Source Pollution Identification:
Colorado/S. Ute Reach
Animas River Watershed
Chester Anderson
Koren Nydick
Mark Oliver
Christopher Peltz
Estella Moore
Carolyn Livensperger
B.U.G.S. Consulting - Mountains Studies Institute - Basin Hydrology
March 2011
For the Animas Watershed Partnership
Contents
Introduction and Background ......................................................................................................... 2
Sampling water for total nitrogen and phosphorus ..................................................................... 2
Management Measures and Best Management Practices ........................................................... 2
Sampling for benthic macroinvertebrates ................................................................................... 7
Animas River Watershed Plan .................................................................................................... 7
Periphyton Biomass and Nitrogen 15 Stable Isotope Analyses .................................................. 9
Rapid Geomorphic Analysis ..................................................................................................... 10
Animas Watershed Group Coordination and Project Management .......................................... 11
Appendix 1. Narrative Description and Interpretation of Chlorophyll-a Data; Mountain Studies
Institute ......................................................................................................................................... 12
Methods......................................................................................................................................... 12
Results:.......................................................................................................................................... 13
Appendix 2. Narrative Description and Interpretation of N15 Data; Mountain Studies Institute 22
Nitrogen Pollution as Threat to Water Sources ........................................................................ 22
Reasoning and Utility of Using 15N to Determine Anthropogenic Sources of Nitrogen ........ 22
Methods..................................................................................................................................... 23
Periphyton sampling ............................................................................................................. 23
Colorado State University Laboratory/Isotope Methods ...................................................... 24
Statistical Analyses ................................................................................................................... 24
Results 15N ................................................................................................................................ 24
Conclusions 15N ........................................................................................................................ 25
References ................................................................................................................................. 36
1
Introduction and Background
This project completes a watershed-wide pollution source identification study of the Animas
River that began in 2006 (B.U.G.S. Consulting 2008).
The Animas River is a complex river. The headwaters are at altitudes greater than 12,000 feet,
beginning in the alpine life zone and within the highly mineralized San Juan Caldera and ending
at 5,500 feet at the confluence with the San Juan River in semi-desert sage-brush scrublands and
highly erosive sedimentary strata. Politically, the Animas River begins in the State of Colorado,
flows through the Southern Ute Indian Tribe Reservation and into the State of New Mexico,
flowing from EPA Region 8 into EPA Region 6.
In 2006 a pollution source identification study was completed in the New Mexico portion of the
river and this report addresses sources of pollution in the Colorado/Southern Ute portion of the
river. The purpose of both studies was to identify: sources of pollution in the watershed; areas of
degraded assimilative capacity for pollutants of the river; management measures and best
management practices (BMPs) to mitigate or remediate sources of pollution and improve
assimilative capacity.
These tasks benefited both consumptive and non-consumptive uses and helped protect beneficial
and designated uses.
In this study, the concentration of total nitrogen and total phosphorus was measured in 18
Animas River sites and 28 inflows. Periphyton biomass (Appendix 1), N15 isotope (Appendix 2)
and benthic macroinvertebrate community data were collected at each of the 18 Animas river
sites. Also completed was a geomorphic assessment that quantified the present-day
characteristics of the river channel and adjoining flood plains through the use of various field
methods (see Attachment 1).
Sample sites were identified by a reconnaissance completed September 2009 where each inflow
to the Animas River (including pipes, field drains, natural drainages, and irrigation ditch return
flows) were identified, photographed, mapped and water chemistry characteristics (specific
conductivity, pH, concentration of nitrate/nitrite) were measured with a water quality sonde.
