U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2010–5164

Prepared in cooperation with the U.S. Environmental Protection Agency

and the Kentucky Energy and Environment Cabinet

Breakpoint Analysis and Relations of Nutrient and Turbidity 

Stressor Variables to Macroinvertebrate Integrity in Streams 

in the Crawford-Mammoth Cave Uplands Ecoregion, Kentucky, 

for the Development of Nutrient Criteria
Cover photograph: Hardins Creek at Stone Hill Fork near Hardinsburg, Kentucky 

(USGS STAID 03303202), August 27, 2007. (Photograph by Kentucky Division of Water.)
Breakpoint Analysis and Relations of Nutrient and 

Turbidity Stressor Variables to Macroinvertebrate 

Integrity in Streams in the Crawford-Mammoth Cave 

Uplands Ecoregion, Kentucky, for the Development 

of Nutrient Criteria

By Angela S. Crain and Brian J. Caskey

Prepared in cooperation with the U.S. Environmental Protection Agency

and the Kentucky Energy and Environment Cabinet

Series Name 2010–5164

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2010

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Suggested citation:

Crain, A.S. and Caskey, B.J., 2010, Breakpoint analysis and relations of nutrient and turbidity stressor variables 

to macroinvertebrate integrity in streams in the Crawford-Mammoth Cave Uplands Ecoregion, Kentucky, 

for the development of nutrient criteria: U.S. Geological Survey Scientific Investigations Report 2010—5164, 29 p.
iii

Contents

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

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

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

Description of Study Area ..........................................................................................................2

Study Design and Methods..........................................................................................................................5

Site Selection.........................................................................................................................................5

Sample Collection and Processing Methods...................................................................................5

Nutrients and Turbidity................................................................................................................5

Macroinvertebrate Communities ..............................................................................................5

Data Analysis.........................................................................................................................................5

Distribution of the Nutrient and Turbidity Variables.................................................................................6

Relations between the Nutrient and Turbidity Stressor Variables ........................................................7

Macroinvertebrate Community Attributes, Relations Between Stressor and 

Response Variables, and Nutrient Breakpoints using

Macroinvertebrate Attributes ........................................................................................................7

Summary of Macroinvertebrate-Community Attributes.................................................................7

Distribution of the Macroinvertebrate Communities and Their Relations 

to Nutrient Stressor Variables...............................................................................................9

Macroinvertebrate-Community Breakpoints of the Nutrient Stressor Variables 

and Implications for Developing Nutrient Criteria in the 

Crawford-Mammoth Cave Uplands Ecoregion ................................................................12

Macroinvertebrate Communities and the Comparison of Trophic Classification 

for Nutrient Concentrations and Turbidity Values ...........................................................12

Summary and Conclusions.........................................................................................................................14

References Cited..........................................................................................................................................14

Appendixes ...................................................................................................................................................17

Figures

1. Map showing the location of the sampling sites used in the 

Crawford-Mammoth Cave Uplands Ecoregion breakpoint 

analysis study in Kentucky, 2007–08..........................................................................................3

2–4. Scatter plots showing:

2. Median total phosphorus concentrations, A, and median total nitrogen 

concentrations, B, to the total number of individuals for the 

Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study .......................10

3. Median total phosphorus concentrations to the number of Ephemeroptera, 

Plecoptera, and Trichoptera genus taxa for the Crawford-Mammoth Cave 

Uplands Ecoregion breakpoint analysis study.......................................................................11

4. Median total phosphorus concentrations to the average tolerance value 

for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study ..........11
iv

Tables

1. Names and locations of sampling sites used in the Crawford-Mammoth Cave 

Uplands Ecoregion breakpoint analysis study in Kentucky, 2007–08 ................................4

2. Summary statistics for nutrient and turbidity variables used in the 

Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis 

study in Kentucky, 2007–08........................................................................................................6

3. Absolute Kendall tau correlations among the median stressor variables 

used in the Crawford-Mammoth Cave Uplands Ecoregion breakpoint 

analysis study, 2007–08..............................................................................................................7

4. Abbreviations, definitions, and summary statistics for macroinvertebrate 

attributes and metrics for the Crawford-Mammoth Cave Uplands Ecoregion 

breakpoint analysis in Kentucky, 2007–08. .............................................................................8

5. Macroinvertebrate breakpoint summary (n=19) for the stressor variables

 used in the Crawford-Mammoth Cave Uplands Ecoregion breakpoint 

analysis study, 2007–08..............................................................................................................9

Conversion Factors

5. Boxplot showing distribution to the median total phosphorus and total 

nitrogen concentrations (presented on a log scale) compared to Dodds 

and others (1998) trophic classifications , and distribution of the median 

turbidity concentrations compared to Klein and others (2008) turbidity biological 

impact classification in selected datasets used in the Crawford-Mammoth Cave 

Uplands Ecoregion breakpoint analysis study, 2007–08.......................................................13

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch (in.)

kilometer (km) 0.6214 inch (in)

mile (mi) 1.609 kilometer (km) 

Area

square kilometer (km2

) 247.1 acre

square kilometer (km2

) 0.3861 square mile (mi2)

Volume

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

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 

25°C).

Concentrations of nutrients in water are given either in milligrams per liter (mg/L) or micrograms 

per liter (μg/L). Concentrations of turbidity are given in Formazin nephelometric units.
v

Abbreviations/Acronyms

CHLa chlorophyll a

EPT Ephemeroptera, Plecoptera, and Trichoptera

FNU Formazin nephelometric units

HBI Hilsenhoff’s Biotic Index

KDOW Kentucky Division of Water

N nitrogen

NO2

+NO3-N nitrite plus nitrate, as nitrogen

P phosphorus

r

2 coefficient of determination

τ Kendall tau statistics

TKN Total Kjeldahl nitrogen

TN total nitrogen

TNI Total Number of Individuals

TP total phosphorus

TR Taxa Richness

TV Tolerance Value

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

Acknowledgments

The authors would like to thank the Kentucky Division of Water (KDOW) 

and U.S. Geological Survey (USGS) scientists and technicians for their 

commitment to collecting and processing the data. We would also like 

to specifically thank Lara Panayotoff and John Brumley of KDOW for 

their help in the study design and technical review of the report; 

Elizabeth Ciganovich (USGS) for her technical editorial review; and 

Jeffrey W. Frey (USGS) and Timothy C. Willoughby (USGS) for their 

technical colleague reviews.
vi
Abstract

To assist Kentucky in refining numeric nutrient criteria 

in the Pennyroyal Bioregion, the U.S. Geological Survey 

and the Kentucky Division of Water collected and analyzed 

water chemistry, turbidity, and biological-community data 

from 22 streams throughout the Crawford-Mammoth Cave 

Upland ecoregion (U.S. Environmental Protection Agency 

Level IV Ecoregion, 71a) within the Pennyroyal Bioregion 

from September 2007 to May 2008. Statistically significant 

and ecologically relevant relations among the stressor (total 

phosphorus, total nitrogen, and turbidity) variables and 

response (macroinvertebrate-community attributes) variables 

and the breakpoint values of biological-community attributes 

and metrics in response to changes in stressor variables were 

determined. 

Thirteen of 18 macroinvertebrate attributes were significantly and ecologically correlated (p-value < 0.10) with 

at least one nutrient measure. Total number of individuals, 

Ephemeroptera-Plecoptera-Trichoptera richness, and average 

tolerance value were macroinvertebrate measures that most 

strongly correlated with the concentrations of nutrients. Comparison of the average macroinvertebrate-breakpoint value for 

the median concentration of total phosphorus (TP, 0.033 mg/L) and 

for median concentration of total nitrogen (TN, 1.1 mg/L) to 

Dodds’ trophic classification for TP and TN indicates streams 

in the Crawford-Mammoth Cave Uplands ecoregion within 

the Pennyroyal Bioregion would be classified as mesotrophiceutrophic. The biological breakpoint relations with median 

concentrations of TP in this study were similar to the U.S. 

Environmental Protection Agency proposed numeric TP criteria (0.037 mg/L), but were 1.5 times higher than the proposed 

numeric criteria for concentrations of TN (0.69 mg/L). No 

sites were impacted adversely using median turbidity values 

based on a 25 Formazin nephelometric turbidity unit biological threshold. The breakpoints determined in this study, in 

addition to Dodds’ trophic classifications, were used as multiple lines of evidence to show changes in macroinvertebrate 

community and attributes based on exposure to nutrients.

Introduction

In 1996, the U.S. Environmental Protection Agency’s 

(USEPA) National Water Quality Inventory identified excess 

amounts of nutrients as the second leading cause of impairment in rivers and streams and as the primary cause of impairments in lakes and reservoirs (U.S. Environmental Protection 

Agency, 1997). The excess amounts of nutrients, primarily 

nitrogen and phosphorus, are commonly cited as principal 

reasons why water bodies in Kentucky do not fully support 

their designated uses (Kentucky Energy and Environment 

Cabinet, 2008a). Many rivers and streams in Kentucky have 

been listed as impaired streams for nutrients in the state’s 

2008 Integrated Report to Congress on the Condition of Water 

Resources in Kentucky (Kentucky Energy and Environment 

Cabinet, 2008a). Recently, a model of the Mississippi River 

Basin found Kentucky was one of nine states that contributes 

the majority of nitrogen (N) and phosphorus (P) to the Gulf of 

Mexico, resulting in hypoxia of those waters (Alexander and 

others, 2008). Although nutrients are necessary for the growth 

of plants and animals, excessive amounts can be detrimental 

to aquatic ecosystems and to the health of organisms living 

in and using water and can limit human uses of a water body. 

For example, elevated concentrations of nutrients can lead to 

excessive aquatic plant growth that can reduce the amount 

of dissolved oxygen in the water and alter the stream habitat, 

both of which are critical for fish and other aquatic life. Excessive aquatic plant growth also can interfere with recreational 

activities, such as fishing, swimming, and boating, and can 

cause unpleasant taste and odors in drinking water.

