Seeking Science-Based Nutrient Standards For Coastal ... · Blackwater streams are the most common...

UNC-WRRI-2002-341

SEEKING SCIENCE-BASED NUTRIENT STANDARDS FOR COASTAL BLACKWATER STREAM SYSTEMS

by Michael A. Mallin Lawrence B. Cahoon Matthew R. Mclver Scott H. Ensign

Center for Marine Science University of North Carolina at Wilmington Wilmington, North Carolina

August 2002

Copies available from: Water Resources Research Institute of The University of North Carolina

North Carolina State University Box 7912 Raleigh, NC 27695-7912

THE UNIVERSITY OF NORTH CAROLINA is composed of the sixteen public senior irrstitutions in North Carolina.

UNC-WRRJ-2002-341

SEEKING SCIENCE-BASED NUTRIENT STANDARDS FOR COASTAL BLACKWATER STlREAM SYSTEMS

by Michael A. Mallin, Lawrence B. Cahoon, Matthew R. McIver, and Scott €4. Ensign

Center for Marine Science University of North Carolina at Wilmington

Wilmington, North Carolina 28409

The research on which this report is based was financed by the Water Resources Research Institute of The University of North Carolina. Contents of this publication do not necessarily reflect the views and policies of the Institute, nor does mention of trade names of commercial products constitute their endorsement by the Institute or the State of North Carolina.

WRRI Project Nos. 701 7 1 & 70 177 August 2002

One hundred thirty copies of this report were printed at a cost of $918.60 or $7.07 per copy.

ACKNOWLEDGMENTS

We thank the Water Resources Research Institute of The University of North Carolina (Projects #70 171 and #70 177) for financial support Additional financial support was provided by the Lower Cape Fear River Program. We thank Jesse Cook, Heather CoVan, Virginia Johnson, Douglas Parsons, Christian Preziosi, G. Chris Shank and Ashley Skeen for field and laboratory assistance. We thank Drs. Alan Lewitus and Eric Koepfler for advice on using the acridine orange bacterial counting technique and Matthew Parrow and Jay Sauber for helpfir1 information. This is Contribution Number 271 of the Center for Marine Science, the University of North Carolina at Wilmington.

... I l l

iv

ABSTRACT

Blackwater streams are the most common lotic systems on the North Carolina Coastal Plain. Despite their abundance, these streams are rarely considered to be sensitive to nutrient loading and are usually not afforded the nutrient protection that estuaries and Piedmont-derived water bodies receive. We used nutrient addition bioassays to assess blackwater stream susceptibility to nutrient loading, with chlorophyll a, biochemical oxygen demand (BOD), and bacterial abundance as response variables. Nutrient additions were nitrate, urea f nitrate (TN), orthophosphate, and orthophosphate + glycerophosphate (TP), with treatment concentrations of 0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg o f N (nitrogen) or P (phosphorus& Experiments were conducted on water from a near-pristine stream (Colly Creek) and a stream draining an area of heavy swine and poultry production (Great Coharie Creek).

Nitrogen, either as nitrate or TN proved to be the nutrient that limited the growth of phytoplankton. In several of the nitrogen treatments, BOD was significantly stimulated over control through the photosynthetic pathway of algal production followed by algal death and decay leading to subsequent BOD increase. Phosphorus did not stimulate the growth of phytoplankton in these experiments. However, significant BOD increases occurred in combined orthophosphate + glycerophosphate addition experiments, through stimulation of heterotrophs (mainly bacteria). Orthophosphate alone did not significantly stimulate BOD. In most cases, chlorophyll a was stimulated to a greater degree in the water from the anthropogenically- impacted creek than water from the pristine creek, and there was stronger coupling between chlorophyll a and BOD. This possibly indicates a microbial community adapted to take advantage of periodic nutrient pulses from rainfall-driven runoff from swine waste sprayfields, poultry litterfields, or fertilized agricultural areas. Significant stimulation of chlorophyll a and BOD occurred with additions of 0.2 - 0.5 mg nitrate-NL, and significant stimulation of chlorophyll and BOD also occurred in Great Coharie Creek with inputs of TN as low as 0.2 mg- N L . Significant stimulation of BOD occurred with additions of either 0.5 or 1.0 mg-PL as TP. Previous experiments conducted on water from the Black and Northeast Cape Fear Rivers using additions of 1 .O mg/L of N or P also showed significant stimulation of chlorophyll a (by N) and adenosine triphosphate (ATP) by P or N.

Thus, in order to prevent biological degradation of blackwater streams we suggest that standards be developed to prevent anthropogenic loading that would increase blackwater stream inorganic nitrogen concentrations above 0.5 mg-N/L and total phosphorus concentrations above 0.5 mg- P L . This should include not only adopting point source nutrient limits but also land-based strategies designed to reduce non-point source nutrient runoff from agricultural fertilizers, poultry littterfields and swine waste lagoon sprayfields as well.

TABLE OF CONTENTS

... ... Acknowledgments .......................................................................................................................... 111

............................................................................................................................................ Abstract v

List of Figures ................................................................................................................................. ix

List of Tables ................................................................................................................................. xi ...

Summary and Conclusions ........................................................................................................... x111

Recommendations .......................................................................................................................... xv

Introduction ..................................................................................................................................... 1 Sources and Magnitudes of Nutrients in Blackwater Rivers ........................................................... 2

Project Design ..................................................................................................................... 3 Specific Objectives .............................................................................................................. 3

Materials and Methods .................................................................................................................... 5

Nutrient Gradient Response Experiments ........................................................................... 5 Response Parameters ........................................................................................................... 6 Statistical Analysis ............................................................................................................... 6 Water Quality Assessments ................................................................................................. 7

Sampling Stations ................................................................................................................ 5

. .

Results .............................................................................................................................................. 9 Water Quality in Colly Creek and Great Coharie Creek ..................................................... 9 Nitrate Addition Experiments ........................................................................................... 12 Total Nitrogen Addition Experiments ............................................................................... 12 Orthophosphate Addition Experiments. ............................................................................ 12

Correlation Analyses ......................................................................................................... 25 Total Phosphorus Addition Experiments ......................................................................... 25

Discussion ...................................................................................................................................... 35

References ...................................................................................................................................... 39

List of Acronyms ........................................................................................................................... 43

vii

viii

LIST OF FIGURES Page

Figure 1. Location of sampling sites in the Black River watershed, southeastern North Carolina .............................................................................................................................................. 4

Figure 2. Post-incubation responses of Colly Creek and Great Coharie Creek water to nitrate-N . . additions, August 1999 ...................................................................................................... 17

Figure 3. Post-incubation responses of Colly Creek and Great Coharie Creek water to nitrate-N additions, JuIy2000 ........................................................................................................... 18

Figure 4. Pre-incubation BOD5 responses of Colly Creek and Great Coharie Creek water to nitrate-N additions, August 1999 and July 2000 ............................................................... 19

Figure 5. Post-incubation responses of Colly Creek and Great Coharie Creek water to combined inorganic+organic N additions, September 1 999.. ........................................................... .20

Figure 6. Post-incubation responses of Colly Creek and Great Coharie Creek water to combined inorganic+organic N additions, August 2000.. .................................................................. 2 1

Figure 7. Pre-incubation BOD5 responses of Colly Creek and Great Coharie Creek water to combined inorganic+organic N additions, September 1999 and August 2000 ................ .22

Figure 8. Post-incubation responses of Colly Creek and Great Coharie Creek water to orthophosphate-P additions, July 1999 ............................................................................. 23

Figure 9. Post-incubation responses of Colly Creek and Great Coharie Creek water to orthophosphate-P additions, May 2000 ........................................................................... .24

Figure 10. Pre-incubation BOD5 responses of Colly Creek and Great Coharie Creek water to orthophosphate-P additions, July 1999 and May 2000 ..................................................... 26

Figure 1 1. Post-incubation responses o f Colly Creek and Great Coharie Creek water to combined inorganic+organic P additions, November 1999. .............................................. 27

Figure 12. Post-incubation responses of Colly Creek and Great Coharie Creek water to combined inorganic+organic P additions, June 2000.. ...................................................... 28

Figure 13. Pre-incubation BOD5 responses of Colly Creek and Great Coharie Creek water to combined inorganic+organic P additions, November 1999 and June 2000 ...................... 29

Figure 14. Bacterial abundance by nutrient addition level for orthophosphate and TP treatments.. ........................................................................................................................ .30

ix

X

LIST OF TABLES

Page

Table 1. Water quality characteristics of Colly Creek and Great Coharie Creek, tributaries of the Black River, 1998-2000 ..................................................................................................... 10

Table 2. Ambient nutrient concentrations (and molar total and inorganic N/P ratios) in bioassay test waters before nutrient additions .................................................................................. 11 . .

Table 3. Statistically significant (p < 0.05) chlorophyll a responses to nutrient additions ........... 13

Table 4. Statistically significant (p < 0.05) BOD5 responses to nutrient additions (post- incubation responses). ...................................................................................................... .14

Table 5. Statistically significant (p < 0.05) BOD20 responses to nutrient additions (post- incubation responses). ...................................................................................................... .14

Table 6. Statistically significant (p < 0.05) BOD5 responses to nutrient additions (pre-incubation responses) .......................................................................................................................... 15

Table 7. Statistically significant (p < 0.05) BOD20 responses to nutrient additions (pre- incubation responses). ....................................................................................................... 15

Table 8. Statistically significant (p < 0.05) bacterial count responses to nutrient additions (post- incubation responses) ........................................................................................................ 16

Table 9. Positive correlations among post-incubation response variables for Colly Creek and Great Coharie Creek waters for all experiments combined .............................................. .3 1

Table 10. Positive correlations among post-incubation response variables for Colly and Great Coharie Creek waters by individual experiment .............................................................. ..32

xi

xi i

SUMMARY AND CONCLUSIONS

Blackwater streams are the most common lotic systems on the North Carolina Coastal Plain. Despite their abundance, these streams are rarely considered to be sensitive to nutrient loading and are usually not afforded the nutrient protection that estuaries and Piedmont-derived water bodies receive. However, recent data from The University of North Carolina at Wilmington’s Lower Cape Fear River Program (LCFRP) have demonstrated that both organic and inorganic nitrogen and phosphorus loading can be high in these systems. Sources include municipal and private point-source discharges and non-point source inputs from concentrated animal feeding operations and traditional agriculture.

