values

USEPA 

values, 

all seasons1

IDEM/

USGS 

values

USEPA 

values, 

all seasons IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 

563

Number of streams 25 nd 23 nd 10 nd nd

Minimum value 3.28 nd 3.28 nd 2.69 nd nd

10th percentile 9.10 nd 9.10 nd 3.08 nd nd

25th percentile 28.5 nd 22.7 nd 4.69 nd nd

50th percentile 46.4 nd 46.4 nd 23.1 nd nd

75th percentile 60.0 nd 60.0 nd 41.2 nd nd

90th percentile 89.2 nd 89.2 nd 92.9 nd nd

Maximum value 143 nd 143 nd 107 nd nd

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.
20 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

Table 10. Mean seston chlorophyll a values collected in 2003 from the Indiana Department of Environmental Management/U.S. 

Geological Survey study and the published U.S. Environmental Protection Agency values for Aggregate Nutrient Ecoregions VI and VII 

and Level III Ecoregions 55 and 56.

[All data except number of streams are mean seston chlorophyll a values in micrograms per liter; IDEM, Indiana Department of Environmental Management; 

USGS, U.S. Geological Survey; USEPA, U.S. Environmental Protection Agency; nd, no data collected or published in U.S. Environmental Protection Agency 

Nutrient Criteria documents; nc, not calculated in U.S. Environmental Protection Agency Nutrient Criteria documents]

Statistic

IDEM/

USGS 

values

USEPA 

values, 

all seasons1 IDEM/

USGS 

values

USEPA 

values, 

all seasons IDEM/

USGS 

values

USEPA values, 

all seasons

Aggregate 

Nutrient

Ecoregion VI2

Level III 

Ecoregion 552

Aggregate 

Nutrient

Ecoregion VII3

Level III 

Ecoregion 563

Number of streams 25 63 23 nd 10 55 21

Minimum value .403 .250 .403 nd .630 .330 .330

10th percentile .543 nc .543 nc .651 nc nc

25th percentile 1.01 2.7 .677 nd .767 1.54 3.50

50th percentile 2.02 nc 2.19 nd 1.68 nc nc

75th percentile 3.97 nc 4.55 nd 2.65 nc nc

90th percentile 10.3 nc 10.3 nd 4.45 nc nc

Maximum value 35.7 47.6 35.7 nd 6.24 36.4 36.4 

1

Values are the median of all seasons for all samples collected from a single stream.

2

U.S. Environmental Protection Agency, 2000a.

3

U.S. Environmental Protection Agency, 2000b.

Conclusions

Excessive inputs of nutrients into streams have humanhealth, economic, and ecological consequences. In 2000, the 

USEPA proposed nutrient water-quality criteria to protect 

streams from excess nutrients. This report is one of several 

reports done as a cooperative effort between IDEM and the 

USGS to collect new data to assist IDEM in determination of 

nutrient criteria. Data were gathered from May through October 2003 at 38 randomly selected sites in the Upper Wabash 

River Basin, Indiana, for algal biomass, habitat, nutrients, 

and biological communities (fish and invertebrates). Basin 

characteristics (land use and drainage area) and biologicalcommunity attributes and metric scores were determined for 

the basin of each sampling site. The yearly Principal Components Analysis site scores were calculated for algal biomass 

(periphyton and seston). The yearly Principal Components 

Analysis site scores for the first axis (PC1) were related using 

Spearman’s rho to the seasonal algal-biomass, basin-characteristics, habitat, seasonal nutrient, and biological-community 

attribute and metric score data.

Of the 43 fish-community attributes and metrics examined, the periphyton PC1 was negatively related to one fishcommunity attribute (tolerant percent) and positively related to 

one fish-community metric score (insectivore percent metric 

score). Of the 21 invertebrate-community attributes and metrics examined the periphyton PC1 site score was negatively 

related to three attributes (EPT count, EPT-to-total ratio, and 

number of taxa) and four metric scores (EPT count metric 

score, EPT-to-total ratio metric score, macroinvertebrate Index 

of Biotic Integrity metric score, and number of taxa metric 

score). This study in the Upper Wabash River in 2003 was 

the first time that invertebrates were found to be significantly 

related to the periphyton PC1 site scores when compared with 

similar studies conducted by IDEM and the USGS in the West 

Fork White River in 2001 and the Whitewater and East Fork 

White River Basins in 2002.