In 1998, a new initiative to the Clean Water Act called 

the Clean Water Action Plan was launched to direct states, in 

conjunction with the USEPA, to develop numeric criteria for 

total phosphorus and total nitrogen (causal variables) along 

with chlorophyll a (CHLa) and turbidity (response variables) 

(U.S. Environmental Protection Agency, 1998). Although individual states are responsible for establishing their own waterquality criteria, the USEPA recommended ecoregional nutrient 

water-quality criteria as a baseline and has provided guidance 

Breakpoint Analysis and Relations of Nutrient and Turbidity 

Stressor Variables to Macroinvertebrate Integrity in Streams 

in the Crawford-Mammoth Cave Uplands Ecoregion, Kentucky, 

for the Development of Nutrient Criteria

By Angela S. Crain and Brian J. Caskey
2 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

to states for establishing more precise numeric water-quality 

criteria for nutrients to protect aquatic life and recreational and 

other uses of rivers and streams on a site-specific or subregionspecific basis (U.S. Environmental Protection Agency, 2000a 

and b). The USEPA-recommended ecoregional nutrient criteria 

are based on USEPA Aggregated Nutrient Ecoregions that are 

areas with similar geographic features including topography, 

soils, geology, land use, and biogeography (U.S. Environmental Protection Agency, 2000c and d). The USEPA established 

recommended nutrient criteria for nitrate as nitrogen (NO3-N), 

total Kjeldahl nitrogen as nitrogen (TKN-N), total nitrogen 

(TN), and total phosphorus (TP) based on either the 25th

percentile of median concentrations at all sampled sites or the 

75th percentile when only reference-site concentration data 

are used. Discussions of the USEPA numeric nutrient criteria 

relevant to this study are limited to TP, TN, and turbidity.

Kentucky is subdivided into seven Level III Ecoregions: 

Southwestern Appalachians, Central Appalachians, Western 

Allegheny Plateau, Interior Plateau, Interior River Valley and 

Hills, Mississippi Alluvial Plain, and Mississippi Loess Plain 

(Woods and others, 2002). Because Kentucky has a diverse 

topography, an empirical regional classification scheme based 

on river basins, physiographic regions, and ecoregions was 

developed by the Kentucky Division of Water (http://www.

epa.gov/wed/pages/ecoregions/ky_eco.htm#Principal%20

Authors, Woods and others, 2002) to help clarify inherent 

biological differences in the state (Pond and others, 2003; 

Pond and McMurray, 2002; and Pond and others, 2000). 

For macroinvertebrates, four bioregions have been established: 

Bluegrass, Mountains, Mississippi Valley-Interior River, and 

Pennyroyal. Only the Crawford-Mammoth Cave Uplands 

(USEPA Level IV Ecoregion, 71a) within the Pennyroyal 

Bioregion was examined in this study.

Although nutrients are nontoxic to biological organisms 

at most ambient concentrations (Miltner and Rankin, 1998), 

relations between nutrient concentrations (causal variables) 

and biological attributes (response variables) are important for 

evaluating the effects of eutrophication on river and stream 

ecosystems. In a typical stressor-response relation, a change in 

a stressor (causal) variable (TP and TN, in the case of nutrients) results in a corresponding change in a response variable. 

Significant relations between nutrients and algal biomass have 

been found in streams (Biggs, 2000; Dodds and others, 2002; 

Stevenson and others, 2006). Robertson and others (2006) 

found significant relations between nutrients and periphyton 

CHLa in Wisconsin. However, other recent studies in nutrient-rich streams have found no significant relations or weak 

relations between nutrient concentrations and algal biomass 

(Figueroa-Nieves and others, 2006; Caskey and others, 2007; 

Frey and others, 2007; Leer and others, 2007; Lowe and 

others, 2008; Royer and others, 2008). The mixed results of 

these studies suggest that relations between nutrient concentrations and CHLa may not be applicable elsewhere and that 

developing nutrient-criteria for rivers and streams in nutrientrich areas may be problematic. In addition, nutrient thresholds 

based on relations between nutrients and algae may not be 

the same as those between nutrients and macroinvertebrates, 

because macroinvertebrates are indirect consumers of nutrients 

and generally are the direct indicators of stream health (Wang 

and others, 2007). Thus, using other biological community 

attributes and metrics (that is, macroinvertebrate communities) 

could be helpful in developing nutrient criteria for rivers and 

streams. 

Statistical analyses that directly relate eutrophication 

(stressor) to biological indicators or attributes can be used to 

develop ecologically meaningful nutrient criteria. Previous 

nutrient and algal biomass studies have shown mixed results 

because linear statistical techniques were applied to nonlinear data. Instead, nonlinear statistical techniques (breakpoint 

analysis) may be better at discerning the relations between 

nontoxic stressor and response variables and identifying 

breakpoints (Qian and others, 2003). Development of defensible nutrient criteria for rivers and streams in nutrient-rich areas 

will be complex and require intensive data analysis.

In 2007, the U.S. Geological Survey (USGS), the 

USEPA, and the Kentucky Energy and Environment Cabinet–Kentucky Division of Water began a cooperative study 

to identify statistically significant and ecologically relevant 

relations between casual variables (TP, TN, and turbidity) and 

response variables (macroinvertebrate-community attributes) 

at 22 sampling sites in the Crawford-Mammoth Cave Uplands 

ecoregion (USEPA Level IV Ecoregion, 71a) within the 

Pennyroyal Bioregion as defined by the Kentucky Division of 

Water (Kentucky Energy and Environment Cabinet, 2003). 

Purpose and Scope

This report summarizes the statistically significant and 

ecologically relevant relations between the causal variables 

(TP, TN, and turbidity) and the biological response variables 

(macroinvertebrate-community attributes) in the CrawfordMammoth Cave Uplands ecoregion within the Pennyroyal 

Bioregion. Breakpoint analysis was used to detect the concentration of the stressor variables where there was a significant 

change in the biological response variables. Results of the 

breakpoint analysis will provide the Kentucky Division of 

Water with relevant information that could be useful for evaluating which potential macroinvertebrates indicators are most 

appropriate for the development of nutrient criteria for rivers 

and streams in the Pennyroyal Bioregion.

Description of Study Area

The 22 sampling sites for this study were selected from 

the Kentucky Division of Water’s Reference Reach Program 

and are located within the Crawford-Mammoth Cave Uplands 

ecoregion (part of the Pennyroyal Bioregion). Thirteen sampling sites were located northeast of Bowling Green, Ky., and 

nine sites were located west and northwest of Bowling Green, 

Ky (fig.1). Sampling-site drainage areas ranged from 6.99 km2

to 101 km2 (table 1). 
Abstract

3Figure 1. Location of the sampling sites used in the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study in Kentucky, 2007–08.

#* #* #* #* #* #* #* #* #* #* #* #* #* #* #*#*#* #* #*#*#* #* HC MC N C CC FC GR BF

WF

YBC

LMC

BRB

BEAR

TWR

ELC

LBDC

UTMF

DC

LC

BDC

B C

NFRR

BRBW

Ohio Riv er

Oh io River

0 20 10 MILES

Base from U.S. Geological Survey digital data, 2004, NAD83, Lambert Conformal Conic

EXPLANATION

#Sampling sites and identifiers

!

!

Frankfort Louisville

KENTUCKY

Paducah

0 25 12.5 KILOMETERS

86° 87° 88°

38°

37°

Study areaRiver or stream
4 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Table 1. Names and locations of sampling sites used in the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study in Kentucky, 2007–08. [Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geo- logical Survey; STAID, station identification; KDOW, Kentucky Division of Water; dd.mm.ss, degrees minutes seconds; KY, Kentucky; UT, unnamed tributary; km2, square kilometers] Sampling site name (refer to figure 1 for location) USGS STAID KDOW stream name County Latitude (dd.mm.ss) Longitude (dd.mm.ss) Site type2 Drainage area (km2

)

BC1 03320300 Buck Creek 0.3 mi below KY 189 below 2nd UT Christian 36 58 57 -87 20 56 Wadeable 16.1

BDC 03311600 Beaverdam Creek at KY 101 and KY 259 Edmonson 37 09 14 -86 13 34 Wadeable 28BEAR 03311800 Bear Creek at St. Augustine Church Road near Clarkson Grayson 37 26 52 -86 14 18 Wadeable 27.5BF 03320100 Buck Fork Pond River above KY 507 off Flat Rock Road Todd 36 55 20 -87 15 18 Wadeable 41.2

BRB 03317940 Big Run Branch at Sunbeam Road near Leitchfield Grayson 37 30 13 -86 20 27 headwater 11.9

BRBW 03317395 Big Run Branch at Watson School Road near Leitchfield Grayson 37 30 28 -86 19 35 headwater 6.99

CC 03384330 Crooked Creek below Turkey Knob Road Crittenden 37 24 58 -88 04 03 Wadeable 101

DC 03303203 Dorridge Creek at KY 1385 near Hardinsburg Breckinridge 37 49 50 -86 26 49 Wadeable 42.3

ELC 03316110 Elk Lick Creek 0.2 miles below Epleys-Stuart Church Road bridge Todd 36 55 21 -86 57 45 Wadeable 59.3FC 03320200 Forbes Creek 0.1 miles above SR 189 bridge Christian 36 58 15 -87 19 23 Wadeable 25.9

GR 03315180 Gasper River 0.2 miles above Bucksville Road bridge Logan 36 56 10 -86 43 33 Wadeable 68.1

HC 03303202 Hardins Creek at Stone Hill Fork near Hardinsburg Breckinridge 37 47 46 -86 27 49 Wadeable 18.4

LBDC 03311700 Little Beaverdam Creek on Pleasant Grove Road Warren 37 08 04 -86 21 13 Wadeable 34.4LC 03316870 Linders Creek at KY 920 near White Mills Hardin 37 37 47 -86 11 19 Wadeable 72.3

LMC 03316900 Little Meeting Creek at Limp Road near Leitchfield Grayson 37 35 40 -86 13 04 Wadeable 31.6

MC1 03316885 Meeting Creek at KY 920 near Big Clifty Grayson 37 34 34 -86 12 59 Wadeable 69.2

NC 03310340 Nosey Creek at Mt. Zion Road near Millerstown Grayson 37 27 48 -86 04 34 Wadeable 23.8

NFRR 03317400 North Fork Rough River at KY 1073 near Hardinsburg Breckinridge 37 42 28 -86 20 49 Wadeable 28.2

TWR1 03382550 Tradewater River at Sparkman Road Christian 36 58 40 -87 30 44 Wadeable 57

UTMF 03320720 UT McFarland Creek 0.1 miles below Flowers Road Christian 37 03 58 -87 26 45 Wadeable 17.3

WF 03437450 Warrens Fork above Vaughns Grove Road Christian 36 51 48 -87 19 31 Wadeable 14.2

YBC 03302500 Yellowbank Creek at Cart-Manning Crossing Road Wildlife Management Area Breckinridge 37 59 44 -86 28 56 Wadeable 40.9

1 Macroinvertebrate community data not collected2 Site type based on Kentucky criteria for the size of drainage area.
Study Design and Methods 5

Nutrients and Turbidity

Nutrient samples (ammonia as nitrogen (NH3-N), total 

Kjeldhal nitrogen as nitrogen (TKN), nitrite plus nitrate as 

nitrogen (NO2+NO3-N), and TP were collected 9–11 times 

per sampling site from September 2007 to May 2008 by 

USGS personnel following approved Kentucky Division of 

Water water-quality monitoring methods (Kentucky Energy 

and Environment Cabinet, 2005). The sample-collection 

method used in this study was the nonisokinetic dip-sampling 

method. All nutrient samples were preserved with sulfuric 

acid, placed on ice, and transported to the Kentucky Division 

of Environmental Services Centralized Laboratory Facility 

for analysis. Nutrient samples were analyzed by colorimetric methods (U.S. Environmental Protection Agency, 1993). 