Blackwater systems normally contain somewhat reduced dissolved oxygen relative to clearwater streams because they have contact with organic sediments in floodplain swamps, are high in bacteria, and high concentrations of dissolved organic carbon are available as substrates for bacterial respiration. Thus, blackwater streams are particularly at risk for pollution-caused hypoxia and anoxia (low dissolved oxygen stress). The Cape Fear River system displays chronic summer hypoxia in the large blackwater rivers and various creeks, lasting from June through September or October This hypoxia problem occurs both in high flow summers and low flow summers; thus, it cannot be accounted for simply as inflow of oxygen-depauperate swamp water during rain events.

Our overall hypothesis for this research was that hypoxia in the Cape Fear basin is exacerbated by inputs of organic and inorganic nutrients. We concentrated our investigations in the Black River basin, a major subwatershed of the lower Cape Fear River system. Experiments were conducted on water from a near-pristine stream (Colly Creek) and a stream draining an area of heavy animal production (Great Coharie Creek). We used nutrient addition bioassays to assess blackwater stream susceptibility to nutrient loading, with chlorophyll a, BOD (biochemical oxygen demand), and bacteria counts as measured response variables to the inputs. Nutrient additions were nitrate, urea + nitrate (TN), orthophosphate, and orthophosphate + glycerophosphate (TP), with treatment concentrations of 0, 0.2, 0.5, 1.0, 2.0, and 5.0 mg o f N or P L . In addition to the experiments, water quality data were collected monthly from the two sites.

The two watersheds differ greatly in anthropogenic usage. Colly Creek is a wetlands-rich system with few CAFOs (concentrated animal feeding operations) while Great Coharie Creek has much less wetlands coverage and many CMOS. These differences are reflected in the water quality data. The pH values of Colly Creek were low, indicating large contributions of organic-acid rich swamp water, while the higher pH of great Coharie Creek likely results from less swamp inputs combined with anthropogenic loading Total nitrogen concentration was similar between creeks, but Great Coharie Creek had 37% of total nitrogen in the inorganic form (primarily nitrate) while only 11% of total nitrogen in Colly Creek was inorganic. Total phosphorus concentrations were about 4X higher in Great Coharie Creek than in Colly Creek, as were orthophosphate concentrations. Mean total and inorganic NP ratios were well above the Redfield ratio for phytoplankton (1 6) in both Colly Creek and Great Coharie Creek for the 24-month period. However, inorganic N/P ratios were at or below the Redfield ratio during the growing season

xi ii

when the experiments were conducted. Chlorophyll a was low in both creeks. Fecal coliform bacteria counts were generally higher in Great Coharie Creek than in Colly Creek.

Nitrogen, either as nitrate or TN, proved to be the nutrient that limited the growth of phytoplankton. In several of the nitrogen treatments, BOD was significantly stimulated over control through the autotrophic pathway of algal production followed by algal death and decay leading to subsequent BOD increase. Phosphorus did not stimulate the growth of phytoplankton in these experiments. However, significant BOD increases occurred in TP addition experiments, through stimulation of heterotrophs (mainly bacteria). Orthophosphate alone did not stimulate BOD. In most cases, chlorophyll a was stimulated to a greater degree in the water from the anthropogenically-impacted creek than water from the pristine creek, and there was stronger coupling between chlorophyll a and BOD. This possibly indicates a microbial community adapted to take advantage of periodic nutrient pulses from rainfall-driven runoff from swine waste sprayfields, poultry litterfields, or fertilized agricultural areas. Significant stimulation of chlorophyll a and BOD occurred with additions of 0.2 - 0.5 mg nitrate-NL, and significant stimulation of chlorophyll a and BOD also occurred in Great Coharie Creek with inputs of TN as low as 0.2 mg-N/L. Significant stimulation of BOD occurred with additions of either 0.5 or 1.0 mg-P/L as TP. Previous experiments conducted on water from the Black and Northeast Cape Fear Rivers using additions of 1 .O mg/L of N or P also showed significant stimulation of chlorophyll a (by N) and ATP (by P or N).

Direct bacterial counts conducted following the outdoor incubations were positively correlated with chlorophyll a yield, and both of these parameters were correlated with BOD. Thus, nitrogen additions to blackwater systems have the potential to increase BOD (and subsequently decrease dissolved oxygen). This is through the indirect photosynthetic pathway of algal bloom formation, algal bloom death and decay, bacterial increase, and increased BOD. Optimal conditions for algal bloom formation are nitrogen inputs into shallow, slow moving streams during spring and summer, with sufficient light availability (ie., relatively open forest canopy). Blooms in shallow streams will eventually enter larger and deeper light-limited blackwater rivers where bloom death will occur with consequent BOD increases.

Phosphorus inputs did not increase chlorophyll a biomass in these freshwater streams. This finding verified our previous experiments using Black and Northeast Cape Fear River water. However, phosphorus loading was found to lead directly to significant increases in BOD. This occurred in BOD tests run using water that had been amended with total phosphorus but not incubated outdoors, where algal growth could occur. Furthermore, direct bacterial counts showed significant increases without corresponding increases in chlorophyll a. Thus, the combined inorganic + organic phosphorus treatments caused direct bacterial growth stimulation that was sufficient to significantly increase BOD as well.

xiv

RECOMMENDATIONS

Currently, waters of the Cape Fear basin are not considered to be nutrient sensitive by the North Carolina Division of Water Quality (NCDWQ). As such, there is limited data available on nutrient discharges from licensed point source dischargers. While some larger dischargers regularly report total nitrogen and total phosphorus data from their effluent, many important dischargers in the 0.1-1 .O MGD (million gallons per day) range report only quarterly TN and TP data, which is useless for computing nutrient loads. Thus, we recommend that NCDWQ begin requiring weekly TN and TP data from point source dischargers exceeding 0.1 million gallons per day (MGD) in order to provide the State of North Carolina with useful point source loading information in the Cape Fear basin.

Our results clearly show the potential impacts of nitrogen and phosphorus loading on blackwater stream quality. To avoid phytoplankton blooms and subsequent (BOD) increases, we recommend that the State of North Carolina adopt an ambient blackwater stream inorganic nitrogen standard of 0.5 mg-NL. To avoid direct increases in BOD caused by stimulation of bacteria and other heterotrophs we recommend an ambient blackwater stream standard of 0.5 mg-P/L for total phosphorus. Strategies to avoid exceeding these stream standards should include limits on N and P dischargers from NPDES permit holders in the Cape Fear watershed. Efforts to reduce non-point source loading of nutrients should include 50 fl vegetated stream buffers basinwide, no fbrther loss of wetlands and increased utilization of constructed wetlands as runoff treatment systems, continued efforts to remove C M O S and sprayfields from river floodplains, and continued efforts to manage fertilizer applications and erosion. As total maximum daily load (TMDL) rules are established for the Cape Fear River basin, these recommendations should be incorporated into those rules as well, recognizing the distinct character of blackwater streams.

xv

INTRODUCTION

Blackwater stream systems are the characteristic type o f water body in the coastal plain region of North Carolina, as well as much of the southeastern United States (Meyer 1992; Smock and Gilinsky 1992). These streams have darkly stained water and rarely host surface-forming blue- green algal scums; thus these streams have historically not been considered to be nutrient-sensitive waters. For example, blackwater streams in North Carolina’s Cape Fear River basin are not protected from excessive nutrient loading (with the exception of ammonium).

Recent research efforts have demonstrated that, contrary to accepted dogma, blackwater streams can and do support phytoplankton blooms under certain conditions. These blooms have occurred under both waste spill and natural conditions in spring and summer (Burkholder et al. 1997; Mallin et al. 1997; 1999b), and after forestry-induced clearcuts (Ensign and Mallin 2001). Blooms have also been induced experimentally by additions of either organic or inorganic nitrogen (Mallin et al. 1998; Mallin et al. 2001). In contrast, experiments have also demonstrated that organic and inorganic phosphorus additions appeared to stimulate the heterotrophic community only (Mallin et al. 1998; Mallin 2000; Mallin et al. 200 1). These previous experiments utilized nutrient addition levels of 1 .O mg/L as N or P. In the field, monthly surveys by the LCFRP have demonstrated a wide range of nutrient concentrations in coastal plain streams, ranging from below analytical detection limits to over 22.5 mg/L of nitrate-N and over 4.7 mg/L of orthophosphate-P (Mallin et al. 1999a). Thus, it is important to determine key nutrient concentrations that stimulate significant biological responses by stream autotrophic and heterotrophic communities.

