EFFECTS OF ACID MINE DRAINAGE ON LEAF CONSUMPTION AND FINE

PARTICULATE ORGANIC MATTER PRODUCTION BY THE CRAYFISH,

ORCONECTES SANBORNII

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Daniel A. Brown

August 2007

This thesis titled

EFFECTS OF ACID MINE DRAINAGE ON LEAF CONSUMPTION AND FINE

PARTICULATE ORGANIC MATTER PRODUCTION BY THE CRAYFISH,

ORCONECTES SANBORNII

by

DANIEL A. BROWN

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Kelly S. Johnson

Associate Professor of Biological Sciences

Benjamin M. Ogles

Dean, College of Arts and Sciences

Abstract

BROWN, DANIEL A., M.S., August 2007, Biological Sciences

EFFECTS OF ACID MINE DRAINAGE ON LEAF CONSUMPTION AND FINE

PARTICULATE ORGANIC MATTER PRODUCTION BY THE CRAYFISH,

ORCONECTES SANBORNII (68 pp.)

Director of Thesis: Kelly S. Johnson

Crayfish feed on a wide range of plant and animal materials and are one of the

dominant macroinvertebrates found in streams of the eastern United States. By consuming large amounts of leaf litter from riparian vegetation, crayfish are an important component of stream ecosystem dynamics. During the feeding process, leaves are shredded into coarse (CPOM) and fine particulate organic matter (FPOM) and become a basal food resource for many other stream-dwelling organisms. Acid mine drainage

(AMD) is a significant ecological issue in southeastern Ohio and other areas where extensive mining for coal has occurred. The focus of this research was to determine the effects AMD has on the feeding rates of the crayfish, Orconectes sanbornii. This research consisted of two main components, a laboratory experiment and a field experiment. The laboratory experiment investigated the effects of changes in pH on crayfish leaf processing rates and FPOM production and compared the quality of leaves conditioned in AMD-impacted streams versus control streams (no AMD impact), through

two laboratory feeding trials. Detrital processing by crayfish in AMD-polluted and

control streams was quantified in situ using field enclosures. Macroinvertebrate colonization of the field enclosures was also recorded and compared between field sites.

The results indicate that while crayfish are tolerant to acidic conditions, crayfish in low pH (4.0) consumed less leaf area and produced less FPOM than those in higher pH conditions (5.0-7.0). When subjected to pH 4.0, crayfish produced approximately 25% the amount of FPOM as those in pH 7.0. Leaf consumption varied in the two trials.

Crayfish consumed more of the leaves conditioned in the control stream during the initial trial, but more of the leaves conditioned in an AMD-impacted stream in the second trial.

This reflects the complex nature of the leaf conditioning process in streams, which can be influenced by many environmental factors. This research may prove to be beneficial as a reference for future research and reclamation projects.

Approved:

Kelly S. Johnson

Associate Professor of Biological Sciences

Acknowledgments

I would like to give special thanks to my advisor, Kelly Johnson for her

encouragement and steadfast support throughout my research. I would also like to extend thanks and much appreciation to my committee members, Donald Miles and Matthew

White, for their valuable input and support. The recirculating tanks, without which the laboratory experiments would not have been possible, were provided by Morgan Vis.

Thanks also to Willem Roosenburg for allowing me the use of his muffle furnace and to

Warren Currie for the use of his pH meter. Roger Thoma provided verification of my crayfish identifications as well as valuable knowledge pertaining to crayfish. Special thanks go to Dave Elliot for his help with transportation to the field sites and his undeniable skill at making repairs to necessary equipment.

I would also like to give thanks to Ashley Smith, Chad Kinney, Tracy Morman,

Levi Rose, Breanna Harris, and Kelli Johnson for their help with various aspects along the way. Last but certainly not least, thanks to my family for their tremendous support.

Thanks to my parents, David E. and Mary E. Brown, and my brother David J. Brown for his help with polishing this manuscript.

6

Table of Contents Page

Abstract...... 3 Acknowledgments...... 5 List of Tables ...... 8 List of Figures...... 9 CHAPTER ONE: LABORATORY EXPERIMENT ...... 10 Introduction...... 10 Research Objectives...... 13 Methods...... 15 Leaf Conditioning ...... 15 Acid Treatments...... 16 Capture and Use of Crayfish in the Laboratory Experiment ...... 16 Quantifying Leaf Consumption ...... 18 Fine Particulate Organic Matter (FPOM) ...... 19 Statistical Analyses of Leaf Consumption and FPOM Production...... 20 Leaf Carbon and Nitrogen Content...... 21 Results...... 22 Water Temperature Data...... 22 Crayfish Body Size and Leaf Area Consumed ...... 24 Cumulative Leaf Consumption...... 26 Leaf Consumption During Intervals ...... 33 FPOM Production...... 38 Holding Period Analysis - Laboratory Trial #2 ...... 41 Notes Concerning pH and Leaf Conditioning Treatments ...... 41 Leaf Analyses...... 42 Discussion...... 42 pH Effects on Leaf Consumption ...... 42 Leaf Conditioning Effects on Leaf Consumption...... 43 CHAPTER TWO: FIELD EXPERIMENT ...... 46 Introduction...... 46 Research Objectives...... 46 Methods...... 47 Calculations and Statistical Analysis...... 50 Results...... 52 Jordan Run (control) ...... 53 Truetown (control)...... 53 Carbon Hill (control)...... 54 Rock Run (AMD-impacted) ...... 54 Job’s Hollow (AMD-impacted) ...... 55 Railroad 13 (AMD-impacted)...... 55 Comparison of Enclosures With and Without Crayfish ...... 56 Comparison Between Field Sites...... 56 7

Discussion...... 60 Leaf Pack Colonization by Macroinvertebrates...... 60 The Role of Crayfish in Stream Ecosystems ...... 60 Future AMD Management...... 62 LITERATURE CITED ...... 63 8

List of Tables

Table Page

1.1: Mean OCL for Orconectes sanbornii used in both Laboratory Trials ...... 24

1.2: Spearman Correlation values (rs) for OCL and leaf consumption...... 25

1.3: ANOVA table - Cumulative leaf consumption - Laboratory Trial #1...... 29

1.4: ANOVA table - Cumulative leaf consumption - Laboratory Trial #2...... 33

1.5: ANOVA table - Leaf consumption during intervals - Laboratory Trial #1...... 35

1.6: ANOVA table - Leaf consumption during intervals - Laboratory Trial #2...... 38

1.7: ANOVA table - FPOM production during Laboratory Trials #1 and #2 ...... 41

1.8: Percentages of N, C, and C/N ratios for conditioned leaves...... 42

1.9: Water chemistry data for the study sites from the fall and winter, 2005-06...... 45

2.1: Five year averages of autumn water chemistry (2001-05) for all field sites ...... 47

2.2: Biological data for field sites...... 48

2.3: Family richness (FT), Diversity (H′), mean number of individuals/enclosure...... 53

2.4: Macroinvertebrates, identified to family level, in the leaf packs...... 59 9

List of Figures

Figure Page

1.1: Water temperature data for Laboratory Trial #1...... 23

1.2: Water temperature data for Laboratory Trial #2...... 23

1.3: Relationship between OCL and leaf consumption for Laboratory Trial #1 ...... 25

1.4: Relationship between OCL and leaf consumption for Laboratory Trial #2 ...... 26

1.5: Cumulative leaf consumption during both Laboratory Trials, by pH treatment .....28

1.6: Cumulative leaf consumption during Laboratory Trial #1, by leaf condition...... 29

1.7: Cumulative leaf consumption during Laboratory Trial #2, by leaf condition...... 32

1.8: Leaf consumption during 4th interval of Laboratory Trial #1, by pH treatment...... 34

1.9: Leaf consumption during intervals in Laboratory Trial #1, by leaf condition ...... 35

1.10: Leaf consumption during 2nd interval of Laboratory Trial #2, by pH treatment ...37

1.11: Leaf consumption during intervals in Laboratory Trial #2 by leaf condition ...... 37

1.12: FPOM produced during both Laboratory Trials, by pH treatment...... 39

1.13: FPOM produced during both Laboratory Trials, by leaf condition...... 40

2.1: Map of all six field study sites...... 51

2.2: Diversity indices for macroinvertebrates in enclosures...... 58 10

CHAPTER ONE: LABORATORY EXPERIMENT

Introduction Acid Mine Drainage (AMD) is a significant ecological issue in areas where there

has been extensive surface mining, in particular for coal. Throughout the Appalachian states, including a substantial portion of southeastern Ohio, AMD has an enormous impact on the health of streams and rivers. In Ohio alone, over 2500 miles of streams and rivers are affected by AMD (ODNR 1997).

Acid drainage impairs streams by lowering the pH and increasing concentrations of aluminum and other metals (Schindler 1988). In streams impacted by AMD, species richness and biological diversity are often significantly reduced, fish and amphibians are often eliminated, aquatic habitats are altered, and leaf litter breakdown rates are often greatly reduced (Starnes 1985, Bermingham et al. 1996, Bauers 2003).

In forested headwater streams, allochthonous leaf litter from riparian vegetation is a major source of energy (Minshall 1967, Hieber and Gessner 2002, Zhang et al. 2004).

