Prepared in cooperation with the National Park Service

Ambient and Potential Denitrification Rates in 

Marsh Soils of Northeast Creek and Bass Harbor 

Marsh Watersheds, Mount Desert Island, Maine

Scientific Investigations Report 2012–5166

U.S. Department of the Interior

U.S. Geological Survey
Cover. Extensive nuisance algal bloom in the Bass Harbor Marsh estuary on June 9, 2008. (Photograph by Thomas G. Huntington)
Ambient and Potential Denitrification Rates 

in Marsh Soils of Northeast Creek and 

Bass Harbor Marsh Watersheds, 

Mount Desert Island, Maine

By Thomas G. Huntington, Charles W. Culbertson, and John H. Duff

Prepared in cooperation with the National Park Service

Scientific Investigations Report 2012–5166

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

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Suggested citation:

Huntington, T.G., Culbertson, C.W., and Duff, J.H., 2012, Ambient and potential denitrification rates in marsh soils of 

Northeast Creek and Bass Harbor Marsh watersheds, Mount Desert Island, Maine: U.S. Geological Survey Scientific 

Investigations Report 2012–5166, 40 p., at http://pubs.usgs.gov/sir/2012/5166/.
iii

Contents

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

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

Description of Study Area ...................................................................................................................3

Purpose and Scope ..............................................................................................................................9

Field Denitrification Methods.......................................................................................................................9

Laboratory Denitrification Methods .........................................................................................................11

Laboratory N2O Analytical Methods and Computation of N2O Release Rate ....................................12

Field Methods for Water Quality Sampling and Measurements..........................................................12

Laboratory Methods for Water Quality Analysis ....................................................................................14

Ancillary Data...............................................................................................................................................14

Laboratory Denitrification Experiments to Determine N2 to N2O Ratios.............................................15

Laboratory Denitrification Experiments to Determine Response to Nitrate and 

Glucose Additions ..........................................................................................................................15

Field Measurements of Denitrification Under Ambient Conditions.....................................................19

Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments ........23

Field Denitrification Rate in Relation to Environmental Variables .......................................................35

Tributary Water Quality...............................................................................................................................36

Denitrification Potential of Marsh Soils at Acadia National Park .......................................................36

Conclusions...................................................................................................................................................37

Acknowledgments.......................................................................................................................................38

References Cited..........................................................................................................................................38

Figures

1. Shaded relief map showing the locations of the study areas, including the 

Northeast Creek (NEC) and Bass Harbor Marsh (BHM) watersheds on Mount 

Desert Island, Maine....................................................................................................................4

2. Map showing Northeast Creek watershed area, Mount Desert Island, Maine, and 

surface-water sampling locations on the major tributaries and the location of a 

groundwater monitoring well .....................................................................................................5

3. Aerial map showing the locations of denitrification measurement sites and 

groundwater sampling wells in Northeast Creek watershed, Mount Desert 

Island, Maine.................................................................................................................................6

4. Map showing the location of Bass Harbor Marsh watershed, Mount Desert 

Island, Maine, and surface-water sampling locations on the major tributaries................7

5. Aerial map showing the locations of denitrification measurement sites and 

groundwater sampling wells in Bass Harbor Marsh watershed, Mount Desert 

Island, Maine.................................................................................................................................8

6. Bar graphs showing production rates of nitrous oxide in the laboratory determined 

after the indicated incubation intervals with A, no glucose added and B, glucose 

added at 3.6 grams per square meter of fresh soil ...............................................................17

7. Boxplots showing laboratory nitrous oxide production rates following nitrate, 

glucose, and acetylene addition treatment additions after the indicated incubation 

intervals for A, Northeast Creek watershed sites GW2, GW3, and GW5 and B, Bass 

Harbor Marsh watershed sites GW1, GS2, and GW3...........................................................18
iv

8. Boxplots showing A, average of the field denitrification rates for sites GW4 and 

GW2, July 2008 in the Northeast Creek watershed. B, Average of the field denitrifica-

tion rates for sites GW1–GW6, July and August 2009 in the Bass Harbor Marsh 

watershed ....................................................................................................................................22

9. Graphs showing A, water levels in the Fresh Meadow Marsh, Northeast Creek 

watershed, Mount Desert Island, Maine, and discharge at Otter Creek U.S. 

Geological Survey (USGS) streamgage 01022840; and B, daily precipitation at the 

McFarland Hill weather station, near Bar Harbor, Mount Desert Island, Maine.............23

10. Graph showing average rate of denitrification over the measurement period from 

3 hours 5 minutes to 20 hours 32 minutes after sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed site GW2 in June 2009 ....................................24

11. Graph showing average rate of denitrification over measurement period from 

4 hours 33 minutes to 22 hours 32 minutes after sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed site GW3 in June 2009 ....................................24

12. Graph showing average rate of denitrification over measurement period from 

4 hours 22 minutes to 22 hours 01 minutes after sealing chamber to marsh sediment 

surface on Bass Harbor Marsh watershed site GW4 in July 2009.....................................25

13. Graph showing average rate of denitrification over measurement period from 

5 hours 8 minutes to 22 hours 43 minutes after sealing chamber to marsh sediment 

surface on Bass Harbor Marsh watershed site GW6 in July 2009.....................................25

14. Graph showing average rate of denitrification over measurement period from 

4 hours 27 minutes to 19 hours 32 minutes after sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed site GW5 in August 2009 ................................25

15. Graph showing average rate of denitrification over measurement period from 

2 hours 51 minutes to 26 hours 16 minutes after sealing chamber to marsh sediment 

surface at Northeast Creek watershed site GW4 in September 2009 ...............................26

16. Graphs showing relation between field denitrification rate and headspace nitrous 

oxide concentration and the varying incubation intervals once the chamber was 

sealed to the marsh surface at the Northeast Creek watershed site GW4 in September 

2009, immediately following the following treatment additions: A, 1.1 grams of nitrogen 

per square meter only; B, 1.1 grams of nitrogen per square meter plus 3.6 grams of 

glucose per square meter; C, 5.5 grams of nitrogen per square meter only; D, 5.5 grams 

of nitrogen per square meter plus 3.6 grams of glucose per square meter; E, 11 grams 

of nitrogen per square meter; F, 11 grams of nitrogen per square meter plus 3.6 grams 

of glucose per square meter; G, 22 grams of nitrogen per square meter; H, 22 grams 

of nitrogen per square meter plus 3.6 grams of glucose per square meter; I, 55 grams 

of nitrogen per square meter; and J, 55 grams of nitrogen per square meter plus 

3.6 grams of glucose per square meter ..................................................................................27

17. Graphs showing relation between field denitrification rate and headspace nitrous 

oxide concentration and the varying incubation intervals once the chamber was 

sealed to the marsh surface at the Bass Harbor Marsh watershed site GW4 in 

September 2009, immediately following the following treatment additions: A, 1.1 grams 

of nitrogen per square meter only; B, 1.1 grams of nitrogen per square meter plus 

3.6 grams of glucose per square meter; C, 5.5 grams of nitrogen per square meter 

only; D, 5.5 grams of nitrogen per square meter plus 3.6 grams of glucose per square 

meter; E, 11 grams of nitrogen per square meter; F, 11 grams of nitrogen per square 

meter plus 3.6 grams of glucose per square meter; G, 22 grams of nitrogen per square 

meter; H, 22 grams of nitrogen per square meter plus 3.6 grams of glucose per square 

meter; I, 55 grams of nitrogen per square meter; J, 55 grams of nitrogen per square 

meter plus 3.6 grams of glucose per square meter; K, 110 grams of nitrogen per square 

meter; and L, 110 grams of nitrogen per square meter plus 3.6 grams of glucose per 

square meter ...............................................................................................................................31
v

Tables

1. Dates of field (in situ) denitrification rate measurements, locations of measurements, 

and treatments applied on measurement dates....................................................................10

2. Surface water and groundwater water-quality monitoring stations in Northeast 

Creek and Bass Harbor Marsh watersheds sampled in 2008 and 2009 ............................13

3. Constituent name, U.S. Geological Survey National Water Information System 

parameter codes and method codes, and minimum reporting limits for temperature, 

specific conductance, dissolved oxygen, and nutrients......................................................14

4. Ratios of N2 to N2O denitrification products measured in laboratory incubation 

experiments using acetylene to block production of N2 ......................................................16

5. Denitrification rates for each month, year, and site under ambient (no treatment 

additions) and following treatments where nitrate was added alone or in combination 

with glucose ................................................................................................................................20

6. Denitrification rates in paired measurements under ambient or glucose only 

addition.........................................................................................................................................35

7. Pearson correlation coefficients between denitrification rate and the noted 

variables under experimental conditions where low levels of nitrate were added and 

no glucose was added...............................................................................................................36

8. Mean and standard errors of nutrient concentrations in tributaries to Northeast 

Creek and Bass Harbor Marsh.................................................................................................37

Conversion Factors, Datum, and Abbreviations

Inch/Pound to SI

Multiply By To obtain

Length

foot (ft) 0.3048 meter (m)

inch (in.) 3.937×10-5 micrometer (μ)

inch (in.) 25.4 millimeter (mm)

inch (in.) 2.54 centimeter (cm)

mile (mi) 1.609 kilometer (km)

Area

acre 4,047 square meter (m2)

square mile (mi2) 2.590 square kilometer (km2)

square foot (ft2) 0.09290 square meter (m2)

Volume

gallon (gal) 3.785 liter (L)

ounce, fluid (fl. oz) 29.57 milliliter (mL)

cubic inch (in3) 16.39 cubic centimeter (cm3)

Mass

ounce, avoirdupois (oz) 28.35 gram (g)

ounce, avoirdupois (oz) 0.02835 milligram (mg)

Application rate

ounce, avoirdupois per square yard 

(oz/yd2)

0.02949 grams per square meter 

(g/m2)
vi

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

 °F=(1.8×°C)+32

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

 °C=(°F–32)/1.8

Vertical coordinate information is referenced to the North American Vertical Datum of 1988 

(NAVD 88).

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

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

25°C).

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

or micrograms per liter (μg/L).

Abbreviations

BMH Bass Harbor Marsh

NADP National Atmospheric Deposition Program

NEC Northeast Creek Estuary

NPS National Park Service

USGS U.S. Geological Survey
Ambient and Potential Denitrification Rates in Marsh Soils 

of Northeast Creek and Bass Harbor Marsh Watersheds, 

Mount Desert Island, Maine

By Thomas G. Huntington, Charles W. Culbertson, and John H. Duff

Abstract

Nutrient enrichment from atmospheric deposition, 

agricultural activities, wildlife, and domestic sources is a 

concern at Acadia National Park on Mount Desert Island, 

Maine, because of the potential problems of degradation of 

water quality and eutrophication in estuaries. Degradation of 

water quality has been observed at Bass Harbor Marsh estuary 

in the park but only minimally in Northeast Creek estuary. 

Previous studies at Acadia National Park have estimated 

nutrient inputs to estuaries from atmospheric deposition and 

surface-water runoff, and have identified shallow groundwater 

as an additional potential source of nutrients. Previous 

studies at Acadia National Park have assumed that a certain 

fraction of the nitrogen input was removed through microbial 

denitrification, but rates of denitrification (natural or maximum 

potential) in marsh soils have not been determined. The U.S. 

Geological Survey, in cooperation with Acadia National Park, 

measured in-place (in-situ) denitrification rates in marsh soils 

in Northeast Creek and in Bass Harbor Marsh watersheds 

during summer 2008 and summer 2009. Denitrification was 

measured under ambient conditions as well as after additions 

of inorganic nitrogen and glucose.

In-place denitrification rates under ambient conditions 

were similar to those reported for other coastal wetlands, 

although they were generally lower than those reported for salt 

marshes having high ambient concentrations of nitrate (NO3

). 

Denitrification rates generally increased by at least an order of 

magnitude following NO3

 additions, with or without glucose 

(as the carbohydrate) additions, compared with the ambient 

treatments that received no nutrient additions. The treatment 

that added both glucose and NO3 resulted in a variety of 

denitrification responses when compared with the addition of 

NO3 alone. In most cases, the addition of glucose to a given 

rate of NO3 addition resulted in higher rates of denitrification. 

These variable responses indicate that the amount of labile 

carbohydrates can limit denitrification even if NO3 is 

present. For most sites in both watersheds, the maximum 

denitrification rates ranged from 150 to 900 micromoles of 

nitrous oxide per square meter per hour. These rates were 

equivalent to the release of 37 to 221 grams of nitrogen 

per square meter per year. Weak positive correlations were 

observed between denitrification rate and soil temperature and 

measured ammonium concentration in groundwater. Weak 

negative correlations were observed between denitrification 

rate and water level and specific conductance.

The rates of denitrification in Bass Harbor Marsh and 

Northeast Creek under ambient conditions, both of which were 

relatively low, indicate that NO3 availability is low in both 

systems. It is evident from the addition of combined treatments of NO3

 and glucose that these marsh soils are capable of 

comparatively high rates of denitrification, therefore, estuarine eutrophication is not a result of nitrogen inputs to marsh 

soils that are in excess of the denitrification capacity in these 

systems. If terrestrial inputs to the estuary are the cause of 

the observed eutrophic condition in Bass Harbor Marsh, then 

these inputs to the estuary must bypass the marsh in channelized surface flow, or perhaps they circumvent the marsh in 

shallow groundwater seepage along subsurface pathways that 

enter the estuary directly.

Introduction

Acadia National Park was created to protect the natural 

beauty of the only rocky headlands along the Atlantic coast of 

the United States. Fundamental to this purpose is protection of 

the ecological, scientific, and scenic attributes of the Acadia 

coastal ecosystems. More than 10 percent of the park’s land 

area is classified as wetland (Calhoun and others, 1994), 

including forested and scrub/shrub wetlands, freshwater 

emergent marshes, peatlands, and salt marshes (Roman and 

others, 2001). The estuaries at the park provide unique habitats 

for fish and wildlife, nursery grounds for coastal fish species, 

and foraging habitat for wetland and aquatic birds. Estuaries 

also act as filters for water that ultimately enters the nearshore 

zone, thus helping to maintain the quality of coastal water 

(Herbert, 1999). Coastal salt marshes and wetlands are among 

the most productive ecosystems in North America, the tidally 

exported productivity of their wetland plant communities 
2 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

being capable of profoundly effecting estuarine productivity. 

Additionally, coastal wetlands are among the most threatened 

ecosystems in North America. Changes in the physiochemical 

conditions of sediment that are related to nutrient enrichment 

or perturbations because of human activity can alter the pattern 

of plant zonation, enabling highly competitive invasive plant 

species to potentially displace native plant species (Burke 

and others, 2002). Shoreline development and agricultural 

runoff also threaten the sustainability of salt marsh ecosystems 

throughout the northeastern United States (Silliman and 

Bertness, 2004; Fitch and others, 2009).