Conditions of low oxygen stress (hypoxia and anoxia) have been said to occur rarely in unpolluted blackwater streams (Smock and Gilinsky 1992). However, other research has noted that blackwater systems contain relatively lower dissolved oxygen than clearwater streams because they have contact with organic sediments in ff oodplain swamps, are high in bacteria, and high concentrations of dissolved organic carbon are available as substrates for bacterial respiration (Meyer 1992). Thus, these factors cause blackwater systems to be particularly at risk to hrther dissolved oxygen decreases due to pollution-caused hypoxia and anoxia. The Cape Fear River system displays chronic summer hypoxia in the large blackwater rivers and various creeks, lasting from June through September or October (Mallin et ai. 1998). Hypoxia occurs both in high flow summers and low flow summers; thus, it cannot be accounted for simply as inflow ofoxygen-poor swamp water during rain events. Low dissolved oxygen problems have been recognized by the NCDWQ as being particularly problematic in the Cape Fear River basin (North Carolina Department of Environmental Health and Natural Resources 1994; North Carolina Department of Environment and Natural Resources 2000).

Based on field and experimental data, we contend that algal blooms do occur in blackwater streams. These blooms occur only in spring or summer, primarily in shallow streams. However, these blooms die upon entering the deep, light-limited main channels and become a source of BOD. Thus, nutrient loading, through the photosynthetic pathway, can indirectly exacerbate low dissolved oxygen problems in blackwater rivers.

Direct heterotrophic responses to nutrient loading are also important. The principal constituents of the heterotrophic plankton community are the naturally occurring bacteria. Fungi are also a

part (Padgett et al. 2000), as are grazers such as protozoans and dinoflagellates that can respond and reproduce rapidly to an increase in bacterial prey biomass (Pace 1982; Beaver and Crisman 1989; Sanders 1991). Because of the high heterotrophic capacity of blackwater systems, growth stimulation of bacteria and protozoans by nutrient loading may hrther stress dissolved oxygen levels. Experimentally derived evidence suggests that phosphorus loading to feeder streams directly increases heterotrophic biomass (Padgett et al. 2000; Mallin et al. 2001), thus increasing BOD and leading to decreased dissolved oxygen concentrations in blackwater streams. This, in combination with the blackwater algal bloom formation potential noted above, leads us to hypothesize that the ultimate net result of nutrient loading in blackwater streams is a significant increase in BOD in the system. Our overall hypothesis for this research was that hypoxia in Cape Fear basin blackwater streams is exacerbated by inputs of organic and inorganic nutrients.

SOURCES AND MAGNITUDES OF NUTRIENTS IN BLACKWATER BASINS

Non-Point Sources. The densest concentrations of industrial hog and poultry operations (called CMOS - concentrated animal feeding operations) in the United States are in Sampson and Duplin Counties, most of which drain into the Cape Fear watershed, which includes the mainstem Cape Fear, Northeast Cape Fear, and Black Rivers. Over 100,000 metric tons of nitrogen and 40,000 tons of phosphorus are imported into North Carolina annually to support this industry (Cahoon et

; Glasgow and Burkholder 2000). These nutrients can be considered “new” nutrients, % of this feed originates out-of-state (but most of the N and P remains in North Carolina

as manure). Sampson and Duplin Counties produce high loads of excess available plant nutrients (Barker and Zublena 1995) and are considered to have high farm animal non-point source

ion potential (Natural Resources Conservation Service 1995). Large quantities of these nts have been shown to enter receiving waters through lagoon breaches and leaks (Mallin et

al. 1997; Burkholder et al. 1997). High nutrient levels have been found in runoff from swine waste spray fields (Westerman et al. 1985), subsurface leakage from waste lagoons (Huffman and Westerman 1995), and in streams draining swine farms (Stone et al. 1995; Gilliam et al. 1996). High loads of organic nutrients can be found in animal wastes as well (Westerman et al. 1990; Mallin et al. 1997). The Cape Fear watershed also has extensive row crop areas that can be

cant non-point nutrient sources to waterways (Natural Resources Conservation Service 1995; Gilliam et al. 1996).

Point Sources. An ongoing comprehensive sampling effort (Mallin et al. 1999a) has demonstrated that point-source discharges can contribute high concentrations of nutrients to blackwater streams in the Cape Fear watershed. For example, in a stream below a textile mill we have documented average total nitrogen concentrations of 3.9 mgL (range 0.4-21.6 m a ) , and total phosphorus averaging 0.8 mg/L (range 0.1-4.9 mg/L). In a stream below a municipal wastewater treatment plant we have recorded average total nitrogen of 5.5 mg/L (range 0.6-24.3 mg/L) and average total phosphorus of 1.0 mg/L (range 0.1-4.0 mg/L). Whether nutrient loading is derived from point or non-point sources, it is clear that the water quality degradation potential of excessive nutrient loading to these widespread blackwater systems requires experimental investigation under controlled circumstances. It is also important to note that these blackwater systems are just upstream of and drain into brackish coastal waters, and impacts to these “sentinel systems” will likely precede and lead to more impacts in estuarine fishery nursery areas.

2

PROJECT DESIGN

We concentrated our investigations in the Black River basin, a major subwatershed of the lower Cape Fear River system (Fig. 1). This system was designated by NCDWQ as an Outstanding Resources Water in 1991, yet since then has had major increases in CAFOs. This river suffered large anoxia-driven fish kills in 1995 and 1996 from anthropogenic BOD loads entering the watershed.

We conducted a series of nutrient addition bioassays using a gradient from no additions to high additions (5.0 mg/L) to determine what nutrient concentrations are “breakpoints” or yield a substantial increase in response variables. Our previous data showed clear responses at 1 .O mg/L as N or P (Mallin et al. 1998); thus, we intended to bracket this concentration. Our bioassays were conducted spring through fall, as previous winter bioassays yielded much lower growth than spring or summer (Mallin et ai. 1998). For pattern verification purposes our goal was to run each complete set of bioassays twice. Our response variables were chlorophyll a (which has a North Carolina State standard of 40 p a ) ; direct bacterial counts, which provide an estimate of the heterotrophic microbial response; and BOD, for which some discharge standards exist, and which is a good measure of potential dissolved oxygen problems.

SPECIFIC OBJECTIVES

Specific objectives of the project were to:

types and nutrient gradients on the growth of phytoplankton in two different subwatersheds of the Black River;

Assess the BOD of the samples after exposure to the nutrient addition gradient to determine potential effects of nutrient loading on dissolved oxygen concentrations, both before and after outdoor bioassay incubations;

these blackwater systems; and

indicative of potential water quality problems in blackwater systems, and then use these concentrations to recommend ambient nutrient and/or discharge nu systems.

Utilize nutrient addition bioassay experiments to determine the ct of different nutrient

Quantify the resident bacterial responses to the different nutrients and nutrient gradients in

Utilize the experimental results to determine breakpoints or key nutrient concentrations

es for blackwater

3

P

Figure 1. Location of sampling sites in the Black River watershed, southeastern North Carolina

Great Coharie Creek watershed

0 Sampling station watersheds 0 Sampling stations

0 40 Kilometers I I __ ____ - .-- __-_ - -1

Atlantic Ocean

MATERIALS AND METHODS

SAMPLING STATIONS

Water for the experiments was collected on eight occasions in 1999 and 2000 at two stations in the targeted region (Fig. 1). One was Colly Creek at NC53 near the Pender-Sampson County line (GPS coordinates N 34 27.900, W 78 15.392); the other was Great Coharie Creek at SR 1214 in Sampson County (N 34 55.114, W 78 23.324). These watersheds offer different water quality characteristics: Colly Creek receives low anthropogenic loading while Great Coharie Creek has numerous CMOS in its watershed (Table 1). Water quality in Great Coharrie Creek declined during the period 1993-1 998 (North Carolina Department of Environment and Natural Resources 2000).

NUTRIENT GRADIENT RESPONSE EXPERTMENTS

We tested the hypotheses that 1) nitrogen loading to blackwater streams can cause spring and summer phytoplankton bloom formation, 2) phosphorus loading will stimulate high levels of heterotrophic activity through increased bacterial production and 3) either nutrient, at a given concentration, will cause significant increases in stream BOD. These hypotheses were tested by the use of nutrient-gradient addition bioassays. The basis of these experiments is to add the experimental nutrient(s) covering a concentration gradient to replicated river water samples and determine if the phytoplankton and heterotrophic communities in the samples show a positive response (Le., a chlorophyll and/or field bacterial production increase and/or BOD increase).

The specific design was as follows. Water was collected on station in 25-L carboys, returned to the University of North Carolina at Wilmington Center for Marine Science, and dispensed into 1- gallon cubitainers (3 L / cubitainer). Nutrients tested were orthophosphate alone, total phosphorus (50% orthophosphate + 50% glycerophosphate-’equimolar’), nitrate alone, and total nitrogen (50% nitrate + 50% urea combination)

During previous blackwater research (Mallin et al. 1998; Mallin et al. 2001) we ran simultaneous N, P, and N + P treatments using a single nutrient concentration. As the objectives of this study were to test several different nutrient concentrations simultaneously, we chose to analyze one nutrient at a time because of space limitation in the pools (they hold a maximum of 36 cubitainers). Thus, each nutrient was tested on two separate occasions over a concentration range (expressed as final concentration as N or P) of no additions (control), 0.2, 0.5, 1.0, 2.0 and 5.0 mgk, with each treatment run in triplicate. Nitrate was tested in August 1999 and July 2000; total nitrogen (TN) in September 1999 and August 2000, orthophosphate in July 1999 and May 2000, and total phosphorus (TP) in November 1999 and June 2000.

Cubitainers were floated on flow-through pools near the Center for Marine Science, University of North Carolina at Wilmington, at ambient water temperatures. The cubitainers were covered by two layers of neutral density screening to allow solar irradiance penetration of about 30% of that reaching the water surface to prevent photostress to the phytoplankton. The cubitainers were kept in motion by constant circular agitation of the pond water using a submerged bilge pump (Mallin et al. 1998). Incubations were run for 6 days and the cubitainers were sampled on days 1,

5

3 and 6 for the response parameters. In previous nutrient addition bioassay research on estuarine systems we obtained strong algal responses in 1 - 3 days (Mallin et al. 1999b). However, preliminary bioassay experiments using water from blackwater streams required 3 - 6 days for a strong response; thus we used 6 days for our final collection. Estuarine experiments (Paerl et al. 1990; Rudek et al. 1991) sometimes showed sharp algal responses on day 1, followed by chlorophyll a decreases on days 2 and 3, possibly due to rapid zooplankton grazing. In contrast, our blackwater experiments always showed the highest chlorophyll a levels on day 6, indicating that zooplankton grazing is not an important factor in the experiments, or the field either.