With leaf litter having such a significant role in stream ecosystems, an understanding of human impacts on ecosystem processes such as leaf litter breakdown is essential. Leaf litter breakdown is driven largely by detritivorous macroinvertebrates (shredders), bacteria, and filamentous fungi, although physical abrasion also plays a role (Hieber and

Gessner 2002). Through feeding activities, macroinvertebrate shredders can play a critical role in stream energetics by accelerating the leaf breakdown process (Schlief and

Mutz 2006). Microbial colonization contributes to the breakdown of coarse particulate organic matter (CPOM), and typically enhances shredding (Smock and Harlowe 1983) by 11

strongly influencing how quickly it is consumed by shredding macroinvertebrates (van

Frankenhuyzen et al. 1985). Fungi enhance the palatability and available energy of leaf litter through degradation (van Frankenhuyzen and Geen 1986). Therefore, decreased rates of breakdown of organic matter in acidic waters may be a result of reduced microbial activity and poor leaf conditioning in addition to a decrease in shredder abundance and feeding activity.

There is some disagreement in the literature as to how conditioning of leaves is affected by AMD. Groom and Hildrew (1989) demonstrated that leaves conditioned in a neutral stream were colonized by a biofilm consisting of more filamentous fungi,

bacteria, and entrapped particles than leaves conditioned in an acid stream. The leaves

conditioned in the acid stream were not only much less colonized, but were also covered

by an amorphous flocculate matter. Their findings suggest that the food quality for collecting and shredding detritivores in acid streams may be reduced. Bermingham et al.

(1996) also reported a reduction in leaf litter breakdown downstream from a coal mine

effluent that carried high metal loads and low pH. In contrast, van Frankenhuyzen and

Geen (1986) suggested acidification may increase the abundance of fungal decomposers

in streams and lakes. They found fungal colonization of leaves increased at low pH, thus

improving the availability of microbial and leaf-derived energy and enabling the

caddisfly Clistoronia magnifica to thrive at lower pH. These disparate results emphasize

the need for additional studies to understand how different components of systems, and in

particular crayfish, respond to acid pollution. 12

Crayfish are omnivores that feed on a wide range of plant and animal materials

(Usio and Townsend 2001). They are among the dominant macroinvertebrates in the

streams of the eastern United States (Pflieger 1996), often accounting for a substantial proportion of invertebrate biomass (up to 30%) and production (Momot et al. 1978,

Creed and Reed 2004). They are also an important component of stream ecosystem dynamics (Whitledge and Rabeni 1997) because their feeding activities significantly impact the abundance of basal food resources (leaf litter, plant and fungal biomass) and populations of small consumers (Rosemond et al. 1998, Usio 2000). As macroconsumers primarily of detritus such as leaf litter, crayfish are strong indicators of ecosystem health/stability in stream communities (Rosemond et al. 1998).

Recently, crayfish have been given the title of “ecosystem engineers” due to their impact on community structure and ecosystem processes (Creed and Reed 2004).

Ecosystem engineers impact the structure of the community and ecosystem processes through the creation, modification, and maintenance of habitats as well as by altering the rates at which resources are made available to other organisms. Crayfish can be considered ecosystem engineers based on their influence on detrital processing rates, alteration of the dominant algal cover, and through bioturbation of both coarse and fine sediments (Creed and Reed 2004). Despite the importance of crayfish as ecosystem engineers, relatively few studies have investigated the significance of crayfish as shredders (Usio and Townsend 2001, Creed and Reed 2004).

Compared to other aquatic organisms such as fish and many insects, crayfish have a higher tolerance for acidification. While increased acidity results in lowered 13

survivorship of the crayfish Cambarus bartonii, commonly found in headwater streams in the eastern United States, they were still quite tolerant, showing a 50% survival rate in

water with a pH of 2.43 (DiStefano et al. 1991). In comparison, one of the most acid-

tolerant fish found in eastern coldwater streams, the Brook Trout (Salvelinus fontinalis)

has a 50% survival rate at a pH of 3.5 (Daye and Garside 1975). Despite their tolerance

to acidity, sublethal effects may affect the rates at which crayfish process leaves.

Changes in leaf processing rates of crayfish could have consequences for both crayfish

fecundity and the energy flow through the rest of the stream ecosystem.

Research Objectives The research described for the laboratory experiment had two main objectives.

The first objective was to compare the quality of leaf litter conditioned in streams

polluted by AMD versus control streams with no AMD impact, using laboratory feeding

trials with O. sanbornii. The second objective was to investigate the effects of changes in

pH on leaf litter processing rates by O. sanbornii in the laboratory.

Prior to the research, several possible interpretations of results were considered.

First, crayfish may process leaves conditioned in a stream affected by AMD more slowly

than leaves conditioned in an unimpacted stream. Such a result could be attributed to low

microbial colonization or the presence of toxic metals from AMD making the leaves less

palatable. An alternate explanation, as indicated by van Frankenhuyzen and Geen

(1986), was that AMD-conditioned leaves may actually be more nutritious due to greater

fungal biomass therefore requiring that they eat less. However, if the opposite result

occurs (leaves processed more rapidly) this could be due to the leaves conditioned in 14

streams with AMD impact being less nutritious. If AMD conditioned leaves are less

nutritious, crayfish may have to process more leaves in order to obtain the necessary

energy for survival.

In addition to effects on leaf conditioning, acid mine polluted water may influence

the rates at which crayfish process leaves due to physiological stress. Physiological stress

in crayfish might result in sluggishness and slower processing rates. Conversely, stress

may increase the metabolic rate of crayfish, which might consequently lead to faster

processing rates. In a recent study of the response of crayfish to acidification in

headwater streams, Seiler and Turner (2004) found that individual crayfish grew more

slowly in acidified streams, and speculated that energetic costs associated with

osmoregulation, ion retention, and respiration become increased with acidification.

However, they did not measure consumption rates or litter processing rates.

Interestingly, they found that although acidification slowed growth, crayfish densities

were sixfold higher in these streams, in part due to the absence of fish predation. Support

for Seiler and Turner’s (2004) explanation involving increased energetic costs is provided

by Rowe et al. (2001), who found that the standard metabolic rate (SMR) of several

invertebrates, including crayfish, was elevated following exposure to a mixture of trace

elements in a polluted habitat. In particular, the SMR of the crayfish Procambarus

acutus in locations contaminated with trace elements from slurried coal ash was up to

30% higher than individuals from uncontaminated habitats. Crayfish may increase their food consumption in an effort to compensate for such an increase in metabolic rate, and thus exhibit faster rates of leaf breakdown under moderate acid stress. 15

Methods The effect of acid stress (pH 4.0, 5.0, 6.0, 7.0) and leaf quality (conditioned in

AMD-impacted vs. control streams) on crayfish processing rates was tested by means of a factorial laboratory experiment. The experiment was repeated twice, once from

December 16-30, 2005, and the second time from January 26 to February 9, 2006.

Leaf Conditioning All leaves used during the course of the laboratory experiments were collected just after abscission on November 2, 2005 from a single silver maple (Acer saccharinum) tree located in Athens County, Ohio (N 39° 16.841′, W 082° 05.611′, elevation 999′).

Silver maple leaves were selected as it is a species commonly found in riparian zones in this region and the leaves condition relatively quickly compared to other riparian species such as the American Sycamore (Platanus occidentalis). All leaves were brought back to the laboratory and air dried.

The leaves for the first laboratory trial were conditioned for 32 days during

November and early December, 2005 by placing them in mesh sacks (commercial cabbage bags purchased from Apex Feed and Supplies, Marietta, Ohio) and submerging the sack in a stream. Half of the leaves were conditioned at an AMD-impacted site

(Railroad 13 (N 39° 35.805′, W 082° 11.247′)), a segment of Sunday Creek south of

Corning, Ohio). The other half of the leaves were conditioned in an unimpacted stream

(Jordan Run, near Desonair Nature Preserve). This stream is designated by the Ohio EPA as a high quality reference site, with no known environmental impacts. For the second 16

laboratory trial, the leaves were conditioned for 34 days, from December 22, 2005 to

January 25, 2006, at the same locations as the first trial.

Acid Treatments Four recirculating tanks with aerating pumps were each partially filled with

56.781 L of distilled water. Once filled, 1.5 grams of Kent Marine salts (Kent Marine,

Inc., Acworth, Georgia 30102) were added to each tank in order to provide a defined and

consistent balance of ions necessary for the health of aquatic organisms.

Sulfuric acid (H2SO4) was added to lower the pH to the desired level for each tank.

The pH was monitored daily using a handheld pH meter (Hanna Instruments USA

Headquarters, Woonsocket, Rhode Island 02895) and Whatman pH Indicator strips

(Whatman International Ltd., Maidstone, England). If the pH showed an increase (for example, from ammonia produced as waste by the crayfish) additional sulfuric acid was added in order to lower the pH back to the desired level. Water temperature of each tank was monitored daily.

Capture and Use of Crayfish in the Laboratory Experiment All crayfish that took part in the laboratory trials were captured from Margaret

Creek near the Ervin Road bridge (N 39° 18.161′, W 082° 08.625′) in Athens, OH. This site has no AMD impact and crayfish are fairly plentiful. For the laboratory trials, the crayfish were collected between December 11-12, 2005 for the first trial and January 19-

22, 2006 for the second trial.

For the first trial, the best method for capturing crayfish was to use a D-ring dip net to scoop up leaf masses and other debris from the middle of pools. Prior to the 17

second trial, a rain event occurred and high water levels resulted. The temperature at this

time was higher than in December 2005, resulting in increased crayfish activity. At this

time, the most efficient method for catching crayfish involved scooping along the bank of

the stream with a D-ring dip net, especially near overhangs and root masses.