The Water Resources Management Plan for the Acadia 

National Park (Kahl and others, 2000) identifies accelerated 

rates of freshwater and of coastal marine eutrophication as a 

priority issue for water quality and as one of the most important challenges for resource management in the Park. Residential development outside the park boundary is increasing at an 

unprecedented rate. For example, between 1981 and 2001 the 

number of residences in the combined drainage basins of Aunt 

Betseys Creek, French Hill Brook, Old Mill Brook, and Stony 

Brook in the Northeast Creek (NEC) watershed increased 

from 83 to 279 (Nielsen, 2002a,b). Recent studies show that 

Bass Harbor Marsh (BHM) estuary is already tending toward a 

eutrophic state (Doering and others, 1995; Kinney and Roman, 

1998). Evidence from many other Atlantic coast estuarine systems indicates that land clearing and subsequent installation of 

septic systems and application of lawn fertilizers are likely to 

increase nutrient loads of both groundwater and surface water 

entering the estuary (Harvey and Odum, 1990; Valiela and others, 1990, 2000; Howes and others, 1996; Bricker and others, 

1999; National Research Council, 2000; Cloern, 2001; Tobias 

and others, 2001; Valiela and Bowen, 2002).

The chief nutrient of concern for eutrophication in the 

Acadia estuaries is nitrogen. Nitrogen inputs to the NEC 

and BHM watersheds and estuaries derive from atmospheric 

deposition, septic effluent, tidal inputs, agricultural runoff, 

and wildlife. Net mineralization of soil organic nitrogen also 

can contribute dissolved inorganic nitrogen species. Previous 

studies have quantified inputs from atmospheric deposition 

and from surface water for NEC (Nielsen, 2002a) and BHM 

(Doering and others, 1995), but inputs from groundwater and 

tidal sources have not been determined.

Previous studies at Acadia National Park have assumed 

that microbial denitrification removed a certain fraction of the 

nitrogen input to the watershed, but rates of denitrification 

(natural or maximum potential) in marsh soils have not been 

determined. Denitrification is the reduction of the aqueous 

nitrogen (N) oxides, nitrate (NO3

-

) and nitrite (NO2

-

), to the 

gases nitric oxide (NO), nitrous oxide (N2O), and dinitrogen 

(N2) (Knowles, 1982). The generalized denitrification reactions are shown in the equations below.

Oxic reactions: 

Organic N NH4 NO NO +

(aq) 2

-

(aq) 3

-

(aq) → → 1 2 →3

(1)

Anoxic reactions:

NO3 NO N O N -

(aq) (g) 2 (g) 2 (g) → → 4 5 →6

(2)

In this reaction pathway, in the oxic zone of marsh soils, 

organically bond nitrogen (organic N) is shown being converted into inorganic aqueous ammonium (NH4

+

; reaction 1) 

that is heterotrophic decomposition of organic matter termed 

ammonification. In reaction 2 (NH4

+

) is converted to NO2

-

(aq)

and in reaction 3 NO2

-

(aq), is converted to NO3

-

(aq) these two 

reactions are collectively termed nitrification. When NO3

-

(aq)

enters the anoxic zone in marsh soils the sequence of denitrification reactions (reactions 4, 5, and 6) occur to produce NO(g), 

N2O(g), and N2(g).

In the course of these microbially mediated reactions 

both N2O and N2 may be released to the atmosphere, although 

N2 is typically the primary product. In wetland soils, such 

as the marshes at Acadia National Park, the amount of NO 

produced is usually very low compared to the amount of N2

and N2O because virtually all NO is converted to N2O before 

it can escape from soils (Davidson and Kingerlee, 1997). The 

final reaction in the denitrification reaction sequence (reaction 6) is the conversion of N2

O to N2, this reaction is also 

termed consumption of N2O. Under certain conditions, and 

during a defined time period, where the consumption of N2O is 

greater than the production of N2O, then net consumption will 

be observed. Under natural conditions some variable fraction 

of the N2O that is produced is released to the atmosphere as 

a gas and the remainder is further reduced to dinitrogen gas 

(N2) which is also released to the atmosphere (Knowles, 1982; 

Groffman and others, 2006). Acetylene blocks reaction 6, the 

conversion of N2O to N2 with the result that virtually all of 

the product of denitrification will be N2O (Tiedje and others, 

1989; Groffman and others, 2006). Most denitrifying bacteria 

in soils are heterotrophs that oxidize organic matter and use 

inorganic nitrogen oxides as electron acceptors.

Denitrification occurs most readily when oxygen 

is depleted, that is, in suboxic conditions of less than 

0.2 milligrams of oxygen per liter (<0.2 mg O2 L-1) that 

usually exist in a relatively thin stratum at the oxic/anoxic 

interface in wetland soils (Seitzinger and others, 2006). A 

variable water table and the presence of marsh vegetation, 

the roots of which can channel oxygen to deeper soil strata, 

can increase the availability of nitrate and suitable organic 

substrates for denitrifying bacteria. In the absence of oxygen, 

the tendency for increasingly negative reduction-oxidation 

potential favors other electron acceptors and inhibits the 

mineralization of organic nitrogen to NO3, thus restricting the 

zone of denitrification to the oxic/anoxic interface. In the NEC 

and BHM watersheds, we assume that most denitrification 

occurs in the marsh soils and in estuarine sediments, as 

Seitzinger and Nixon (1985), Seitzinger (1988), and Day and 

others (1989) have observed in other coastal wetlands.

Denitrification rates increase with increasing nitrogen 

loading (Seitzinger and others, 1984; Seitzinger and Kroeze, 

1998; Wigand and others, 2004), and such rates have been 
Introduction 3

shown to be seasonally and diurnally dependent, governed 

principally by temperature, supply of NO3, and availability of 

dissolved organic carbon (Smith and others, 1985; Jorgensen 

and Sorenson, 1988; Koch and others, 1992; Lee and others, 

1997; Tobias and others, 2001). Other environmental factors—

such as the organic carbon content in sediment (Puckett 

and others, 2002), salinity, dissolved oxygen concentration 

(Koch and others, 1992), and hydrogen sulfide concentration 

(Sorenson and others, 1980)—also affect denitrification 

potential. Denitrification rates also are related to tidal 

flooding regime (Koch and others, 1992), pH, and sediment 

reduction-oxidation (redox) potential (Patrick and Delaune, 

1977; Knowles, 1982; Smith and others, 1983). Because 

many factors influence denitrification rates and because 

reported rates are highly variable (for example, Greene, 2005; 

Seitzinger and others, 2006), it is important to determine 

ambient rates and maximum potential rates in areas of interest.

Description of Study Area

Northeast Creek (NEC) is a small microtidal estuary in 

the northeastern part of Mount Desert Island, Maine, that is 

experiencing increasing residential development in the watershed (fig. 1). The NEC watershed, 26.02 square kilometers 

(km2) in areal extent, comprises several subwatersheds (fig. 2). 

A partial flow restriction near the estuary outlet results in tidal 

inundation of most marsh sediments only during high spring 

tides. Recent studies indicate that deep groundwater in the 

underlying fractured bedrock is not substantial in the freshwater budget for NEC (Nielsen, 2002b). Shallow groundwater 

in the NEC watershed is known to contain elevated levels of 

inorganic nitrogen, but the pathways by which this groundwater reaches the estuary are not known. We have assumed 

that some fraction of this shallow groundwater interacts with 

marsh soils before reaching the estuary and that some of the 

entrained inorganic nitrogen is denitrified in these soils. Shallow groundwater discharge is a potential mechanism for eutrophication, because it can deliver dissolved nitrogen species 

from domestic septic systems and from farming or other uses 

of fertilizer to estuaries (Valiela and others, 1990; Reay and 

others, 1992; Portnoy and others, 1998; Valiela and Bowen, 

2002). Shallow groundwater can enter the marsh, and eventually the estuary, through seepage zones at the upland/wetland boundary, or groundwater can enter the estuary directly 

through the bottom and banks of creeks (Howes and others, 

1996; Portnoy and others, 1998). Culbertson and others (2007) 

identified groundwater seeps in NEC and BHM by using aerial 

thermal imagery and continuous and discrete measurements 

of temperature and specific conductance in selected seeps, but 

the relative contribution of seeps to total inputs is unknown. 

Information is also lacking on current rates of denitrification, 

and, most importantly on whether these soils have the capacity to denitrify increasing rates of nitrogen addition that would 

very likely accompany increasing residential development.

Denitrification measurement sites in the NEC watershed 

were located in the marsh between the base of the hillslope 

and the estuary (fig. 3). Based on the classifications in a recent 

vegetation map (Lubinski and others, 2003) graminoid mix 

(Calamagrostis canadensis-Scirpus spp.–Dulichium arundinaceum) dominated the vegetation at sites GW1, GW3, and 

GW5; cranberry, sweet gale, leatherleaf, sphagnum shrub fen 

transitioning to willow scrub/shrub fen dominated the vegetation at site GW2; cattail marsh dominated the vegetation at site 

GW4; and sweet gale graminoid fen dominated the vegetation 

at site GW6. The soils were peat-dominated histosols, and the 

depth of the peat varied from about 1 meter (m) near the base 

of the hillslopes to greater than 2 m near the estuary. During 

most of the field denitrification measurements the water table 

was at the land surface or less than 5 to 10 centimeters (cm) 

below land surface. Although these sites did not flood tidally, they were flooded with freshwater for most of the period 

during June through November 2009 due to unusually high 

rainfall events and very slow drainage. The specific conductance in the shallow groundwater typically ranged from 400 to 

3,000 microsiemens (μS) (Huntington and others, 2011).

The BHM watershed, draining 21.78 km2, in the southwestern part of Mount Desert Island, comprises several 

subwatersheds (fig. 4), the largest of which, Marshall Brook, 

lies outside park boundaries and is substantially affected 

by residential and commercial development and a former 

landfill. Signs of eutrophication in BHM estuary, the largest 

tidal marsh estuary on the island, were documented more than 

20 years ago (Doering and others 1995). Evidence of a continuing eutrophication in BHM in response to input of internal 

and external sources of nitrogen is noted in more recent 

studies (Kinney and Roman, 1998; Farris and Oviatt, 1999). 

A 1999 to 2000 study (Nielsen and others, 2002) corroborated the findings of Doering and others (1995) that dissolved 

inorganic nitrogen from freshwater sources comprises a substantial part of the total nitrogen load to BHM from Marshall 

and Heath Brooks; organic nitrogen comprised most of the 

total nitrogen from other tributaries feeding the BHM estuary 

(Doering and others, 1995).

Denitrification measurement sites in the BHM watershed 

were located in the marsh between the base of the hillslope 

and the estuary (fig. 5). Based on the classifications in a recent 

vegetation map (Lubinski and others, 2003) the vegetation 

at these sites was dominated by saltmeadow cordgrass and 

mixed gramioid forb saltmarsh. The vegetation adjacent to 

the estuary (where sites GW4 and GW6 were located) was 

much more dense compared with the sparse vegetation at the 

other sites. The soils were peat-dominated histosols, and the 

peat was of variable depth from about 1 m near the base of the 

hillslopes to greater than 2 m near the estuary. During most of 

the field denitrification measurements, the water table was at 

the land surface or less than 5 cm below land surface. These 

sites flooded tidally during high tide during most of the lunar 

tidal cycle. Our field measurements were conducted during the 

neap tides when the marsh surface did not flood. The specific 

conductance in the shallow groundwater typically ranged from 

10,000 to 30,000 μS (Huntington and others, 2011).
4 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Northeast Creek

t

Figure 2

0 2 4

0 1 2 3 4 5 MILES

1 3 5 KILOMETERS

MAINE

Mount Desert Island

Bar

Harbor

Bass

Figure 4 Harbor

Atlantic Ocean

Atlantic Ocean

Lake or pond

Stream 

Road

Somes

Sound

Eagle

Lake

Bass Harbor

Marsh

Base from U.S. Geological Survey digital line graph, 1:100,000 Modified from Culbertson and others, 2007

Bar Harbor, 1985

Study area shown in figures 2 and 3

EXPLANATION

68°25'

44°25'

68°10'

44°15'

Figure 1. The locations of the study areas, including the Northeast Creek (NEC) and Bass Harbor Marsh (BHM) watersheds 

on Mount Desert Island, Maine.
Introduction 5

Fresh

Meadow

Marsh

Extent of

Figure 3

Old Mill

Brook

Stony

Brook

French 

Hill

Brook

Salsbury 

Cove

Hulls Cove

Hamilton Pond

Somes Sound

Thomas

Bay

Eagle Lake

NORWAY ROAD

CROOKED

ROAD

ROUTE 198/102

ROUTE 233

BAR HARBOR

ROUTE 3

N ortheast Creek

44°20'56"

44°25'14"

68°21'02"

Aunt

Betseys

Brook

EXPLANATION

Surface-water sample site 

Groundwater monitoring well

0

0

1

1

2 MILES

2 KILOMETERS

Northeast Creek watershed boundary

Base from U.S. Geological Survey

Digital line graph Salsbury Cove,

Maine quadrangle 1:24,000

MAINE

Study area

Mount Desert 

Island

68°14'36"

Figure 2. Northeast Creek watershed area, Mount Desert Island, Maine, and surface-water sampling locations on the 

major tributaries and the location of a groundwater monitoring well.
6 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

MAINE

Study area

figure 2

Mount Desert 

Island

GW5

GW6 GW1

GW3

GW4

GW2

Groundwater monitoring

wells and denitrification

measurement sites and

identifier

EXPLANATION

44°24'40"

44°24'50"

44°25'

44°25'10"

68°18'50" 68°18'40"

GW3

0 75 150 300 METERS

0 250 500 1,000 FEET

Base from U.S. Geological Ortho images,

Coordinate System, GCS, North American Datum 1983

Figure 3. The locations of denitrification measurement sites and groundwater sampling wells in 

Northeast Creek watershed, Mount Desert Island, Maine.
Introduction 7

MAINE

Study area

Mount Desert 

Island

EXPLANATION

Surface-water sample site 

Northeast Creek watershed boundary

Extent of

Figure 5

0

0

0.5

0.5

1 MILE

1 KILOMETER

Buttermilk

Brook

Southwest 

Harbor

Bass Harbor

Bass Harbor

Lurvey Brook

Adams

Brook

Bass

Harbor

Marsh

Base from U.S. Geological Survey

Digital line graph Salsbury Cove, Maine

quadrangle 1:24,000

Tremont BASS HARBOR ROAD

HIO FIRE ROAD

ROUTE 102A

ROUTE 102

Southwest

Harbor

FIRE ROAD

Marshall Brook

Heath Brook

SEAL COVE ROAD

44°14'08"