RESPONSE PARAMETERS

Samples for chlorophyll a analysis were filtered through 1 .O pm pore sized glass fiber filters and analyzed with a Turner Model IO-AU fluorometer following Welschmeyer (1994). The response of the heterotrophic community was measured by direct bacterial counts, following the acridine orange fluorescence microscopy technique pioneered by Francisco et al. (1973) as modified by Hobbie et al. (1977). Stained bacteria were enumerated at lOOOX using a fluorescence-equipped Olympus BX50 compound microscope equipped with the following filter combination: Olympus cube UM 566 (460-490 excitation filter, 500 beam splitter, and 5 15 barrier filter). Sufficient random fields were counted to quantify at least 400 cells. One slide per cubitainer was counted for a total of three replicates per treatment. Blanks were enumerated by counting the total number of bacteria in 40 random fields. Blanks were subtracted from counts to obtain final values (as bacteridml). The BOD5 analyses were performed according to Standard Methods (American Public Health Association 1995). The BODzo analyses were also conducted to gain insight into longer-term responses. The BOD analyses (in duplicate) were conducted on test water after being amended with nutrients but before outdoor incubation (to test for heterotrophically generated BOD) as well as after the outdoor incubations on day 6 (to account for combined heterotrophically and photosynthetically generated BOD).

STATISTICAL ANALYSIS

Statistical analyses of nutrient limitation test results were accomplished using the Statistical Analysis System (SAS) procedure of Analysis of Variance (ANOVA). This test utilizes the means and standard deviations of the response data and determines if a significant difference exists (p < 0.05) between the response means of the various nutrient treatments. Pfa difference in response means exists among the treatments, the ANOVA test was followed by treatment ranking by the least significant difference (LSD) procedure. This statistical test compares each treatment response mean with the others, shows which of the different treatments (nutrient additions) elicited the greatest response, and ranks the treatment responses in descending order. This provides a sound statistical basis for reporting which nutrient concentrations lead to significant phytoplankton or heterotrophic growth in the system being tested. This statistical treatment is recommended in Day and Quinn (1989) and examples of its successfid use in studies in North Carolina are provided in Paerl et al. (1990), Rudek et al. (1991), and Mallin et al. (1999b; 2001). Correlation analyses were conducted among the four response variables for each experiment individually and for all experiments combined using SAS (Schlotzhauer and Littell 1987).

6

WATER QUALITY ASSESSMENT

Water temperature, pH, dissolved oxygen, turbidity, and conductivity data were collected on site using a YSI 6920 multiparameter water quality monitor. The water used for the bioassays was assessed for a suite of nutrient parameters. Total Kjeldahl nitrogen (TKN), nitrate, ammonium, orthophosphate and total phosphorus (TP) were measured using Standard Methods (American Public Health Association 1995). Total nitrogen (TN) was computed as TKN plus nitrate. Chlorophyll a was measured using a fluorometric method (Welschmeyer 1994) on a Turner Model 10-AU fluorometer. Fecal coliform bacteria were quantified using the membrane filtration method (mFC) and BOD by Standard Methods (American Public Health Association 1995). For regional comparative purposes, in 2000 and 2001 stream flow and depth data were collected monthly from these two streams plus four others in the Black River watershed that are sampled regularly by the LCFRP (Little Coharrie Creek, Six Runs Creek, Hammond Creek and Browns Creek). Depth was measured every three meters from bank to bank and averaged, and flow was measured with a Marsh McBirney Flo-Mate Model 2000 meter at mid depth in the middle of the channel.

7

RESULTS

WATER QUALITY IN COLLY CREEK AND GREAT COHARIE CREEK

Relevant land use data for the two watersheds, along with water quality data for the two creeks from January 1998 through December 2000, are presented in Table 1. The two watersheds differ greatly in anthropogenic usage. Colly Creek watershed contains two small National Pollution Discharge Elimination System discharges with an average combined discharge of about 0.42 MGD. Great Coharie Creek watershed contains the Clinton Wastewater Treatment Plant (average discharge 2.8 MGD) and the Newton Grove Wastewater Treatment Plant (average discharge 0.05 MGD). Colly Creek is a wetlands-rich system with few CMOS while Great Coharie Creek has much less wetlands coverage and many CAFOs. These differences are reflected in the water quality data. The pH values of Colly Creek were low, indicating large contributions of organic-acid rich swamp water, while the higher pH of Great Coharie Creek likely results from less swamp inputs combined with anthropogenic loading. The nutrient compositions of the two systems differ as well (Tables 1 and 2). Total nitrogen (TN) concentration was similar between creeks, but Great Coharie Creek had 38% of TN in the inorganic form (primarily nitrate) while only 11% of TN in Colly Creek was inorganic. Total phosphorus (TP) concentrations were about four times as high in Great Coharie Creek as in Colly Creek, as were orthophosphate concentrations (Table 1). Average total and inorganic N/P were well above the Redfield ratio for phytoplankton (1 6) in both Colly Creek and Great Coharie Creek for the 24-month period (Table 1). However, inorganic N/P ratios for the spring and summer growing season were at or below the Redfield ratio (Table 2). Chlorophyll n was low in both creeks (Table 1). Fecal coliform bacteria counts were generally higher in Great Coharie Creek than in Colly Creek. Based on the water quality data, Great Coharie Creek is quite representative of other regional streams (Mallin et al. 1999a), whereas Colly Creek is unusual in being relatively pristine. Hydrological data from 2000-2001 differed little between the two streams (Table 1).

The largest meteorological event to occur during the study period was Hurricane Floyd beginning September 15, 1999 (Bales et al. 2000). Massive flooding occurred throughout the study area, as in other areas of the state. Stream sampling conducted 5 days after Hurricane Floyd showed depressed pH, likely caused by inputs of swamp water during flooding. There was unusually low nitrate in Great Coharie Creek, probably a result of the post hurricane hypoxic conditions (Table 1). Total nitrogen concentration in both creeks remained within 20% of the 3-year average, but post storm total phosphorus concentrations in Great Coharie Creek were six times higher than those in Colly Creek and orthophosphate concentrations were twelve times higher (Table 1). Water collected for the September 1999 experiment was influenced by heavy rains that were associated with Hurricane Dennis during the first week in September.

9

Table 1. Water quality characteristics (mean and range) of Colly Creek and Great Coharie Creek, tributaries of the Black River, January 1998-December 2000. Flow and depth data are from May 2000 to April 2001.

Parameter Colly Creek Great Coharie Creek

Watershed area (km2) Percent wetlands coverage CMOS in watershed S t r eadow (m3/s) Depth (m)

Mean values for 1998-2000

PH conductivity (pS) nitrate-N ( p a ) ammonium-N ( p a )

orthophosphate-P (&L)

Total N/P Inorganic N/P Chlorophyll a ( p a ) Fecal coliforms/lOO niL,

Five days after Hurricane Floyd

(P@>

TP (EL&)

PH nitrate-N (pg/L) ammonium-N ( p a ) Il*N (PLg/L) ortho p ho sp hate-P (pzgL) TP (PLg/L> Fecal coliforms/ 100 mL

326 55.2

6 7.2 (0.6-21.0) 1.0 (0.4-2.0)

3.8 (3.1-5.1) 60 (40-140) 7 (5-20)

91 (5-540) 895 (680-1870)

10 (1-59) 33 (5-140) 90 31 1.4 (0.2-18.1) 65 (5-250)

3.1 5

50 980

5 20

9

554 11.8

95 6.4 (1.4-16.7) 0.9 (0.3-1.7)

6.1 (4.9-7.0) 130 (60-3 10) 297 (5-930)

69 (10-170) 977 (580-1750)

46 (13-142) 143 (40-570) 44 33 1.3 (0.1-9. 1)

205 (6-2168)

4.9 5

70 780

60 120

9

10

Table 2. Ambient nutrient concentrations (and molar total and inorganic N/P ratios) in bioassay test waters before nutrient additions.

Colly Creek ( p a )

Date TN N03--N m + - N TP PO~"-P TN/TP

July 1999 990 10 170 50 10 43.8

Aug, 1999 1010 10 11 50 10 44.8

Sep. 1999 1030 10 80 20 10 114.0

Nov. 1999 920 10 60 30 10 67.9

May 2000 840 10 60 30 20 62.0

June 2000 910 10 50 40 10 50.4

July 2000 1020 10 150 50 30 45.2

Aug. 2000 810 20 70 20 10 89.7

IN/IP

39.9

4.7

19.9

15.5

7.8

13.4

11.8

19.9

Great Coharie Creek ( p a )

Date TN N03--N "-N TP p0i3-p TN/TP ~ / I P

July 1999 1 140

Aug. 1999 920

Sep. 1999 1160

Nov. 1999 730

May 2000 620

June 2000 720

July 2000 620

Aug. 2000 840

230 50 170

80 120 23 0

170 110 23 0

120 50 120

40 50 80

20 60 180

10 90 140

10 110 210

90

100

80

50

40

60

60

70

14.8

8.8

11.2

13.5

17.1

8.9

9.8

8.9

6.9

4.4

7.8

7.5

5.0

2.9

3.7

3.8

11

NITRATE ADDITION EXPERIMENTS

Nitrate addition experiments were run in August 1999 and July 2000. While there were differences among creeks and magnitude of responses, a similar pattern emerged from both experiments. In all cases nitrate additions of 0.5 mg/L and greater, and in most cases additions of 0.2 mg/L, or more led to significant chlorophyll a increases over control (Table 3 ) , with generally higher responses in the 2000 experiment than in the 1999 experiment (Figs. 2 and 3). These chlorophyll a increases were reflected by significant post-incubation BOD increases as well, beginning at either 0.2 or 0.5 mg/L (Tables 4 and 5). It is notable that there were greater chlorophyll a and BOD responses during both experiments to water from Great Coharie Creek (the anthropogenically impacted system) than Colly Creek water. The BOD analyses run on test waters after nutrient additions, but before outdoor incubations, did not show significant increases in most cases (Tables 6 and 7; Fig. 4). For Great Coharie Creek in August, additions of 0.5, 1 .O, and 2.0 mg-N/L led to statistically significant increases over control, but these increases were of - <20% (Fig. 4). Fluorescence microscopy indicated that post-incubation bacterial counts were significantly greater than control for nitrate-N additions of either 1 .O or 2.0 mg/L for both creeks during both experiments (Table 8).