Sampling water for total nitrogen and phosphorus
At each inflow water samples were collected and analyzed for concentration of total nitrogen and
total phosphorus. Total nitrogen and total phosphorus concentrations were also measured in the
Animas River upstream of each return flow. Discharge was measured for each inflow. Discharge
in the Animas and was estimated using the USGS gauges at Tall Timber Resort, Durango, CO
and at the Colorado/New Mexico State Line, and subtracting depletions of flows from the
Animas due to diversions for irrigation ditches and adding return flows from measured inflows
and tributaries. The load of total nitrogen and total phosphorus in the Animas and the carrying
capacity of total nitrogen and total phosphorus in the Animas was calculated. The loading of total
nitrogen and total phosphorus from each inflow into the Animas was also calculated (see New
Mexico Environment Department 2006 for methods). Samples were collected and preserved
following EPA approved standard operating procedures (B.U.G.S. Consulting 2005) and
shipped to and processed by ACZ Labs, an accredited lab in Steamboat Springs, CO. Sample
sites and data are shown in Table 1.
Management Measures and Best Management Practices
Based on loading data of total nitrogen and total phosphorus, inflow sites were prioritized and
the best management practice for the site was determined and described along with an estimated
cost for each best management practice (Table 2).
2
Table 1. Colorado/Southern Ute Reservation loading data, Baker’s Bridge to NM State Line.
Animas River Sample Sites
Site Name
KOA
Effluent
Hot Springs
Trimble
Lane
Hermosa San
Effluent
Sherer Creek
Inflow
Falls Creek
Inflow
Animas
River Right
Down Falls
Creek
Inflow
House Right
S.
Campground
Inflow
City
Diversion
Inflow
Riverview
Storm Drain
High School
Latitude
Longitude
TN
Conc. TN
Discharge (ug/l) Load
TN
Carrying
Capacity
Inflows
TP
Conc. TP
(ug/l) Load
TP
Carrying
Capacity
TN
Conc.
Discharge (ug/l) TN Load
TP
Conc.
(ug/l) TP Load
37.44917
37.44758
107.80125
107.80237
823
600
0.4 1775.58
0.28 906.13
1863.08
1358.26
0.08
0.07
355.12
226.53
310.51
226.38
0.09
0.02
38.48
0.46
18.16
0.05
9
4.25
0.00
37.38541
107.83654
700
0.04
151.02
1584.63
0.09
339.80
264.11
1.9
0.14
1.43
0.12
1.23
37.38519
107.84279
700
0.18
679.60
1584.63
0.02
75.51
264.11
0.71
84.66
324.20
4.9
18.76
37.37906
107.84881
1.13
0.05
0.30
0.02
0.12
37.3633
107.8471
29.39
0
0.00
0.04
6.34
37.34977
107.8448
37.33076
107.84297
1.2
0.04
0.26
0.01
0.06
37.3183
107.84993
886
0
0.00
0.02
95.57
37.2962
107.87024
1.08
0.07
0.41
0.03
0.17
1.90
0.2
0.78
0.00
0.84
0.13
0.00
0.14
37.28865 107.870029
37.28715 107.87187
732
545
0.16
631.70
1.5 4409.28
1657.07
1233.75
0.025
0.24
98.70
705.48
276.18
205.63
0.00
0.00
3
Animas River Sample Sites
Site Name
Junction
Creek
Fish
Hatchery
Main Street
Pipe Inflow
Main Street
Spring
Inflow
Skate Park
Lightner
Creek
Durango
Effluent
High Bridge
Grandview
Inflow
Inflow Up S.
Durango
Effluent
S. Durango
Effluent
Animas @
Basin Creek
Animas @
Weasel Skin
Powerline
Return Flow
Latitude
Longitude
TN
Conc. TN
Discharge (ug/l) Load
TN
Carrying
Capacity
Inflows
TP
Conc. TP
(ug/l) Load
TP
Carrying
Capacity
TN
Conc.
Discharge (ug/l) TN Load
TP
Conc.