44°16'45"

68°22'44" 68°18'29"

Figure 4. The location of Bass Harbor Marsh watershed, Mount Desert Island, Maine, and surface-water sampling 

locations on the major tributaries.
8 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

MAINE

Study area

figure 3

Mount Desert 

Island

0 50 100 200

Groundwater monitoring

wells and denitrification

measurement sites and

identifier

EXPLANATION

GW3

GW5

GW6

GW4

GW3

GW2

GW1

44°15'15"

44°15'30"

44°15'45"

68°20'45" 68°20'30"

0 50 100 200 METERS

0 287.5 575 1,150 FEET

Base from U.S. Geological Ortho images,

Coordinate System, GCS, North American Datum 1983

Figure 5. The locations of denitrification measurement sites and groundwater sampling wells in 

Bass Harbor Marsh watershed, Mount Desert Island, Maine.
Field Denitrification Methods 9

Purpose and Scope

This report provides estimates of in-place (in situ) rates 

of denitrification in marsh soils under ambient conditions and 

after treatment with NO3 alone, glucose alone, and combinations of NO3

 and glucose in Northeast Creek and Bass Harbor 

Marsh watersheds, Mount Desert Island during the summers 

of 2008 and 2009. The data were collected by the U.S. Geological Survey (USGS), in cooperation with Acadia National 

Park, as part of a study to determine current in-place and 

maximum potential denitrification rates. Laboratory incubation 

experiments also are described that report the ratio of N2 to 

N2O produced during denitrification under ambient conditions 

(no treatment additions) and under treatments in which various 

rates of NO3 were added, with or without glucose, and with or 

without acetylene. The purpose of determining ratios of N2 to 

N2O produced in these laboratory experiments was to apply 

these ratios in field measurements during which no acetylene 

block was used and in which only N2O was measured. Ratios 

from the laboratory experiments also informed the design of 

the field experimental additions of NO3 and glucose additions to assure that appropriate rates of these additions would 

be applied. The more controlled conditions of the laboratory 

experiments, in conjunction with the field experiments assisted 

in the study’s understanding of denitrification responses to 

treatments that included glucose only, acetylene only, and various rates of nitrogen additions with and without glucose.

Ancillary measurements that were made during this study 

are reported to help explain variations in measured denitrification rates. Nutrient data in surface water and groundwater 

and specific conductance, temperature, and dissolved oxygen 

data are included in this report. Surface water chemistry of the 

tributaries draining to these estuaries is discussed in relation to 

previously published measurements for these sites. This report 

evaluates whether the entire record of surface water chemistry 

support detectable temporal trends in various nutrient concentrations. This report also provides data on groundwater level 

that was recorded continuously during the growing season 

in Fresh Meadow Marsh in the NEC watershed. Finally, this 

report also provides data on soil temperature, precipitation, 

and discharge at Otter Creek, on air temperature, and on tides. 

Additional details on methods and quality assurance to ensure 

data quality are reported in Huntington and others (2011).

Field Denitrification Methods

In-place denitrification measurements were made by 

using a static chamber technique in which denitrification 

was estimated from the increase in N2O concentration in the 

headspace of a chamber placed on the surface of the marsh 

sediment (Oremland and others, 1984; Meding and others, 

2001). The flux chambers were constructed from white opaque 

plastic buckets having an internal diameter of 28.4 cm and 

were equipped with battery-operated fans to mix the gas in 

the headspace and with rubber septa for sampling the gas. 

The chambers were pressed into the soil surface to a depth of 

10 cm, thus insuring an effective seal so that the N2O released 

from the soil into the headspace could not escape or be mixed 

with ambient air. The volume of the headspace was recorded 

during each incubation. Gas samples for N2O analysis were 

collected from the chambers through rubber septa using glass 

syringes at various time intervals following installation and 

sealing of the chambers. Gas samples were stored in glass 

vacutainers (vials) sealed with Teflon-lined butyl rubber stoppers. Huntington and others (1998) describe the static chamber 

technique for measuring soil respiration.

Denitrification measurements (based on measured N2O 

release rates) were made in marsh soils in NEC and BHM. 

Table 1 lists the dates and sites of the flux measurements, 

whether the measurements were made under ambient 

conditions, or whether NO3 was added and at what rate or 

whether glucose, or both were added. Nitrate, glucose, and 

acetylene (in a limited number of experiments) additions 

were made after the chambers had been installed into the soil 

to ensure that the additions were confined to the soil beneath 

the chambers. Treatment solutions of NO3 and glucose were 

made from concentrates prepared in the laboratory and diluted 

in the field with shallow groundwater that was pumped from 

piezometers at the denitrification measurement sites. One 

liter (L) of a solution of the indicated concentration of NO3

or NO3 and glucose was added through an opening in the 

top of the chamber by using a funnel. These additions of 0, 

5, 15, 25, 50, 75, 100, 200, 250, 500, and 1,000 millimolar 

(mM) NO3 solutions were equivalent to nitrogen addition 

rates of 0, 1.1, 3.3, 5.5, 11, 17, 22, 44, 55, 110, and 221 

grams of nitrogen per square meter (g N m-2) respectively. 

This report refers to “ambient” treatments as those receiving 

zero NO3 and zero glucose. For treatments in which glucose 

was added, the 1-L treatment solutions contained 75 mM 

glucose, equivalent to 213 g glucose m-2 when the glucose 

was applied to each chamber. After the treatment additions, 

a rubber septum was installed in the top of each chamber to 

ensure a gas-tight seal, and the fans were started in order to 

mix the gases in the chamber headspace. In most cases, two 

or three replicate chambers were used in order to determine 

average denitrification rates at a given location and for a given 

treatment. One exception was treatments in which both NO3

and glucose were added; in these cases, one chamber usually 

received this treatment at a given site.

Preliminary measurements indicated that little to no N2O 

was released under ambient conditions. If denitrification were 

occurring under ambient conditions in the field, it was possible 

that virtually all of the N2O was being converted to N2. To rule 

out this possibility in a limited number of initial field experiments we used acetylene inhibition, which prevents N2

O from 

being converted to N2 so that we would detect denitrification 

if it were occurring under ambient conditions. Acetylene was 

added in the field, following the method described by Tiedje 

and others (1989) by bubbling acetylene gently into the 1-L 

solutions for 2 minutes to saturate the solutions before introducing these saturated solutions into the chambers.
10 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Table 1. Dates of field (in situ) denitrification rate measurements, locations of measurements, and treatments applied on 

measurement dates.

[g N m-2, grams of nitrogen per square meter; NEC, Northeast Creek watershed; GW, groundwater site; FC, flux chamber; BHM, Bass Harbor Marsh watershed. 

An “X” indicates that the treatment was included in the experiment on that date. A “Y” indicates that measurements on this date included the addition of acetylene. A “Z” indicates that measurements on this date included the addition of nitrate plus glucose; --, additive was not included in measurements on this date]

Date Start

month/day Site name(s) Ambient Nitrate1(g N m-2) Acetylene Glucose1(g glucose m-2)

Nitrate plus

glucose

May 2008 5/29 NEC GW1-2 X -- -- -- --

5/30 NEC GW3-4 X -- -- -- --

June 2008 6/10 NEC GW1-3 X -- Y -- --

6/11 NEC GW4,6 X -- -- -- --

6/12 NEC GW5 X -- -- -- --

July 2008 7/15 NEC GW1, GW3 X -- -- -- --

7/16 NEC GW1, GW3 -- 1.1 -- -- --

7/16 NEC GW4 X -- -- -- --

7/17 NEC GW2 FC1-3 X -- -- -- --

7/17 NEC GW2 FC4-6 X 1.1 -- -- --

7/17 NEC GW4 FC4-6 -- 1.1 -- -- --

7/17 NEC GW5 FC1-3 X -- -- -- --

7/17 NEC GW5 FC5-6 -- 1.1 -- -- --

7/18 NEC GW6 FC1-4 X -- -- -- --

7/18 NEC GW6 FC5-6 -- 1.1 -- -- --

Aug. 2008 8/13 NEC GW4, GW2 X 1.1 Y -- --

8/14 NEC GW4 -- 11 Y -- --

Sept. 2008 9/10 BHM GW3 X -- -- -- --

9/11 BHM GW3 -- 5.5,11; 16.5; 22 -- -- --

9/12 BHM GW3, GW2 -- 5.5 Y -- --

9/15 BHM GW1 -- 5.5 Y -- --

9/15 BHM GW4 -- 5.5 Y -- --

9/16 BHM GW5 -- 3.3 -- -- --

Oct. 2008 10/30 BHM GW6 -- 22; 55; 110; 220 Y -- --

June 2009 6/15 BHM GW1-6 X -- -- -- --

6/16 BHM GW3 -- 1.1; 5.5; 11; 22; 55; 110 -- 213 Z

6/17 BHM GW2 -- 1.1; 5.5; 11; 22; 55; 110 -- 213 Z

July 2009 7/13 BHM GW1-6 X -- -- -- --

7/14 BHM GW4 -- 1.1; 5.5; 11; 22; 55; 110 -- 213 Z

7/15 BHM GW6 -- 1.1;5 .5; 11; 22; 55; 110 -- 213 Z

7/16 NEC GW1-6 X -- -- 213 --

Aug. 2009 8/25 BHM GW1-6 X -- -- 213 --

8/26 BHM GW5 X 1.1; 5.5; 11; 22; 44 -- 213 Z

Sept. 2009 9/21 NEC GW1-6 X -- -- 213 --

9/22 NEC GW4 X 1.1; 5.5; 11; 22; 44 -- 213 Z

1

Nitrate and glucose were added in 1 liter of solution through the top of the chamber after sealing to the marsh surface immediately before sealing the chamber and thus initiating the incubation time.
Laboratory Denitrification Methods 11

Repeated measurements were made during the growing 

seasons in 2008 and 2009 at six denitrification sites in NEC 

(fig. 3) and six in BHM (fig. 5). Flooding during both years 

limited the number of denitrification measurements that could 

be made in NEC. Standing water greater than 5 cm in altitude 

over the marsh soil surface prevented denitrification measurements with the type of chamber used.

Laboratory Denitrification Methods

During denitrification, N2 and N2O are produced, but 

N2 is usually the primary by product (Knowles, 1982). 

The ratio of N2 to N2O can vary, depending on landscape 

position (Meding and others, 2001), sediment properties, 

and availability of NO3 (Weier and others, 1993; Parton and 

others, 1996; Lee and others, 1997). To determine these 

site-specific ratios we conducted classic laboratory acetylene 

inhibition denitrification assays (Oremland and others, 1984; 

Duff and others, 1996) on sediment samples collected at each 

of the denitrification measurement sites. We did not expect 

differences in ratios based on moisture content because 

these soils were saturated at all times. We ran experiments to 

determine ratios under conditions of no NO3 addition and two 

levels of NO3 addition to account for potential differences in 

ratios that might depend on NO3 availability. We attempted 

to address the issue of differences in soil properties by 

determining ratios using soils from many different sites 

rather than applying a single ratio for all sites. We were not 

able to control for potential differences in ratios that might 

have resulted from differences in temperature between our 

laboratory experiments and the field. We estimated the ratio 

of N2 to N2O produced by comparing N2O produced in the 

absence of acetylene and in the presence of acetylene (Meding 

and others, 2001). Acetylene blocks the microbial conversion 

of N2O to N2 (Tiedje and others, 1989; Groffman and others, 

2006), therefore in the presence of acetylene all of the gaseous 

nitrogen released will be in the form of N2O. Denitrification 

rate was calculated by applying the appropriate ratio of N2 to 

N2O as follows:

Denitrification rate = ( ) N O2 PR 2 × ( ) N :N O2 2 N OPR 

  + ( ) (3)

where

N2OPR is N2O production rate, and

N2: N2O is the ratio of N2 produced to N2O produced.

All rates are reported in units of micromoles of N2O per square 

meter per hour.

There are important limitations to this approach to estimating in-place denitrification rates. Because of differences 

in temperature, sediment structure, and redox status, we do 

not know whether the laboratory determined N2 to N2O ratios 

are comparable to the true field ratios. To our knowledge, 

no methods can yet let us determine the “true” field N2:N2O

ratios because all approaches that have been attempted are 

accompanied by serious limitations and simplifying assumptions (Groffman and others, 2006). Therefore we are not able 

to assess the error associated with this method. The main 

limitation to the acetylene inhibition approach is that acetylene can also inhibit nitrification (Groffman and others, 2006). 

This would be most problematic when the NO3 denitrified 

during the field measurement is derived mostly from nitrification taking place during that “incubation period” rather than 

being derived from ambient nitrogen within pore waters. The 

advantage of this in-place method is that the primary incubation is done in the field, thereby avoiding the compromising 

of variables that occurs when soils or sediments are brought to 

the laboratory and assayed under conditions very unlike those 

in the field. This method is particularly advantageous for this 

project’s objective of determining maximum potential denitrification rates because, in conditions in which NO3

 levels are 

high (as in our treatments that add NO3), the inhibitory effect 

of acetylene on nitrification is no longer a relevant issue.

Soils from all of the measurement sites were assayed to 

determine N2 to N2O ratios and in these experiments moderate 

amounts of NO3 were added to stimulate denitrification and to 

make the conditions comparable to in-place treatments where 

NO3 was added. Soils from half of the measurement sites were 

assayed to determine N2 to N2O ratios under ambient conditions (no treatment additions) and under conditions where NO3 

and glucose were added together. Preliminary observations of 

N2O production rates in these laboratory experiments indicated 

that production rates were very low; indeed, N2O consumption 

was occasionally observed when NO3 was not added. It was 

therefore decided that using average ratios determined from 

two or three sites under ambient conditions would provide a 

reasonable estimate of the N2 to N2O ratios without performing these experiments on the soils from all locations. If we 

observed N2O consumption in individual replicates in these 

experiments, we did not use the N2 to N2O ratios from these 

replicates to determine average N2 to N2O ratios for use in the 

computation of rates of N2O production.