TOTAL NITROGEN ADDITION EXPERIMENTS

Total nitrogen (50% nitrate-N + 50% urea-N) addition experiments were run in September 1999 and August 2000. There was no chlorophyll a response to the additions in Colly Creek in September 1999 (Fig. 5) , but there were significant chlorophyll n increases in Great Coharie Creek water for the 1.0 and 5.0 mg-N/L treatments (there was a lot of variability among replicates as well). This experiment occurred after rains from Hurricane Dennis, so increased swamp water inputs into Colly Creek likely led to high water color but very low chlorophyll a concentrations. There were no significant post incubation BOD increases during September 1999 (Tables 4 and 5; Fig. 5 ) . The August 2000 experiment yielded very high chlorophyll a production in the Great Coharie Creek water, with statistically significant increases in treatments of 0.2, 0.5, 1.0, 2.0, and 5.0 mg/L of TN (Fig. 6). This chlorophyll production was reflected in post incubation BOD increases as well, all of which were statistically significant at concentrations of 0.2 mg-N/L and above (Fig. 6). There was low chlorophyll n yield in Colly Creek, with no statistically significant increases over control Pre-incubation BOD increases were not significant for August 2000 (Fig. 7), indicating that post incubation BOD increases were primarily driven through the photosynthetic pathway. There were significant pre-incubation BOD20 increases at the 2.0 and 5.0 mg-N/L levels for Great Coharie Creek in September 1999. There were no significant post- incubation bacterial increases over control for either creek during the September 1999 experiment. However, bacterial increases were significant at 1 .O mg-N/L and above for Great Coharie Creek and at 1 .O mg-N/L for Colly Creek in the August 2000 experiment (Table 8).

ORTHOPHOSPHATE ADDITION EXPERZMENTS

Orthophosphate addition experiments were run in July 1999 and May 2000. In almost all cases there were no significant chlorophyll a increases due to inorganic phosphorus additions (Table 4; Figs. 8 and 9). In Colly Creek in July 1999 the I .O mg/L treatment led to a small but statistically

12

Table 3. Statistically significant (p < 0.05) chlorophyll a responses to nutrient additions, relative to control.

Treatment Colly Creek Great Coharie Creek

Nitrate August 1999 2.0, 1.0,0.5,0.2 July 2000 5.0,2.0, 1.0,0.5,0.2

Nitrate + Urea Sep. 1999 Aug. 2000

Orthophosphate July 1999 May 2000

1 .o

Orthophosphate + Glycerophosphate

Nov. 1999 June 2000

5.0,2.0, 1.0, 0.5,0.2 5.0,2.0, 1.0,0.5

5.0, 1.0 5.0,2.0, 1.0,0.5,0.2

1 .o

13

Table 4. Statistically significant (p < 0.05) BODS responses to nutrient additions (post-incubation responses), relative to control.


Nitrate Aug. 1999 July 2000




Nov. 1999 June 2000

5.0, 2.0, 1.0, 0.5, 0.2 5.0, 2.0, 1.0, 0.5, 0.2

5.0, 2.0, 1.0, 0.5, 0.2 5.0, 2.0, 1.0, 0.5

5.0, 2.0, 1.0, 0.5, 0.2

5.0, 2.0, 1.0 5.0

Table 5 . Statistically significant (p < 0.05) BOD2o responses to nutrient additions (post-incubation responses), relative to control.


Nitrate Aug. 1999 5.0, 2.0, 1.0, 0.5, 0.2 na July 2000 5.0, 2.0, 1.0, 0.5, 0.2 5.0, 2.0, 1.0, 0.5




Nov. 1999 June 2000

1.0, 0.2

5.0 5.0, 2.0

5.0 5.0, 2.0, 1.0, 0.5, 0.2

na 5.0

5.0, 2.0 5.0

14

Table 6. Statistically significant (p < 0.05) BOD5 responses to nutrient additions (pre-incubation responses), relative to control.






Nov. 1999 June 2000 5.0, 2.0, 1.0

5.0, 2.0, 1.0, 0.5

2.0, 1.0, 0.5

5.0, 2.0, 1.0, 0.5 5.0, 2.0

Table 7. Statistically significant (p < 0.05) BODzo responses to nutrient additions (pre-incubation responses), relative to control.






Nov. 1999 June 2000

na

5.0, 2.0

na

5.0, 2.0, 1.0, 0.5 5.0, 2.0, 1.0

5.0, 2.0, 1.0, 0.5 5.0, 2.0, 1.0

15

Table 8. Statistically significant (p < 0.05) bacterial count responses to nutrient additions (post- incubation counts), relative to control.


Nitrate Aug. 1999 5.0, 2.0 July 2000 5.0, 2.0, 1.0

Nitrate + Urea Sep. 1999 Aug. 2000 1 .o

Orthophosphate July 1999 May 2000 5.0


Nov. 1999 June 2000

5.0, 2.0, 1.0, 0.5, 0.2

5.0, 2.0, 1.0 5.0, 2.0, 1.0

5.0, 2.0, 1.0

2.0

5.0 5.0

16

Figure 2. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to nitrate-N additions, August 1 999. *denotes significantly greater than control.

* * 18

16

2 1 4

-12 0 3

(D z: 10

E 8 >.

0 g 6 0 $ 4

2

0

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

9

8

7

6 cq

$ 5

; 4

111

0 3

2

a

0

*

* T

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0 NUTRIENT TREATMENT (mg-NIL)

17

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-NIL)

1.8

1.6

1.4

2 0.8 0 m 0.6

0.4

Figure 4. Pre-incubation BOD5 responses of Colly Creek (left) and Great Coharie Creek (right) water to nitrate-N additions, August 1999 (top), July 2800 (bottom). *denotes significantly greater than control.

T T

0.2

0 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

1.4

1.2

1 .o

ua

M 0.6

T

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-N/L)

19

Figure 5. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic+organic N additions, September 1999. *denotes significantly greater than control.

1 10

9

8

7

6

5

4

3

2

1

0 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

18

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0


20

Figure 6. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic+organic N additions, August 2000. *denotes significanlty greater than control.

100

90

80

70

60

50

40

30

20

I O

0 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

12.0

10.0

- 8.0 i

- 6.0 n 0 m

F y?

4.0

2.0

0.0

* *

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-N/L)

21

Figure 7. Pre-incubation BOD, responses of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic + organic nitrogen additions, September 1999 (top), August 2000 (bottom). *denotes significantly greater than control.

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

1.6 I I


22

Figure 8. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to orthophosphate-P additions, July 1999. *denotes significantly greater than control.

4.5

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

5.0

4.5

4.0

3.5 n

3.0

- 2.5 B 0 2.0

E" ua

I .5

1 .o 0.5

0.0

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-PIL)

23

I

Figure 9. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to orthop May 2000. *denotes significantly greater than control.

3.0

2.5 T T

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

3.0

2.5

2.0 n

r!

'1.5 Q 0

F v)

m 1 .o

0.5

0.0 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-P/L)

24

significant chlorophyll a increase over control. However, none of the post-incubation BOD5 increases were significant (Fig. 8). There were some significant post-incubation BOD2' responses, but without a clear pattern (Table 5) . None of the pre-incubation BOD5 tests showed a significant increase over control (Table 6; Fig. 10). There were no significant bacterial increases over control for the July 1999 experiment (Table 8). The May 2000 experiment showed significant bacterial increases at 5.0 mg-P/L for Colly Creek and at 2.0 mg-P/L for Great Coharie Creek (Table 8).

TOTAL PHOSPHORUS ADDITION EXPERIIVENTS

Total phosphorus (50% orthophosphate-P + 50% glycerophosphate P) addition experiments were run in November 1999 and June 2000. None of the treatments yielded significant chlorophyll a increase except for the 1 .O mg-P/L treatment in the June 2000 Great Coharie Creek experiment (Table 3; Figs. 11 and 12). However, in November 1999 the post-incubation BOD5 assays showed statistically significant increases over control for 1.0, 2.0, and 5.0 mg-P/L in Colly Creek, and for 5.0 mg-P/L in Great Coharie Creek (Table 4; Fig. 11). Post incubation BOD5 increases in the June 2000 experiment were not significant (Fig. 12). However, post-incubation BOD20 responses were significant for both creeks at either 2.0 or 5.0 mg-P/L (Table 5). The pre- incubation BOD assays showed significant increases over control in November 1999 to all treatments from 0.5 mg-P/L and above (Fig. 13). In June 2000 there were significant pre- incubation BODS and BOD20 increases in Colly Creek water for 1.0, 2.0, and 5.0 mg-P/L and in Great Coharie Creek water for 2.0 and 5.0 mg-P/L treatments (Fig. 13). The BOD increases ranged from 15% at the low additions to more than doubling the BOD at the high P additions. For the November 1999 experiment bacterial counts were significantly greater than control at 0.2 mg-P/L and above for Colly Creek and at 5.0 mg-P/L for Great Coharie Creek (Table 8).