(ug/l) TP Load
37.2856
107.87218
545
0.08
235.16
1233.75
0.03
88.19
205.63
7.51
0.87
35.22
0.19
7.69
37.28096
107.87344
545
0.07
205.77
1233.75
0.03
88.19
205.63
5.93
0.82
26.22
0.06
1.92
37.28106
107.87872
0.4
0.74
1.60
0.04
0.09
37.28102
37.27768
107.87872
107.88274
545
0.08
235.16
1233.75
0.03
88.19
205.63
0.3
2.1
0.56
2.31
0.91
26.16
0.02
0.00
0.23
37.26819
107.88609
545
0.08
235.16
1233.75
0.03
88.19
205.63
2.57
0.38
5.27
0.09
1.25
37.25908
37.23419
107.8773
107.86833
545
545
0.32
0.24
940.65
705.48
1233.75
1233.75
0.09
0.12
264.56
352.74
205.63
205.63
13.44
14.74
1068.51
3
217.47
37.21769
107.85383
0.99
0.15
0.80
0.04
0.21
107.84727
107.84733
0.8
0.13
0.56
0.04
0.17
2.10
2.48
28.03
4.6
51.99
0.56
1.32
3.99
0.08
0.24
37.20361
800
0.08
345.19
1811.01
0.03
129.45
301.83
107.87916
985
0.33 1753.19
2229.81
0.09
478.14
371.63
37.15221 107.888286
992
0.235 1257.36
2245.65
0.135
722.31
374.28
37.13327
992
0.61 3263.78
2245.65
0.14
749.07
374.28
37.18503
107.88906
4
Animas River Sample Sites
Site Name
Trumble
Trumble
Spring
Inflow
Spring River
Right Inflow
Florida
River
Bondad
Return
Inflow
Inflows
TN
TN
TP
TP
TN
TP
Conc. TN
Carrying Conc. TP
Carrying
Conc.
Conc.
Latitude Longitude Discharge (ug/l) Load
Capacity (ug/l) Load
Capacity Discharge (ug/l) TN Load
(ug/l) TP Load
37.10082 107.88776
992
0.42 2247.20
2245.65
0.31 1658.64
374.28
7.99
1.05
45.26
0.1
4.31
37.10082
107.88776
0.5
2.38
6.42
37.05584
107.88065
1.10
0.63
3.74
0.01
0.06
37.05003
107.87278
4.5
1.07
25.97
0.215
5.22
37.04494
107.87598
Average
13.11
0.28
19.80
Sum=1644.11
0.13
9.19
Sum=426.70
992
0.735 3932.59
1328.33
2245.65
1650.41
0.25 1337.62
435.91
374.28
275.07
0.00
5
Table 2. Suggested best management practices and estimated cost for Colorado/S. Ute
priority sites (costs are for design and construction only).
Estimated
Site Name
BMP or next step
Cost
Durango
Tertiary treatment system
$500,000
Effluent
Hermosa San
Tertiary treatment system
$200,000
Effluent
Sprinkler Irrigation and landowner education about flood
Trumble Inflow
$350,000
irrigation
Junction Creek Recon upstream to identify sources of nutrients
$4.500
S. Durango
Tertiary treatment system
$200,00
Effluent
Fish Hatchery
Improving existing tertiary treatment
$50,000
Skate Park
Recon upstream to identify sources of nutrients
$3,500
Sprinkler Irrigation and landowner education about flood
Florida River
$1,000,000
irrigation
Bondad Return Sprinkler Irrigation and landowner education about flood
$350,000
Flow
irrigation
KOA Effluent
Tertiary treatment
$200,000
Trumble Spring Sprinkler Irrigation and landowner education about flood
$350,000
Inflow
irrigation
Lightner Creek Sediment reduction (Lightner Creek Task Force)
$350,000
Powerline
Sprinkler Irrigation and landowner education about flood
$350,000
Return Flow
irrigation
Spring River
Sprinkler Irrigation and landowner education about flood
$150,000
Right
irrigation
S. Campground
Recon upstream to identify sources of nutrients
$3,500
Inflow
TOTAL COST $3,857,204.500
6
Sampling for benthic macroinvertebrates
Benthic macroinvertebrates are excellent indicators of the long term health of a river and
were collected at each Animas sample site and analyzed using standard operating
procedures. The benthic macroinvertebrate data was incorporated into the Animas BMI
Database and the Hilsenhoff Biotic Index, an index for hyper-enrichment of surface
waters, was calculated for each macroinvertebrate sample collected. Sampling, sample
handling and sample processing for benthic macroinvertebrates followed EPA approved
Standard Operating Procedures (B.U.G.S. Consulting 2009). The results are shown in
Figure 1 and the data can be found at:
http://www.bugsconsulting.com/Clients/AnimasWatershedPartnership.aspx.