Marsh soil was collected using a hand auger to a depth 

of 1.25 meters (m) and the material was stored at 4 degrees 

Celsius (°C) until use. Groundwater was also obtained from 

shallow piezometers at NEC and at BHM for use in the laboratory incubations. This groundwater was also stored at 4°C 

until use. Large root fragments were removed and the soil 

material was ground up using a hand-operated food grinder 

to facilitate mixing and to allow subsequent subsampling of 

representative fractions of the original sample. Approximately 

20 g of fresh soil was weighed into each 135 cubic centimeter 

(cm3) glass flask. The groundwater from each location was 

used to make up solutions containing various concentrations 

of NO3 on the same day that these treatments were used in the 

laboratory incubation experiments. Twenty milliliters (mL) of 

each appropriate solution was added to each flask, and then 

the flasks were sealed. These 20-mL solutions of 5, 10, 50, 

75, 100, 200, 500, 750 and 1,000 mM NO3 were equivalent 

to nitrogen addition rates of 0.01, 0.14, 0.7, 1.05, 1.4, 3.5, 7, 
12 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

10.5, 14 mg N g-1 fresh soil in the incubation flask respectively). Deionized water was used to make a stock solution of 

1-molar (M) glucose. Syringes were used to add 0.4 mL of the 

1-M glucose solution to each flask that received the glucose 

treatment bringing the solution to a final glucose concentration 

of 19.6 mM (equivalent to 3.6 mg glucose per gram of fresh 

soil). The screw cap lids for the flasks were Teflon lined and 

were equipped with a rubber septum to facilitate additions of 

glucose by syringe, sparging with helium (He), addition of 

acetylene, or simply gas sampling using syringes.

After sealing, all flasks were sparged with ultrahigh 

purity (UHP) He for 20 minutes to establish anoxic conditions. 

For treatments that received acetylene, 14 cm3

 of headspace 

was first removed by syringe and then 14 cm3

 of acetylene was 

added to achieve a final headspace concentration of approximately 15 percent acetylene by volume. All flasks were then 

placed on a mechanical shaker for 5 minutes. After this initial 

equilibration, 4-mL gas samples were withdrawn from each 

flask and transferred to 3-mL vacutainers to determine N2

O 

concentration at time zero (T0

). Two gas bags were prepared: 

one contained UHP He, and the other contained 15 percent 

acetylene (by volume) in UHP He. Four mL of “make-up” gas 

from the appropriate bag was added to all flasks after collecting T0

 samples to restore the original headspace volume 

and pressure. After adding the appropriate make-up gas for 

each treatment, the flasks were shaken continuously until the 

subsequent collection of gas samples (for example, T1

,T2,T3, 

orT4

). During each subsequent sampling, 4 mL of gas was 

collected from the headspace by syringe and transferred to a 

3-mL vacutainer, and 4 mL of make-up gas was added back to 

each flask. The gas samples in vacutainers were shipped to a 

laboratory for analysis.

Subsamples of fresh, ground, and homogenized marsh 

soils were analyzed to determine the water fraction and the 

fraction of organic matter (OM). Fresh soil samples were 

weighed and then oven-dried at 105°C until they reached a 

constant weight; they were then reweighed to gravimetrically 

determine the water fraction (grams of water per gram of 

oven-dry soil). These samples were ashed in a muffle furnace 

at 500°C until they reached a constant weight, whereupon they 

were reweighed to gravimetrically determine the OM content (grams OM per gram oven-dry soil) (Davies, 1974). The 

results of the gravimetric analysis were used to present the 

denitrification data on a per dry weight or per organic matter 

weight basis.

Laboratory N2O Analytical Methods 

and Computation of N2

O Release Rate

The gas samples were shipped to a USGS laboratory 

in Menlo Park, California, for determination of N2

O. Gas 

samples were analyzed by nickel-63 electron capture detection gas chromatography using standard methods (Oremland 

and others, 1984; Duff, 1996; Aelion and Shaw, 2000). This 

laboratory maintains quality assurance and quality control 

protocols that include duplicate analyses, blanks, and multiple 

calibration standards during all sample runs. The laboratory 

reported the N2

O concentrations from the vacutainers (headspace at each sampling time) in units of nanomoles N2

O mL-1. 

In-place rates of N2

O release were determined as nanomoles 

N2

O m-2 hr-1 using the volume of the chamber headspace, the 

initial and subsequent headspace concentrations, the area of 

the marsh surface covered by the chamber (m-2), and the duration of the incubation (hr):

N2O release rate = 

T +T

area

incubation time

2 1 Conc.×vol. Conc.×vol.

, (4)

where

T1 Conc.×vol. = is concentration of N2O multiplied by 

the chamber volume at the start of the 

incubation period, and

T2 Conc.×vol. = is concentration of N2O multiplied by 

the chamber volume at the end of the 

incubation period.

Field Methods for Water Quality 

Sampling and Measurements

Samples were collected from tributaries that flow into the 

NEC and BHM estuaries (figs. 2, 4; table 2) on an approximately monthly schedule during the 2008 and 2009 growing seasons. Table 3 lists the constituents analyzed in these 

samples which were collected from shallow groundwater in 

NEC and BHM marsh soils at the sites where in-place denitrification measurements were made (figs. 3, 5). A field meter 

was used to determine specific conductance and temperature at 

these surface-water and groundwater sampling locations.

A peristaltic pump was used for sampling surface water 

pumping water directly from a fast-flowing section of the 

tributary through a cartridge filter (0.45-micrometer (μm) 

polyether sulfone membrane filters), and into a 125-mL amber 

polyethylene bottle at each measuring site. All groundwater samples were collected by pumping water from shallow 

piezometers that were installed in the NEC and BHM marsh 

soils at the measurement sites. The piezometers were 5-cmdiameter commercial PVC, 1 meter in length, and screened 

over the entire length, except within 15 cm of each end. The 

screen size was 0.0254 cm, and the bottoms of the piezometers were equipped with a conical 5-cm well point. Holes 

were augured with a hand auger by using a 3.75-cm diameter bit to ensure a tight seal between the piezometers and 

the surrounding marsh soil. Piezometers were installed with 

unscreened pipe 10 cm in altitude above land surface, and with 

caps installed to prevent precipitation from entering. Samples 

were pumped with a peristaltic pump or a vacuum pump into 
Field Methods for Water Quality Sampling and Measurements 13

Table 2. Surface water and groundwater water-quality monitoring stations in Northeast Creek and Bass Harbor Marsh watersheds 

sampled in 2008 and 2009.

[Latitude and longitude determined with Global Positioning System (±2 to 5 meters); USGS, U.S. Geological Survey; mi2, square miles; NEC, Northeast Creek 

watershed; BHM, Bass Harbor Marsh watershed; GW, groundwater site]

Site name USGS 

station number Latitude Longitude

Drainage 

area1

(mi2)

Number of 

samples

Surface-water sites

Aunt Betseys Brook near Bar Harbor, Maine 01022815 44°24′22.0" 68°19′10.0" 0.63 11

French Hill Brook near Bar Harbor, Maine 01022817 44°24′23.0" 68°18′44.0" 0.56 11

Old Mill Brook at Old Norway Drive near Bar Harbor, Maine 01022800 44°23′55.0" 68°17′14.0" 1.55 12

Marshall Brook near Southwest Harbor, Maine 01022890 44°16′29.0" 68°21′05.0" 1.97 7

Lurvey Brook near Southwest Harbor, Maine 01022892 44°16′44.0" 68°21′28.0" 0.105 6

Heath Brook near Tremont, Maine 01022895 44°16′40.0" 68°22′05.0" 0.91 8

Buttermilk Brook near Southwest Harbor, Maine 44°15′59.6" 68°20′38.5" 1

Stony Brook below Hamilton Pond near Bar Harbor, Maine 01022810 44°25′28.0" 68°17′29.0" 2.66 1

Adams Brook near Southwest Harbor, Maine 44°14′52.3" 68°20′22.4" 1

Adams Brook near Southwest Harbor, Maine 44°14′35.3" 68°19′53.0" 1

Adams Brook near Southwest Harbor, Maine 44°14′37.4" 68°19′58.3" 1

Adams Brook near Southwest Harbor, Maine 44°14′45.0" 68°20′09.2" 1

Aunt Betseys Estuary near Bar Harbor, Maine 44°24′38.7" 68°18′45.8" 1

Bass Harbor Marsh Estuary, near Southwest Harbor, Maine2 44°15′35.2" 68°20′42.4" 1

Bass Harbor Marsh Estuary, near Southwest Harbor, Maine3 44°15′35.2" 68°20′42.4" 1

Intermittent stream draining to Aunt Betseys Bridge near GW2 44°24′40.0" 68°18′32.0" 1

Groundwater sites4

NEC GW1 442439068184501 44°24′39.1" 68°18′45.2" 9

NEC GW2 442437068184201 44°24′36.5" 68°18′42.6" 7

NEC GW3 442439068184601 44°24′38.7" 68°18′45.8" 8

NEC GW4 442437068184401 44°24′37.0" 68°18′44.3" 8

NEC GW5 442504068184401 44°25′03.6" 68°18′44.7" 7

NEC GW6 442439068184602 44°24′39.2" 68°18′46.2" 3

BHM GW1 441533068202701 44°15′32.5" 68°20′27.7" 4

BHM GW2 441532068202901 44°15′32.0" 68°20′29.8" 4

BHM GW3 441532068203101 44°15′32.1" 68°20′31.1" 7

BHM GW4 441532068203301 44°15′32.1" 68°20′33.2" 4

BHM GW5 441538068204701 44°15′37.9" 68°20′47.4" 4

BHM GW6 441535068204201 44°15′35.2" 68°20′42.4" 6

1

Drainage areas not available for surface-water sites without USGS sites identification numbers.

2

Sample collected at water surface.

3

Sample collected 20 centimeters below water surface.

4

For groundwater sites, only pretreatment (ambient) samples were collected. Drainage areas are not applicable to groundwater sites.
14 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Table 3. Constituent name, U.S. Geological Survey National Water Information System parameter codes and method 

codes, and minimum reporting limits for temperature, specific conductance, dissolved oxygen, and nutrients.

[°C, degrees Celsius; na, not applicable; μS/cm, microsiemens per centimeter; mg/L, milligrams per liter; N, nitrogen; P, phosphorus]

Water-quality constituent

USGS and NWIS codes

Minimum or laboratory reporting limit Parameter Method

Temperature, water (°C) 00010 na 0.01°C

Specific conductance, field (μS/cm at 25°C) 00094 na 1.0 μS/cm

Oxygen, dissolved (mg/L) 00300 na 0.1 mg/L

Ammonium (mg/L as N) 00608 00623 0.01 mg/L

Silica, dissolved (mg/L as SiO2

) 00955 CL151 0.5 mg/L

Phosphorus, orthophosphate, dissolved (mg/L as P) 00671 00048 0.006 mg/L

Nitrate plus nitrite (mg/L as N) 00631 CL050 0.10 mg/L

Nitrogen, total, dissolved (mg/L and N) 62854 CL063 0.01 mg/L

250-mL polyethylene bottles, all of which were prewashed 

and rinsed profusely prior to sample collection. Groundwater 

samples were filtered on site with syringe cartridge filters, or 

were filtered in a laboratory within 6 hours by using a peristaltic pump and a cartridge filter or a syringe and a cartridge 

filter. All filters were 0.45-μm pore size, and all samples were 

filtered into 125-mL amber polyethylene bottles that had 

been acid washed and rinsed three times with filtered sample 

prior to sample collection. Samples were chilled on ice until 

delivered to the USGS Maine Water Science Center (ME 

WSC) laboratory in Augusta, Maine, within 36 to 48 hours 

of collection. After arriving at the ME WSC laboratory, all 

samples were frozen and stored before shipment to the Woods 

Hole Oceanographic Institution Nutrient Analytical Facility 

laboratory for analysis.

Field values for specific conductance and water 

temperature were measured at the time of sample collection 

by using an Orion model 122 meter. The meter was calibrated 

before each use by using standard solutions for specific 

conductance. For surface water measurements, the Orion 

probe was placed directly in a fast-flowing section of the 

stream and allowed to equilibrate before recording the value. 

If the peristaltic pump was used groundwater samples were 

pumped into a beaker, and the Orion probe was placed into 

the beaker while water was being pumped in and allowed to 

equilibrate before readings were recorded. If the hand vacuum 

pump was used, water was first pumped into a vacuum flask 

and then poured into a beaker, the Orion probe was placed into 

the beaker, and readings were recorded within 30 seconds. 

Dissolved oxygen was determined by using a Chemetrics 

field test kit that uses the indigo carmine colorimetric method 

(Gilbert and others 1982; American Society of Testing and 

Materials International, 2012).

Laboratory Methods for Water Quality 

Analysis

Ammonia was analyzed colorimetrically by the indophenol method, Lachat QuickChem Method 31–107–060–1–B 

(Scheiner, 1976; American Public Health Association, 1998, 

method 4500–NH3

–F, p. 4.108–4.109). Silicate was analyzed colorimetrically by using Lachat QuickChem method 

31–114–27–1–C (American Public Health Association, 

1998, method 4500–SiO2

–F, p. 4.160–4.161). Phosphate was 

analyzed colorimetrically by Lachat QuickChem Method 

31–115–01–1–H (Murphy and Riley, 1962; American Public 

Health Association, 1998, method 4500–P–E, p. 4.146–4.147). 

Nitrate and NO2

 were analyzed colorimetrically by using 

Lachat QuickChem Method 31–107–04–1–E (Wood and others 1967; American Public Health Association, 1998, method 

4500–NO3

–F, p. 4.118–4.119). The concentration of NO2 in 

these waters is expected to be below detection based on a previous study at Acadia National Park (Culbertson and others, 

2007), therefore, throughout this report this analysis is referred 

to as NO3

. Total dissolved nitrogen was analyzed colorimetrically following alkaline persulfate digestion (D’Elia and others, 1977; American Public Health Association, 1998, method 

4500–Norg, p. 4.102–4.103).

Ancillary Data

Water level was recorded continuously at one site (USGS 

streamgage 442439068184502) in Fresh Meadow Marsh, NEC 

(fig. 2), using standard USGS methods. The well was equipped 

with a Design Analysis DH21 submersible pressure transducer 
Laboratory Denitrification Experiments to Determine Response to Nitrate and Glucose Additions 15

programmed to record water level and water temperature 

every 15 minutes. Soil temperature and water level relative to 

land surface was recorded periodically at each of the denitrification measurement sites. Soil temperature was determined 

using a glass thermometer at a soil depth of 15 cm below the 

surface. Water level was determined by measuring the depth 

to the free water surface from the top of the 5-cm-diameter 

piezometers, described in the preceding “Field Methods for 

Water Quality Sampling and Measurements” section, that were 

installed at each of the 12 monitoring sites and corrected for 

the altitude of the piezometers above land surface. Daily precipitation data were obtained from the National Park Service 

(NPS) at Acadia National Park. The NPS maintains a National 

Atmospheric Deposition Program (NADP) monitoring site that 

uses a Belfort tapping bucket rain gage to record daily precipitation data (http://nadp.sws.uiuc.edu/) at McFarland Hill on 

Mount Desert Island. The NADP program is administered by 

the Illinois State Water Survey in Champaign, Ill. Tidal data 

for Frenchmans Bay, Bar Harbor, Maine, on Mount Desert 

Island were obtained from National Oceanographic 

and Atmospheric Administration (NOAA) data files at 

http://tidesandcurrents.noaa.gov/. Streamwater discharge 

records were obtained for Otter Creek near Bar Harbor, Maine 

(USGS streamgage 01022840).