Concentrations of nutrients discussed in this report represent 

their concentrations expressed as either nitrogen or phosphorus. For example, a concentration of nitrate expressed at 

10 milligrams per liter (mg/L) refers to a concentration of 

nitrate as 10 mg/L as nitrogen. In addition, turbidity was measured during each site visit at the time of nutrient sampling by 

use of a YSI Inc., Model 6920 multi-parameter sonde outfitted 

with a YSI Inc. 6136 turbidity sensor. The multiparameter 

sondes were calibrated daily. A statistical summary of the 

concentration of nutrients and turbidity at each site is found in 

appendix 1.

Macroinvertebrate Communities

The macroinvertebrate communities were assessed by the 

Kentucky Division of Water in May 2008 at 19 of the 22 sites 

(Kentucky Energy and Environment Cabinet, 2008b). Community attributes and metric scores were calculated for the 

macroinvertebrate-community data, and metric scores were 

determined by Kentucky Division of Water personnel upon 

completion of the assessments (Kentucky Energy and Environment Cabinet, 2008b).

Data Analysis

Stressor variables (TP, TN, and turbidity) were analyzed 

to determine if a gradient (range) was observed, and then the 

observed data gradients were compared to published trophiclevel boundaries (Dodds and others, 1998), in the case of 

nutrients, and published biological impacts (Klein and others, 

2008), in the case of turbidity. The stressor variables were then 

correlated to the response variable (macroinvertebrate attributes) to identify the statistically and ecologically significant 

relations among the stressor and response variables. By use of 

the statistically significant correlations observed as a guide, 

scatterplots were created between the relations of interest to 

determine if the relations of interest were ecologically significant. Finally, breakpoints were computed for the ecologically 

significant relations to determine the concentration of the 

nutrient variables where the greatest change (breakpoint) was 

observed in the response variable.

The Crawford-Mammoth Cave Uplands ecoregion is 

composed of hilly uplands containing cliffs and wide karst 

valleys (Woods and others, 2002). Sinkholes, caverns, springs, 

and subterranean drainage are common; however, surface 

drainage can be significant. The mean monthly temperature ranges from -6.11°C to 7.78°C in winter (December to 

March) and 18.33°C to 32.78°C in summer (June to September); the mean annual precipitation in this area ranges from 

107 to 130 centimeters (Woods and others, 2002). Soils in the 

study area are nutrient-rich and fertile. A mixture of forests, 

pasture land, and crop land occur; farming is widespread 

throughout the bioregion. 

Study Design and Methods

 Nutrient, turbidity, and macroinvertebrate-community 

data were collected at sampling sites on first- to fourth-order 

streams in the Crawford-Mammoth Cave Uplands Ecoregion, 

Kentucky (USEPA Level IV Ecoregion, 71a). The sampling 

sites (fig.1 and table 1) were selected by the Kentucky Division of Water. Nutrient samples were collected at 22 sites from 

September 2007 to May 2008. Macroinvertebrate-community 

samples were collected at 19 sites (table 1). These samples 

were collected in the fall of 2007 and spring of 2008.

Site Selection

Twenty-two sites were selected in the Crawford-Mammoth Cave Uplands ecoregion within the Pennyroyal Bioregion to determine how the biotic integrity of the streams 

responds to changes in nutrient concentrations and turbidity 

values. The sampling sites were specifically selected by the 

Kentucky Division of Water as part of their Reference Reach 

Program (Kentucky Energy and Environment Cabinet, 2003). 

This approach is based on the range of natural conditions 

found in streams with similar physical characteristics and 

minimal human impact. A typical reference reach watershed 

contains a high proportion of natural vegetation and has minimal point-source discharges, agricultural land, mining, and 

urban development. 

Sample Collection and Processing Methods

Nutrient and turbidity data were collected by USGS personnel from September 2007 to May 2008 at all 22 sampling 

sites. Nutrient samples were collected following Kentucky 

Division of Water Water-Quality Monitoring Standard Operating Procedures (Kentucky Energy and Environment Cabinet, 

2005). Turbidity was measured using a multiparameter sonde 

at the time of nutrient sampling. The macroinvertebrate-community data used in this report was collected by the Kentucky 

Division of Water. Macroinvertebrate-community data were 

collected in the spring of 2008 at 19 sampling sites. 
6 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

Boxplots of the median stressor variables (TP, TN, and 

turbidity) were created (TIBCO Spotfire S+® 8.1 for Windows® User’s Guide, 2008) for the macroinvertebrate sites. 

The published trophic-level boundaries (Dodds and others, 

1998), in the case of nutrients, and published biologicalimpact value (Klein and others, 2008), in the case of turbidity, 

were overlaid on the boxplots. Next, the stressor variables 

were correlated to the response variable using Kendall tau (τ) 

rank correlation. Environmental data are not normally 

distributed, so a nonparametric procedure, such as Kendall tau 

(τ) is the preferred method for determining relations between 

variables (Helsel and Hirsch, 2002). 

In this report, for a correlation to be considered statistically significant, the Kendall tau rank correlation was required 

to have, at most, a 10-percent significance level (α = 0.10). 

Although a | τ | with a significance level of 0.10 is considered 

significant, there is a possibility of introducing a Type I error 

in which the relation is declared present when the relation is 

not present (Helsel and Hirsch, 2002). 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 at a specific 

alpha level. Although useful for reducing Type I error, this 

technique increases the chance of producing a Type II error, 

in which no relation is declared when a relation is present. 

Because ecological significance was weighted higher than statistical significance and the data set was small, no Bonferroni 

corrections were applied to the data analysis for this report.

Bootstrap regression-tree analysis was used in TIBCO 

Spotfire S+ (TIBCO Spotfire S+® 8.1 for Windows® user’s 

guide, 2008) to determine the concentration where the greatest 

change was observed for each ecologically significant stressorresponse relation. For the nutrient stressor variables (TP and 

TN), the mean breakpoints for the ecologically significant 

attributes from the macroinvertebrate dataset were determined. Confidence intervals for the bootstrap regression-tree 

analysis were used to determine the confidence intervals at 

the 90th percentile of the median breakpoints identified in the 

regression-tree analysis. Bootstrapping simulates the results of 

repeated experiments based on the observed data by randomly 

selecting subsets of the observed data. The approach used 

in the bootstrap regression-tree preserved sampling along a 

gradient and allowed for the variables of the distribution to 

change along that gradient. This was done by defining groups 

of data along the environmental gradient and forcing the 

bootstrap to resample within those groups, thus preserving the 

gradient and also the variability of the distribution.

Distribution of the Nutrient and 

Turbidity Variables

Mueller and Spahr (2006) found that streams within 

the Midwest, including Kentucky, have some of the highest 

nutrient loadings in the United States. Although streams for 

this study mostly were sampled during periods of stable lower 

flow conditions, which typically have lower nutrient levels 

(Lowe and others, 2008), many streams within the study were 

nutrient enriched, based on the trophic classification by Dodds 

and others (1998). The median values for each of the three 

stressor variables were calculated from the 9–11 samples collected at each site from September 2007 to May 2008. Stressor 

variables, specifically TP, and TN (NO2+NO3-N plus TKN), 

were censored to one-half the reporting limit for each variable. 

The detection limit for TP was 0.01 mg/L with about 6 percent 

of the samples having values less than the detection limit. 

For TKN, 88 of 230 of the values recorded (about 37 percent 

of the samples) had values less than a reporting limit of 

0.20 mg/L. For NO2+NO3-N, only one percent of the samples 

had values less than a reporting limit of 0.01 mg/L. 

The summary statistics of the nutrient stressor variables used in the macroinvertebrate data-breakpoint analysis 

are shown in table 2. Within the macroinvertebrate dataset, 

median concentrations of stressor variables ranged from 

0.016 to 0.182 mg/L for TP, and from 0.39 to 4.9 mg/L 

for TN (table 2). Median turbidity values ranged from 

2.4 FNU to 14 FNU (table 2).

Table 2. Summary statistics for nutrient and turbidity variables 

used in the Crawford-Mammoth Cave Uplands Ecoregion 

breakpoint analysis study in Kentucky, 2007–08.

[TP, total phosphorus; mg/L; milligrams per liter; TN, total nitrogen; FNU, 

Formazin nephelometric units; n, number of samples]

Statistic Median TP 

(mg/L)

Median TN 

(mg/L)

Median turbidity 

(FNU)

(n=19)

Minimum 0.016 0.39 2.4

25th percentile .023 .79 4.0

Median .032 1.2 5.4

Mean .050 1.6 6.4

Maximum .182 4.9 14
Macroinvertebrate Community Attributes, Relations Between Stressor and Response Variables and Nutrient Breakpoints 7

Macroinvertebrate Community 

Attributes, Relations Between 

Stressor and Response Variables, 

and Nutrient Breakpoints using 

Macroinvertebrate Attributes

The macroinvertebrate-community data were evaluated 

in three ways to aid in the understanding of the distribution 

of the nutrient concentrations and relations between nutrient 

concentrations and the macroinvertebrate response variables. 

First, the stressor variables were (1) examined for distribution, 

and (2) examined for relations among the stressor variables. 

Second, the stressor variables were examined for relations 

between the stressor and macroinvertebrate response variables, 

and the ecological significant relations were further examined 

to determine the breakpoints. Third, the computed breakpoints 

were compared to Dodds’ trophic classification levels for 

nutrients and Klein and others (2008) biological impact classification for turbidity.

Summary of Macroinvertebrate-Community 

Attributes

Eighteen macroinvertebrate attributes were used to 

characterize the macroinvertebrate data (Appendix 2). These 

attributes included taxa richness (TR, 1 attribute), total number 

of individuals (TNI, 1), relative abundance or total number 

of insect orders (6), insect families (3), and insect genera (2), 

feeding classifications (1), and pollution tolerance (3) (table 

4). The Hilsenhoff Biotic Index (HBI, Hilsenhoff, 1988), the 

Hilsenhoff Family Level Biotic Index (Family HBI), and the 

average pollution tolerance value (Average TV) represent 

the stress response of the macroinvertebrate assemblage to 

organic pollution. Both the HBI and Family HBI are based 

on a 1 to 10 scale, with 1 being the least tolerant and 10 

being the most tolerant. Average tolerance values (Average 

TV) are based on the same scale as the HBI and Family HBI. 