For both trials, the crayfish were brought back to the lab and held in a cold

storage room at 40˚F for at least four days. The time required to capture crayfish for the

second laboratory trial was prolonged due to elevated water levels and as a result, there

was more variation in the length of time the crayfish spent in cold storage. The number

of days the crayfish were held in cold storage prior to the second trial ranged from 4 to 7

days. A one-way analysis of variance (ANOVA) was run in order to determine whether

the time spent in cold storage impacted leaf consumption. All data were analyzed using

SPSS 14.0 statistical software (SPSS Inc. Headquarters, 233 S. Wacker Drive, Chicago,

IL 60606).

Prior to being subjected to the experiment the crayfish were removed from the

cold storage room and gradually brought up to ambient room temperature. The orbital carapace length (OCL) of each crayfish was measured. This measurement, used by Usio

and Townsend (2002), was taken from the back of the eye socket to the center of the

posterior end of the carapace and was recorded in order to account for the variation in

sizes of the crayfish used in the trials. For each trial, a Spearman’s rho (rs) correlation

analysis (see Table 3.4) was run in order to determine whether crayfish OCL had an

effect on the amount of leaf area consumed. Containers were made semi-permeable by

cutting two 3 x 7 cm holes on opposite sides and covering the holes with fine mesh 18 organza fabric (Wal-Mart, Athens, Ohio 45701). This modification allowed for water movement in and out of the containers while preventing the loss of Fine Particulate

Organic Matter (FPOM) produced by the crayfish. The crayfish were then placed into individual containers (Rubbermaid deep square 42 oz. containers, Rubbermaid, Inc.,

Huntersville, NC 28078-1801) with lids and individuals were assigned randomly to the pH treatments. Each container was given a unique number identification. Twelve containers were submerged below the water line in each of the four recirculating tanks, for a total of 48 crayfish per laboratory trial.

At the conclusion of the laboratory experiment, all crayfish were preserved in

70% ethanol. Individuals were later identified to species with the aid of A Key to the

Crayfish of Ohio (Thoma 1999). Further verification was made through a series of photographs sent via email to the author of this guide. Voucher specimens from both field and laboratory experiments are located in the Ohio University Zoological Museum.

Quantifying Leaf Consumption The rate of leaf breakdown by individual crayfish was quantified by scanning individual leaves with a Microtek ScanMaker E6 flatbed digital scanner and using Kodak

Imaging for Windows Software to determine the surface area before and after placement into the containers with the crayfish. Leaves were re-scanned after 4, 7, 10, and 14 days.

If all of the initial leaf area was consumed, new leaves were scanned and placed into the containers. The decrease in surface area provided an indication of the amount of leaf broken down by the crayfish. 19

The scans of the leaves were then filled and leaf area (in pixels) was measured

using Jandel Sigma Scan Image Management Software v2.0. Pixels were converted to

square millimeters (sq. mm) by taking the total number of pixels along one axis of a

standard image and dividing by the length (mm) of that axis. The resulting value was

squared. Finally, each pixel count was divided by this value in order to obtain a value in

sq. mm.

Image Dimensions 1224 x 1224 (pixels) 126.5mm x 126.5mm

1224/126.5 = 9.676 (9.676)2 = 93.623 = # of pixels in each sq. mm

To ensure the accuracy of these calculations, 10 x 10 mm pieces of paper

similarly colored to the leaves were cut out and scanned. The resulting pixel counts had a

percentage error of less than 0.3% from the expected value.

Fine Particulate Organic Matter (FPOM) In addition to leaf area consumed, fine particulate organic matter (FPOM) produced by each crayfish was measured. As mentioned previously, the containers holding each individual crayfish in the recirculating tanks were semi-permeable due to mesh covered holes. The mesh was fine enough (‹1 mm) to contain most FPOM. At the 20

end of each two week trial, the FPOM from each container was harvested by straining

through Glass Fiber Filter Circles (Fisherbrand G6 1.6µm, item #09-804-110A, heat

stable to 500ºC/932ºF). After the FPOM was strained, the filters with FPOM were dried

at room temperature for 10 days. Once the filters and FPOM were completely dried, they

were each weighed and their dry mass (DM) recorded. Ash mass (AM) was obtained by

heating each individual sample at 450°C for 2.5 hours in a muffle furnace (Thermolyne

1300 Model F-B1315M, Thermolyne Corporation, subsidiary of Sybron Corporation,

Dubuque, Iowa), and weighed to the nearest 0.01 mg. Obtaining the ash free dry mass

(AFDM) for analysis was necessary because the deposition of silt and metals in aquatic

environments can alter the mass of the organic matter. The muffle furnace oxidized the

carbon, leaving only silt and metals (AM), the weight of which is then subtracted from

the DM in order to obtain AFDM. During the analyses, AFDM was referred to as

FPOM.

AFDM = {DMsample – AMsample}

Statistical Analyses of Leaf Consumption and FPOM Production The amount of leaf area broken down as well as FPOM produced by the crayfish

inhabiting water with varying levels of pH were analyzed separately by a two-way

ANOVA, with water pH (4.0, 5.0, 6.0, 7.0) and conditioning of leaves (AMD impacted

vs. unimpacted) as main effects. These analyses evaluated differences in the rates of

processing by crayfish of leaves conditioned in an AMD impacted stream versus leaves 21

conditioned in an unimpacted stream, differences in crayfish feeding rates due to changes in pH, and any interactions between the two effects.

Leaf Carbon and Nitrogen Content Ten samples of leaves from each laboratory trial were analyzed for carbon (C) and nitrogen (N) content. Each sample consisted of 3-4 leaves. Of the 10 samples from each laboratory trial, 1 sample from each stream (Jordan Run and Railroad 13) used for conditioning was frozen just after the conditioning period. The remaining 8 samples consisted of leaves used in the laboratory trial. For each laboratory trial, there were samples representing the four pH levels of 4.0, 5.0, 6.0, and 7.0 for both the Control and

AMD conditioning locations.

At the conclusion of each laboratory trial, leaves were placed in storage bags and frozen. Prior to analyses, all samples were placed in paper bags and dried for seven days at 69˚C in a Fisher Scientific Isotemp Oven, Model 630F (Fisher Scientific International).

When the leaf samples were completely dried, they were pulverized using a Spex

CertiPrep 8000M Mixer/Mill (Spex CertiPrep, Inc., Metuchen, New Jersey 08840). This

mixer/mill can be best described as a high-energy ball or shaker mill. The samples were

placed inside a steel vial containing four steel balls: two one-half inch in diameter and

two one-quarter inch in diameter. Once the samples were secured, the Spex CertiPrep

8000M Mixer/Mill shook the steel vial back and forth in a figure-8 motion at approximately 1080 cycles per minute. Each sample underwent this process for 90 seconds. Following this procedure, each sample was placed in a glass vial and labeled 22

before being sent off to the Ohio State University’s STAR Lab (Wooster, Ohio), for

analysis of C and N using an elemental analyzer (Elementar Vario Max CN). From the percentages of C and N, the C:N ratios were calculated (Table 3.10). The data were then

analyzed using an independent samples t-test in order to determine whether there were differences between leaves conditioned at each of the two sites as well as whether there were differences between the leaves conditioned for the first laboratory trial and those conditioned for the second laboratory trial.

Results

Water Temperature Data The original plan involved keeping the water temperature of each tank constant for the duration of each trial. However, fluctuations in the temperature inside Wilson

Research Hall occurred. As a result, the temperature of the water in each tank also fluctuated throughout the duration of each trial. Temperature was monitored at least once daily and throughout both trials all four tanks maintained a temperature within 1.0˚C of

the others and the duration of the differences in temperature lasted no longer than 1 day

(Figures 1.1 and 1.2).

23

24.0

22.0

o 20.0 pH 7 18.0 pH 6 16.0 pH 5 pH 4

Temperature, C Temperature, 14.0

12.0

10.0 14-Dec 16-Dec 18-Dec 20-Dec 22-Dec 24-Dec 26-Dec 28-Dec 30-Dec Date (2005)

Figure 1.1. Water temperature data for Laboratory Trial #1.

24.0

22.0

o 20.0 pH 7 18.0 pH 6 16.0 pH 5 pH 4

Temperature, C Temperature, 14.0

12.0

10.0 24-Jan 26-Jan 28-Jan 30-Jan 1-Feb 3-Feb 5-Feb 7-Feb 9-Feb Date (2006)

Figure 1.2. Water temperature data for Laboratory Trial #2.

24

Crayfish Body Size and Leaf Area Consumed

Laboratory Trial #1 The means for OCL from the first laboratory trial are listed in Table 1.1. There was no correlation between crayfish OCL and leaf consumption after 14 days (rs = 0.019, p = 0.898). In fact, no correlations were observed between crayfish OCL and leaf consumption throughout the entire duration of the first laboratory experiment (see Table

1.2). Figure 1.3 depicts the relationship between OCL and leaf area consumed observed at 14 days, which at that time was the closest OCL and leaf consumption came to being correlated.

Laboratory Trial #2 The means for OCL from the second laboratory trial are listed in Table 1.1. There was a correlation between crayfish OCL and leaf consumption after 4 days (rs = 0.399, p

= 0.006) (see Figure 1.4). No other correlations were observed between crayfish OCL

and leaf consumption during the remainder of the second laboratory trial (see Table 1.2).

Table 1.1. Mean OCL for Orconectes sanbornii used in both laboratory trials.