Laboratory Denitrification Experiments 

to Determine N2 to N2O Ratios

Laboratory incubation experiments using marsh soils 

from the NEC and BHM field measurement sites were 

conducted in order to determine the appropriate ratios of N2

to N2O to apply to measurements of N2O release in the field 

in order to obtain denitrification rates. The results of these 

experiments under ambient conditions (no treatment additions) 

and under various levels of NO3 additions with and without 

glucose are provided in a separate report (Huntington and others, 2011, summarized in table 4). For the experiments receiving NO3

 additions, the ratios of N2 to N2O varied from 0.09 to 

4.0, averaging 0.43 at NEC and 1.33 at BHM (table 4). These 

ratios are within the range of ratios reported in the literature 

for terrestrial and coastal marsh environments (Seitzinger, 

1988; Weier and others, 1993; Parton and others, 1996; Lee 

and others, 1997; Meding and others, 2001). For example, 

Seitzinger (1988) reported ratios of N2 to N2O in the range of 

0.18 to 2.3 for estuarine and coastal marine sediments.

The observed ratios of N2 to N2O varied considerably 

among sites and were generally higher in BHM than NEC 

(table 4). The ratios of N2 to N2O generally were substantially 

higher under ambient conditions or under glucose only conditions than when NO3

 or NO3 plus glucose was added (table 4). 

However, the limited number of analyses and the high variability for these treatments indicates a high uncertainty in the 

actual ratios under these conditions. One of the reasons for 

higher variability in the ratios under ambient or glucose-only 

treatments is that the absolute rates of N2O release were very 

low under these conditions. If any substantial N2O consumption had occurred in one treatment but not in another during 

the incubation, that increase would have skewed the results. 

We did not quantify N2O consumption; rather, we measured 

net N2O release during the incubation period.

Laboratory Denitrification Experiments 

to Determine Response to Nitrate and 

Glucose Additions

In one experiment using soil from NEC site GW4, NO3

was added at 10 rates, ranging from 0 to 14 mg N g-1 fresh soil 

with and without the addition of 3.6 mg glucose per g fresh 

soil, and all treatments received acetylene. N2O production 

rates increased with increasing incubation time from 1 hour 

44 minutes to 19 hours 46 minutes for all treatments (figs. 6A 

and B). The largest increases in rates of denitrification (by 

factors of approximately 4 to 10) were between the third 

(3 hours 45 minutes to 5 hours 47 minutes) and fourth 

(5 hours 47 minutes to 19 hours 46 minutes) time intervals. 

These responses indicate a lag in the microbial denitrification 

that occurred following the treatment additions both with 

and without glucose additions in this NEC marsh soil. The 

initial rates (from T0 to 1 hour 44 minutes) were very low 

(<10 nanomoles (nmol) N2O g-1 hr-1) in all cases. Surprisingly, 

no increase in denitrification rate occurred with higher NO3

concentrations at sites where no glucose was added (fig. 6A). 

Denitrification rates decreased linearly from about 55 to 

8 nmol N2O g-1 hr-1 in response to linearly increasing NO3

additions ranging from 1.05 to 14 mg N g-1 fresh soil (fig. 6A) 

indicating that NO3 addition rates greater than 1 mg N g-1

fresh soil probably inhibited denitrification or other microbial 

pathways that produce N2O. Inhibition of denitrification 

(to the end products N2O and N2) under conditions of high 

NO3 concentrations has been reported (Francis and Mankin, 

1977; Glass and Silverstein, 1998). The mechanism for this 

inhibition may be related to toxicity from the accumulation 

of NO2 or of nitrous acid (Glass and Silverstein, 1998). 

It is unlikely that acetylene inhibition of nitrification 

affected production of NO3, since we typically observed no 

measureable rates of N2O production in field experiments 

when no acetylene was added. Denitrification rates at sites 

where glucose was added were substantially higher than at 

sites where no glucose was added for all NO3 treatments and 

for all time periods except during the initial incubation period 

(T0 to 1 hour 44 minutes) (fig. 6B). The addition of glucose 

increased the rate of denitrification by a factor of about 4 to 

5, to 200 to 300 nmol N2O g-1 hr-1 for the lower rates of NO3

addition (fig. 6B). Denitrification rates with added glucose 

decreased with increasing NO3 additions above 0.7 mg N g-1

fresh soil, again indicating an inhibition of denitrification at 

the higher rates of NO3 addition.
16 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Table 4. Ratios of N2 to N2O denitrification products measured in laboratory incubation experiments using acetylene to block 

production of N2

.

[Where experiments were not conducted, the values shown are the means of the measured values for the soils in that estuary. N, number of replicates in the 

experimental trials; NEC, Northeast Creek watershed; GW, locations 1 through 6 in each watershed; --, no measurements for that watershed site; BHM, Bass 

Harbor Marsh watershed; Standard deviations were not calculated if the number of replicates was less than three]

Site name Average N2

/N2O

standard deviation N

Ambient (no treatment additions)

NEC GW1 2.58 2

NEC GW2 2.33 2

NEC GW3 4.55 1

NEC GW4 3.15 --

NEC GW5 2.39 2

NEC GW6 3.15 --

BHM GW1 10.38 8.01 4

BHM GW2 1.49 1

BHM GW3 5.94 --

BHM GW4 5.94 --

BHM GW5 5.94 --

BHM GW6 5.94 --

Nitrate added

NEC GW1 0.09 0.07 3

NEC GW2 0.12 2

NEC GW3 0.32 0.38 3

NEC GW4 0.30 2

NEC GW5 0.58 0.33 4

NEC GW6 1.16 1

BHM GW1 1.10 0.70 5

BHM GW2 0.72 0.24 6

BHM GW3 0.64 0.33 4

BHM GW4 1.33 --

BHM GW5 0.21 2

BHM GW6 4.00 2

Site name Average N2

/N2O

standard deviation N

Nitrate and glucose added

NEC GW1 1.66 --

NEC GW2 0.21 2

NEC GW3 1.66 --

NEC GW4 1.66 --

NEC GW5 3.10 2

NEC GW6 1.66 --

BHM GW1 0.70 --

BHM GW2 0.69 0.06 4

BHM GW3 0.94 2

BHM GW4 0.70 --

BHM GW5 0.46 1

BHM GW6 0.70 --

Glucose added

NEC GW1 11.92 --

NEC GW2 1.36 --

NEC GW3 11.92 --

NEC GW4 11.92 --

NEC GW5 22.48 2

NEC GW6 11.92 --

BHM GW1 36.71 --

BHM GW2 1.13 1

BHM GW3 72.29 1

BHM GW4 36.71 --

BHM GW5 36.71 --

BHM GW6 36.71 --

In another laboratory experiment using soils from NEC 

sites (GW1, GW2, GW3, and GW5) and from BHM sites 

(GW1, GW2, GW3 and GW5), NO3

 was added at three rates 

(0, 1.05, and 7 mg N g-1 fresh soil). For each rate of added 

NO3

 there were four combinations of treatments involving 

acetylene and 3.6 mg glucose g-1 fresh soil: (1) no acetylene, 

no glucose; (2) no acetylene, with glucose; (3) with acetylene, 

no glucose; and (4) with acetylene and with glucose. Complete data for these experiments have been previously reported 

(Huntington and others, 2011). When no NO3

 was added, with 

or without glucose addition, in the NEC soils there was little 

to no N2

O production unless acetylene was added (fig. 7A). 

When acetylene was added in these zero NO3

 treatments N2O 

production increased to 5 to 7 nmol N2

O g-1 dry soil hr-1. In 

contrast, in the BHM sediments little to no N2

O production 

was observed unless NO3

 was added, regardless of whether 

acetylene or glucose were added (fig. 7B).

When NO3

 was added, N2O production increased markedly in all cases (typically to between 10 and 50 nmol N2

O 

g-1 dry soil hr-1) depending on the site and on whether glucose 

or acetylene had been added (figs. 7A and B). The rate of 

N2

O production was substantially higher during the second 

incubation period (3 hours 53 minutes to 7 hours 54 minutes) 

after initiation of the incubation compared to the first incubation period (T0

 to 3 minutes 53 minutes) for most treatments, 

although the differences between these sequential periods 

were smaller in NEC soils than in BHM soils (fig. 7). Generally, the rates of N2

O production were higher in response to 
Laboratory Denitrification Experiments to Determine Response to Nitrate and Glucose Additions 17

0:00 to 1:44 1:44 to 3:45 3:34 to 5:47 5:47 to 19:46

EXPLANATION

Incubation intervals, in hours:minutes

Treatment addition, in milligrams of nitrogen (added as nitrate) per gram of fresh soil

Nitrous oxide production rate, in nanomoles per gram per hour

0.01

0.1

1

10

100

1,000

0 0.01 0.14 0.7 1.05 1.4 3.5 7 10.5 14

0.1

1

10

100

1,000

A. No glucose added

B. Glucose added

Figure 6. Production rates of nitrous oxide in the laboratory determined after the indicated 

incubation intervals with A, no glucose added and B, glucose added at 3.6 grams per square meter 

of fresh soil. Soil samples are from Northeast Creek watershed site GW4. Location of site shown in 

figure 3.

the addition of 1.05 mg N g-1 fresh soil than after adding 7 mg 

N g-1 fresh soil for both BHM and NEC soils. The reduction 

in N2O production at the higher rate of N addition was more 

pronounced in the NEC soils than in the BHM soils. It is likely 

that the higher rates of N addition resulted in some inhibition 

of denitrification.

The addition of acetylene resulted in an increase in 

the rate of N2O production for all treatments during which 

NO3 was added and in the zero NO3 treatments in NEC soils 

(fig. 7), as expected, because acetylene blocks the conversion of N2

O (produced during the first step in denitrification) 

to N2. The fact that little to no N2O production was observed 

in the treatments in which no NO3 was added, but that N2O 

was produced when acetylene was added, confirms that net 

N2O production did not occur at measurable rates under these 

laboratory conditions unless NO3 was added. There was either 

little to no production of N2O, or the production of N2O was 

balanced by N2O consumption during these incubations. The 

proportional increase in N2O production in the treatments that 

added acetylene compared with adding no acetylene was not 

influenced by the addition of glucose in any of the treatments 

in which NO3 was added (figs. 7A and B).

The addition of 3.6-mg glucose g-1 fresh soil increased 

N2O production in all treatments in which NO3 was added and 

in the zero NO3 treatment on NEC soils in which acetylene 

was added (fig. 7A). The glucose-induced increase in N2O 

production was greater during the second incubation period 

(3 hours 53 minutes to 7 hours 54 minutes) during which NO3
18 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine Nanomoles of nitrous oxide per gram of dry soil per hour

Neither acetylene nor glucose added

Glucose added, acetylene not added

Acetylene added, glucose not added

Both acetylene and glucose added

-10

0

10

20

30

40

50

60

70

80

0:00 to 3:53

0 mg N

0:00 to 3:53

1.05 mg N

0:00 to 3:53

7.0 mg N

3:53 to 7:54

0 mg N

3:53 to 7:54

1.05 mg N

3:53 to 7:54

7.0 mg N

Incubation interval, in hours:minutes, and

mass of nitrogen (N) added, in milligrams (mg)

EXPLANATION

A. Northeast Creek watershed

B. Bass Harbor Marsh watershed

-5

0

5

10

15

20

25

30

35

40

45

Figure 7. Laboratory nitrous oxide production rates following nitrate, glucose, and acetylene addition 

treatment additions after the indicated incubation intervals for A, Northeast Creek watershed sites 

GW2, GW3, and GW5 and B, Bass Harbor Marsh watershed sites GW1, GS2, and GW3. Location of site 

shown in figure 3.
Field Measurements of Denitrification Under Ambient Conditions 19

was added, compared to the first period (T0 to 3 hours 53 minutes) (fig. 7A). The increasing N2

O production in response to 

added glucose suggests that denitrification at these marsh soils 

may be limited by the availability of labile carbohydrates as 

well as by the availability of NO3. Alternatively, the addition 

of glucose could have stimulated bacterial growth that, in turn, 

lowered the oxygen concentration, resulting in conditions 

more favorable for denitrification.

The fact that measured N2O production generally was 

higher during the second incubation period (3 hours 53 minutes to 7 hours 54 minutes) compared with the first period (T0

to 3 hours 53 minutes) probably indicates a lag in the microbial response to the NO3

 addition treatments. The addition of 

glucose did not appear to affect whether a lag occurred or how 

pronounced the lag was. In one experiment using soils from 

one NEC site, the incubations were extended an additional 

15 hours and 15 minutes. In this experiment, in all cases in 

which NO3 was added (1.05 mg N g-1 and 7 mg N g-1 fresh soil 

with and without glucose and with and without acetylene), 

the N2O production rates during the third incubation period 

(7 hours 54 minutes to 23 hours 9 minutes) were substantially 

higher than those observed in the second period (data reported 

in Huntington and others, 2011). In this extended incubation period experiment, for the treatments where no NO3

 was 

added, N2O production rates decreased from the first through 

the third incubation periods regardless of the addition of 

glucose or of acetylene, thus emphasizing the lack of denitrification activity at sites where no NO3

 was added rather than 

attributing the decrease to a simple lag in activity.

Field Measurements of Denitrification 

Under Ambient Conditions

Preliminary measurements at NEC in May and June 2008 

indicated that there was a lag in the release of a detectable 

amount of N2O in the chambers so that, during most of the 

experiments samples were not collected until 2 to 4 hours 

after the chambers were sealed. During 2008, preliminary 

measurements at NEC indicated that the addition of acetylene 

alone (without NO3) resulted in no measurable release of N2O 

(Huntington and others, 2011). Therefore, acetylene was not 

used in any further field experiments. To calculate the denitrification rate during the initial period following the sealing 

of the chambers and the collection of the first gas samples, 

the study assumed the initial headspace concentration of N2O 

to be zero. All the field measurements of denitrification rates 

reported in this section are the result of measured N2O release 

and of applying the laboratory-determined, site-specific ratio 

of N2:N2O. At NEC and BHM watersheds, average initial 

denitrification rates (following the sealing of chambers to the 

marsh surface) under ambient conditions were relatively low 

(between 0 and 26 micromoles (μmol) N2O per square meter 

per hour (m-2 hr-1)) (table 5). After the initial periods, average 

measured denitrification rates under ambient conditions were 

typically between 0 and 3 μmol m-2 hr-1 (table 5). The apparent initial rates generally were not sustained after the first few 

hours during these incubations, suggesting that the minimal 

N2O production was balanced by microbial consumption of 

N2O such that N2O concentration in the chamber headspace 

did not continue to increase.