CORRELATION ANALYSES

Correlation analyses were run among all four response variables for post outdoor incubation data. For all experiments combined, bacterial counts were positively correlated with BOD5, BOD20, and chlorophyll n (Table 9). Chlorophyll a was likewise positively correlated with BOD5 and BODzo. These correlations differed according to individual treatments, however (Table IO). For most nitrate experiments, chlorophyll a concentrations and bacterial counts were significantly correlated. Both chlorophyll a and bacterial counts were significantly correlated with BOD5 and BOD20 in most nitrate experiments, but more consistently in Great Coharie Creek water than in Colly Creek water (Table 10). In the August 2000 TN experiment ther between both bacterial counts and chlorophyll a concentrations and the two BOD tests. There were no significant positive correlations between chlorophyll a and either bacterial counts or BOD for the inorganic phosphorus experiments (we note that there was a considerable amount or variability in some of the bacterial counts-Fig. 14). However, bacterial counts were strongly correlated with BODS and BOD20 in the November 1999 TP experiment, and displayed a near- significant correlation among these parameters in the June 2000 TP experiment (Table 10). In the August 2000 TN experiment there were strong correlations between both bacterial counts and chlorophyll a concentrations and the two BOD tests. There were no significant positive correlations between chlorophyll n and either bacterial counts or BOD for the inorganic phosphorus experiments (we note that there was a considerable amount of variability in some of

ere strong correlations

25

Figure I O . Pre-incubation BOD5 responses of Colly Creek (left) and Great Coharie Creek (right) water to orthophosphate-P additions, July 1999 (top), May 2000 (bottom). *denotes s i g n if ica n t I y greater than con t ro I.

1.2

1 .o

0.8 E!

- 0.6 L=r 0 c[II 0.4

0.2

0.0

T T T T

E" v)

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0


26

0.4

Figure 11. Post-incubation response of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic +

*denotes significantly greater than control. , November 1999.

0.0 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

4.0 I * -r 3.5 I *

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0


27

Figure 12. Post-incubation responses of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic + organic phosphorus additions, June 2000. *denotes significantly greater than control.

4.0

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

NUTRIENT TREATMENT (mg-P/L) ~

3.5

$ 3.0

tu 2.5

2.0 a 2 1.5 0 -I 1.0 0

0.5

n

3 Y

-I -I

*

T

I 0.0

I 0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

3.0

28

4.0

3.5

3.0

=! 2.5

- 2.0 c3 0 1.5 m

1 .o 0.5

0.0

n

2 v)

Figure 13. Pre-incubation BOD5 responses of Colly Creek (left) and Great Coharie Creek (right) water to combined inorganic + organic P additions, November 1999 (top), June 2000 (bottom). *denotes significantly greater than control.

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0

3.0

2.5

2.0 n

2

- 1.5 c9 0

F rc)

a 1 .o

0.5

0.0 I t I t I I r- I I I 1

0.0 0.2 0.5 1.0 2.0 5.0 0.0 0.2 0.5 1.0 2.0 5.0


29

Figure 14. Bacterial abundance by nutrient addition level for orthophosphate treatments (top) a n d TP treatments (bottom). Includes o n e s tandard error of the mean.

3,500,000

3,000,000 -I - 5 2,500,000 cn 0

- z 2,000,000 - ,E 1,500,000 L Q) 2 1,000,000 m

500,000

T

I I I I I 0 ON 0.2 0.5 I .o 2.0 5.0

ORTHOPHOSPHATE REATMENT (mg-P/L)

1,800,000 I

30

Table 9. Positive Great Coharie Cr probability (p).

correlations among post-incubation response variables for Colly Creek and -eek waters for all experiments combined. Pearson correlation coefficient (r) /

BODS BOD20 Chlorophyll a

Bacterial counts 0.556 0.49 1 0.605 0.001 0.001 0.001

Chlorophyll a 0.752 0.001

0.648 0.001

31

L- e n m a 6)-

c, cd

SI

5 0 p3

c,

G E .e L a,

o l m r -0

0 0 Q.9

r - m cod-

0 0 ?9

r - - m o 0 0 Q.9

r - - m o 0 0 ".9

Q.3 m o l

0 0 Q.9

ol"

0 0 0 0

;?;s \Do Q.9 '79

t; 3 0

m d - m d - 0 0 6.9

d-- \Do 0 0 0\9

m - m o 0 0 0\9

B B " m o 0 0 ".9

IC)" M O

0 0 099

m " 0 0 0

0 0 "9

c-4" 0 0

0 0 0\9

0 0 - r -0

0 0 0\9

cd 1 3

cd

a, 0

.& t; c, 8 t;

m" s m" 0

L c,

3 - 0

0 0 0 m h 5 h

3

M " 0 0

0 0 ?9

0 0 m r - - 0 0 '19

cd L a, c, 0

.&

m"

c1

" 0 0 c o t -

0 0 '19

m a - 0 0

0 0 '19

v)

v) .e

h cd 2 z: 0

cd a,

.e c, 3

0 L- e -2

%

a, 3

3 .& cd

cd cd a 0 r: I I cd r:

c,

32

the bacterial counts) (Fig. 14). However, bacterial counts were strongly correlated with BOD5 and BOD20 in the November 1999 TP experiments and displayed a near-significant correlation among these parameters in the June 2000 TP experiment (Table 10).

33

DISCUSSION

The results clearly show nitrogen limitation of phytoplankton growth in both of the blackwater creeks investigated. There were strong positive responses to all nitrate addition experiments and positive responses to the TN additions in some experiments. This verifies and complements the results of earlier nitrogen addition bioassays conducted on water from the Black and Northeast Cape Fear Rivers (Mallin et al. 1998; Mallin et al. 2001). During the nitrogen addition experiments inorganic N/P ratios were either at or well below the phytoplankton N/P Redfield ratio of 16 (Table 2). In general, N/P ratios in Great Coharrie Creek were lower than in Colly Creek (Table 2). Bacterial counts taken following the outdoor incubations were positively correlated with chlorophyll a yield and both of these parameters were correlated with biochemical oxygen demand. Thus, nitrogen additions to blackwater systems have the potential to increase BOD (and subsequently decrease dissolved oxygen) through the indirect autotrophic pathway of algal bloom formation, algal bloom death and decay, bacterial increase, and increased BOD. Optimal conditions for algal bloom formation are nitrogen inputs into shallow, slow moving streams during spring and summer, with sufficient light availability (i.e., relatively open forest canopy). Blooms in shallow streams will eventually enter larger and deeper light-limited blackwater rivers where bloom death will occur with consequent BOD increases (Mallin et al. 200 1).

How hospitable is the solar irradiance field of blackwater streams in this region for phytoplankton growth? The major factor limiting light availability would be canopy shading of lower order streams, which does occur and is especially common in pristine, forested regions. However, the modern mid-Atlantic and southeastern Coastal Plain (Maryland, Virginia, North Carolina, and increasingly South Carolina) has been completely transformed by logging and agricultural activities, both traditional crop and large-scale CAFO operations, which have decimated much of the forest cover. Also, CAFO-rich watersheds such as the Cape Fear River system have no required vegetated buffer zones, either for agriculture or forestry operations. Thus, most lower order streams pass through a landscape mosaic consisting of farm fields, CAFO spray or poultry litter fields, canopied forests, and numerous road crossings, allowing much opportunity for significant solar irradiance to impact stream waters.

Water color is certainly a major factor attenuating light in deep, higher order streams. This is a factor that is central to our hypothesis that blooms in shallow streams enter larger rivers, rapidly die and stimulate BOD. In lower order blackwater streams water column light attenuation is much less of a problem to phytoplankton than traditionally believed. Based on the LCFRP stream monitoring program, the median summertime (May through August, years 2000 and 2001) depth z of a selection of six second to third order streams was 0.9 m (n = 47). The median summer light attenuation coefficient k for the nearby Black River was 3.66/m. Considering that the median summer surface irradiance (IO) for the Black River was 15 15 pE m-2 s‘l, we determined that the mean irradiance I, for a 0.9 m water column (by the equation I, = Io[ 1-e’””] / kz) is 443 pE m s , which is approximately the 30% irradiance level that yields maximum photosynthesis, Pmax (Mallin and Paerl 1992). Even the bottom would receive approximately 56 pE m=L s-’, still well within the euphotic zone. First order streams are even shallower; thus, under non-pristine conditions, the summer irradiance field of lower order blackwater streams can and does provide hospitable conditions for bloom formation.