Trial 1 Trial 2 pH N Mean OCL Std. Dev. N Mean OCL Std. Dev. 4.0 12 13.91 4.03 12 12.18 2.40 5.0 12 13.90 3.87 12 12.11 2.13 6.0 12 13.79 3.53 12 12.16 2.16 7.0 12 14.00 3.94 12 12.26 2.21

Condition AMD 24 13.72 3.11 24 12.05 1.82 Control 24 14.08 4.31 24 12.30 2.48 Total 48 13.90 3.72 48 12.18 2.16 25

Table 1.2. Spearman Correlation values (rs) and p-values from analyses of OCL and leaf consumption by Orconectes sanbornii, during both laboratory trials. Bonferroni correction cut-off for significance is p ≤ 0.02.

Trial 1 Trial 2

Days rs prs p 4 0.143 0.332 0.399 0.006* 7 0.119 0.420 0.115 0.440 10 0.093 0.527 0.067 0.655 14 0.019 0.898 0.021 0.889 0-4 0.059 0.332 0.399 0.006* 4-7 0.143 0.692 0.060 0.685 7-10 -0.204 0.164 0.030 0.840 10-14 -0.104 0.483 0.034 0.820

5.0

4.0

3.0

2.0

1.0

0.0 CONDITION

Log Leaf Area Consumed (sq. mm) -1.0 Control

-2.0 AMD .9 1.0 1.1 1.2 1.3 1.4 1.5

Log OCL (mm)

Figure 1.3. Scatterplot illustrating the relationship between Orconectes sanbornii carapace length and leaf area consumed after 14 days in laboratory trial #1.

26

5.0

4.0

3.0

2.0

1.0

0.0 CONDITION

Log Leaf Area Consumed (sq. mm) -1.0 Control

-2.0 AMD .9 1.0 1.1 1.2 1.3 1.4 1.5

Log OCL (mm)

Figure 1.4. Scatterplot illustrating the relationship between Orconectes sanbornii carapace length and leaf area consumed after 4 days in laboratory trial #2.

Cumulative Leaf Consumption

Laboratory Trial #1 The amount of leaf area consumed by crayfish was compared among four pH levels (n = 12 for each pH) and two types of leaf conditioning (n =24 for each treatment) using a two-way ANOVA with pH and leaf conditioning treatment as the main effects.

The data failed to meet assumptions of normal distribution and therefore were log transformed. Statistical analyses were completed using SPSS 14.0 statistical software

(Chicago, IL 60606).

For the first trial, there was no significant difference between total leaf area consumed by crayfish after 4 days in varying pH levels (F = 0.513, p = 0.675) (Table

1.3). However, crayfish consumed more leaves conditioned at Jordan Run (high quality 27 stream) than leaves conditioned at Railroad 13 (AMD impacted) (F = 7.921, p = 0.008)

(Figure 1.6).

After seven days there was a significant pH effect on the total leaf area consumed by crayfish (F = 4.180, p = 0.012). A Bonferroni post hoc test showed that the consumption rates of individuals in pH of 4.0 was significantly less than those in pH of

7.0 (p = 0.021) and nearly significant compared to crayfish in pH of 6.0 (p = 0.057)

(Figure 1.5). Also after seven days, the crayfish had still consumed more of the leaves conditioned at Jordan Run than leaves conditioned at Railroad 13 (F = 7.185, p = 0.011)

(Figure 1.6).

After ten days there continued to be a significant difference in the total leaf area consumed by crayfish based on pH (F = 6.227, p = 0.001). A Bonferroni post hoc test indicated that the consumption rates of the individuals in pH 4.0 were significantly lower than those in pH 6.0 (p = 0.043) and 7.0 (p = 0.001) (Figure 1.5). The crayfish consumed more total leaf area of the leaves conditioned at Jordan Run than leaves conditioned at

Railroad 13 after ten days (F = 13.176, p = 0.001) (Figure 1.6).

At the completion of the first laboratory trial (after fourteen days), there was a significant difference in the total amount of leaf area consumed by crayfish based on pH

(F = 10.746, p = <0.001). The consumption rates of individuals in pH 4.0 were significantly lower than those in pH 5.0 (p = 0.004), 6.0 (p = 0.002), and 7.0 (p = <0.001)

(Figure 1.5). There was no difference between the consumption rates of individuals in pH 5.0 compared to those in pH 6.0 (p = 1.000) and 7.0 (0.512). Also at the completion 28

of the first trial, the crayfish had consumed more of the leaves conditioned at Jordan Run

than leaves conditioned at Railroad 13 (F = 5.655, p = 0.022) (Figure 1.6).

Trial 4.000 Trial 1 Trial 2

3.500

3.000 q. mm) for both trials

2.500

2.000 Log leaf area consumed (s

1.500

pH 4 pH 5 pH 6 pH 7 pH

Figure 1.5. Mean (±SE) log leaf area consumed at the completion of both laboratory trials by Orconectes sanbornii at the four pH treatments.

29

4.000

3.500

3.000

2.500 nsumed (sq. mm) - Trial 1 2.000

1.500

1.000

AMD 0.500 CONTROL Cumulative Log Leaf Area Co

0.000 4 7 10 14 Days

Figure 1.6. Mean (± SE) cumulative log leaf area consumed by Orconectes sanbornii during laboratory trial #1, based on location of leaf conditioning.

Table 1.3. ANOVA table for cumulative leaf area consumption during laboratory trial #1, with pH and location of leaf conditioning as treatments.

Treatment SS F p

4 days pH 1.448 0.513 0.675 Leaf Condition 7.446 7.921 0.008 pH x Leaf Condition 0.263 0.093 0.963

7 days pH 6.775 4.180 0.012 Leaf Condition 3.882 7.185 0.011 pH x Leaf Condition 0.146 0.090 0.965

10 days pH 7.458 6.227 0.001 Leaf Condition 5.260 13.176 0.001 pH x Leaf Condition 0.522 0.436 0.728

14 days pH 10.763 10.746 <0.001 Leaf Condition 1.888 5.655 0.022 pH x Leaf Condition 1.639 1.636 0.196 30

Laboratory Trial #2 The amount of leaf area consumed by crayfish for the second laboratory trial was

compared among the same four pH treatments and two leaf conditioning treatments as the

first experiment using a two-way ANOVA with pH and leaf conditioning treatments as

the main effects. However, from the beginning of the trial until the end of the 3rd interval

(10 days) there was a discrepancy in sample sizes. The treatments of pH 7.0 (n = 11) and

AMD conditioned leaves (n = 23) had one less individual than the other pH treatments (n

= 12) and the leaves conditioned in the control stream (n = 24) during that time period.

This was a result of a scanning error for the initial scan of the AMD conditioned leaf fed to an individual in pH 7.0. During the initial scan, a portion of the leaf was unknowingly folded under itself. For the next three scans (after 4, 7, and 10 days) this portion was not folded under and negative values for leaf consumption were thus obtained for this individual. Rather than analyze the data with this particular individual’s erroneous negative values, data from this individual were omitted from the analyses until doing so for the last interval (10-14 days) and the total leaf area consumed (after 14 days) when the data were positive. Similarly to the first trial, data for the second trial failed to meet assumptions of normal distribution and were log transformed.

After 4 days of the second trial there was no significant difference between leaf area consumed by crayfish in varying pH treatments (F = 1.575, p = 0.211) (Table 1.4).

There was also no significant difference between leaf area consumed by crayfish depending on where leaf conditioning occurred (F = 0.511, p = 0.479).

After 7 days, there was a significant difference between leaf area consumed by crayfish in varying pH treatments (F = 5.789, p = 0.002). A Bonferroni post hoc revealed 31

that crayfish in pH 4.0 consumed significantly less than those in pH 6.0 (p = 0.025) and

7.0 (p = 0.002) and nearly so than those in pH 5.0 (p = 0.053). There was no significant

difference in leaf consumption based on where the leaves were conditioned (F = 3.259, p

= 0.79). This result was strengthened by the fact that a one-way ANOVA also indicated

no significant difference in the amount of leaf area consumed based on location of leaf

conditioning (F = 2.287, p = 0.137).

After 10 days, there was a significant difference in total leaf area consumed by

crayfish based on pH treatment (F = 6.350, p = 0.001). A Bonferroni post hoc test

indicated that crayfish in pH 4.0 consumed significantly less leaf area than those in pH

6.0 (p = 0.039) and 7.0 (p = 0.001). There was no significant difference in leaf area consumed by crayfish in pH 4.0 and those in pH 5.0 (p = 0.227). There was a significant

difference observed for the total amount of leaf area consumed by crayfish based on

where leaf conditioning took place. The crayfish fed leaves from the AMD site consumed more total leaf area than those that were fed leaves conditioned at the control

site (F = 4.837, p = 0.034) (Figure 1.7).

After 14 days, at the completion of the second laboratory trial, there was a

significant difference in the total amount of leaf area consumed by crayfish based on pH

treatment (F = 9.689, p = <0.001). A Bonferroni post hoc showed that crayfish in pH 4.0

consumed significantly less total leaf area than those in pH 6.0 (p = 0.002) and 7.0 (p =

<0.001) but not those in pH 5.0 (p = 0.264) (Figure 1.5). Also at the completion of the second laboratory trial, there was a significant difference between the total leaf area consumed by the crayfish based on where the leaves were conditioned. The crayfish 32

consumed more leaves conditioned at Railroad 13, the AMD-impacted site, than leaves

conditioned at Jordan Run, the control site (F = 4.338, p = 0.044).

4.000

3.500

3.000

2.500

2.000

1.500

1.000 AMD CONTROL 0.500 Cumulative Log Leaf Area Consumed (sq. mm) in Trial 2

0.000 4 7 10 14 Days

Figure 1.7. Mean (±SE) cumulative log leaf area consumed during laboratory trial #2 by Orconectes sanbornii, based on location of leaf conditioning.