The macroinvertebrate attributes used are summarized in 

table 4.

Relations between the Nutrient and 

Turbidity Stressor Variables

The strongest and most frequently significant correlations 

(| τ |) between the stressor variables were between concentrations of TP and TN (table 3). This strong correlation suggests 

the relatively even distribution of nutrient concentrations measured in the streams in the Crawford-Mammoth Cave Uplands 

ecoregion within the Pennyroyal Bioregion. An additional factor that may help understand the trophic status and functioning 

of streams is turbidity. Turbidity is an optical property defined 

as the measurement of light commonly scattered at 90 degrees 

to the incident light by suspended particles in an aqueous 

medium (Uhrich and Bragg, 2003). Turbidity can be used as 

a surrogate for suspended sediment (Rasmussen and others, 

2009), and it potentially can be used as an indicator of increasing algal biomass caused by nutrient enrichment. Median 

and maximum turbidity measurements were compared to 

25 Formazin nephelometric units (FNU), a turbidity biological impact classification (Klein and others, 2008). Part of the 

reason for the low median turbidity values in this study was 

because samples generally were collected during stable, lowerflow conditions. Of the stressor variables studied, turbidity 

could be the most misleading when assessing the trophic level 

of streams, because headwater and wadable streams generally 

have lower turbidity. This is especially true for study designs 

that sample only during stable, low-flow conditions, but the 

alternative design of collecting samples only during elevated 

flow would overestimate turbidity. 

Table 3. Absolute Kendall tau correlations among the median 

stressor variables used in the Crawford-Mammoth Cave Uplands 

Ecoregion breakpoint analysis study, 2007–08.

[TP, total phosphorus; mg/L, milligrams per liter; TN, total nitrogen; FNU, 

Formazen nephelometric units; n, number of samples; Bold text, p < 0.10]

Stressor Median TP 

(mg/L)

Median TN 

(mg/L)

Median turbidity 

(FNU)

(n=19)

TP -- 0.579 0.223

TN 0.579 -- 0.129

Turbidity 0.223 0.129 --
8 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Table 4. Abbreviations, definitions, and summary statistics for macroinvertebrate attributes and metrics for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis in Kentucky, 2007–08. Attribute Abbreviation Definition Sample number Minimum Median Maxi- mum Standard deviation Total Taxa Richness TR Total number of distinct taxa present in a sample 19 48.0 75.0 102 14.5 Total Number of Individuals TNI Total number of individuals present in a sample 19 1,410 3,202 9,494 2,388 Ephemeroptera, Plecoptera, and Trichoptera Richness EPT Total number of distinct taxa in the orders Ephemeroptera, Plecop- tera, and Trichoptera 19 4.00 18.0 35.0 8.98 Percentage of Orders Ephemeroptera, Plecoptera, and Trichoptera EPT Relative abundance in the orders Ephemeroptera, Plecoptera, and Trichoptera 19 3.65 26.7 58.7 18.9 Hilsenhoff Biotic Index HBI Index that summarizes overall pollution tolerance of a benthic arthropod community with a single value 19 4.66 5.59 6.54 0.666 Percentage of Dominant Family % 5 Dom Relative abundance of the numerically dominant family relative to total number of organisms in sample 19 38.0 61.8 89.3 12.6 Percentage of Primary Clingers % ClngP Relative abundance of organisms that need hard, silt-free substrates to "cling" to 19 28.2 51.4 85.8 17.7 Percentage of Ephemeroptera % Ephem Relative abundance of mayflies 19 0.631 15.7 47.6 14.6 Percentage of Chironomidae % Chiro Relative abundance of midges 19 8.58 41.9 69.9 17.5 Number of Ephemeroptera, Plecoptera, and Trichoptera Genus Taxa EPT GenusTax Total number of distinct taxa at the genus level in the orders Ephemeroptera, Plecoptera, and Trichoptera 19 4.00 18.0 31.0 7.80 Number of Total Genus Taxa GenusTax Total number of distinct taxa at the genus level in a sample 19 40.0 65.0 90.0 13.1 Number of Ephemeroptera, Plecoptera, and Trichoptera at the family level Family EPT Total number of distinct taxa at the family level in the orders Ephemeroptera, Plecoptera, and Trichoptera 19 3.00 12.0 17.0 4.00 Total Number of Individuals at the Family Level Family Richness Total number of family-level macroinvertebrate communities in a sample. Measures the diversity of the macroinverebrate com- munity. 19 19.0 35.0 45.0 7.07 Average Tolerance Value Average TV Average of the tolerance values of all species within the benthic arthropod community 19 5.09 5.71 6.64 .485 Modified Percentage of Ephemeroptera, Plecop- tera, and Trichoptera Abundance m% EPT Abundance of the generally pollution-sensitive insect orders Ephemeroptera, Plecoptera, and Trichoptera 19 .677 22.9 57.5 17.1 Hilsenhoff Family Level Biotic Index Family HBI Average-weighted pollution tolerance value of all arthropod identi- fications at the family level in a sample. 19 5.35 6.11 7.03 0.58 Percentage of Nutrient Tolerance % Nutrient Tol 1Percent of the composite of 14 nutrient tolerant taxa 19 27.2 58.7 90.2 18.1 Percentage of Oligochaeta % Oligo Relative abundance of earthworms 19 .822 4.99 61.3 15.1

1Fourtteen taxa include three EPT genera (Chemumatophyche, Baetis, and Stenacron), one crustacean genus (Lirceus), two snail genera (Physella and Elimia), two beetle genera (Psephenus and Stenelmis), 

one black fly genus (Simulium), four midge genera (Polypedilum, Pheotanytarsus, Cricotopus and Chironomus), and aquatic worms (Oligochaeta).
Macroinvertebrate Community Attributes, Relations Between Stressor and Response Variables and Nutrient Breakpoints 9

correlated with both the median concentrations of TP and 

TN and showed an increasing trend with increasing nutrient 

concentrations (fig.2). Two additional attributes, EPT richness 

and Average TV, were also strongly correlated with median 

concentrations of TP (table 5). The EPT richness decreased as 

median concentrations of TP increased (fig.3). The EPT taxa 

are generally intolerant of nutrients, so the richness of EPT 

taxa would be expected to decrease with higher concentrations 

of nutrients. A consequence of decreasing diversity and quality 

of the macroinvertebrate community is the potential negative 

impact on the fish community. The Average TV increased as 

median concentrations of TP increased (fig.4). This relation 

suggests the presence of more tolerant benthic arthropods and 

poorer water quality.

Distribution of the Macroinvertebrate 

Communities and Their Relations to Nutrient 

Stressor Variables

Many of the macroinvertebrate-community attributes 

(13 of 18 attributes) were significantly and ecologically correlated with at least one of the TP or TN statistical measures 

(table 5). Three attributes, TNI, Ephemeroptera, Plecoptera, 

and Trichoptera (EPT) richness, and Average TV, were chosen 

as the best macroinvertebrate measures and used for additional 

detailed investigation. Correlation analysis and scatterplots 

indicated that these attributes were the most responsive to 

differences in nutrient concentrations. The TNI index was 

more strongly correlated with nutrient concentrations than 

the other attributes. The TNI index was most significantly 

Table 5. Macroinvertebrate breakpoint summary (n=19) for the stressor variables used in the Crawford-Mammoth Cave Uplands 

Ecoregion breakpoint analysis study, 2007–08.

[TP, total phosphorus; TN, total nitrogen; mg/L, milligrams per liter; p-value <0.10]

Biological response variable

Stressor 

variable to 

biological 

attribute 

Kendal tau

Mean 

bootstrap 

breakpoint 

of stressor 

variable

Lower 

bootstrap 

confidence 

interval

Upper 

bootstrap 

confidence 

interval

Median TP (mg/L)

Total Taxa Richness -0.231 0.032 0.026 0.040

Total Number of Individuals 0.488 0.033 0.026 0.040

Number of Ephemeroptera, Plecoptera, and Trichoptera -0.300 0.032 0.026 0.040

Percentage of Dominant Family 0.275 0.033 0.026 0.040

Percentage of Ephemeroptera -0.251 0.034 0.026 0.040

Number of Ephemeroptera, Plecoptera, and Trichoptera Genus Taxa -0.273 0.033 0.026 0.040

Number of Ephemeroptera, Plecoptera, and Trichoptera at the family level -0.397 0.032 0.026 0.040

Total Number of Individuals at the Family Level -0.252 0.034 0.026 0.040

Average Tolerance Value 0.380 0.032 0.026 0.040

Modified Percentage of Ephemeroptera, Plecoptera, and Trichoptera Abundance -0.287 0.035 0.026 0.040

Percentage of Nutrient Tolerance 0.228 0.034 0.026 0.040

Median TN (mg/L)

Total Number of Individuals 0.571 1.1 0.850 1.23

Number of Ephemeroptera, Plecoptera, and Trichoptera at the family level -0.287 1.1 0.850 1.23

Percentage of Nutrient Tolerance 0.228 1.0 0.850 1.23
10 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

Figure 2. Scatter plot of the A, median total phosphorus concentrations and B, median total nitrogen concentrations to the 

total number of individuals for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007–08. (Trophic 

classifications from Dodds and others, 1998.)

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

MEDIAN TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER, AS PHOSPHORUS

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

9,000

10,000

Mean bootstrap

breakpoint = 0.033 mg/L

Dodds mesotropic-eutrophic

boundary = 0.075 mg/L

Dodds oligotrophic-mesotropic

boundary = 0.025 mg/L

TOTAL NUMBER OF INDIVIDUALS

A

0 1 2 3 4 5 6

MEDIAN TOTAL NITROGEN, IN MILLIGRAMS PER LITER, AS NITROGEN

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

9,000

10,000

Mean bootstrap

breakpoint = 1.1 mg/L

Dodds mesotropic-eutrophic

boundary = 1.5 mg/L

Dodds oligotrophic-mesotropic

boundary = 0.7 mg/L

B
Macroinvertebrate Community Attributes, Relations Between Stressor and Response Variables and Nutrient Breakpoints 11

Figure 3. Scatter plot of the median total phosphorus concentrations to the number of Ephemeroptera, Plecoptera, and Trichoptera 

genus taxa for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007–08. (Trophic classifications from Dodds 

and others, 1998.)