Although methodological differences caution against 

interpretations based on comparisons with denitrification 

rates reported in other studies (Groffman and others, 

2006; Seitzinger and others, 2006), that broader context of 

rates determined in other environments provides a useful 

framework to assess results from studies in environments 

that have never been monitored. The observed rates under 

ambient conditions at NEC and BHM are somewhat lower 

than those that Greene (2005) reported (39 to 95 μmol m-2

hr-1) in a review of freshwater wetland, mudflat, and coastal 

wetland studies. The denitrification rates under ambient 

conditions in NEC and BHM are comparable to, or somewhat 

higher than, rates that Tobias and Neubauer (2009) reported 

for salt marsh ecosystems having low to moderate ambient 

NO3 concentrations (0 to 50 mM NO3). However, these rates 

at NEC and BHM are somewhat lower than the rates the 

same authors reported for salt marshes having high ambient 

NO3 concentrations (>50 μM NO3), which ranged was from 

0 to 100 μmol m-2 hr-1 (Tobias and Neubauer, 2009). The 

same review of denitrification in salt marshes reported that, 

in spite of large variability among sites, an overall positive 

relationship exists between ambient NO3 concentrations in 

pore waters and denitrification rates. In the context of the 

reports of these other studies, the marsh soils in NEC and 

BHM are in the low to moderate ranges for denitrification and 

for NO3 concentration.

During these experiments under ambient conditions, 

subsequent sampling of chamber headspace gas at periodic 

intervals up to about 24 hours after chamber sealing generally 

did not result in higher measured denitrification rates; in fact, 

rates usually declined over time (table 5). The data on N2O 

production rate for each measurement date and site are published in Huntington and others (2011). Denitrification rates 

during successive time intervals following chamber sealing 

(T0) are presented for NEC sites GW4 and GW2 in July 2008, 

illustrating the average rate during time intervals T0 to 4 hours 

36 minutes, 4 hours 36 minutes to 7 hours 20 minutes, 7 hours 

20 minutes to 11 hours 20 minutes, and 11 hours 20 minutes to 

22 hours 59 minutes (fig. 8A). The rates over time were either 

relatively constant (NEC GW4) or, more typically, decreased 

to near zero (NEC GW2) (fig. 8A). Similar results, in that rates 

either did not increase or decreased over time, were observed 

for sequential time periods following chamber sealing during 

July and August 2009 at BHM, where results were averaged 

for sites GW1 through GW6 (fig. 8B). We assume that the 

N2:N2O ratios do not change over time. If these ratios changed 

over time that could account for some of the changes in rates 

that we observed.
20 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Table 5. Denitrification rates for each month, year, and site under ambient (no treatment additions) and following treatments where 

nitrate was added alone or in combination with glucose.—Continued

[For the ambient measurements, the chamber headspace concentrations were assumed to be zero at time zero (T0). NEC, Northeast Creek watershed, GW, locations 1 through 6 in each watershed, BHM, Bass Harbor Marsh; g, grams; N, nitrogen, μmole, micromole; hr, hour; yr, year]

Month Year Site name

Period after 

chamber sealing1

(hh:mm)

Nitrate

(g N m-2)

Glucose

(g glucose m-2)

Denitrification rate

(μmol N2O m-2 hr-1) (g N m-2 yr-1)

No treatment additions

June 2008 NEC GW1, GW2, GW4, GW5, GW6 Variable 0 0 Below detection Below detection

July 2008 NEC2 T0 to 4:00 0 0 7.2 1.8

4:00 to 14:17 0 0 1.8 0.44

Sept. 2008 BHM GW3 T0 to 15:12 0 0 2.4 0.59

No second time period

June 2009 BHM2 T0 to 3:20 0 0 26 0.64

3:20 to 7:30 0 0 0 0

July 2009 BHM2 T0 to 3:33 0 0 7.5 1.8

3:33 to 7:58 0 0 8.2 2

July 2009 NEC2 T0 to 3:35 0 0 13 3.2

3:35 to 6:43 0 0 2.4 0.59

Aug. 2009 BHM2 T0 to 4:07 0 0 16 3.9

4:07 to 8:19 0 0 0.45 0.11

Sept. 2009 NEC2 T0 to 2:07 0 0 14 3.4

2:07 to 15:19 0 0 2.5 0.61

Lowest level of nitrate added

July 2008 NEC GW1 3:58 to 8:36 1.1 0 93 23

2008 NEC GW2 6:34 to 16:32 1.1 0 158 39

2008 NEC GW3 2:58 to 6:11 1.1 0 86 21

2008 NEC GW4 35:05 to 45:22 1.1 0 292 72

2008 NEC GW5 0:29 to 6:01 1.1 0 65 16

2008 NEC GW6 4:58 to 23:36 1.1 0 125 31

Sept. 2008 BHM GW1 5:34 to 21:59 5.5 0 123 30

2008 BHM GW2 2:58 to 4:20 5.5 0 10 2.5

2008 BHM GW3 7:58 to 21:08 5.5 0 21 5.2

2008 BHM GW4 0:00 to 3:59 5.5 0 31 7.6

2008 BHM GW5 2:45 to 4:53 3.3 0 20 4.9

2008 BHM GW6 2:15 to 4:38 3.3 0 41 10

June 2009 BHM GW3 4:33 to 6:47 1.1 0 80 20

2009 BHM GW2 3:05 to 6:43 1.1 0 171 42

July 2009 BHM GW4 4:22 to 6:43 1.1 0 75 18

2009 BGM GW6 5:08 to 9:51 1.1 0 111 27

Aug. 2009 BHM GW5 4:27 to 7:27 1.1 0 75 18

Sept. 2009 NEC GW4 20:23 to 26:23 1.1 0 82 20
Field Measurements of Denitrification Under Ambient Conditions 21

Table 5. Denitrification rates for each month, year, and site under ambient (no treatment additions) and following treatments where 

nitrate was added alone or in combination with glucose.—Continued

[For the ambient measurements, the chamber headspace concentrations were assumed to be zero at time zero (T0). NEC, Northeast Creek watershed, GW, locations 1 through 6 in each watershed, BHM, Bass Harbor Marsh; g, grams; N, nitrogen, μmole, micromole; hr, hour; yr, year]

Month Year Site name

Period after 

chamber sealing1

(hh:mm)

Nitrate

(g N m-2)

Glucose

(g glucose m-2)

Denitrification rate

(μmol N2O m-2 hr-1) (g N m-2 yr-1)

Maximum observed rate of denitrification

July 2008 NEC GW1 3:58 to 8:36 1.1 0 93 23

2008 NEC GW2 6:34 to 16:32 1.1 0 158 39

2008 NEC GW3 2:58 to 6:11 1.1 0 86 21

2008 NEC GW4 35:05 to 45:22 1.1 0 292 72

2008 NEC GW5 0:29 to 6:01 1.1 0 65 16

2008 NEC GW6 4:58 to 23:36 1.1 0 125 31

Sept. 2008 BHM GW1 5:34 to 21:59 5.5 0 123 30

2008 BHM GW2 2:58 to 4:20 5.5 0 10 2.5

2008 BHM GW3 4:40 to 7:55 22 0 99 24

2008 BHM GW4 0:00 to 3:59 5.5 0 63 15

2008 BHM GW5 2:45 to 4:53 3.3 0 111 27

2008 BHM GW6 2:15 to 4:38 3.3 0 75 18

Oct. 2008 BHM GW6 22:39 to 24:25 110 0 129 32

June 2009 BHM GW3 6:09 to 16:52 55 0 258 63

2009 BHM GW3 9:53 to 22:03 55 213 477 120

2009 BHM GW2 6:10 to 27:02 110 0 327 80

2009 BHM GW2 2:41 to 6:19 110 213 475 120

July 2009 BGM GW4 4:12 to 6:33 110 0 522 130

2009 BGM GW4 4:04 to 6:25 110 213 581 140

2009 BGM GW6 5:00 to 9:43 55 0 785 190

2009 BGM GW6 9:33 to 21:28 110 213 541 130

Aug. 2009 BHM GW5 7:19 to 19:211 44 0 168 41

2009 BHM GW5 4:07 to 7:33 5.5 213 680 170

Sept. 2009 NEC GW4 20:23 to 26:18 5.5 0 133 33

2009 NEC GW4 20:24 to 26:19 44 213 489 120

1

The periods are bounded by the sampling times, after sealing chambers ,over which the rates were calculated based on the increase in concentration of N2O 

in the headspace and application of the appropriate N2 to N2O ratio. The times are averages for all replicates for each treatment.

2

GW1 through GW6 averaged together.
22 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Error bars—Show standard error of the mean

-10

-5

0

5

10

15

20

25

30

35

July 2008 GW4 July 2008 GW2

Measurement date and site

T0 to 4:36

4:36 to 7:20

7:20 to 11:20

11:20 to 22:59

Denitrification rate, in micromoles of nitrous

oxide production per square meter per hour

EXPLANATION

A. Northeast Creek watershed

Incubation intervals, in hours:minutes

Error bars—Show standard error of the mean

July 2009 GW1 through GW6 Aug. 2009 GW1 through GW6

Measurement date and sites

EXPLANATION

T0 to 3:33

3:33 to 7:58

7:58 to 20:59

Incubation intervals, in hours:minutes

B. Bass Harbor Marsh Watershed

Denitrification rate, in micromoles of nitrous

oxide production per square meter per hour

-10

-5

0

5

10

15

20

25

Figure 8. A, Average of the field denitrification rates for sites GW4 and GW2, July 2008 in the Northeast Creek watershed. B, Average 

of the field denitrification rates for sites GW1–GW6, July and August 2009 in the Bass Harbor Marsh watershed. For both figures

denitrification rates were for ambient conditions during sequential periods following the time chambers were sealed to the marsh soil 

surface. Initial rates were calculated assuming sealing time (T0) as being zero nanomoles of nitrous oxide per milliliter. Location of site 

shown in figure 3.
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 23

Field Measurements of Denitrification 

Following Nitrate and Glucose 

Addition Treatments

Denitrification rates generally increased by an order of 

magnitude or more following NO3 additions, with or without 

glucose additions, compared with the ambient treatments 

that received no nutrient additions (table 5). Denitrification 

rates generally increased with increasing rates of added NO3, 

although the response was not linear, and in some cases rates 

were lower at the highest NO3 addition rates compared with 

low to moderate NO3 addition rates. Flooded conditions during 

most of 2009 (fig. 9A) limited our ability to make denitrification measurements in response to variable rates of NO3 addition on NEC until September 2009. In Fresh Meadow Marsh, 

where our denitrification measurement sites were located, the 

water level in Northeast Creek was at or above the ground 

surface nearly continuously from June through December in 

2009 (fig. 9A). These persistent flooded conditions in Fresh 

Meadow Marsh had never been observed before by Acadia 

National Park resource managers or by USGS scientists working in this area for many years. The timing and amplitude of 

changes in water level in Fresh Meadow Marsh corresponded 

closely to the measured discharge at Otter Creek (USGS 

streamgage 01022840). Mount Desert Island received an 

unprecedented amount of rainfall during this period (fig. 9B).

In June 2009 the BHM sites GW2 and GW3 showed 

large increases in denitrification rates in the range of 1.1 to 

11 g N m-2, in response to NO3 additions (when no glucose 

was added) but these sites showed no increase in denitrification rates in response to NO3

 additions greater than 55 g N m-2

Date

0

20

40

60

80

100

120

-0.5

0

0.5

1.0

1.5

2.0

2.5

3.0

5/30/09 6/29/09 7/30/09 8/29/09 9/29/09 10/29/09 11/29/09

Discharge at Otter Creek USGS station

01022840, in cubic feet per second

Water level, in feet above land surface

Water level in Northeast Creek watershed

Otter Creek discharge

A

EXPLANATION

B

Date

5/1/09 6/1/09 7/1/09 8/1/09 9/1/09 10/1/09 11/1/09 12/1/09

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Precipitation, McFarland Hill,

in inches

Figure 9. A, Water levels in the Fresh Meadow Marsh, Northeast Creek watershed, Mount Desert Island, Maine, and discharge 

at Otter Creek U.S. Geological Survey (USGS) streamgage 01022840; and B, daily precipitation at the McFarland Hill weather 

station, near Bar Harbor, Mount Desert Island, Maine.
24 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

(figs. 10 and 11). The BHM site GW4 in July 2009 illustrates 

large increases in denitrification in response to moderate rates 

of NO3

 addition (1.1, 5.5, and 11 g N m-2) followed by proportionately smaller increases in denitrification rate at higher 

rates of NO3

 addition (22, 55 and 110 g N m-2) (fig. 12). At the 

BHM site GW6 in July 2009, denitrification rates increased 

initially from near zero to about 190 μmol N2

O m-2 hr-1 in 

response to the addition of 5.5 g N m-2 , did not increase above 

200 μmol N2

O m-2 hr-1 for additions of 11 and 22 g N m-2 , then 

increased to nearly 800 μmol N2

O m-2 hr-1 with the addition 

of 55 g N m-2, but then decreased to 300 μmol N2

O m-2 hr-1

in response to (110 g N m-2), the highest rate of addition of 

nitrate (fig. 13). In August 2009, denitrification at the BHM 

GW5 site increased from near zero to greater than 150 μmol 

N2

O m-2 hr-1 in response to NO3 additions of as much as 11 g N 

m-2 and did not increase further in response to NO3 additions of 

22 or 44 g N m-2 (fig. 14). In September 2009, denitrification 

rates on site NEC GW4 increased from near zero to 65 μmol 

N2O m-2 hr-1 in response to NO3 additions of as much as 

5.5 g N m-2, then gradually declined to 40 μmol N2O m-2 hr-1

in response to increasing NO3 additions from 11 to 44 g N m-2

(fig. 15).

The addition of glucose at a fixed rate of 213 g m-2

in association with the added NO3 resulted in a variety of 

responses in denitrification rate compared with the addition 

of NO3 alone. In some cases the addition of glucose resulted 

0 20 40 60 80 100 120

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

EXPLANATION

-50

0

50

100

150

200

250

300

350

Figure 10. Average rate of denitrification 

over the measurement period from 

3 hours 5 minutes to 20 hours 32 minutes 

after sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed 

site GW2 in June 2009.