33

Table 1.4. ANOVA table for cumulative leaf area consumption during laboratory trial #2, with pH and location of leaf conditioning as treatments.

Treatment SS F p

4 days pH 3.107 1.575 0.211 Leaf Condition 0.336 0.511 0.479 pH x Leaf Condition 1.177 0.597 0.621

7 days pH 7.860 5.789 0.002 Leaf Condition 1.475 3.259 0.079 pH x Leaf Condition 1.010 0.744 0.533

10 days pH 7.794 6.350 0.001 Leaf Condition 1.979 4.837 0.034 pH x Leaf Condition 1.100 0.896 0.452

14 days pH 6.665 9.689 <0.001 Leaf Condition 0.995 4.338 0.044 pH x Leaf Condition 0.378 0.549 0.651

Leaf Consumption During Intervals

Laboratory Trial #1 For the first trial, there was no effect of pH on leaf area consumed by crayfish in

the interval of 0-4 days (F= 0.513, p = 0.675) (Table 1.5). However, in the 2nd (4-7 days)

(F = 4.414, p = 0.009), 3rd (7-10 days) (F = 4.886, p = 0.005), and 4th intervals (10-14

days) (F = 7.224, p = 0.001) (Figure 3.7), pH did have a significant effect on the amount

of leaf area consumed by crayfish. Bonferroni post hoc tests revealed that crayfish in a

pH of 4.0 processed leaves more slowly than crayfish in pH levels of 6.0 (p = 0.028) and

7.0 (p = 0.025) during the 2nd interval (4-7 days). During the 3rd interval (7-10 days), individuals in pH 4.0 consumed leaves more slowly than those in pH 7.0 (p = 0.005).

During the same interval, individuals in pH 5.0 also consumed leaves at a lower rate than those in pH 7.0 (p = 0.041). During the 4th interval (10-14 days), crayfish in pH 4.0 34 consumed leaves more slowly than those in pH 5.0 (p = 0.004), pH 6.0 (p = 0.012), and pH 7.0 (p = 0.001) (Figure 1.8). There was no difference between the consumption rates of individuals in pH 5.0 and those in pH 6.0 (p = 1.000) or in pH 7.0 (p = 1.000) during this interval.

A conditioning effect on the rate of leaf consumption by crayfish was observed during the 1st (F= 7.921, p = 0.008) and 3rd intervals (F = 9.928, p = 0.003) (Figure 1.9).

During both of these intervals, crayfish consumed leaves conditioned in the high quality stream (Jordan Run) more quickly than those conditioned in the AMD-impacted stream

(Railroad 13). However, during the 2nd (F = 3.509, p = 0.068) and 4th (F = 0.009, p =

0.924) intervals there was no observed effect of leaf treatment on consumption rates by crayfish.

4.000

3.500

3.000

2.500

2.000

1.500

1.000

0.500

Log Leaf Area Consumed (sq. mm) between 10-14 days 10-14 between mm) (sq. Consumed Area Leaf Log 0.000

pH 4 pH 5 pH 6 pH 7 pH

Figure 1.8. Mean (± SE) log leaf area consumed during the 4th interval (10-14 days) of laboratory trial #1 by Orconectes sanbornii in varying pH treatments.

35

4.000

3.500

3.000

2.500

2.000

1.500

1.000

AMD 0.500 CONTROL Log Leaf Area Consumed (sq. mm) in Intervals - Trial 1

0.000 0-4 4-7 7-10 10-14 Interval Days

Figure 1.9. Mean (±SE) log leaf area consumed during intervals in laboratory trial #1 by Orconectes sanbornii, based on the location of leaf conditioning.

Table 1.5. ANOVA table for leaf area consumption during intervals in laboratory trial #1, with pH and location of leaf conditioning as treatments.

Treatment SS F p

0-4 days pH 1.448 0.513 0.675 Leaf Condition 7.446 7.921 0.008 pH x Leaf Condition 0.263 0.093 0.963

4-7 days pH 12.676 4.414 0.009 Leaf Condition 3.359 3.509 0.068 pH x Leaf Condition 1.032 0.359 0.783

7-10 days pH 8.806 4.886 0.005 Leaf Condition 5.965 9.928 0.003 pH x Leaf Condition 1.791 0.994 0.406

10-14 days pH 15.663 7.224 0.001 Leaf Condition 0.007 0.009 0.924 pH x Leaf Condition 3.926 1.811 0.161 36

Laboratory Trial #2 For the second trial, there was no effect of pH on leaf area consumed by crayfish during the interval of 0-4 days (F = 1.575, p = 0.211) (Table 1.6) as well as the 3rd interval of 7-10 days (F = 2.514, p = 0.072). There was a significant effect of pH on leaf area consumed by crayfish during the 2nd interval of 4-7 days (F = 5.808, p = 0.002) and

also during the final interval of 10-14 days (F = 7.085, p = 0.001). A Bonferroni post hoc indicated that during the 2nd interval (4-7 days) the crayfish in pH 4.0 consumed significantly less leaf area than the crayfish in pH 5.0 (p = 0.014), 6.0 (p = 0.025), and 7.0

(p = 0.003) (Figure 1.10). During the 4th and final interval (10-14 days) the crayfish in

pH 4.0 consumed significantly less leaf area than crayfish in pH 6.0 (p = 0.009) and 7.0

(p = 0.004). Also during the 4th interval, there was no difference in the amount of leaf

area consumed by crayfish in pH 5.0 compared to that of crayfish in pH 4.0 (p = 1.000)

and 5.0 (p = 0.062). However, crayfish in pH 5.0 consumed significantly less leaf area than crayfish in pH 7.0 during this interval (p = 0.026).

No leaf conditioning effects on the rate of leaf consumption were observed during any of the intervals of the second laboratory trial. There was no significant difference in the rate at which leaves conditioned in the control stream and leaves conditioned in the

AMD-impacted stream were consumed by crayfish during the 1st (F = 0.511, p = 0.479),

2nd (F = 1.284, p = 0.264), 3rd (F = 1.715, p = 0.198) or 4th (F = 1.933, p = 0.172)

intervals. Figure 1.11 shows the comparison of leaf area consumed during the 4th interval

by crayfish based on where the leaves were conditioned. This was the closest the

treatment of leaf conditioning came to having an effect during any of the intervals. 37

3.500

3.000

2.500

2.000

1.500

1.000

0.500 Log Leaf Area Consumed (sq. mm) between 4-7 days 0.000

pH 4 pH 5 pH 6 pH 7 pH

Figure 1.10. Mean (±SE) log leaf area consumed during the 2nd interval (4-7 days) of laboratory trial #2 by Orconectes sanbornii in varying pH treatments.

4.000

3.500

3.000

2.500 mm) in Intervals- Trial 2 2.000

1.500

1.000 AMD CONTROL 0.500 Log Leaf Area Consumed (sq.

0.000 0-4 4-7 7-10 10-14 Interval Days

Figure 1.11. Mean (±SE) log leaf area consumed by Orconectes sanbornii during intervals in laboratory trial #2 based on location of leaf conditioning. 38

Table 1.6. ANOVA table for leaf area consumption during intervals in laboratory trial #2, with pH and location of leaf conditioning as treatments.

Treatment SS F p

0-4 days pH 3.107 1.575 0.211 Leaf Condition 0.336 0.511 0.479 pH x Leaf Condition 1.177 0.597 0.621

4-7 days pH 14.571 5.808 0.002 Leaf Condition 1.074 1.284 0.264 pH x Leaf Condition 3.234 1.289 0.291

7-10 days pH 7.032 2.514 0.072 Leaf Condition 1.600 1.715 0.198 pH x Leaf Condition 2.959 1.058 0.378

10-14 days pH 10.704 7.085 0.001 Leaf Condition 0.974 1.933 0.172 pH x Leaf Condition 0.332 0.220 0.882

FPOM Production At the conclusion of the first laboratory trial, there was a significant difference between the amount of FPOM produced by the crayfish based on the pH (F = 14.067, p =

<0.001) (Table 1.7). A Bonferroni post hoc indicated that crayfish in pH of 4.0 produced significantly less FPOM than those crayfish in pH 5.0 (p = 0.002), 6.0 (p = 0.001), and

7.0 (p = <0.001) (Figure 1.12). There was no difference in the amount of FPOM produced by the crayfish in this experiment based on location of leaf conditioning (F =

0.335, p = 0.566) (Figure 1.13).

At the conclusion of the second trial, there was strong difference between the amount of FPOM produced by the crayfish based on pH (F = 11.332, p = <0.001). A

Bonferroni post hoc revealed that crayfish in pH 4.0 produced significantly less FPOM 39

than crayfish in pH 6.0 (p = 0.007) and pH 7.0 (p = <0.001) but did not produce less

FPOM than those in pH 5.0 (p = 0.288) (Figure 1.12). There was a significant difference

in the amount of FPOM produced by the crayfish in this trial based on location of leaf

conditioning (F = 11.317, p = 0.002) (Figure 1.13).

Trial -1.0000 Trial 1 Trial 2 -1.2000

-1.4000

-1.6000

-1.8000 Log AFDM (g)

-2.0000

-2.2000

-2.4000

pH 4 pH 5 pH 6 pH 7 pH

Figure 1.12. Mean (±SE) log ash free dry mass (AFDM) of fine particulate organic matter (FPOM) produced by the crayfish, Orconectes sanbornii at pH treatments of 4.0, 5.0, 6.0 and 7.0 for both laboratory trials.