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

MEDIAN TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER, AS PHOSPHORUS

2

4

6

8

10

12

14

16

18

NUMBER OF EPHEMEROPTERA, PLECOPTERA, AND

TRICHOPTERA GENUS TAXA

Mean bootstrap

breakpoint = 0.032 mg/L

Dodds mesotropic-eutrophic

boundary = 0.075 mg/L

Dodds oligotrophic-mesotropic

boundary = 0.025 mg/L

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

MEDIAN TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER, AS PHOSPHORUS

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

6.8

AVERAGE TOLERANCE VALUE

Mean bootstrap

breakpoint = 0.032 mg/L

Dodds mesotropic-eutrophic

boundary = 0.075 mg/L

Dodds oligotrophic-mesotropic

boundary = 0.025 mg/L

Figure 4. Scatter plot of the median total phosphorus concentrations to the average tolerance value for the Crawford-Mammoth Cave 

Uplands Ecoregion breakpoint analysis study, 2007-08. (Trophic classifications from Dodds and others, 1998.)
12 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

Macroinvertebrate-Community Breakpoints of 

the Nutrient Stressor Variables and Implications 

for Developing Nutrient Criteria in the CrawfordMammoth Cave Uplands Ecoregion

Regression-tree analyses were performed to define 

specific breakpoints (thresholds) in the stressor-response relations. The strongest and most ecologically significant of these 

relations and their breakpoints are listed in table 5. All of the 

breakpoint values for TP and TN were statistically significant 

at p-value < 0.10. 

The breakpoint values identified by the regression-tree 

analysis in the responses to changes in median concentrations 

of TP were 0.032 mg/L to 0.035 mg/L (table 5). The ranges in 

the significant breakpoint values in the response to changes 

in median concentrations of TN were 1.0 mg/L to 1.1 mg/L 

(table 5). The positive response of the total number of individuals (TNI) index, which is most strongly correlated with 

nutrients, is shown with respect to median concentrations of 

TP and TN in figure 2. In general, TNI increased as nutrient 

concentrations increased. Other strong responses based on 

correlation analysis and scatterplots were between median 

concentrations of TP and EPT richness and Average TV 

attributes. The median concentration of TP increased as EPT 

richness decreased, with a mean breakpoint of 0.032 mg/L 

(fig.3). This relation suggests that as the diversity and quality 

of the macroinvertebrate community decreases, the impact on 

the fish community potentially becomes more negative. The 

breakpoint value found for Average TV (0.032 mg/L) matched 

the EPT richness index. However, the response was positive 

between Average TV and the median concentration of TP, so 

as the median concentration of TP increased, the Average TV 

index increased (fig.4).

The average median concentration of TP (0.033 mg/L) 

associated with the biological breakpoints in this study 

(table 5) were lower than the TP breakpoint values of 

0.091 mg/L and 0.053 mg/L within Wisconsin (Robertson 

and others, 2006) and Indiana (Caskey and others, 2010), 

respectively. In general, the biological breakpoint relations 

with median concentrations of TP in this study were similar 

to the USEPA proposed numeric TP criteria (0.037 mg/L) for 

Aggregated Ecoregion IX (central and western Kentucky) for 

rivers and streams.

The biological breakpoint values for the median TN 

concentration (1.1 mg/L) in this study (table 5) are similar 

with the TN breakpoint value of 1.1 mg/L within Wisconsin 

(Robertson and others, 2006), and three times lower than the 

TN breakpoint value of 3.3 mg/L within Indiana (Caskey 

and others, 2010). The biological breakpoint relations with 

median concentrations of TN in this study were not similar 

to the USEPA proposed numeric TN criteria for Aggregated 

Ecoregion IX (central and western Kentucky) for rivers and 

streams (0.69 mg/L). 

Macroinvertebrate Communities and the 

Comparison of Trophic Classification for 

Nutrient Concentrations and Turbidity Values

Trophic state is a classification system designed to “rate” 

rivers and streams based on the amount of biological productivity occurring in the water. To assess the trophic state 

of each stream, the breakpoints of the median stressor concentrations for TP and TN were compared to Dodds’ trophic 

classification. Dodds and others (1998) proposed classifying 

streams into trophic-state conditions similar to those developed for lakes and reservoirs (U.S. Environmental Protection Agency, 2000). The approach used by Dodds and others 

(1998) was based upon establishing statistical distributions of 

TP, TN, and periphyton and seston CHLa using existing data. 

Three trophic-state conditions are classified as oligotrophic, 

mesotrophic, and eutrophic. Dodds and others (1998) classified the oligotrophic-mesotrophic boundaries of Midwest 

streams as 0.025 mg/L for TP and 0.70 mg/L for TN, and the 

mesotrophic-eutrophic boundaries as 0.075 mg/L for TP and 

1.5 mg/L for TN. 

Based on Dodds’ trophic-state conditions, streams in 

this study were most often classified as eutrophic, based on 

the distribution of median TP concentrations (44 percent of 

values), and mesotrophic-eutrophic, based on the distribution of maximum TP concentrations (94 percent of values) 

(fig.5). Comparison of the average biological breakpoint 

for the median concentration of TP (0.033 mg/L) to Dodds’ 

trophic classification for TP indicates streams in the Pennyroyal Bioregion would be classified as mesotrophic-eutrophic. 

The average biological breakpoint for the maximum concentration of TP of 0.156 mg/L found in this study was more than 

two times higher than the trophic levels Dodds and others 

(1998) describe as hypereutrophic.

The trophic-state stream classifications based on median 

and maximum TN concentrations were similar to those for the 

median and maximum TN concentrations, with 56 percent of 

TN values considered eutrophic and 69 percent of TN values 

considered mesotrophic-eutrophic (fig.5). The average biological breakpoint for the median concentration of TN (0.93 mg/L) 

indicates that streams in the Pennyroyal Bioregion would 

be classified as mesotrophic-eutrophic according to Dodds’ 

trophic classification for TN. The average biological breakpoint for the maximum concentration of TN (1.8 mg/L) is 

slightly higher than Dodds’ mesotrophic-eutrophic boundary 

(1.5 mg/L).

An additional factor that may aid understanding of the 

trophic status and functioning of streams is turbidity. Turbidity 

can be used as a surrogate for suspended sediment (Rasmussen and others, 2009), and it could be used as an indicator of 

increasing algal biomass caused by nutrient enrichment. No 

biological breakpoints in relation to turbidity measurements 

were reported in this study, because no significant correlations were shown. However, median turbidity measurements 

were compared to 25 FNU, a turbidity biological impact 
Macroinvertebrate Community Attributes, Relations Between Stressor and Response Variables and Nutrient Breakpoints 13

Figure 5. Distribution to the median total phosphorus and total nitrogen concentrations (presented on a log scale) compared 

to Dodds and others (1998) trophic classifications , and distribution of the median turbidity concentrations compared to Klein 

and others (2008) turbidity biological impact classification in selected datasets used in the Crawford-Mammoth Cave Uplands 

Ecoregion breakpoint analysis study, 2007–08. (FNU, Formazin nephelometric units.) TOTAL PHOSPHORUS, IN MILLIGRAMS PER LITER

Dodds oligotrophic-mesotrophic

boundary = 0.025 mg/L

Dodds mesotrophic-eutrophic

boundary = 0.075 mg/L

0.01 0.02 0.03 0.05 0.10 0.20

19

Dodds mesotrophic-eutrophic

boundary = 1.5 mg/L

Dodds oligotrophic-mesotrophic

0.3 0.5 1.0 2.0 3.0 5.0

boundary = 0.7 mg/L

19

TOTAL NITROGEN, IN MILLIGRAMS PER LITER

30 Number of 

 values

EXPLAN ATION

 Sc hematic box plot

75th percentile

Median

25th percentile

TURBIDITY, IN FORMAZIN NEPHELOMETRIC UNITS

1 5 10 50 100

19

Turbidity biological impact classification = 25 FNU
14 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

classification (Klein and others, 2008) (fig.5). No sites were 

impacted adversely using median turbidity values for macroinvertebrates, but more than 89 percent of the sites were 

impacted using maximum turbidity values for macroinvertebrates based on the 25 FNU biological threshold used by 

Klein and others (2008). Part of the reason for the low median 

turbidity values in this study was that samples generally were 

collected during stable, lower flow conditions. Of the stressor 

variables studied, turbidity could be the most misleading when 

assessing the trophic level of streams, because headwater 

and wadable streams generally have lower turbidity. This is 

especially true for study designs that sample only during 

stable, low-flow conditions, but the alternative design of collecting samples only during elevated flow would overestimate 

turbidity. 

Summary and Conclusions

Nutrients are essential to the development, health, and 

diversity of plants and animals in surface waters, yet excessive 

inputs of nutrients into streams have potential human-health, 

economic, and ecological consequences. Kentucky is part 

of the nutrient-rich Midwest, which leads the nation in corn 

and soybean production; these row-crops require the use of 

significant amounts of nutrients—primarily from fertilizer and 

manure for their production. Within the United States, streams 

sampled within the Midwest have some of the highest nutrient 

concentrations and loading, which contributes to the hypoxia 

issue in the Gulf of Mexico. Studies using breakpoint analysis in Wisconsin and Indiana found that indicators, such as 

biological-community attributes and metrics, could be helpful 

for developing nutrient criteria for streams in such nutrientrich regions.

The USGS and the KDOW collected water-quality 

and biological data in 22 wadable streams throughout the 

Crawford-Mammoth Cave Uplands ecoregion within the Pennyroyal Bioregion from September 2007 to May 2008. These 

samples were collected to determine if the changes among 

macroinvertebrate-community attributes and metrics were 

statistically and ecologically related to changes in stressor 

(TP, TN, and turbidity) variables. Breakpoint analysis was 

used for the most statistically significant and ecologically 

relevant relations to find the concentration of the stressor variables where the greatest change occurred with the biological 

species and community attributes and metrics.

Median concentrations of stressor variables ranged from 

0.016 mg/L to 0.182 mg/L for TP and 0.39 mg/L to 4.9 mg/L 

for TN within the macroinvertebrate dataset. Median turbidity 

values ranged from 2.4 FNU to 14 FNU.

Many macroinvertebrate-community (13 of 18) attributes 

were statistically significant (p-value < 0.10) and ecologically relevant in relation with concentrations of TP and TN, 

suggesting that nutrients have direct or indirect links with 

those biological communities in wadable streams in the 

Crawford-Mammoth Cave Uplands ecoregion within the 

Pennyroyal Bioregion. Correlation analysis and scatterplots 

indicated three macroinvertebrate-community attributes (Total 

Number of Individuals, Ephemeroptera-Plecoptera-Trichoptera richness, and Average Tolerance Value) were the most 

responsive to differences in nutrient concentrations. 

Breakpoints for the macroinvertebrate-community 

attributes were generally consistent with a mesotrophiceutrophic status when using median nutrient concentrations. 

The macroinvertebrate attributes breakpoint values in the 

responses to changes in median concentrations of TP range 

from 0.032 mg/L to 0.035 mg/L. The ranges in the significant 

breakpoint values in response to changes in median concentrations of TN were 1.0 mg/L to 1.1 mg/L.