There were no statistically significant relations between 

the periphyton PC1 site score and the five basin characteristics, nine habitat, and 12 nutrient variables examined. The lack 

of relations between nutrients and periphyton algal biomass 

can possibly be explained by several factors including scouring of algal growth by increased streamflow that causes algal 

growth to restart in the stream, annual and seasonal differences 

in storm events that can allow for “wet” and “dry” years with 

high levels of algal biomass corresponding to high and low 

nutrients depending upon the season, shading from canopy 

cover and turbidity, and grazing by snails, invertebrates, and 

fish.
Conclusions, Acknowlegments, and References 21

The seston PC1 site score, which was most influenced by 

July CHLa, had no significant relations among the five basin 

characteristics, nine habitat, and 12 nutrient variables examined, or the 43 fish- and 21 invertebrate-community attributes 

and metric scores examined. The lack of relations among 

seston and all environmental and biological-community 

attributes and metric scores may be attributed to the lack of 

large streams in this study. Additionally, the lack of relations 

between nutrients and seston and periphyton algal biomass in 

this study and similar studies conducted by IDEM and USGS 

in 2001 and 2002 suggests that biological communities may be 

useful in measuring nutrient enrichment in streams. Periphyton algae may be more important than seston algae in relating algal biomass with biological communities because the 

periphyton algae had more significant relations with fish and 

invertebrate attributes and metric scores than seston algae.

To understand how the choice of sampling sites might 

have affected the results, an analysis of the drainage area and 

land use was done. The random site selection in 2003 in the 

Upper Wabash River Basin was skewed to small streams. 

Forty-seven percent of the basins were less than 50 km2

(headwater streams); 45 percent ranged between 501 and 

1,000 km2 (wading streams). The lack of large streams in the 

Upper Wabash River Basin could account for the few relations 

among the seston and the biological-community data. Every 

site except one in the Upper Wabash River Basin had basins 

that consisted of more than 75 percent agricultural land use. 

This lack of an agricultural-land use gradient potentially could 

mask relations between the PC1 (periphyton and seston) and 

the environmental variables and biological-community (fish 

and invertebrates) attributes and metric scores.

The values for nutrients (nitrate, TKN, TN, and TP) and 

CHLa (periphyton and seston) were compared to published 

USEPA values for the respective ecoregions. CHLa (periphyton and seston) values either were greater than 25th-percentile 

published USEPA values or extended data ranges in Aggregate 

Nutrient and Level III Ecoregions. If the values for the 25th

percentile proposed by the USEPA were adopted as nutrient 

water-quality criteria, the percentage of samples in the Upper 

Wabash River Basin that would have exceeded these criteria 

ranged from 37.8 percent in Ecoregion VI for seston CHLa to 

100 percent in Ecoregion VII for TKN and TN.

Acknowledgments

The authors thank Indiana Department of Environmental Management and U.S. Geological Survey personnel for 

collecting and processing the data. We also thank Patricia H. 

Long of the USGS for editorial support and James Petersen 

(USGS), Jill Frankforter (USGS), and Dennis Clark (IDEM) 

for their technical reviews of the manuscript. Projects of this 

type are a team effort and could not have been completed 

without the efforts of the IDEM and USGS personnel, which 

are sincerely appreciated.

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24 Relations of PCA Site Scores in the Upper Wabash River Basin, Indiana, 2003

Appendix 1. Metrics used by Indiana Department of Environmental Management for habitat, fish, and invertebrates (Dufour, 2002).

Metrics1 Definition

Habitat 

SubstrateScore A metric to evaluate substrate type, origin, silt cover, and embeddedness.

InstreamCoverScore Instream cover types and the amount (availability) of instream cover.

ChannelMorphologyScore Quality of the stream channel related to the creation and stability of instream habitat (channel 

sinuosity, channel development, channelization, stability, and modifications).

RiparianZoneandBankErosionScore Quality of the riparian buffer zone and flood-plain vegetation, looking at riparian width, 

predominant surrounding land uses, and bank-erosion status.

PoolGlideQualityScore Quality of pool/glide taking into account maximum pool depth, morphology, and velocity.

RiffleRunQualityScore Quality of riffle/run, taking into account riffle/run depth, substrate, and embeddedness.

GradientScore A measure of the influence of gradient and stream size on the biological community and available 

habitat.