40

Trial -1.2000 Trial 1 Trial 2

-1.4000

-1.6000

-1.8000 Log AFDM (g)

-2.0000

-2.2000

AMD Control Location of Leaf Conditioning

Figure 1.13. Mean (±SE) log ash free dry mass (AFDM) of fine particulate organic matter (FPOM) produced by the crayfish, Orconectes sanbornii based on the location of leaf conditioning.

41

Table 1.7. ANOVA table for FPOM production during both laboratory trials, with pH and leaf conditioning location as treatments.

Trial 1 Treatment SS F p

14 days pH 2.653 14.067 <0.001 Leaf Condition 0.021 0.335 0.566 pH x Leaf Condition 0.161 0.856 0.472

Trial 2 Treatment SS F p

14 days pH 3.224 11.332 <0.001 Leaf Condition 1.073 11.317 0.002 pH x Leaf Condition 0.008 0.027 0.994

Holding Period Analysis - Laboratory Trial #2 During the second laboratory trial, the amount of time crayfish spent in the cold

storage had no significant effect on their leaf consumption rates. After 4 days no

difference was observed in the amount of leaves consumed by crayfish based on how

long they were in cold storage (F = 1.513, p = 0.225). There were also no differences in

the amount of leaves consumed based on time spent in cold storage after 7 days (F =

0.437, p = 0.728), 10 days (F = 0.147, p = 0.931), and 14 days (F = 0.040, p = 0.989).

Notes Concerning pH and Leaf Conditioning Treatments During the course of both laboratory trials, there were no observed interactions

between pH treatments and leaf conditioning treatments on either leaf consumption or

FPOM production by O. sanbornii. 42

Leaf Analyses The leaves conditioned at Jordan Run and Railroad 13 for the first laboratory trial

contained similar percentages of nitrogen. However, the leaves conditioned at Jordan

Run for the first laboratory trial had a lower percentage of nitrogen than the leaves

conditioned at the same location for the second trial (Table 1.8).

Table 1.8 Percentages of nitrogen (N), carbon (C), and C/N ratio for leaves conditioned at Jordan Run (Control) and Railroad 13 (Heavy impact), prior to each laboratory trial.

Location Date %N %C C:N Ratio Jordan Run 12/16/2005 0.832 38.605 46.400 Railroad 13 12/16/2005 0.809 42.890 53.016 Jordan Run 1/26/2006 1.150 45.502 39.567 Railroad 13 1/26/2006 0.892 36.786 41.240

Discussion pH Effects on Leaf Consumption During the first laboratory trial, pH treatment had a strong effect. By the completion of the trial, there appeared to be a pH threshold effect on leaf consumption by

O. sanbornii. In pH 4.0, O. sanbornii was sluggish (based on personal observations) and did not consume as much leaf matter, nor produce as much FPOM as O. sanbornii in pH

5.0 and above. O. sanbornii in pH 5.0 consumed leaves and produced FPOM at levels comparable to those in pH 6.0 and 7.0. The only period during the second trial in which a threshold effect was observed was during the 2nd interval (4-7 days). At other times in

the second trial there appeared to be differences in the amount of leaf area consumed by

crayfish in pH 4.0 vs. pH 5.0, however, the differences were not significant statistically. 43

Such a suggested threshold effect would indicate that if the pH of a stream is at

least 5.0, the crayfish should be able to consume leaf litter and produce a basal food

resource at rates similar to that found in streams with desired pH levels. This could

potentially have important implications in remediation efforts by promoting more

available food resources and thus higher abundances and diversity of macroinvertebrates

in AMD impacted streams.

When looking at the leaf consumption by crayfish during the intervals, it is apparent that as the trials progressed, the pH effect got stronger. This suggests that O.

sanbornii initially showed resistance to low pH. Such a response should not be

considered unexpected since crayfish would be more likely to show resistance to acidic

conditions at initial exposure rather than after chronic exposure.

Leaf Conditioning Effects on Leaf Consumption An increase in the nitrogen compound concentration of plant material occurs

during decomposition and most likely is a result of the microbial colonization of the decomposing leaf litter. Therefore, reductions in microbial colonization can result in

reduced palatability of leaves to shredders such as crayfish (Kok and Van der Velde

1994). Adams et al. (2005) attribute leaf nutritional value according to the percentages of

C and N, as well as C/N ratios. Leaves having higher %N and lower C/N ratios are more

nutritious. Usio (2007) stated that there is a positive relationship between leaf processing

rate and %N and a negative relationship between C/N ratio and leaf processing rates. 44

Several recent studies have shown that various species of crayfish exhibit a preference for leaves with higher percentages of N and lower C/N ratios (Adams et al.

2003, Adams et al. 2005, and Usio 2007). Adams et al. (2003) showed that Orconectes virilis preferred leaves with high percentages of N and lower C/N ratios. Similar preferences were observed for Procambarus clarkii (Adams et al. 2005) and Japan’s only native crayfish, Cambaroides japonicus (Usio 2007).

The previously mentioned studies showed that when given a choice, crayfish prefer leaves with higher %N and lower C/N ratios. However, during the course of this research, O. sanbornii did not have a choice between location where leaf conditioning took place and their degree of nutritional value. The results from the second trial showed that O. sanbornii given leaves conditioned at the heavy AMD-impacted site consumed more than those given leaves conditioned at the control site. When given only one option, it seems reasonable that crayfish will consume more leaf area when the leaves are of lower nutritional quality in order to obtain the necessary nutrients.

Stream water quality, including pH levels, tend to show seasonal trends. During the late summer, pH levels in our study basins tend to be at the lowest and gradually increase until late winter, when the highest pH values are usually recorded. Therefore, water quality tends to be better during the winter than other times of the year (Dills and

Rogers 1974). Comparison of the chemical data from the heavy AMD-impacted Railroad

13 site between fall (October 2005) and late winter (February 2006), parallels the findings of Dills and Rogers (1974), showing a slight increase in pH and noticeable

-2 reductions in specific conductivity (SC), sulfates (SO4 ), and total dissolved solids (TDS) 45 from fall to late winter (see Table 1.9). As a result, it is clear that the water quality was improved in the winter, corresponding with the time period of leaf conditioning for the second trial. Therefore, improvement of water quality may account for the differences observed in the second laboratory trial, where leaves conditioned at Railroad 13 during that time were more readily consumed by O. sanbornii.

Table 1.9. Water chemistry data for the study sites from the fall and winter, 2005-06. Data from the late winter of 2006 for Truetown and RR 13 indicate the changes in water chemistry between autumn and winter. Water chemistry measures include pH, Specific -2 Conductivity (SC), Sulfate (SO4 ), and Total Dissolved Solids (TDS). All data, with the exception of Jordan Run, were obtained from http://www.watersheddata.com/.

-2 Site name Watershed AMD pH SC (μs/cm) SO4 (mg/L) TDS (mg/L)

Jordan Run Hocking River low 7.00 * 335 n/a n/a Carbon Hill Monday Creek low 6.55 3 858 314 592 Truetown Sunday Creek low 6.95 1 1260 493 910 6.95 2 372 84.3 243

Rock Run Monday Creek heavy 6.80 4 758 294 493 Job's Hollow Monday Creek heavy 8.76 5 1130 587 910 RR 13 Sunday Creek heavy 6.03 6 1217 550 912 6.33 7 576 186 351 4 5 6 7 * data recorded 11-23-05 ¹ 10-14-05; ² 2-8-06; ³ 10-19-05; 3-14-05; 10-18-05; 10-11-05; 2-9-06 46

CHAPTER TWO: FIELD EXPERIMENT

Introduction

The conventional approach to studying the effects of shredders and

microorganisms on leaf litter breakdown is through litter bag studies (Hieber and Gessner

2002). The litter bags, filled with leaf packs, closely resemble the way leaf litter forms discrete resource patches in streams. However, most litter bag surveys only measure activity of small invertebrates because the mesh excludes macroconsumers. As a result, many studies have focused on how acidification influences smaller macroinvertebrate shredders, such as stoneflies (Plecoptera), caddisflies (Trichoptera), amphipods

(Amphipoda) and microbial breakdown of leaf litter (van Frankenhuyzen and Geen 1986,

Groom and Hildrew 1989, Griffith and Perry 1993). Currently, little is known about the effects pollution has on crayfish and their role as shredders. The role of crayfish as shredders, however, is being increasingly recognized and it is now realized that any disruption of crayfish abundance or activity could have consequences extending through the ecosystem (Huryn and Wallace 1987, Usio and Townsend 2001, Seiler and Turner

2004).

Research Objectives

The main objective of the field experiment was to quantify detrital processing by a key detritivore, the crayfish (Orconectes sanbornii (Faxon, 1854)), in AMD polluted streams and control streams (with no AMD impact) in situ using leaf litter bags as 47

enclosures. At the conclusion of the experiment, differences in macroinvertebrate

composition in the enclosures were also investigated.

Methods

The field experiment took place at six sites in the Sunday Creek, Monday Creek,

and Hocking River watersheds, located in Athens, Hocking, and Perry Counties in

southeastern Ohio (Tables 2.1, 2.2, and Figure 2.1). A total of six segments of stream

were used during the course of the field study: three that are heavily impacted by AMD

input, and three control segments with no known AMD impact.

Table 2.1. Five year averages of autumn water chemistry (2001-2005) for all field sites. -2 Water chemistry measures include pH, Specific Conductivity (SC), Sulfate (SO4 ), and Total Dissolved Solids (TDS). All data, excluding Jordan Run data, were obtained from http://www.watersheddata.com/. Jordan Run data collected by the author.