The goal of the study was not to develop numeric nutrient 

criteria, but to demonstrate the breakpoint analysis approach 

between nutrient concentrations and some aspects of macroinvertebrate attributes and metrics. Although the sample size was 

small, this study found meaningful relations between nutrient 

concentrations and changes in macroinvertebrate attributes 

and metrics in the Pennyroyal Bioregion. The average biological breakpoint value for concentrations of TN (1.1 mg/L) in 

this study was not similar to the USEPA proposed numeric 

TN criteria for rivers and streams (0.69 mg/L). This finding 

suggests that setting the TN criteria to that criteria proposed 

by the USEPA would be conservative and that many rivers 

and streams in the Pennyroyal Bioregion would be considered 

impaired, despite the use of biological-community attributes 

that indicate acceptable water-quality conditions. The average biological breakpoint in relation with median concentrations of TP (0.033 mg/L) was similar to the USEPA proposed 

numeric TP criteria (0.037 mg/L) for rivers and streams in the 

study area.

Results from this study demonstrate that macroinvertebrate attributes and metrics can be used to evaluate relations 

between concentrations of nutrients and macroinvertebrate 

communities. In addition, breakpoint values in nutrient concentrations can be identified. With additional biological data 

(such as diatoms, CHLa and fish), the biological assessment 

of macroinvertebrate communities has a greater potential for 

success in developing and refining numeric nutrient criteria in 

the Pennyroyal Bioregion. 

References Cited

Alexander, R.B., Smith, R.A., Schwarz, G.E., Boyer, E.W., 

Nolan, J., and Brakebill, J.W., 2008, Differences in 

phosphorus and nitrogen delivery to the Gulf of Mexico and 

the Mississippi River Basin: Environmental Science and 

Technology, v. 42, no. 3, p. 822–830.
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200605.pdf.
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htm#Principal%20Authors.
Appendixes
18 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams
Appendix 1

19

Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued[Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; 

CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Sampling site 

name 

(refer to figure 

1 for location)

USGS station 

number

KDOW site 

number

Sample-collection 

date

Ammonia, in 

milligrams per 

liter, as nitrogen

Total Kjehldal 

nitrogen, in 

milligrams per 

liter

Nitrite plus 

nitrate, in milligrams per liter, 

as nitrogen

Total 

Phosphorus, 

in milligrams 

per liter

Total nitrogen, 

in milligrams 

per liter

Turbidity, 

in Formazin 

nephelometric 

units

BC 03320300 DOW03002019 9/24/2007 E0.031 0.72 0.02 0.045 0.74

10/22/2007 <0.025 E0.44 E0.01 0.082 0.46 27.0

11/27/2007 <0.025 <0.2 2.01 0.025 2.21 2.612/18/2007 <0.025 <0.2 1.87 0.024 2.07 1.21/14/2008 <0.025 <0.2 1.29 E0.017 1.492/14/2008 <0.025 <0.2 1.11 0.027 1.31 4.6

3/19/2008 E0.049 1.00 0.35 0.164 1.35 125.04/8/2008 1.34/29/2008 <0.025 <0.2 0.02 <0.01 0.22 1.5

5/13/2008 <0.025 <0.2 0.19 E0.011 0.39 05/19/2008 <0.025 E0.25 0.32 E0.016 0.57 0.6

BDC 03311600 DOW03016002 9/26/2007 <0.025 E0.28 1.08 0.065 1.36 4.7

10/25/2007 <0.025 0.69 6.11 0.150 6.80 8.411/28/2007 <0.025 E0.28 3.70 0.057 3.98 9.1

12/18/2007 <0.025 E0.25 3.25 0.055 3.50 8.1

1/15/2008 <0.025 E0.29 2.83 0.040 3.12 10.1

2/12/2008 0.118 1.14 0.89 0.325 2.03 105.03/11/2008 <0.025 0.38 2.14 0.045 2.52 4.9

4/7/2008 E0.038 0.58 2.00 0.063 2.58 3.84/24/2008 <0.025 <0.2 1.87 0.038 2.07 1.75/6/2008 E0.033 E0.42 1.88 0.054 2.30 4.85/27/2008 <0.025 E0.32 2.03 0.051 2.35 6.7

BEAR 03311800 DOW03002019 9/25/2007 E0.034 E0.24 0.09 0.021 0.32

10/25/2007 <0.025 0.52 2.05 0.081 2.57 5.611/28/2007 <0.025 E0.25 1.63 0.040 1.88 3.4

12/19/2007 <0.025 <0.2 1.43 0.033 1.63 2.4

1/15/2008 <0.025 <0.2 1.11 0.025 1.31 5.3
20 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued [Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water] Sampling site name (refer to figure 1 for location) USGS station number KDOW site number Sample-collection date Ammonia, in milligrams per liter, as nitrogen Total Kjehldal nitrogen, in milligrams per liter Nitrite plus nitrate, in milli- grams per liter, as nitrogen Total Phosphorus, in milligrams per liter Total nitrogen, in milligrams per liter Turbidity, in Formazin nephelometric units BEAR 03311800 DOW03002019 2/12/2008 0.110 0.85 0.48 0.155 1.33 40.8 3/11/2008 <0.025 <0.2 0.64 0.034 0.84 26.0 4/7/2008 <0.025 E0.27 0.42 0.022 0.69 1.8 4/24/2008 <0.025 <0.2 0.06 E0.019 0.26 1.4 5/6/2008 E0.030 E0.24 0.14 E0.013 0.37 0.8 5/27/2008 E0.044 E0.2 0.25 <0.01 0.45 0.3 BF 03320100 DOW03004020 9/24/2007 <0.025 0.76 0.02 0.064 0.79 10/22/2007 0.205 0.92 1.00 0.155 1.92 42.0 11/28/2007 <0.025 E0.39 3.06 0.038 3.45 3.0 12/18/2007 <0.025 E0.46 1.90 0.072 2.36 8.8 1/14/2008 E0.033 E0.47 1.73 0.071 2.20 2/14/2008 0.0673 E0.41 1.46 0.071 1.87 15.0 3/19/2008 E0.049 E0.43 0.64 0.121 1.07 98.0 4/8/2008 E0.027 E0.22 0.71 0.071 0.93 15.3 4/29/2008 E0.030 E0.28 0.28 0.028 0.56 2.5 5/13/2008 0.0612 0.53 0.32 0.041 0.85 4.2 5/20/2008 E0.045 0.54 0.62 0.042 1.16 4.9 BRB 03317940 DOW03008026 9/25/2007 <0.025 E0.31 0.95 0.044 1.25 10/25/2007 <0.025 E0.45 1.66 0.081 2.11 8.6 11/28/2007 <0.025 E0.22 1.06 0.041 1.28 21.7 12/18/2007 <0.025 <0.2 1.18 0.031 1.38 4.4 1/15/2008 <0.025 <0.2 1.13 0.022 1.33 5.2 2/12/2008 0.089 0.88 0.52 0.168 1.40 122.0 3/11/2008 <0.025 <0.2 0.80 0.032 1.00 12.0 4/8/2008 <0.025 E0.24 0.74 0.023 0.98 1.5 4/24/2008 <0.025 <0.2 0.24 0.027 0.44 2.1 5/6/2008 <0.025 E0.31 0.30 0.026 0.60 0.5 5/27/2008 0.0566 <0.2 0.61 0.034 0.81 1.3
Appendix 1

21

Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued[Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; 

CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Sampling site 

name 

(refer to figure 

1 for location)