-2 -1

35

There is also documented phytoplankton bloom activity in these systems caused by nutrient loading. Mallin et al. (1997) described a massive nutrient load from a 1,000,000 gallon CAFO leak into a set of first to third order well-canopied streams that led to chlorophyll a blooms up to 120 pg/L for over two weeks. Routine LCFRF' monitoring activities have also shown algal blooms occurring at blackwater stream stations (Mallin et al. 2000). Ensign and Mallin (2001) described nutrient loading following forest clearcutting operations in which phytoplankton blooms exceeding 160 pg/L as chlorophyll a occurred as a response. Ensign and Mallin (2001) fixther documented that the demise of each bloom led to severe hypoxia or anoxia in the water column, likely caused by the large BOD load created by the decomposing algal bloom

In several instances nitrogen inputs as low as 0.2 mg-N/L were sufficient to produce statistically significant chlorophyll a and BOD increases over control. While the effect of this concentration was statistically significant, the 0.5 tol.0 mg-N/L levels appeared to produce notably greater responses that would be indicative of water quality degradation in terms of algal blooms, bacterial increases, and/or BOD loading (Figs. 2 and 3). Our previous investigations utilized 1.0 mg-N/L as our test concentrations and found large and statistically significant increases in chlorophyll a over controls (Mallin et al. 1998; Mallin et al. 2001). Chlorophyll a levels in our experiments appeared to reach a maximum at 1.0 mg-N/L, with little hrther biomass accumulation (Figs. 2, 3, and 5). This may have been due to self-shading by the algae. Since pristine blackwater streams normally maintain chlorophyll a levels < 5.0 pgA, we think that in order to maintain reasonably natural conditions chlorophyll a biomass should be kept to no more than one half of the state standard of 40 p a . Therefore, we suggest that the State of North Carolina adopt ambient standards designed to maintain inorganic nitrogen at or below 0.5 mg-N/L to protect water quality in blackwater stream systems.

Based on various types of experiments, phosphorus is generally considered to be the principal nutrient limiting the growth of phytoplankton in freshwaters (Hecky and Kilham 1988). Some reasons for this include the high N/P ratios in river water, the high N/P ratio in water draining fertilized agricultural catchments, the larger amounts of nitrogen in rainfall relative to phosphorus, and the often abundant quantities of nitrogen-fixing blue-green algae in lakes (Paerl 1982; Hecky and Kilham 1988). However, we have demonstrated nitrogen limitation of phytoplankton growth in two 5* order rivers (Mallin et al. 2001) and two 3rd order streams in southeastern North Carolina. There are some potential reasons for this nitrogen limitation. The inorganic N/P ratio in these streams during the growing season is generally near or below the Redfield ratio (Table 2). This may be due to the potential sources of nutrient loading to these blackwater systems. As mentioned, the agricultural landscape is dominated by CAFOs. Animal waste has a generally low N/P ratio compared with fertilizers (Cahoon et al. 1999). Thus, the sources of nutrients to these waters may be instrumental in forcing the nitrogen limitation of phytoplankton. It is notable that the TN/TP ratio in Colly Creek is high, in contrast to the inorganic N/P ratio (Table 2) . However, much of the organic nutrient fraction of this material is swamp derived and may be quite refractory and unavailable to phytoplankton. As for nitrogen fixation, while blue-green algal blooms have been seen in disturbed blackwater streams in this region (Ensign and Mallin 200 l), normally blue-green algae comprise only a minor component of the phytoplankton community in these streams (Mallin et al. 2001). Thus, blue-green algal nitrogen fixation is likely not a significant nutrient source to these waters.

36

Phosphorus inputs did not increase chlorophyll a biomass in these freshwater streams. This finding verified our previous experiments using Black and Northeast Cape Fear River water (Mallin et al. 1998; Mallin et al. 2001). However, phosphorus loading was found to directly lead to significant increases in BOD. This occurred in BOD tests run using water that had been amended with total phosphorus but not incubated outdoors. Direct bacterial counts conducted after outdoor incubations showed significant increases without corresponding increases in chlorophyll a (Table 8). Thus, the combined inorganic + organic phosphorus treatments caused direct bacterial growth stimulation that was sufficient to significantly increase BOD as well. Although our previous work in the rivers showed ATP stimulation by orthophosphate alone, in the present experiments the inorganic phosphorus alone caused few significant BOD or bacterial increases. It required organic phosphorus to do so consistently. Our previous work showed that organic phosphorus inputs almost always led to significant ATP increases (Mallin et al. 1998; Mallin et al. 2001).

These experiments showed significant BOD increases occurring either at the 0.5 or 1 .O mg-P/L concentrations (Figs. 11 and 13). Our previous work utilized 1.0 mg-P/L for the inorganic and organic P treatments, which both yielded significant ATP increases (Mallin et al. 1998; Mallin et al. 2001). Other experiments have also shown notable BOD increases at the 1 .O mg-P/L level (Mallin 2000; Mallin et al. 2000). Therefore we recommend that the State of North Carolina adopt an ambient standard designed to keep total phosphorus concentrations below 0.5 mg-P/L in blackwater stream systems to avoid further increases in BOD and subsequent decreases in dissolved oxygen.

Our experiments indicated phosphorus limitation of bacterial growth in these blackwater streams and rivers, despite the rather low growing season NP ratios. Phosphorus has been experimentally determined to limit or stimulate bacterial growth in other situations as well (Morris and Lewis 1992; Chrzanowski et al. 1995; Cotner et al. 2000). Structurally, bacteria have a much higher PIC ratio than do phytoplankton (Valdstein et al. 19S8), This is primarily due to the phosphorus in membrane phospholipids and nucleic acids (Valdstein et al. 1988: Kirchman 1994). This suggests that the phosphorus requirements of bacteria should be considerably higher than their nitrogen requirements (Kirchman 1994). In our experiments addition of inorganic phosphorus alone led to increases in bacterial growth in some situations (Fig. 14), but only a few of these were statistically significant (Table 8). Since phosphorus uptake requires energy, bacteria might, in some cases, be phosphorus and carbon co-limited (Currie 1990). This could explain the stronger responses of bacteria to glycerophosphate (Table 8; Fig. 14), and significant increases in BOD to the organic P treatment (Tables 4-7). Dissolved organic carbon is abundant in blackwater streams, but much of it is refractory (Meyer 1990). Therefore, it would be less available as an energy source for bacteria to use in inorganic phosphorus uptake.

The data from our experiments showed similar general patterns between the impacted stream (Great Coharie Creek) and the pristine system (Colly Creek). However, Great Coharie Creek clearly showed stronger responses to nutrient loading in terms of chlorophyll a (Table 3), as well as stronger coupling among chlorophyll, bacteria, and BOD (Table 10). This may reflect the presence of a plankton community that is adapted to utilize the periodic pulses of nutrients that Great Coharie Creek receives during rainfall-driven runoff events. Great Coharie Creek has much

37

less wetlands coverage and many more CAFOs than Colly Creek, thus elevated inorganic and labile organic nutrients are more likely to enter receiving streams after storm events in this watershed (Mallin 2000) and Table 2.

As mentioned, most blackwater streams in eastern North Carolina lack nutrient protections, with the exception of ammonium standards. Many point source dischargers in the Cape Fear basin are not required to collect even monthly TN and TP samples from their emuents. Thus, NCDWQ cannot obtain reasonable estimates of point source nutrient inputs into the Cape Fear system. We recommend that NCDWQ require weekly TN and TP samples from NPDES dischargers exceeding 0.1 MGD in order to acquire accurate data for nutrient loading in this system.

Broad-based field monitoring by the Lower Cape Fear River Program has found high levels of TN, nitrate, TP, and orthophosphate below some public and private sewage treatment facilities (Mallin et al. 1999b). Additionally, spills from swine and poultry C M O S have contributed large quantities of nutrients to these stream systems (Burkholder et al. 1997; Mallin et al. 1997; Mallin et al. 1999~). Chronically, CAFOs load nitrogen and phosphorus into receiving streams well in excess of our suggested standards for maintaining stream water quality (Stone et al. 1995; Gilliam et al. 1996). Daily, vast quantities of nutrients in the form of animal feed are imported into the Coastal Plain to feed the huge numbers of swine and poultry raised in CAFOs (Cahoon et al. 1999; Glasgow and Burkholder 2000). Nutrients and organic matter derived from human and animal wastes will be largely labile; thus having a particularly high potential to cause algal blooms, increased bacterial loads, and elevated BOD. Blackwater streams on the Coastal Plain have naturally lower dissolved oxygen levels than clearwater streams, particularly during summer (Meyer 1990; Meyer 1992; Mallin et al. 2001). Thus, any fkrther inputs ofBOD will decrease dissolved oxygen levels to concentrations stresshl to aquatic life. We recommend that the State of North Carolina take legal action to reduce nitrogen and phosphorus inputs to Coastal Plain blackwater systems. Strategies to achieve this include placing limits on nitrogen and phosphorus discharges for NPDES permit holders. Strategies for reduction of non-point source nutrient loading should include a basinwide 50 R vegetated buffer along streams in the Cape Fear watershed, no fkrther loss of wetlands and increased utilization of constructed wetlands for stormwater runoff treatment, continued efforts by the state to remove CAFOs and sprayfields from river floodplains, and continued efforts to manage fertilizer applications and erosion

38

REFERENCES CITED

American Public Health Association . 1995. StandardMethods for the Examination of Wuter and Wastewater. 19th ed., Washington, D.C.

Bales, J.D., C.J. Oblinger and A.H. Sallenger, Jr. 2000. Two months of flooding in eastern North Carolina, September-October 1999, Water Resources Investigations Report 00-4093, U. S. Geological Survey, Raleigh, N.C.

Barker, J.C. and J.P. Zublena. 1995. Livestock manure nutrient assessment in North Carolina. Proc. of the 7th International Symposium on Agriculture and Food Processing Wastes, American Society of Agricultural Engineers, St. Joseph, Michigan.

Beaver, J.R. and T.L. Crisman. 1989. The role of ciliated protozoa in pelagic freshwater systems Microb. Ecol. 17:lll-136.

Burkholder, J.M., M.A. Mallin, H.B. Glasgow, Jr., L.M. Larsen, M.R. McIver, G.C. Shank, N. Deamer-Melia, D.S. Briley, J. Springer, B.W. Touchettte and E. K. Hannon. 1997. Impacts to a coastal river and estuary from rupture of a swine waste holding lagoon J. Environ. Qual. 26: 145 1- 1466.