-2 Site name Watershed AMD impact Drainage Area (sq.mi.) pH SC (μs/cm) SO4 (mg/L) TDS (mg/L)

Jordan Run * Hocking River low n/a 7.00 335.0 n/a n/a Carbon Hill Monday Creek low 25.0 6.35 723.4 241.4 473.4 Truetown Sunday Creek low 120.0 7.03 882.6 304.8 583.2

Rock Run Monday Creek heavy 18.0 6.22 892.2 374.4 653.2 Job's Hollow Monday Creek heavy 2.9 4.65 1012.4 425.8 681.0 RR 13 Sunday Creek heavy 11.2 6.51 1120.0 465.8 764.8 * data collected 11-23-05

48

Table 2.2. Biological data for field sites. MAIS scores (a rapid bioassessment index based on macroinvertebrates) obtained from http://www.watersheddata.com/

Site name Site Code Watershed AMD MAIS Field Notes

Jordan Run n/a Hocking River low n/a Exceptional Warmwater Habitat - Ohio EPA Carbon Hill MC 00300 Monday Creek low 13 Biological condition Good - KSJ Truetown SC 073 Sunday Creek low 11 Biological condition Poor - CK

Rock Run MC 00830 Monday Creek heavy 11 Biological condition Poor - KSJ Job's Hollow JH 00500 Monday Creek heavy 7 Biological condition Very Poor - CK RR 13 SC 076 Sunday Creek heavy 11 Biological condition Poor - CK * MAIS data collected between September-October, 2005

At each location, eight 91 x 61 cm bags with 3 mm mesh (commercial cabbage

bags purchased from Apex Feed and Supplies, Marietta, Ohio) served as enclosures and were placed as pairs in pools with iron rebar stakes securing them in place. Each enclosure contained 2 leaf packs of dry silver maple leaves that were pre-weighed at approximately 5 grams each (±1.00 g). Each leaf pack was held together at the base with a 1.25 inch metal binder clip, as described by Usio (2000). The enclosures, containing only leaf packs, were transported to the sites inside a large plastic container to minimize leaf breakage. At each field site, 4 pairs of bags were placed in pools (to minimize leaf breakage due to rapid water flow) and left for 14 days to allow for the conditioning of the leaves and colonization by resident shredder assemblages. After 14 days, a single crayfish (with OCL between 12-18 mm) was added to four of the enclosures at each site so that the enclosures were paired (one with crayfish, one without) in each pool. The four enclosures at each site containing only leaf packs and no crayfish served as controls for the amount of leaf breakdown resulting from water currents and smaller shredders. The crayfish (Orconectes sanbornii) used in this field experiment were captured from the 49

Jordan Run (Control) (N 39° 15.580′, W 081° 49.130′) field site on November 14, 2005.

Individuals were captured primarily through the use of a kick seine.

All of the enclosures were checked 5 days after the crayfish were added in order to ensure that the enclosures as well as crayfish were not lost or left dry by changes in stream water levels. After the first 5 days, all enclosures and crayfish were present.

Unfortunately, two pairs of enclosures were lost between days 5 and 14, one at Truetown

(N 39° 27.659′, W 082° 12.563′) and one at Rock Run (N 39° 36.277′, W 082° 21.109′).

These disappearances coincided with a significant rain event and elevated water levels. It should also be noted that the enclosures lost at Rock Run were nearest to the road and disruption of field studies by children has occurred there in the past.

At the end of the field study, each enclosure was removed from the stream and placed in a large plastic container for transport. In the laboratory, crayfish were removed and the leaf packs were examined for the presence of invertebrates. All invertebrates were collected, identified to family (except for Oligochaetes, which were identified to order), and preserved in 70% ethanol. The remaining leaf mass was dried and weighed in order to quantify the amount shredded. The difference was assumed to have been shredded as it was not possible to distinguish between leaf material shredded by feeding activity or movement of water through the enclosures.

All crayfish were preserved in 70% ethanol at the conclusion of the field study.

Voucher specimens from both field and laboratory experiments are located in the Ohio

University Zoological Museum. 50

Calculations and Statistical Analysis To characterize communities of macroinvertebrates that colonized the bags,

Family Richness (FT) and diversity (H′) were calculated using the Shannon-Weiner Index of Diversity. Maximum diversity (H′max) was also calculated. Evenness (J′), an indicator

of how evenly individuals are distributed among families, was obtained by dividing H′ by

H′max.

A two-sample T-test was used to test the differences between the two diversity

indices within each site for macroinvertebrate colonization in enclosures without crayfish

(control) and in enclosures with crayfish (Zar 1999). The family richness and mean

individuals/enclosure were also compared based on the presence or absence of crayfish

with a two-sample T-test. For these analyses, field sites were grouped together by level

of AMD impact (control and AMD-impacted).

The diversity, total number of families, and mean individuals/enclosure at each

site were tested separately using two-sample T-tests, with presence or absence of AMD

impact as the treatment 51

Figure 2.1. Map of all six field study sites, located in Athens, Hocking, and Perry counties in SE Ohio. Jordan Run and Railroad 13 (RR 13) also served as leaf conditioning sites for leaves used in the laboratory experiment.

52

Results Prior to the field experiment there was a discrepancy in the weighing of the leaf packs, which resulted in a substantial amount of error. Consequently, it was not possible to accurately determine how much of the leaf packs had been shredded, and thus the data regarding detrital processing by crayfish were not analyzed. As a result, the most useful information from this study related to family richness and diversity at impacted sites and between enclosures with and without crayfish. The family richness at each of the three high quality/control sites was relatively high with the highest family richness at Truetown with 13 families, 11 families at Carbon Hill, and 10 families at Jordan Run (Table 2.3A).

Truetown had the highest overall family richness, despite the loss of two enclosures.

Rock Run, a site with AMD impact had the lowest family richness of all the sites with only 3 families represented (Table 2.3B), but also had a loss of two enclosures. The two other sites with AMD impact, Railroad 13 and Job’s Hollow had relatively low family richness at 5 and 6 families respectively (Table 2.3B).

53

Table 2.3. Family Richness (FT), Shannon-Weiner Diversity values (H′), maximum diversity values (H′max), Evenness (J′), and mean number of individuals per enclosure, without crayfish (control) and with crayfish, at all field sites.

A. Control Streams Jordan RunCarbon Hill Truetown

Control Crayfish Total Control Crayfish Total Control Crayfish Total # Families 7 7 10 10 7 11 9 10 13 H' 0.688 0.654 0.725 0.740 0.624 0.727 0.721 0.524 0.628

H'max 0.845 0.845 1.000 1.000 0.845 1.041 0.954 1.000 1.114 J' 0.814 0.774 0.725 0.740 0.738 0.698 0.756 0.524 0.564 Ind./enclosure 4.750 7.000 5.875 9.250 5.250 7.250 11.330 25.000 18.167

B. AMD-Impacted Streams Rock Run Job's Hollow Railroad 13 Control Crayfish Total Control Crayfish Total Control Crayfish Total # Families 223636255 H' 0.301 0.118 0.251 0.754 0.452 0.726 0.244 0.539 0.481

H'max 0.301 0.301 0.477 0.778 0.477 0.778 0.301 0.699 0.699 J' 1.000 0.391 0.527 0.968 0.946 0.933 0.811 0.771 0.688 Ind./enclosure 1.333 4.333 2.833 2.250 1.000 1.625 3.000 5.750 4.375

Jordan Run (control) There were 47 individuals representing 10 families overall at Jordan Run. The overall diversity (H′ = 0.725) and evenness (J′ = 0.725) were relatively high at this site.

There was no obvious difference between the invertebrates found in the control leaf packs (without crayfish) and the leaf packs with crayfish. Two families in the Order

Ephemeroptera (Ephemerellidae and Caenidae) and one family in the Order Plecoptera

(Leuctridae) were represented. Also of interest, two Fantail Darters (Etheostoma flabellare) of the Family Percidae were collected from the leaf packs.

Truetown (control) During the course of the field experiment, a pair of enclosures at Truetown was lost during a rain event. Despite this, Truetown had the highest family richness and 54

number of individuals of any site with 109 individuals representing 13 families. The

control leaf packs were inhabited by 9 families and 34 individuals, while the leaf packs

with crayfish had 10 families and 75 individuals. The overall diversity at this site was

relatively high (H′ = 0.628), but the evenness was low (J′ = 0.564). Evenness was lower

largely due to the fact that of the 109 individuals, 50 were from the Family

Chironomidae. Two families in the Order Ephemeroptera (Caenidae, Leptophlebiidae),

one family in the Order Plecoptera (Leuctridae), and one family in the Order Trichoptera

(Polycentropididae) were represented at this site (see Table 2.4).

Carbon Hill (control) Carbon Hill displayed a relatively high overall family richness with 11 families

represented by 58 individuals. Diversity (H′ = 0.727) and evenness (J′ = 0.698) were also

relatively high. Control leaf packs had 10 families represented by 37 individuals, while

leaf packs with crayfish had 7 families represented by 21 individuals. Three families in

the Order Ephemeroptera (Caenidae, Leptophlebiidae, and Isonychiidae), one family in

the Order Plecoptera (Leuctridae), and two families in the Order Trichoptera

(Limnephilidae, Polycentropididae) were present in the leaf packs at this site. Carbon

Hill and the other two control sites were the only sites that had representatives in the order Plecoptera present in leaf packs (see Table 2.4).