0 20 40 60 80 100 120

EXPLANATION

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

-50

0

50

100

150

200

250

300

350

400

450

Figure 11. Average rate of denitrification 

over measurement period from 4 hours 

33 minutes to 22 hours 32 minutes after 

sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed 

site GW3 in June 2009.
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 25

0 20 40 60 80 100 120

EXPLANATION

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

-50

0

50

100

150

200

250

300

350

Figure 12. Average rate of denitrification 

over measurement period from 4 hours 

22 minutes to 22 hours 01 minutes after 

sealing chamber to marsh sediment 

surface on Bass Harbor Marsh watershed 

site GW4 in July 2009.

100

300

500

700

900

0 20 40 60 80 100 120

EXPLANATION

-100

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

Figure 13. Average rate of denitrification 

over measurement period from 5 hours 

8 minutes to 22 hours 43 minutes after 

sealing chamber to marsh sediment 

surface on Bass Harbor Marsh watershed 

site GW6 in July 2009.

0 10 20 30 40 50

EXPLANATION

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

-100

0

100

200

300

400

500

600

Figure 14. Average rate of denitrification 

over measurement period from 4 hours 

27 minutes to 19 hours 32 minutes after 

sealing chamber to marsh sediment 

surface at Bass Harbor Marsh watershed 

site GW5 in August 2009.
26 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

-100

0

100

200

300

400

500

0 10 20 30 40 50

EXPLANATION

Nitrate added

Nitrate plus glucose added

Grams of nitrogen per square meter of soil added as nitrate

Denitrification rate, in micromoles of nitrous

oxide per square meter per hour

Figure 15. Average rate of 

denitrification over measurement 

period from 2 hours 51 minutes to 

26 hours 16 minutes after sealing 

chamber to marsh sediment surface 

at Northeast Creek watershed site 

GW4 in September 2009.

in higher rates of denitrification in response to a given rate 

of NO3 addition (for example, figs. 10, 12, 14, and 15). In 

some cases, the largest increases in denitrification rate in 

response to the added NO3

 plus glucose occurred at the highest 

rates of NO3

 addition (for example, figs. 10, 12, and 13). In 

August 2009 at the BHM GW5 site the largest enhancement 

from glucose was in response to the 5 and 11 g N m-2 rates 

of NO3

 addition, but at the highest NO3 addition rate (44 g N 

m-2), denitrification rates were higher when NO3 was added 

without glucose (fig. 14). At the NEC GW4 site in September 

2009, enhanced denitrification rates (by a factor of about 6) in 

response to glucose additions were consistently evident at all 

rates of NO3

 addition from 1.1 to 44 g N m-2 (fig. 15). These 

variable responses to the addition of glucose indicate that the 

amount of labile carbohydrates can limit denitrification even if 

NO3

 is present. However, there are obviously other conditions 

in which the addition of glucose did not result in an increase 

in denitrification rates, indicating that glucose was not limiting 

under those conditions or that glucose had no effect on oxygen 

concentration. For example, glucose had relatively little effect 

on denitrification rate in June 2009 at BHM GW2 (fig. 10) and 

in July 2009 at BHM GW4 (fig. 12) at the lower levels of NO3 

addition, but adding glucose at the highest rate of NO3 resulted 

in an increased rate of denitrification. This variable response 

could indicate that insufficient NO3

 limited denitrification only 

until NO3

 was supplied at rates greater than or equal to 55 g 

N m-2 after which, denitrification was limited by both NO3 

and glucose.

Denitrification rates varied with time during the course 

of each measurement after sealing the chambers to the marsh 

surface. These variations were apparent among sites in the 

same watershed, between watersheds, among levels of NO3

addition, and between treatments with and without glucose 

additions. The largest differences in these time series were 

between the BHM and NEC marsh systems. In general, for all 

levels of NO3

 addition, with and without glucose, denitrification rates increased linearly with time during measurements 

in NEC at site GW4 in September 2009, between measurement intervals that were bounded by the times of sampling 

headspace gas (fig. 16). The initial rates during the first 2 to 

3 hours were quite low but they increased to maximum rates 

of 758 μmol N2O m-2 hr-1 at sites where glucose was added and 

increased to 132 μmol N2

O m-2 hr-1 at sites where glucose was 

not added when NO3

 was added at 5.5 g N m-2 (fig. 16). The 

linearly increasing rate of denitrification with time at the NEC 

site is matched with an exponentially increasing headspace 

concentration (fig. 16). The reason for the substantially higher 

denitrification rates under the relatively low NO3

 addition rate 

compared with the denitrification rates at higher NO3

 addition 

levels is not known. It may be related to variation in marsh 

soil properties even within this relatively small area of uniform 

marsh vegetation, or it may be related to the fact that high NO3

concentrations can inhibit the denitrification pathway (to the 

N2

O and N2 end products), possibly because of toxicity from 

an accumulation of NO2

 (Glass and Silverstein, 1998).

At BHM sites, the time series illustrating the denitrification rate response to NO3

 additions was more complicated 

than at NEC sites because of variations between the two sites 

studied as well as variations under different rates of NO3

addition with and without glucose. At the BHM GW4 site in 

July 2009, denitrification rates peaked between 4 hours and 

30 minutes and 6 hours and 30 minutes after chamber sealing, 
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 27

A

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

B

0 5 10 15 20 25 30

Hours after sealing chamber to marsh surface

Denitrification rate

Headspace concentration

EXPLANATION

C

0

5

10

15

20

25

30

0

2

4

6

8

10

12

14

16

0

10

20

30

40

50

60

70

80

90

0

50

100

150

200

250

300

350

400

0

2

4

6

8

10

12

14

16

0

20

40

60

80

100

120

140

Figure 16. Relation between field denitrification rate and 

headspace nitrous oxide concentration and the varying incubation 

intervals once the chamber was sealed to the marsh surface 

at the Northeast Creek watershed site GW4 in September 2009, 

immediately following the following treatment additions: A, 1.1 

grams of nitrogen per square meter only; B, 1.1 grams of nitrogen 

per square meter plus 3.6 grams of glucose per square meter; 

C, 5.5 grams of nitrogen per square meter only; D, 5.5 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; E, 11 grams of nitrogen per square meter; F, 11 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; G, 22 grams of nitrogen per square meter; H, 22 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; I, 55 grams of nitrogen per square meter; and J, 55 grams 

of nitrogen per square meter plus 3.6 grams of glucose per square 

meter. Horizontal bars on the x axis show the period at which the 

denitrification rate begins to apply. The secondary y axis shows 

instantaneous concentrations of nitrous oxide in the headspace at 

the sampling times indicated.
28 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

0 5 10 15 20 25 30

Denitrification rate

Headspace concentration

EXPLANATION

D

Hours after sealing chamber to marsh surface

F

E

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

0

10

20

30

40

50

60

0

100

200

300

400

500

600

700

800

0

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

0

100

200

300

400

500

600

Figure 16.—Continued.
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 29

0 5 10 15 20 25 30

Denitrification rate

Headspace concentration

EXPLANATION

G

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

H

Hours after sealing chamber to marsh surface

I

0

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

80

0

5

10

15

20

25

30

35

40

45

0

100

200

300

400

500

600

0

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

80

Figure 16.—Continued.
30 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

0 5 10 15 20 25 30

Hours after sealing chamber to marsh surface

Denitrification rate

Headspace concentration

EXPLANATION

Headspace concentration,

in nanomoles per milliliter

Denitrification rate, in micromoles of

nitrous oxide per square meter per hour

J

0

10

20

30

40

50

60

0

50

100

150

200

250

300

350

400

450

500

Figure 16.—Continued.

then declined substantially between 6 hours and 30 minutes 

and 22 hours, when the measurement period ended (fig. 17). 

The shapes of these curves were consistent with or without the addition of glucose. These initially increasing and 

subsequently decreasing rates with time were matched with 

headspace concentrations that follow a logistic or sigmoid 

curve implying diminishing availability of substrates during 

the measurement period (fig. 17). The fact that these curves 

were similar independently of the addition of glucose suggests that NO3

 was the primary limiting substrate at this site 

and at this time. At the BHM GW5 site in August 2009, time 

series of the denitrification rate response were more variable, 

headspace concentrations increased quasilinearly over time, 

but the patterns of changes in N2

O production rates over time 

were highly variable and not consistently related to glucose 

additions or to rates of NO3

 addition (Huntington and others, 

2011). The variable responses among sites and dates indicate 

that different factors probably limited denitrification rates 

under the distinctive set of conditions during those measurement periods.

On several measurement dates, glucose was added as a 

treatment without adding NO3

 in order to determine whether 

denitrification in these marsh soils could be limited by the 

availability of a labile carbon substrate alone. In almost 

all cases, both at NEC and at BHM during June through 

September 2009, glucose additions resulted in an increase in 

measured denitrification. The increases ranged from factors 

of 1.05 to 15, median 4.0 (table 6). The denitrification rates in 

these experiments with glucose-only additions were similar 

to most of the rates observed for the lowest levels of NO3

addition (table 5). However, the initial rates of denitrification 

with these glucose-only additions were not sustained beyond 

3 to 4 hours after the chambers were sealed, compared with 

the rates at sites where NO3

 alone or NO3 together with 

glucose were added. In the experiments during which NO3

or NO3

 and glucose were added, denitrification rates either 

increased with time (NEC) or increased and then decreased 

but maintained a high rate compared with the ambient or 

glucose only experiments (BHM) (figs. 16 and 17). The data 

for these ambient and glucose- only experiments for sampling 

periods following the initial periods are reported in Huntington 

and others (2011). The glucose-induced enhancement of 

denitrification indicates the availability of a modest amount of 

NO3

 but a lack of labile carbon.

The maximum denitrification rates observed during 

2008 and 2009 for NEC and BHM for each site at which 

measurements were made following additions of NO3

 or 

NO3

 and glucose are shown in table 5. For most sites in both 

watersheds, the maximum denitrification rates were in the 

range of about 75 to 680 μmol N2

O per square meter per 

hour (m-2 hr-1; 18 to 170 grams of nitrogen per square meter 

per year (g N m-2 yr-1)). The maximum rates were higher for 

the higher rates of nitrogen addition, and they were highest 

at sites where glucose was also added. These rates are higher 

than the maximum reported for salt marshes under ambient 

conditions even under very high ambient NO3

 conditions 

(Tobias and Neubauer, 2009), indicating that these marsh soils 

are capable of denitrifying high rates of nitrogen additions, 

at least one to two orders of magnitude higher than that to 

which they are apparently exposed under ambient conditions. 

Tobias and Neubauer (2009) reported results from several 

studies using several different methods than the methods 

we used so the methods are not directly comparable and 

therefore comparisons between the rates they reported and 
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 31

0

1

1

2

2

3

3

4

4

5

0

10

20

30

40

50

60

70

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

0

20

40

60

80

100

120

Hours after sealing chamber to marsh surface

Denitrification rate

Headspace concentration

EXPLANATION

B

A

C

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

0

50

100

150

200

250

0 4 8 12 16 20 24

Figure 17. Relation between field denitrification rate and 

headspace nitrous oxide concentration and the varying incubation 

intervals once the chamber was sealed to the marsh surface at 

the Bass Harbor Marsh watershed site GW4 in September 2009, 

immediately following the following treatment additions: A, 1.1 

grams of nitrogen per square meter only; B, 1.1 grams of nitrogen 

per square meter plus 3.6 grams of glucose per square meter; 

C, 5.5 grams of nitrogen per square meter only; D, 5.5 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; E, 11 grams of nitrogen per square meter; F, 11 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; G, 22 grams of nitrogen per square meter; H, 22 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; I, 55 grams of nitrogen per square meter; J, 55 grams of 

nitrogen per square meter plus 3.6 grams of glucose per square 

meter; K, 110 grams of nitrogen per square meter; and L, 110 grams 

of nitrogen per square meter plus 3.6 grams of glucose per square 

meter. Horizontal bars on the x axis show the period at which the 

denitrification rate begins to apply. The secondary y axis shows 

instantaneous concentrations of nitrous oxide in the headspace at 

the sampling times indicated.
32 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

0

1

2

3

4

5

6

0

20

40

60

80

100

120

140

160

0

2

4

6

8

10

12

14

16

18

0

2

4

6

8

10

12

14

0

50

100

150

200

250

300

350

Denitrification rate

Headspace concentration

EXPLANATION

D

E

Hours after sealing chamber to marsh surface

F

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

0

50

100

150

200

250

0 4 8 12 16 20 24

Figure 17.—Continued.
Field Measurements of Denitrification Following Nitrate and Glucose Addition Treatments 33

I

H

0

5

10

15

20

25

0

50

100

150

200

250

Denitrification rate

Headspace concentration

EXPLANATION

G

0

5

10

15

20

25

0

50

100

150

200

250

300

350

400

450

500

0 4 8 12 16 20 24

0

50

100

150

200

250

300

350

0

5

10

15

20

25

30

35

40

Hours after sealing chamber to marsh surface

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

Figure 17.—Continued.
34 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

Denitrification rate

Headspace concentration

EXPLANATION

J

0

10

20

30

40

50

60

0 4 8 12 16 20 24

L

K

0

5

10

15

20

25

0

50

100

150

200

250

300

350

0

5

10

15

20

25

30

0

50

100

150

200

250

300

350

400

450

0

200

400

600

800

1000

1,200

Hours after sealing chamber to marsh surface

Headspace concentration, in nanomoles per milliliter

Denitrification rate, in micromoles of nitrous oxide per square meter per hour

Figure 17.—Continued.
Field Denitrification Rate in Relation to Environmental Variables 35

Table 6. Denitrification rates in paired measurements under ambient or glucose only addition.