Figure 1. Collecting macroinvertebrate samples.
Animas River Watershed Plan
Data from this study was incorporated into the Animas River Watershed Plan, a plan
completed by B.U.G.S. Consulting for the Animas Watershed Partnership and based on
the EPA’s Handbook for Watershed Planning (United States Environmental Protection
Agency, Office of Water, Nonpoint Source Control Branch 2008). All documents can be
found at: www.bugsconsulting.com\clients\awp.
7
Figure 1. Hilsenhoff Biotic Index for benthic macroinvertebrates. The HBI value represents the tolerance of benthic
macroinvertebrates to pollution. High values indicate high tolerance to pollution; therefore, low values are the desirable condition.
Values from the Piedra and San Juan Rivers are shown for reference. It is recommended that the Stakeholders establish a goal of less
than 2 throughout the river.
8
9
Periphyton Biomass and Nitrogen 15 Stable Isotope Analyses
Periphyton samples were collected at each Animas River sample site and analyzed for biomass
measured as chlorophyll-a and nitrogen 15 stable isotope signatures. Periphyton is the algae,
bacteria, and fungi growing on surfaces in an aquatic environment. Nitrogen 15 stable isotope is
an indication of areas where untreated or poorly treated sewage enters the river either through
poorly maintained or poorly engineered treatment plants, septic systems or sources of animal
waste. Periphyton sampling, sample handling and sample processing followed an EPA approved
Standard Operating Procedure (B.U.G.S. Consulting 2009).
Four rocks were collected from four locations within a riffle at each Animas sample site scraped
and scrubbed to remove the periphyton from a standard area of 10 cm2. The dislodged periphyton
from each rock was rinsed into a labeled WhirlPack©, covered in foil, and stored on ice. The
longest axis of each rock was measured.
Chlorophyll-a is a commonly used index of algal biomass. Within 24 hours of collection, the
periphyton samples were filtered on glass fiber filters and stored frozen. Periphyton that
consisted of clumps or filaments were blended prior to filtration. Within 30 days of collection,
the frozen periphyton samples were analyzed for chlorophyll-a. Chlorophyll-a in the periphyton
on the filters was extracted with ethanol for 24 hours in the dark. The concentration of
chlorophyll-a in each sample was determined by the acidification method using a
spectrophotometer. Data and results are explained in Appendix 1.
The nitrogen 15 isotopic signature is a measure of the amount of heavy nitrogen 15 compared to
the more common isotope nitrogen 14. It is reported as the delta nitrogen 15 (δN15). Different
sources of nitrogen often have distinctive isotope signatures that can provide a better
understanding of the system than simply measuring concentrations of nitrogen. An increase in
δ15N usually signifies an increasing anthropogenic source. For example, in Toda et al. (2002)
δ15N in periphyton increased with increasing distance from headwaters. The increase in
periphyton δ15N also correlated with increasing relative contribution of nitrate from sewage and
animal waste. A similar relationship was found between increasing agricultural use of
watersheds and increasing δ15N by Luecke and Messner (2005). Nitrate from human and animal
wastes generally has a higher δ15N than nitrate derived soil nitrogen or from synthetic fertilizers,
however. In the 2006 nutrient source identification study conducted on the New Mexico portion
of the Animas River, a striking increase in δ15N occurred downstream of the Aztec Wastewater
Treatment Plant and continued for several miles through the more urbanized lower portion of the
river (B.U.G.S. Consulting 2008)
Within 14 days of collection, the frozen filter samples were dried at 60°C. The filters were then
encapsulated in tin capsules. δ15N was determined by mass spectrometry at Colorado State
University. The work was completed by Mountain Studies Institute, Silverton, CO and data and
results are explained in Appendix 2.