Rock Run (AMD-impacted) Similar to Truetown, Rock Run had a pair of enclosures lost during the course of this study. Rock Run had the lowest family richness of all the sites with only 3 families represented by 17 individuals. The overall diversity (H′ = 0.251) and evenness (J′ = 55

0.527) were the lowest values for any of the sites. The control leaf packs included 2 families represented by 4 individuals while the leaf packs with crayfish had 2 families represented by 13 individuals. The three families present in the leaf packs at this site included two families in the Order Diptera (Chironomidae, Tipulidae) and one in the

Order Megaloptera (Sialidae). Of the 17 individuals, 14 were from the family

Chironomidae (Table 2.4).

Job’s Hollow (AMD-impacted) Job’s Hollow had the fewest total number of individuals (13) present in the leaf packs of any site. The overall family richness was low at 6, yet the diversity (H′ = 0.726) and evenness (J′ = 0.933) were relatively high. The control leaf packs had 6 families represented by 9 individuals, while the leaf packs with crayfish had 3 families represented by 4 individuals. Two families from this site were from the Order

Trichoptera (Polycentropididae, Hydropsychidae) (Table 2.4).

Railroad 13 (AMD-impacted) Railroad 13 had a relatively low family richness in the leaf packs with 5 families represented by 35 individuals. The overall diversity (H′ = 0.481) and evenness (J′ =

0.688) were moderate compared to the other sites. The control leaf packs only had 2 families represented by 12 individuals, while the leaf packs with crayfish had 5 families represented by 23 individuals. One family in the Order Ephemeroptera (Caenidae) was represented (Table 2.4). 56

Comparison of Enclosures With and Without Crayfish Four of the six field sites had differences in macroinvertebrate species diversity based on the presence or absence of crayfish in the enclosures. Rock Run (t = 2.297, p =

0.0389), and Railroad 13 (t = 3.108, p = 0.00370) had higher macroinvertebrate diversity

in enclosures with a crayfish than enclosures without crayfish. Job’s Hollow (t = 3.397, p

= 0.0115) and Truetown (t = 2.0250, p = 0.0461) showed a higher diversity of

macroinvertebrates in enclosures without crayfish. There were no differences in the

macroinvertebrate diversities of enclosures with and enclosures without crayfish at

Jordan Run (t = 0.217, p = 0.830) and Carbon Hill (t = 1.027, p = 0.310).

There were no observed differences between the family richness of enclosures

with crayfish and enclosures without crayfish at the control sites (t = 0.392, p = 0.715)

and AMD-impacted (t = 0.000, p = 1.000) field sites. There were also no observed

differences between the mean number of individuals per enclosure with crayfish and per

enclosure without crayfish at the control sites (t = 0.611, p = 0.600) and AMD-impacted

(t = 1.001, p = 0.402) field sites.

Comparison Between Field Sites The field sites were grouped together based on AMD impact and for each site, the

diversity indices, family richness, and mean number of individual macroinvertebrates per

enclosure were compared separately using two-sample t-tests. For these tests, enclosures

with and without crayfish were combined for each field site. There were no observed

differences in diversity (t = 1.0130, p = 0.313) and mean number of individuals per

enclosure (t = 2.359, p = 0.0595) between the control and AMD-impacted sites. 57

However, there was a significant difference of family richness (t = 4.427, p = 0.00130) between control and AMD-impacted sites, with the control sites having increased family richness.

Macroinvertebrate diversity was also compared between field sites based on presence or absence of crayfish within the enclosures. Significant differences were observed for the diversity of enclosures with crayfish (t = 2.028, p = 0.0453) between the control and AMD-impacted sites and the diversity of enclosures without crayfish (t =

3.542, p ≤0.001) between the control and AMD-impacted sites (Figure 2.2). In both cases, the diversity at the control sites was higher than that of the AMD-impacted sites.

There was no difference in the mean number of individuals in enclosures with crayfish (t

= 1.349, p = 0.300) between the control and AMD-impacted sites. Also, there was no difference in the mean number of individuals in enclosures without crayfish (t = 3.132, p

= 0.0768) between the control and AMD-impacted sites. Similarly to the total family richness calculated between sites, significant differences were observed for family richness of enclosures with crayfish (t = 2.907, p = 0.0486) between the control and

AMD-impacted sites and family richness of enclosures without crayfish (t = 2.780, p =

0.0500) between the control and AMD-impacted sites. In both cases, family richness was greater among the control sites. 58

Figure 2.2. Mean diversity indices for macroinvertebrates in enclosures with and without crayfish at control and AMD-impacted field sites.

59

Table 2.4. Macroinvertebrates, identified to family level, found in the leaf packs at the conclusion of the field experiment.

Control AMD-impacted Jordan RunCarbon Hill Truetown Rock Run Job's Hollow Railroad 13 Order Family Control Crayfish Control Crayfish Control Crayfish Control Crayfish Control Crayfish Control Crayfish Diptera Ceratopogonidae 1 1 Chironomidae 9 12 15 10 16 50 2 12 2 2 9 13 Tipulidae 1 1 2 1 1 2 Ephemeroptera Caenidae 1 4 1 1 4 Ephemerellidae 2 Isonychiidae 1 Leptophlebiidae 1 2 Plecoptera Leuctridae 3 8 10 6 6 12 Trichoptera Hydropsychidae 1 Limnephilidae 1 1 Polycentropodidae 2 1 3 2 1 Odonata Coenagrionidae 1 1 Coleoptera Dryopidae 2 1 Elmidae 1 Megaloptera Corydalidae 1 Sialidae 1 1 2 Lumbriculida Lumbriculidae 33 "Oligochaeta" 1 2 3 Isopoda Asellidae1 1121 Basommatophora Ancylidae 1 Physidae 2 3 4 Neotaenioglossa Hydrobiidae 1 Veneroida Corbiculidae 1 Total1827372134754 139 4 1223

60

Discussion

Leaf Pack Colonization by Macroinvertebrates The diversity of macroinvertebrates that colonized leaf packs within the enclosures was significantly greater when in the presence of a crayfish at two of the six field sites. While the abundances were fairly small and duration of the experiment relatively short, there is a possibility that presence of crayfish in leaf packs may result in an increased diversity of colonizing macroinvertebrates. If true, then this is likely a result of the benefits of a food base created by the crayfish outweighing the negative possibility of being predated by such crayfish. Larger individual crayfish tend to feed chiefly on detritus and plant matter as opposed to younger and smaller individuals that feed primarily on macroinvertebrates (Englund and Krupa 2000). Crayfish tend to retreat to leaf packs during the cold temperatures of winter (personal observation). This, in combination with an abundance of leaf matter in forested headwater streams during the winter months may result in crayfish focusing more on leaves as a food source than macroinvertebrates. Therefore, the FPOM produced by crayfish would likely act as an important source of nutrients for macroinvertebrates (Hoffman 2005). The effect of crayfish presence on macroinvertebrate diversity of leaf packs is a potential area of future research efforts that may provide valuable insight regarding the structuring of stream food webs.

The Role of Crayfish in Stream Ecosystems Crayfish have been called keystone species (Creed 1994) and ecosystem engineers (Creed and Reed 2004). Clearly crayfish are an integral component of stream 61 ecosystems. Generally, crayfish are the largest macroinvertebrates in forested headwater streams, making up a large proportion (~30%) of animal biomass and consequently contributing a large proportion of the benthic biomass (Momot et al. 1978).

Crayfish play an important role by creating FPOM in streams through their feeding activities. Once in the system, the FPOM can then be consumed by various collector-gatherers. As a result, crayfish potentially can have a strong influence on the benthic community assembly (Zhang et al. 2004). Even in areas where crayfish density is relatively low, they are able to have a significant impact on leaf decay. Therefore, slight alterations in crayfish assemblages can have significant impacts on ecosystem processes (Schofield et al. 2001).

Crayfish have even been implicated as players in trophic cascades, based on how their feeding activities can influence species abundances at lower levels (Morin 1999).

Lodge et al. (1994) suggested that in lakes, the consumption of snails by Orconectes rusticus results in increased levels of periphyton. Crayfish have broad diets and since they feed from multiple trophic levels, they can potentially play a significant role in the determination of community structure (Dorn and Wojdak 2004).

Malmqvist and Hoffsten (1999) speculated that the loss of keystone taxa or ecosystem engineers due to heavy metal pollution would likely lead to strong impacts on ecosystem processes. Furthermore, low pH and water quality can lead to decreases in microbial activity and detritivore population size (Kok and Van der Velde 1994). While crayfish are not likely to be lost in AMD-impacted streams, reductions in the amount of leaf litter consumption and FPOM production could have strong negative impacts on stream ecosystems. For example, if shredding is inhibited, a reduction in the available 62 food resources for gatherers, filter feeders, and microorganisms would be expected (Kok and Van der Velde 1994).

Future AMD Management It is difficult to pinpoint causes of community structure change in AMD-impacted sites (Carpenter et al. 1983), as waters impacted by AMD typically are not degraded in a homogeneous manner (Stoertz et al. 2002). While low pH inhibited the ability of O. sanbornii to breakdown leaf matter in the laboratory, there was some variability among the leaves conditioned in the field; this was especially dependent on time of the conditioning process. Carpenter et al. (1983) suggested that changes in breakdown rates can be attributed to microbial community structure. Furthermore, changes in water quality including the pH, increased concentrations of metals, and sedimentation, can also have an influence in microbial community structure. Laboratory experiments are beneficial because they can independently identify variables in the community structure affected by AMD. However, additional field studies will not only show the effects of

AMD on crayfish, but will also indicate how much impact crayfish can have as shredders on headwater stream communities.

63

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