[Glucose-only addition was at 213 grams of glucose per cubic meter. T0 corresponds to the time of chamber sealing]

Start time End time

T1

Ambient denitrification

(μmol N2

 m-2 hr-1)

Start time

T0

End time

T1

75 mM glucose denitrification

(μmol N2

O m-2 hr-1 Location T ) 0

BHM GW2 6/15/09 12:21 6/15/09 15:40 8.60 6/17/09 10:32 6/17/09 13:23 9.01

BHM GW3 6/15/09 12:41 6/15/09 15:48 25.5 6/16/09 16:27 6/16/09 20:03 135

NEC GW1 7/16/09 9:15 7/16/09 12:53 8.38 7/16/09 9:15 7/16/09 12:52 33.3

NEC GW2 7/16/09 9:40 7/16/09 13:02 7.85 7/16/09 9:40 7/16/09 13:01 59.3

NEC GW3 7/16/09 9:10 7/16/09 12:56 24.4 7/16/09 9:10 7/16/09 12:55 57.9

NEC GW4 7/16/09 9:25 7/16/09 12:59 11.9 7/16/09 9:25 7/16/09 12:58 181

NEC GW5 7/16/09 9:11 7/16/09 13:16 12.3 7/16/09 9:14 7/16/09 13:15 74.4

NEC GW6 7/16/09 10:02 7/16/09 13:05 13.1 7/16/09 10:03 7/16/09 13:04 56.2

BHM GW1 8/25/09 11:16 8/25/09 15:25 27.2 8/25/09 11:16 8/25/09 15:24 73.0

BHM GW2 8/25/09 11:13 8/25/09 15:22 12.6 8/25/09 11:14 8/25/09 15:21 1.61

BHM GW3 8/25/09 11:10 8/25/09 15:19 7.39 8/25/09 11:11 8/25/09 15:18 65.9

BHM GW4 8/25/09 11:07 8/25/09 15:16 19.0 8/25/09 11:08 8/25/09 15:15 52.2

BHM GW5 8/25/09 11:04 8/25/09 15:09 15.1 8/25/09 11:05 8/25/09 15:08 46.2

BHM GW6 8/25/09 11:01 8/25/09 15:04 12.3 8/25/09 11:02 8/25/09 15:03 60.1

NEC GW2 9/21/09 16:12 9/21/09 18:01 14.0 9/21/09 16:12 9/21/09 18:00 11.8

NEC GW3 9/21/09 15:52 9/21/09 18:11 18.1 9/21/09 15:52 9/21/09 18:10 38.4

NEC GW4 9/21/09 16:05 9/21/09 18:21 14.0 9/21/09 16:05 9/21/09 18:20 43.8

NEC GW5 9/21/09 15:00 9/21/09 18:17 9.65 9/21/09 15:00 9/21/09 18:16 114

NEC GW6 9/21/09 15:35 9/21/09 18:31 14.1 9/21/09 15:35 9/21/09 18:30 38.1

what we measured should be made cautiously. Nielsen 

(2002a) estimated that, for an 18-month period beginning 

April 1999, the NEC estuary received 5,900 kilograms (kg) 

N in tributary inputs. If that mass of nitrogen were to be 

distributed on the 1.85-km2

 Fresh Meadow Marsh fringing 

the NEC estuary, it would be equivalent to an input of 

2.1 g N m-2 yr-1. Groundwater inputs to NEC and BHM have 

not been quantified, but, on the basis of estimates from Nielsen 

(2002b), a similar amount of nitrogen (2.2 g N m-2 yr-1) may 

have been released during 2001 from the septic systems 

of the estimated 279 residences in the combined drainage 

basins of Aunt Betseys Creek, French Hill Brook, Old Mill 

Brook, and Stony Book that drain into NEC. Denitrification 

in the uplands along the flowpaths from septic systems to the 

fringing marsh and estuary probably removes a substantial 

fraction of this estimated nitrogen input (Ritter and Eastburn, 

1988). Similarly, tidal nitrogen inputs have not been quantified 

for NEC, but a preliminary estimate for BHM indicated that, 

in that estuary, tidal inputs could be comparable to surface 

water inputs (Doering and others (1995). Because bedrock 

sills restrict tidal inflow in both estuaries, tidal inputs (flood 

tide) are limited to substantially less than half that of the tidal 

period. Rising sea level, however, is increasing inputs and the 

residence time of tidal inputs. The denitrification capacity of 

the marsh soils in NEC and BHM (table 5) is clearly greater 

than the potential combined inputs of nitrogen by surface 

water and groundwater.

Field Denitrification Rate in Relation to 

Environmental Variables

Correlation analyses were performed to relate in-place 

denitrification rates to several measured variables. The denitrification rates selected for this analysis were those observed 

for the lowest rates of NO3

 additions because the measured 

denitrification rates under ambient conditions were very low. 

Unfortunately the long-term inundation of the NEC soils 

limited the number of occasions that we could undertake 

comparable experiments on these sites and, in BHM, we had 

no measurements until September 2008. For NEC the lowest 

NO3

 addition rate was 1.1 g N m-2; for BHM the lowest NO3

addition rate was usually 3.3 to 5.5 g N m-2 in 6 cases and 

22 g N m-2 in one case. The data used in this analysis are 

reported in Huntington and others (2011).
36 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

A positive statistically significant (p<0.050) correlation 

was observed between denitrification rate and soil temperature 

(table 7). A positive correlation was expected between soil 

temperature and denitrification, based on previous investigations. There was a weak, but not statistically significant 

(p>0.05), negative correlation between denitrification rate and 

water level in the marsh soils. When the water table was closer 

to the surface, the thickness of the oxic/anoxic interface may 

have been restricted or raised into a zone where NO3

 was less 

available. Such changes in the thickness or relative position 

of the oxic/anoxic interface could explain a tendency towards 

lower denitrification rate with higher water levels. There were 

no significant relationships between denitrification rate and 

concentrations of ammonium, NO3

 and NO2, total dissolved 

nitrogen, or dissolved oxygen in groundwater. The generally 

weak, or lack of, correlations probably reflects differences 

in marsh soil properties that we did not measure such as the 

chemical properties at the oxic/anoxic interface or the thickness of the zone at which denitrification occurred.

The tidal level recorded at Bar Harbor, Maine, at the 

midpoint of the in-place denitrification rate during the measurement period in the field was weakly but not significantly 

(p>0.05) negatively correlated with the denitrification rate 

(table 7). The water level in NEC did not respond directly to 

tidal influences; instead, it was most responsive to surfacewater runoff (fig. 9A). The water level in BHM was not 

recorded continuously but was observed to respond directly to 

the diurnal variation in tide level. The marsh surface at BHM 

would flood regularly for a brief period around high tide during most days in the approximately 2-week period between 

consecutive neap tides.

Tributary Water Quality

Water-quality data for all tributaries to NEC and BHM 

estuaries were presented for all samples collected for this 

study and are reported in Huntington and others (2011). Periodic monitoring of water quality in these tributaries is important for assessing changes in water quality as a result of ongoing changes in land use within these watersheds (for example, 

Nielsen and Kahl, 2007). Residential development outside 

the park boundary has increased rapidly in recent years. For 

example, during 1981 to 2001 the number of residences in 

the combined drainage basins of Aunt Betseys Creek, French 

Hill Brook, Old Mill Brook, and Stony Book in the Northeast 

Creek (NEC) watershed increased from 83 to 279 (Nielsen, 

2002a,b). This increase in residential development may have 

increased nitrogen inputs to the NEC estuary and could have 

increased nutrient concentrations in the tributaries draining 

into the estuaries. However, the concentration of nitrogen 

species determined on multiple samples collected during 

2008 and 2009 for this study does not indicate an increase in 

concentration compared with previous studies for samples 

collected during 1990 to 1992 (Doering and others, 1995) 

or in 1999 and 2000 (table 8; Nielsen and Kahl, 2007). The 

fate of nitrogen inputs in these watersheds, that is presumed 

to be increasing due to the increasing numbers of households 

and septic system effluents, is not known. Denitrification in 

uplands or along groundwater flowpaths may remove most 

of the nitrogen before it reaches the tributaries. Alternatively, 

septic-derived nitrogen may follow flowpaths that lead directly 

to groundwater seepage into the estuaries themselves. Alternatively, again, it is possible that nitrogen derived from increases 

in households has generally not yet arrived at the estuary 

because of long and circuitous pathways and sorption along 

flowpaths. Groundwater seepage into NEC and BHM estuaries 

has been suggested in an earlier study that used aerial thermal 

imagery (Culbertson and others, 2007).

Denitrification Potential of Marsh Soils 

at Acadia National Park

Denitrification requires NO3, suboxic (but not anoxic) 

conditions, and organic substrates (electron donors) for heterotrophic bacteria (Seitzinger and others, 2006). The marshes 

fringing estuaries where marsh soils are high in organic matter 

and subject to variable water levels due to strong tidal forcing (as at BHM), or subject to much weaker tidal forcing and 

periodic surface freshwater flooding (as at NEC) meet two of 

the three criteria. The question at Acadia concerns whether the 

first criterion, availability of NO3

, is met. Given the eutrophic 

state of the BHM estuary, the fringing marsh soils in that 

estuary would probably have higher rates of denitrification 

under ambient conditions than would the marsh soils in the 

comparatively unimpacted NEC marshes. That the rates of 

denitrification in BHM and NEC under ambient conditions 

Table 7. Pearson correlation coefficients between 

denitrification rate and the noted variables under experimental 

conditions where low levels of nitrate were added and no 

glucose was added.

Variable Pearson correlation 

coefficients p-value

Soil organic matter fraction 0.065 0.781

Soil temperature 0.439 0.046

Water level -0.407 0.067

GW ammonia 0.234 0.321

GW nitrate plus nitrite -0.166 0.486

GW total dissolved nitrogen 0.151 0.525

GW specific conductance -0.346 0.135

GW temperature 0.034 0.895

GW dissolved oxygen 0.130 0.632

Bar Harbor tide elevation1 -0.377 0.091

1

Bar Harbor, Maine, tide level at midpoint during the in situ field denitrification measurement period.
Conclusions 37

Table 8. Mean and standard errors of nutrient concentrations in tributaries to Northeast Creek and Bass Harbor Marsh.

[Concentrations are from samples during this study (2008 to 2009, number of samples (n) = 6 to 11) and as reported in previous studies (1999 to 2009, n=20: 

Nielsen, 2002a; Nielsen and Kahl, 2007; and 1990 to 1992, n=6: Doering and others, 1995. For some analyses, nitrate or NH4 concentrations were reported as 

“< the laboratory minimum reporting limit (mrl)”, in these cases values of half the mrl were used in the calculation of the mean values. mg/L, milligrams per 

liter; N, nitrogen; nitrate, nitrate; NH4, ammonium; --, no data; <, less than]

1990 to 1992 1999 to 2000 2008 to 2009

Nitrate 

(mg/L)

NH4 

(mg/L)

Total N 

(mg/L)

Nitrate 

(mg/L)

NH4

(mg/L)

Total N 

(mg/L)

Nitrate 

(mg/L)

NH4

(mg/L)

Total N 

(mg/L)

01022815 Aunt Betseys Brook near Bar Harbor, Maine

Mean -- -- -- 0.03 0.05 0.36 0.03 0.02 0.29

Stderr -- -- -- 0.01 0.01 0.03 0.01 0.01 0.06

01022817 French Hill Brook near Bar Harbor, Maine

Mean -- -- -- 0.04 0.04 0.36 0.02 0.02 0.24

Stderr -- -- -- 0.01 0.01 0.03 0.01 0.01 0.04

01022800 Old Mill Brook at Old Norway Drive near Bar Harbor, Maine

Mean -- -- -- 0.02 0.03 0.41 0.01 0.01 0.15

Stderr -- -- -- 0.01 <0.01 0.07 0.01 <0.01 0.02

01022890 Marshall Brook near Southwest Harbor, Maine

Mean 0.18 0.03 0.48 0.19 0.06 0.46 0.07 0.02 0.23

Stderr -- -- -- 0.03 0.02 0.04 0.02 <0.01 0.03

01022895 Heath Brook near Southwest Harbor, Maine

Mean 0.041 0.021 0.421 0.02 0.03 0.57 0.01 0.02 0.29

Stderr -- -- -- <0.01 <0.01 0.07 0.01 <0.01 0.06

01022892 Lurvey Brook near Southwest Harbor, Maine

Mean 0.041 0.021 0.421 -- -- -- 0.01 0.02 0.22

Stderr -- -- -- -- -- -- <0.01 <0.01 0.03

1

Doering and others (1995) sampled downstream of the confluence of Lurvey and Heath Brooks so that these values are not directly comparable to those 

reported for these tributaries that were sampled separately upstream of their confluence in this study, but are shown here for relative comparison.

measured throughout two consecutive growing seasons are 

similarly relatively low indicates that NO3

 availability is relatively low in both marsh systems. It is evident from the addition of NO3

 together with glucose that these marsh soils are 

capable of comparatively high rates of denitrification (Tobias 

and Neubauer, 2009). The observed estuarine eutrophication 

in BHM is therefore not a result of nitrogen inputs to marsh 

soils that exceed the denitrification capacity in these systems. 

To the extent that eutrophication and nuisance algal blooms 

may be a result of nutrient enrichment derived from human 

activities in the watersheds and from elevated atmospheric 

deposition of nitrogen, it is more probable that these inputs do 

not interact with marsh soils through surface runoff, flooding, 

or shallow groundwater seepage into the marsh soils. If these 

terrestrial inputs to the estuary are the cause of the observed 

eutrophic condition in BHM, they must bypass the marsh in 

channelized surface flow or they must circumvent the marsh in 

shallow groundwater seepage along subsurface pathways that 

enter the estuary directly. Taken together, these observations 

of estuarine eutrophication in BHM, in spite of low rates of 

denitrification under ambient conditions and high denitrification capacity, are consistent with a conceptual model whereby 

nitrogen derived from terrestrial inputs probably bypasses the 

marsh soils (Tobias and Neubauer, 2009).

Conclusions

Denitrification measurements in marsh soils fringing 

the estuaries at Bass Harbor Marsh and Northeast Creek at 

Acadia National Park indicated that, under ambient conditions, denitrification rates were low in both marshes. The low 

denitrification rates, in spite of substantial evidence for eutrophication as demonstrated by extensive nuisance algal blooms 

in the Bass Harbor Marsh estuary, indicated that these marsh 

soils probably do not receive large inputs of nitrogen from the 
38 Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine

surrounding terrestrial landscape. The relatively high rates of 

denitrification observed following the addition of nitrate indicate that these soils have the capacity to denitrify substantially 

more nitrogen than they currently receive. In most cases, the 

addition of glucose plus nitrate resulted in higher rates of denitrification than the addition of nitrate alone. The results of this 

study indicate that limited denitrification capacity of fringing 

marsh soils can be ruled out as a cause to explain eutrophication in these estuaries.

Acknowledgments

The authors are grateful for the support they received 

from several people that contributed to the successful completion of this study. Staff at Acadia National Park (David Manski 

and Bill Gawley) helped facilitate this study from the initial 

conceptualization through its completion. Staff at the U.S. 

Geological Survey, Maine Water Science Center (James 

Caldwell, Charles Schalk, Laura Flight, and Greg Stewart) 

provided assistance with groundwater level data processing 

and interpretation, overall data review, and data uploading into 

the Survey’s National Water Information System database. 

Jeffrey Barbaro, Richard Smith, and Leslie DeSimone, all with 

the U.S. Geological Survey, provided technical reviews that 

improved the report.

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Huntington and others—Ambient and Potential Denitrification Rates in Marsh Soils on Mount Desert Island, Maine—Scientific Investigations Report 2012–5166