Fish2

SpeciesCount Number of species, excluding hybrid species (exclude gizzard shad if in the Wabash River 

mainstem and drainage area is greater than 5,180 square kilometers).

DMS_SpeciesCount Number of darter, madtom, and sculpin species, excluding hybrid species.

Darter_SpeciesCount Number of darter species, excluding hybrid species.

Headwater_Percent Percent of headwater individuals.

LargeRiver_Percent Percent of large river individuals (exclude gizzard shad if in the Wabash River mainstem and 

drainage area greater than 5,180 square kilometers).

Sunfish_SpeciesCount Number of sunfish species, excluding hybrid species.

Centrarchid_SpeciesCount Number of centrarchidae species, excluding hybrid species. 

Minnow_SpeciesCount Number of minnow species, excluding hybrid species.

Sucker_SpeciesCount Number of sucker species, excluding hybrid species. 

RoundBodySucker_SpeciesCount Number of round-body sucker species, excluding hybrid species.

Salmonid_SpeciesCount Number of salmonid species, excluding hybrid species.

Sensitive_SpeciesCount Number of sensitive species, excluding hybrid species.

Tolerant_Percent Percent of tolerant individuals (exclude gizzard shad if in the Wabash River mainstem and drainage 

area greater than 5,180 square kilometers).

Omnivore_Percent Percent of omnivore individuals (exclude gizzard shad if in the Wabash River mainstem and 

drainage area greater than 5,180 square kilometers).

Insectivore_Percent Percent of insectivore or invertivore individuals (exclude gizzard shad if in the Wabash River 

mainstem and drainage area greater than 5,180 square kilometers).

Pioneer_Percent Percent of pioneer individuals.

Carnivore_Percent Percent of carnivore or piscivore individuals (exclude gizzard shad if in the Wabash River 

mainstem and drainage area greater than 5,180 square kilometers).

CatchPerUnitEffort Catch per unit effort (CPUE) or total number of individuals.

CPUElessShads Catch per Unit Effort (CPUE), excluding the number of gizzard shad individuals if in the Wabash 

River mainstem and drainage area greater than 5,180 square kilometers).

SimpleLithophil_Percent Percent of simple lithophilic species (exclude gizzard shad if in the Wabash River mainstem and 

drainage area greater than 5,180 square kilometers).

DELT_Percent

Percent of individuals with deformities, eroded fins, lesions, and tumors (DELT), including 

multiple DELTs (exclude gizzard shad if in the Wabash River mainstem and drainage area 

greater than 5,180 square kilometers).
Appendix 1 25

Appendix 1. Metrics used by Indiana Department of Environmental Management for habitat, fish, andinvertebrates (Dufour, 2002).—

Continued

Metrics1 Definition

Invertebrates

Family Level HBI Summation of the tolerance value times the number of individuals for a specific family divided 

by the total count of individuals for families with a tolerance value.

Number of Taxa Number of families identified in the subsample.

Number of Individuals Total number of individuals for all families identified in the subsample.

Percent Dominant Taxa Highest number of individuals for a given family divided by the total number of individuals in 

the subsample.

EPT Index Total number of families represented in the orders Ephemeroptera, Plecoptera, and Trichoptera.

EPT Count Total number of individuals for orders Ephemeroptera, Plecoptera, and Trichoptera.

EPT Count to Total Number of Individuals Total number of individuals for orders Ephemeroptera, Plecoptera, and Trichoptera divided by 

the total number of individuals in the subsample.

EPT Count to Chironomid Count Total number of individuals for orders Ephemeroptera, Plecoptera, and Trichoptera divided by 

the total number of chironomidae.

Chironomid Count Total number of chironomids in the subsample.

Total Number of Individuals to Number of 

Squares Sorted

Total number of individuals in the subsample divided by the number of squares needed to reach 

the total number of individuals.

1

Each of the fish- and invertebrate community metrics also has a corresponding attribute with the same name but consists of the raw data. 

2 Specific fish species associated with each metric can be found in Dufour, 2002. 

Leer, Frey, Caskey, and Lowe—Relations of Principal Components Analysis Site Scores to Algal-Biomass, Habitat, Basin-Characteristics, Nutrient, and 

Biological-Community Data in the Upper Wabash River Basin, Indiana, 2003—Scientific Investigations Report 2007–5231

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