Cahoon, L.B., J. Mickucki, and M.A. Mallin. 1999. Nutrient imports to the Cape Fear and Neuse River basins to support animal production. Environ. Sci. Tech.

33 14 10-4 15.

Chrzanowski, T.H., R.W. Sterner, and J.J. Elser. 1995. Nutrient enrichment and nutrient regeneration stimulate bacterioplankton growth. Microb. Ecol. 29:22 1-230.

Cotner, J.B., R.H. Sada, H. Bootsma, T. Johengen, J.F. Cavaletto and W.S. Gardner. 2000. Nutrient limitation of bacteria in Florida Bay. Estuaries 23 :611-620.

Currie, D. J. 1990. Large-scale variability and interactions among phytoplankton, bacterioplankton, and phosphorus. Limnol. Oceunogr. 35: 1437-1455.

Day, R.W. and G.P. Quinn. 1989. Comparisons of treatments after an analysis of variance in ecology. Ecol. Monog. 59:433-463.

Ensip, S.E. and M.A. Mallin. 2001. Stream water quality following timber harvest in a Coastal Plan swamp forest. Wat. Res. 35:3381-3390.

Francisco, D.E., R.A. Mah and A.C. Rabin. 1973. Acridine orange epi-fluorescent technique for counting bacteria in natural waters. Trans. Am. Microsc. SOC. 92:416-42 1.

Gilliam, J.W., R.L. Huffman, R.B. Daniels, D.E. Buffington, A.E. Morey and S.A. Leclerc. 1996. Contarnination of surficial aquifers with nitrogen applied to agricultural land. Report No. 306. Water Resources Research Institute of The University of North Carolina, Raleigh, North Carolina.

39

Mallin, M.A., L.B. Cahoon, M.R. McIver, D.C. Parsons, and G.C. Shank. 1999b. Alternation of factors limiting phytoplankton production in the Cape Fear Estuary. Estuaries 22:985-996.

Mallin, M.A., M.H. Posey, G.C. Shank, M.R. McIver, S.H. Ensign, and T.D. Alphin. 1 9 9 9 ~ Hurricane effects on water quality and benthos in the Cape Fear watershed: Natural and anthropogenic impacts. Ecol. Appl. 9:350-362.

Mallin, M.A., J.M. Burkholder, L.B. Cahoon, and M.H. Posey. 2000. The North and South Carolina coasts. Mar. Poll. Bull. 41:56-75.

Mallin, M.A., L.B. Cahoon, D.C. Parsons, and S.H. Ensign. 2001. Effect of nitrogen and phosphorus loading on plankton in Coastal Plain blackwater rivers. J. Freshwater Ecol. 161455-466.

Glasgow, H.B. and J.M. Burkholder. 2000. Water quality trends and management implications from a five-year study of a eutrophic estuary. Ecol. Appl. 10: 1024-1 046.

Hecky, R.E. and P. Kilham. 1988. Nutrient limitation of phytoplankton in freshwater and marine environments: A review of recent evidence on the effects of enrichment. Limnol. Oceanogr. 33 :796-822.

Hobbie, J.E., R.J. Daley, and S . Jasper. 1977. Use of nucleopore fdters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33 : 1225-1228.

Huffman, R.L. and P.W. Westerman. 1995. Estimated seepage losses from established swine waste lagoons in the lower coastal plain of North Carolina. Trans. ASAE 38:449-453.

Kirchman, D.L. 1994. The uptake of inorganic nutrients by heterotrophic bacteria. Microb. Ecol. 28 :25 5-27 1.

Mallin, M.A. 2000. Impacts of industrial-scale swine and poultry production on rivers and

Mallin, M.A., and H.W. Paerl. 1992. Effects of variable irradiance on phytoplankton productivity

estuaries. Am. Sci. 88:26-37.

in shallow estuaries. Limnol. Oceanogr. 37:54-62.

Mallin, M.A., J.M. Burkholder, G.C. Shank, M.R. McIver, H.B. Glasgow, Jr., B. Touchette, and J. Springer. 1997. Comparative effects of poultry and swine waste lagoon spills on the quality of receiving streamwaters. J. Environ. Qual. 26: 1622-163 1.

Mallin, M.A., L.B. Cahoon, D.C. Parsons, and S.H. Ensign. 1998. Effect of organic and inorganic nutrient loading on photosynthetic and heterotrophic plankton communities in blackwater rivers. Report No. 3 15. Water Resources Research Institute of The University of North Carolina, Raleigh, North Carolina.

Mallin, M.A., M.H. Posey, M.L. Moser, L.A. Leonard, T.D. Alphin, S.H. Ensign, M.R. McIver, G.C. Shank, and J.F. Merritt. 1999a. Environmental Assessment of the Lower Cape Fear fiver System, 1998-1999. CMSR Report No. 99-01. Center for Marine Science Research, University of North Carolina at Wilmington, Wilmington, North Carolina.

40

Meyer, J.L. 1990. A blackwater perspective on riverine ecosystems. BioScience 40:643-65 1

Meyer, J.L. 1992. Seasonal patterns of water quality in blackwater rivers of the coastal plain, southeastern United States. In Water Quality in North American River Systems. Becker, C.D. and D.A. Neitzel, eds. Batelle Press, Columbus.

Morris, D.P. and W.M. Lewis, Jr. 1992. Nutrient limitation of bacterioplankton growth in Lake Dillon, Colorado. Limnol. Oceanogr. 3 7: 1 179- 1 192.

Natural Resource Conservation Service. 1995. Eastern North Carolina Cooperative River Basin Study. Natural Resource Conservation Service, USDA, Raleigh, North Carolina.

North Carolina Department of Environment, Health and Natural Resources 1994. Water Quality Progress in North Carolina 1992- 1993. 305(b) Report. Report No. 94-07. North Carolina Department of Environment, Health and Natural Resources, Division of Environmental Management, Raleigh, North Carolina.

North Carolina Department of Environment and Natural Resources. 2000. Cape Fear River Basinwide Water Quality Management Plan., North Carolina Department of Environment and Natural Resources, Division of Water Quality, Raleigh, North Carolina.

Pace, M.L. 1982. Planktonic ciliates: their distribution, abundance, and relationship to microbial resources in a monomictic lake. Can. J Fish. Aquat. Sci. 39: 1 106-1 116.

Padgett, D.E., M.A. Mallin and L.B. Cahoon. 2000. Evaluating the use of ergosterol as a bioindicator for assessing water quality. Emiron. Monitor. Assess. 65 :547-558.

Paerl, H. W. 1982. Factors limiting productivity of freshwater ecosystems. Adv. Microb. Ecol. 6175-1 10.

Paerl, H.W., J. Rudek and M.A. Mallin. 1990. Stimulation of phytoplankton production in coastal waters by natural rainfall inputs: nutritional and trophic implications. Mar. Biol. 107:247-254.

Rudek, J., H.W. Paerl, M.A. Mallin and P.W. Bates. 1991. Seasonal and hydrological control of phytoplankton nutrient limitation in the lower Neuse River Estuary, North Carolina. Mar. Ecol. Prog. Ser. 75:133-142.

Sanders, R. W. 199 1. Trophic strategies among heterotrophic flagellates. In The Biology of Free- Living Heterotrophic Flagellates. D.J. Patterson and J. Larsen, eds. Systematics Association Special Volume, No. 45, pp 21-38. Oxford: Clarenden Press.

Schlotzhauer, S.D. and R.C. Littell. 1987. SAS system for elementary statistical analysis. SAS Institute, Inc., Cary, North Carolina.

41

Smock, L.A. and E. Gilinsky. 1992. Coastal Plain blackwater streams. In Biodiversity of the Southeastern UnitedStates. Hackney, C.T., S.M. Adams and W.H. Martin, eds. New York: John Wiley and Sons, Inc.

Stone, K.C., P.G. Hunt, S.W. Coffey, and T.A. Matheny. 1995. Water quality status of a USDA water quality demonstration project in the eastern coastal plain. J. Soil Wat. Cons. 40:567- 571.

Vadstein, O., A. Jensen, Y. Olsen, and H. Reinertsen. 1988. Growth and phosphorus status of limnetic phytoplankton and bacteria. Limnol. Oceanogr. 33:489-503.

Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and phaeopigments. Limnol. Oceanogr. 39: 1985-1 993.

Westerman, P.W., M.R. Overcash, R.O. Evans, L.D. King, J.C. Burns, and G.A. Cummings. 1985. Swine lagoon effluent applied to coastal bermudagrass:III. Irrigation and rainfall runoff J. Environ. Qual. 14:22-25.

Westerman, P.W., L.M. Safely, Jr., and J.C. Barker. 1990. Lagoon liquid nutrient variation over four years for lagoons with recycle systems. In Agricultural and foodprocessing wastes, Proc. of the Sixth International Symposium on Agricultural and Food Processing Wastes, Chicago, Illinois. ASAE Publication 05-90. American Society of Agricultural Engineers, St. Joseph, Michigan.

42

LIST OF ACRONYMS

ANOVA - Analysis of variance

ATP - Adenosine triphosphate

BOD - Biochemical oxygen demand

CAFO - Concentrated animal feeding operation

LCFRP - Lower Cape Fear River Program

LSD - Least significant difference

mFC - Membrane filtration method

MGD - Million gallons per day

NCDWQ - North Carolina Division of Water Quality

NPDES - National Pollution Discharge Elimination System

SAS - Statistical Analysis System

TKN - Total Kjeldahl nitrogen

TN - Total nitrogen

TP - Total phosphorus

43

Date post:	10-Aug-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Seeking Science-Based Nutrient Standards For Coastal ... · Blackwater streams are the most common...

Documents