USGS station 

number

KDOW site 

number

Sample-collection 

date

Ammonia, in 

milligrams per 

liter, as nitrogen

Total Kjehldal 

nitrogen, in 

milligrams per 

liter

Nitrite plus 

nitrate, in milligrams per liter, 

as nitrogen

Total 

Phosphorus, 

in milligrams 

per liter

Total nitrogen, 

in milligrams 

per liter

Turbidity, 

in Formazin 

nephelometric 

units

BRBW 03317395 DOW03008024 11/28/2007 <0.025 <0.2 0.39 0.020 0.59 59.3

12/18/2007 0.105 <0.2 0.45 0.022 0.65 7.11/15/2008 <0.025 E0.27 0.32 E0.012 0.59 6.5

2/12/2008 0.055 0.67 0.30 0.091 0.97 4.8

3/11/2008 <0.025 <0.2 0.29 0.028 0.49 13.0

4/8/2008 <0.025 E0.24 0.11 E0.016 0.35 1.8

4/24/2008 <0.025 <0.2 <0.01 <0.01 0.21 2.7

5/6/2008 <0.025 E0.32 <0.01 <0.01 0.33 0.25/27/2008 0.0575 <0.2 0.12 <0.01 0.32 0.7

CC 03384330 DOW08013004 9/24/2007 E0.028 0.91 0.06 0.121 0.96

10/22/2007 E0.040 0.77 0.22 0.168 0.99 3.7

11/27/2007 0.087 0.71 1.40 0.293 2.11 26.0

12/18/2007 0.164 E0.46 1.14 0.107 1.60 13.8

1/14/2008 0.067 E0.32 1.03 0.097 1.352/13/2008 0.237 0.68 0.72 0.110 1.40 27.8

3/18/2008 0.592 3.26 0.61 0.461 3.87 319.0

4/7/2008 0.334 0.65 0.54 0.077 1.19 14.9

4/28/2008 0.061 E0.303 0.14 0.057 0.44 5.25/12/2008 0.201 0.52 1.00 0.118 1.52 7.3

5/19/2008 0.538 1.14 0.95 0.163 2.09 8.6

DC 03303203 DOW08035013 9/25/2007 E0.040 E0.32 0.12 E0.016 0.44

10/24/2007 <0.025 0.54 1.23 0.104 1.77 6.2

11/29/2007 <0.025 <0.2 0.96 0.028 1.16 1.9

12/18/2007 <0.025 <0.2 1.13 0.030 1.33 4.0

1/15/2008 <0.025 <0.2 0.87 E0.018 1.07 2.52/14/2008 E0.028 <0.2 0.94 0.026 1.14 9.7

3/11/2008 <0.025 <0.2 0.56 0.033 0.76 17.1
22 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued [Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water] Sampling site name (refer to figure 1 for location) USGS station number KDOW site number Sample-collection date Ammonia, in milligrams per liter, as nitrogen Total Kjehldal nitrogen, in milligrams per liter Nitrite plus nitrate, in milli- grams per liter, as nitrogen Total Phosphorus, in milligrams per liter Total nitrogen, in milligrams per liter Turbidity, in Formazin nephelometric units DC 03303203 DOW08035013 4/8/2008 <0.025 0.54 0.52 0.023 1.06 3.4 4/24/2008 <0.025 <0.2 0.06 E0.019 0.26 0.7 5/6/2008 E0.026 <0.2 0.29 E0.011 0.49 3.9 5/27/2008 <0.025 <0.2 0.21 <0.01 0.41 1.8 ELC 03316110 DOW03012008 9/25/2007 0.080 E0.33 0.18 0.027 0.51 10/23/2007 <0.025 0.94 1.30 0.250 2.24 42.4 11/28/2007 E0.048 E0.41 1.49 0.051 1.90 8.4 12/19/2007 E0.030 0.56 1.25 0.088 1.81 14.5 1/15/2008 <0.025 E0.42 1.32 0.050 1.74 2/15/2008 <0.025 E0.27 1.44 0.047 1.71 10.7 3/20/2008 E0.027 E0.44 0.91 0.089 1.35 128.0 4/8/2008 E0.047 0.60 0.56 0.078 1.16 21.6 4/29/2008 E0.049 E0.26 0.32 0.020 0.58 3.6 5/13/2008 E0.049 0.52 0.30 0.047 0.82 5.4 5/20/2008 0.0586 0.50 0.34 0.033 0.85 6.5 FC 03320200 DOW03004006 9/24/2007 E0.0411 E0.43 0.12 0.032 0.55 10/22/2007 <0.025 E0.26 0.05 0.042 0.31 1.5 11/27/2007 <0.025 E0.24 1.05 0.027 1.29 7.1 12/18/2007 <0.025 <0.2 1.26 0.024 1.46 3.1 1/14/2008 <0.025 <0.2 1.01 E0.015 1.21 2/14/2008 <0.025 <0.2 0.87 0.025 1.07 6.1 3/19/2008 0.0546 0.61 0.41 0.093 1.02 117.0 4/8/2008 E0.035 <0.2 0.39 E0.017 0.59 2.8 4/29/2008 <0.025 <0.2 0.09 E0.014 0.29 3.2 5/13/2008 <0.025 <0.2 0.20 <0.01 0.40 1.1 5/20/2008 <0.025 E0.22 0.23 <0.01 0.45 1.8
Appendix 1

23

Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued[Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; 

CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Sampling site 

name 

(refer to figure 

1 for location)

USGS station 

number

KDOW site 

number

Sample-collection 

date

Ammonia, in 

milligrams per 

liter, as nitrogen

Total Kjehldal 

nitrogen, in 

milligrams per 

liter

Nitrite plus 

nitrate, in milligrams per liter, 

as nitrogen

Total 

Phosphorus, 

in milligrams 

per liter

Total nitrogen, 

in milligrams 

per liter

Turbidity, 

in Formazin 

nephelometric 

units

GR 03315180 DOW03018011 9/25/2007 E0.046 E0.39 1.82 0.105 2.21

10/23/2007 0.0824 1.51 3.97 0.397 5.48 211.011/28/2007 <0.025 <0.2 9.63 0.059 9.83 5.912/19/2007 <0.025 <0.2 7.80 0.054 8.00 12.41/15/2008 <0.025 <0.2 6.79 0.051 6.99

2/15/2008 <0.025 <0.2 6.08 0.049 6.28 9.9

3/20/2008 E0.027 E0.38 3.51 0.068 3.89 31.74/8/2008 <0.025 <0.2 4.70 0.043 4.90 10.94/29/2008 <0.025 <0.2 4.42 0.036 4.62 3.95/13/2008 <0.025 <0.2 4.33 0.045 4.53 2.45/20/2008 <0.025 <0.2 4.84 0.042 5.04 2.9

HC 03303202 DOW08035004 9/25/2007 0.0613 <0.2 22.10 3.110 22.30

10/25/2007 0.318 1.28 1.01 0.493 2.29 10.8

11/29/2007 <0.025 E0.48 2.19 0.197 2.67 7.312/18/2007 0.552 1.07 1.12 0.234 2.19 4.01/15/2008 0.241 1.01 2.08 0.182 3.09 8.72/14/2008 0.549 0.77 1.06 0.175 1.83 13.43/11/2008 0.302 0.77 0.67 0.169 1.44 1302.04/8/2008 E0.031 0.64 1.21 0.094 1.85 1.84/24/2008 E0.045 E0.41 2.62 0.110 3.03 1.45/6/2008 E0.040 1.03 1.21 0.154 2.24 3.25/27/2008 0.0765 0.58 3.66 0.462 4.24 3.2

LBDC 03311700 DOW03016001 9/26/2007 E0.034 E0.33 0.20 0.050 0.53

10/25/2007 <0.025 0.76 4.43 0.154 5.19 6.711/28/2007 <0.025 E0.34 2.72 0.057 3.06 8.0

12/18/2007 0.0731 E0.25 2.55 0.041 2.80 5.11/15/2008 <0.025 E0.31 2.03 0.047 2.34 12.12/12/2008 0.0921 1.12 0.59 0.243 1.71 105.0
24 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued [Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water] Sampling site name (refer to figure 1 for location) USGS station number KDOW site number Sample-collection date Ammonia, in milligrams per liter, as nitrogen Total Kjehldal nitrogen, in milligrams per liter Nitrite plus nitrate, in milli- grams per liter, as nitrogen Total Phosphorus, in milligrams per liter Total nitrogen, in milligrams per liter Turbidity, in Formazin nephelometric units LBDC 03311700 DOW03016001 3/11/2008 <0.025 <0.2 1.38 0.032 1.58 10.2 4/7/2008 E0.025 E0.30 1.22 0.041 1.52 4.4 4/24/2008 <0.025 <0.2 0.90 0.027 1.10 8.7 5/6/2008 E0.026 E0.23 0.83 0.032 1.06 7.6 5/27/2008 E0.034 E0.41 1.56 0.046 1.97 5.2 LC 03316870 DOW03008011 9/25/2007 E0.034 E0.31 0.12 <0.01 0.43 10/24/2007 <0.025 0.68 0.62 0.084 1.30 57.7 11/29/2007 <0.025 E0.28 0.63 0.023 0.90 7.8 12/18/2007 E0.029 <0.2 0.56 0.023 0.76 13.8 1/15/2008 <0.025 <0.2 0.50 E0.018 0.70 10.0 2/12/2008 <0.025 0.53 0.41 0.093 0.94 30.3 3/11/2008 <0.025 <0.2 0.37 0.033 0.57 25.1 4/8/2008 E0.029 E0.40 0.39 0.037 0.79 24.6 4/24/2008 <0.025 0.93 0.23 0.041 1.16 3.2 5/6/2008 <0.025 E0.28 0.22 E0.014 0.50 10.7 5/27/2008 E0.032 E0.2 0.29 <0.01 0.49 4.6 LMC 03316900 DOW03008025 9/25/2007 E0.037 E0.34 0.05 <0.01 0.39 10/24/2007 <0.025 0.57 0.96 0.107 1.52 19.0 11/29/2007 <0.025 <0.2 0.94 0.023 1.14 0.0 12/18/2007 <0.025 <0.2 1.05 0.031 1.25 5.6 1/15/2008 <0.025 E0.23 0.95 E0.017 1.18 5.2 2/14/2008 E0.028 <0.2 0.91 0.021 1.11 38.8 3/11/2008 <0.025 <0.2 0.66 0.032 0.86 13.1 4/8/2008 E0.031 <0.2 0.74 0.027 0.94 6.8 4/24/2008 E0.034 <0.2 0.16 0.023 0.36 1.7 5/6/2008 E0.029 E0.28 0.39 E0.014 0.67 3.1 5/27/2008 0.141 <0.2 0.55 <0.01 0.75 2.1
Appendix 1

25

Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued[Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; 

CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Sampling site 

name 

(refer to figure 

1 for location)

USGS station 

number

KDOW site 

number

Sample-collection 

date

Ammonia, in 

milligrams per 

liter, as nitrogen

Total Kjehldal 

nitrogen, in 

milligrams per 

liter

Nitrite plus 

nitrate, in milligrams per liter, 

as nitrogen

Total 

Phosphorus, 

in milligrams 

per liter

Total nitrogen, 

in milligrams 

per liter

Turbidity, 

in Formazin 

nephelometric 

units

MC 03316885 DOW03004018 9/25/2007 E0.027 E0.41 0.12 0.026 0.53

10/24/2007 <0.025 0.72 1.11 0.168 1.83 43.4

11/29/2007 <0.025 E0.27 0.98 0.043 1.24 5.1

12/18/2007 <0.025 <0.2 1.17 0.037 1.37 4.61/15/2008 E0.036 E0.27 1.06 0.025 1.33 6.12/12/2008 0.189 1.65 0.57 0.236 2.22 310.03/11/2008 <0.025 E0.28 0.72 0.051 1.00 28.04/8/2008 E0.027 E0.32 0.77 0.031 1.10 10.24/24/2008 <0.025 <0.2 0.28 0.026 0.48 1.95/6/2008 0.0723 E0.29 0.46 0.023 0.75 7.515/27/2008 E0.043 <0.2 0.58 0.029 0.78

NC 03310340 DOW03026003 9/25/2007 E0.034 E0.28 0.43 <0.01 0.71

10/24/2007 E0.030 E0.36 1.08 0.045 1.44 11.0

11/28/2007 <0.025 <0.2 0.75 E0.018 0.95 8.912/19/2007 <0.025 <0.2 0.81 0.022 1.01 4.3

1/15/2008 E0.032 <0.2 0.71 E0.014 0.91 5.3

2/12/2008 0.065 0.73 0.45 0.147 1.17 189.0

3/11/2008 <0.025 <0.2 0.55 0.023 0.75 7.6

4/7/2008 E0.026 E0.20 0.51 E0.019 0.71 4.54/24/2008 <0.025 <0.2 0.36 E0.015 0.56 0.6

5/6/2008 <0.025 E0.22 0.36 E0.123 0.59 4.95/27/2008 E0.041 <0.2 0.72 E0.016 0.92 1.8

NFRR 03317400 DOW03008014 9/25/2007 0.062 E0.27 0.10 0.027 0.37

10/24/2007 <0.025 E0.45 1.00 0.066 1.45 14.3

11/29/2007 <0.025 <0.2 0.99 0.028 1.1912/18/2007 <0.025 <0.2 0.99 0.028 1.19 4.8

1/15/2008 <0.025 <0.2 0.82 E0.017 1.02 10.32/14/2008 <0.025 <0.2 0.77 E0.019 0.97 13.9
26 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued [Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water] Sampling site name (refer to figure 1 for location) USGS station number KDOW site number Sample-collection date Ammonia, in milligrams per liter, as nitrogen Total Kjehldal nitrogen, in milligrams per liter Nitrite plus nitrate, in milli- grams per liter, as nitrogen Total Phosphorus, in milligrams per liter Total nitrogen, in milligrams per liter Turbidity, in Formazin nephelometric units NFRR 03317400 DOW03008014 3/11/2008 <0.025 <0.2 0.57 0.026 0.77 15.6 4/8/2008 <0.025 <0.2 0.58 E0.018 0.78 1.7 4/24/2008 E0.042 E0.42 0.35 0.055 0.77 6.1 5/6/2008 E0.035 E0.26 0.46 E0.013 0.72 2.6 5/27/2008 <0.025 <0.2 0.58 0.025 0.78 3.3 TWR 03382550 9/24/2007 0.066 0.90 0.06 0.097 0.96 10/22/2007 <0.025 E0.41 0.34 0.044 0.75 9.0 11/27/2007 <0.025 E0.25 1.08 0.034 1.33 5.4 12/18/2007 <0.025 E0.24 1.15 0.029 1.39 5.3 1/14/2008 <0.025 <0.2 0.85 E0.019 1.05 2/13/2008 <0.025 <0.2 0.76 0.037 0.96 12.4 3/18/2008 <0.025 <0.2 0.36 0.020 0.56 2.9 4/7/2008 <0.025 E0.30 0.48 0.026 0.77 2.5 4/28/2008 <0.025 <0.2 0.13 E0.015 0.33 1.6 5/12/2008 <0.025 <0.2 0.16 <0.01 0.36 0.6 5/19/2008 <0.025 E0.21 0.12 E0.016 0.34 1 UTMF 03320720 DOW03004018 9/24/2007 <0.025 0.51 E0.02 0.114 0.53 10/22/2007 <0.025 E0.44 0.23 0.107 0.67 2.4 11/27/2007 0.116 0.75 5.16 0.168 5.91 5.3 12/18/2007 0.203 E0.39 4.77 0.115 5.16 4.1 1/14/2008 E0.048 E0.27 3.93 0.116 4.20 2/14/2008 0.144 E0.43 2.63 0.096 3.06 7.6 3/19/2008 0.673 2.22 0.77 0.284 2.99 124.0 4/8/2008 <0.025 E0.44 2.28 0.076 2.72 1.2 4/29/2008 E0.028 E0.23 0.62 0.038 0.85 1.2 5/12/2008 <0.025 E0.23 1.39 0.067 1.62 0 5/19/2008 <0.025 E0.41 2.62 0.108 3.03 0.7
Appendix 1

27

Appendix 1. Station number, sample-collection date, nutrient, and turbidity sample results for samples collected for the Crawford-Mammoth Cave Uplands Ecoregion breakpoint analysis study, 2007-08.—Continued[Sampling site name abbreviations: BC, Buck Creek; BDC, Beaverdam Creek; BEAR, Bear Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at Watson School Road; 

CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeeting Creek; MC, Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; TWR, Trade Water River; UTMF, Unnamed tributary to McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. 

USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Sampling site 

name 

(refer to figure 

1 for location)

USGS station 

number

KDOW site 

number

Sample-collection 

date

Ammonia, in 

milligrams per 

liter, as nitrogen

Total Kjehldal 

nitrogen, in 

milligrams per 

liter

Nitrite plus 

nitrate, in milligrams per liter, 

as nitrogen

Total 

Phosphorus, 

in milligrams 

per liter

Total nitrogen, 

in milligrams 

per liter

Turbidity, 

in Formazin 

nephelometric 

units

WF 03437450 DOW20014005 9/25/2007 0.071 <0.2 8.54 0.058 8.74

10/23/2007 <0.025 1.26 2.88 0.695 4.14 94.3

11/28/2007 <0.025 <0.2 7.57 0.041 7.77 3.512/19/2007 <0.025 <0.2 7.22 0.040 7.42 3.81/15/2008 <0.025 <0.2 5.70 0.021 5.902/15/2008 <0.025 <0.2 3.66 0.036 3.86 5.03/20/2008 E0.039 <0.2 2.12 0.044 2.32 14.74/8/2008 4.04/29/2008 <0.025 <0.2 3.07 E0.017 3.27 1.95/13/2008 <0.025 <0.2 3.27 0.024 3.47 2.4

YBC 03302500 DOW08037001 10/25/2007 <0.025 0.60 0.92 0.155 1.52 14.6

11/30/2007 <0.025 E0.21 0.80 0.030 1.00 2.212/18/2007 <0.025 <0.2 1.03 0.043 1.23 311.8

1/15/2008 <0.025 E0.39 0.79 0.027 1.17 13.2

2/11/2008 0.028 <0.2 0.84 0.024 1.04 4.43/11/2008 <0.025 E0.23 0.48 0.051 0.71 14.04/8/2008 E0.048 E0.28 0.40 0.026 0.68 1.84/24/2008 <0.025 <0.2 0.02 0.028 0.22 0.65/6/2008 E0.038 E0.23 0.54 0.027 0.77 3.15/27/2008 E0.036 E0.22 0.07 0.022 0.29 3.6
28 Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams

Appendix 2. Macroinvertebrate community attributes and metrics calculated by site for the Crawford-Mammoth Cave Uplands

[Sampling site name abbreviations: BDC, Beaverdam Creek; BC, Buck Creek; BF, Buck Fork Pond River; BRB, Big Run Branch; BRBW, Big Run Branch at 

Little Beaverdam Creek; LC, Linders Creek; LMC, Little Meeting Creek; NC, Nosey Creek; NFRR, North Fork Rough River; UTMF, Unnamed tributary to

Sampling 

site name 

(refer 

to figure 1 

for 

location)

USGS 

station 

number

KDOW 

site number

Attribute (refer to table 3 for definitions of attributes)

Taxa 

Richness

Total 

Number 

of 

Individuals

Ephemeroptera, 

Plecoptera, 

and 

Trichoptera 

Richness

Percentage 

of Orders 

Ephemeroptera, 

Plecoptera, and 

Trichoptera

Hilsenhoff 

Biotic 

Index

Percentage of 

Dominant 

Family

Percentage of 

Primary 

Clingers

Percentage 

of 

Ephemeroptera

BDC 03311600 DOW03016002 102 5573 35 29.66 4.684 59.19 51.37 17.2

BC 03311800 DOW03002019 62 1811 18 9.663 5.992 59.41 28.21 3.423

BF 03320100 DOW03004020 75 9494 14 3.654 6.242 89.3 28.82 3.096

BRB 03317940 DOW03008026 48 1742 12 7.921 5.778 61.82 34.67 6.027

BRBW 03317395 DOW03008024 62 2404 14 26.74 6.1 50.08 46.5 23.71

CC 03384330 DOW08013004 60 4434 6 4.127 6.476 56.76 57.55 0.631

DC 03303203 DOW08035013 87 2545 21 47.03 5.027 57.05 60.51 19.01

ELC 03316110 DOW03012008 81 4339 26 29.49 5.585 80.36 85.8 7.743

FC 03320200 DOW03004006 86 3202 23 58.71 4.954 75.48 36.22 47.62

GR 03315180 DOW03018011 80 3192 22 57.51 4.909 70.58 57.08 27.13

HC 03303202 DOW08035004 49 6273 4 14.69 6.419 75.83 29.66 0.813

LBDC 03311700 DOW03016001 79 4727 28 47.93 4.66 51.38 54.36 32.3

LC 03316870 DOW03008011 73 1811 27 27.27 4.713 63.55 73.1 18.27

LMC 03316900 DOW03008025 87 3348 31 18.84 5.394 59.37 77.24 7.974

NC 03310340 DOW03026003 86 2645 20 26.01 5.525 38.03 35.99 15.65

NFRR 03317400 DOW03008014 67 1410 17 54.82 5.148 65.74 47.02 37.23

UTMF 03320720 DOW03004018 58 6273 4 4.065 6.539 76.96 72.86 1.163

WF 03437450 DOW20014005 60 9080 9 44.69 6.384 72.01 33.74 40.92

YBC 03302500 DOW08037001 77 2145 15 17.06 6.078 50.16 61.16 7.692
Appendix 2 29

Ecoregion breakpoint analysis study, 2007-08.

Watson School Road; CC, Crooked Creek; DC, Dorridge Creek; ELC, Elk Lick Creek; FC, Forbes Creek; GR, Gasper River; HC, Hardins Creek; LBDC, 

McFarland Creek; WF, Warrens Fork; YBC, Yellowbank Creek. USGS, U.S. Geological Survey; KDOW, Kentucky Division of Water]

Attribute (refer to table 3 for definitions of attributes)

Percentage 

of 

Chironomidae

Number of 

Ephemeroptera, 

Plecoptera, and 

Trichoptera 

Genus Taxa

Number 

of 

Total 

Genus 

Taxa

Number of 

Ephemeroptera, 

Plecoptera, and 

Trichoptera at the 

Family Level

Total Number 

of Individuals 

at the 

Family Level

Average 

Tolerance 

Value

Modified 

Percentage of 

Ephemeroptera, 

Plecoptera, 

and 

Trichoptera 

Abundance

Hilsenhoff 

Family 

Level Biotic 

Index

Percentage 

of Nutrient 

Tolerance

Percentage 

of 

Oligochaeta

41.91 31 90 17 45 5.17 23.15 5.39 46.53 5.65

39.53 18 57 15 35 5.95 6.35 6.11 60.46 32.69

69.91 14 67 12 32 6.18 3.42 6.50 29.23 0.82

62.74 11 40 10 22 5.54 7.92 6.49 39.21 3.50

47.25 13 54 10 27 5.91 25.08 6.46 66.51 7.24

47.9 6 53 6 25 6.64 0.68 7.03 80.36 19.82

22.71 19 75 14 38 5.71 25.34 5.60 62.55 2.51

49.08 23 70 13 38 5.68 8.71 6.02 82.92 2.14

34.94 23 78 14 41 5.77 57.46 5.75 27.17 1.72

21.86 20 69 10 36 5.52 33.21 5.40 58.74 2.82

13.5 4 40 4 19 6.61 0.97 6.87 90.24 61.33

32.7 27 75 14 37 5.20 38.38 5.37 43.62 1.73

54.16 21 59 14 32 5.09 26.12 5.51 63.11 2.98

61.02 27 75 16 36 5.40 16.43 5.97 68.07 4.99

33.08 19 73 14 39 5.67 22.95 6.24 50.93 25.82

8.581 17 61 12 35 5.67 51.21 5.35 36.95 2.20

53.22 4 51 3 21 6.56 1.16 6.92 74.53 5.52

15.37 9 54 7 32 6.47 40.94 6.84 41.55 12.05

42.65 14 65 9 32 6.04 14.17 6.48 53.47 6.95


Crain and Caskey—Breakpoint Analysis and Relations of Nutrient and Turbidity Stressor Variables to Macroinvertebrate Integrity in Streams—SIR 2010–5164

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