*
9
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Rapid Geomorphic Analysis
(Attachment 1, www.bugsconsulting.com/clients/awp)
The geomorphic assessment quantified characteristics of the river channel and adjoining flood
plains through the use of various field methods in order to evaluate the condition of the channel
relative to its stable forms, cause(s) of instability, sources and indicators of increased sediment,
causes of degraded assimilative/functioning capacity, and potential improvement opportunities
and stabilization-restoration techniques. This information was compiled, summarized and
presented on a reach-by-reach basis.
The river was divided into reaches that exhibited similar geomorphic features, based on aerial
photography and channel/flood plain characteristics. Geomorphic assessments performed on a
reach by reach basis were determined by stream type, channel stability, channel bank erosion
potential and adjoining flood plain characteristics. Geomorphic assessments performed at
representative riffle sections, quantified channel bankfull dimensions, wetted-channel water
depth and velocity, and channel bed composition (e.g., cobble, gravel, sand).
In the report (see Attachment 1) a summary of geomorphic findings were presented with a
narrative for each reach describing assessment methods and findings along with interpretations
The methods for each geomorphic parameter are provided below and were completed by Basin
Hydrology, Inc.
Performed on a reach-by-reach basis
 Stream Type
o Determined stream type for each reach using criteria presented by Rosgen in
Applied River Morphology (1996, see attached Classification Key).
 Channel Stability Rating
o Evaluated each reach’s condition at upper bank, lower bank and bottom of channel
using Pfankuch evaluation criteria (see attached Channel Stability (Pfankuch)
Evaluation and Stream Classification Summary form).
o Determined overall channel stability rating based on stream type.
 Channel Bank Erosion Potential
o Evaluated representative banks within each reach using Bank Erosion Potential
evaluation criteria (see attached Bank Erosion Potential form)
o Identified on maps highly eroded and degraded bank sections
o Identified on maps where bank stabilization measures have occurred and their
condition
o Determined overall bank erosion rating for each reach
 Flood Plain Composition
o Identified general land use(s) on active flood plain and any potential adverse impacts
o Identified dominant riparian vegetation, condition of vegetation and estimated
percent ground cover
o Summarized flood plain characteristics for each reach
10
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Performed at representative riffle sections
 Channel Bankfull Dimension
o Measured bankfull width at representative riffle sections
o Estimated bankfull depth at same locations
o Determined bankfull width-to-depth ratio at each sampled riffle section
 Water Depth and Velocity Measurements
o Measured water depth at several locations across wetted channel at representative
riffle sections
o Measured water velocity at several locations across wetted channel at representative
riffle sections
o Provided mean and range of measured water depths and velocities.
 Channel Bed Composition
o Perform Wolman pebble count at representative riffle sections
o Plot pebble count data to quantify channel bed sizes and distribution
o Determine the measure of embeddedness at riffles using methods described in BedMaterial Characteristics of the San Juan River and Selected Tributaries, New
Mexico: Developing Protocols for Stream-Bottom Deposits (Hein, et al, 2004)
.
Animas Watershed Group Coordination and Project Management
The role of the Animas Watershed Project Coordinator in the project was to ensure that
communication between the project manager and the Animas Watershed Project stakeholders
and other river-related community groups occured. There were regular updates on the project
status and outcomes to the stakeholders during each monthly meeting. The AWP Coordinator
also worked with the project manager to ensure the successful execution of this project. The
Coordinator also wrote a grant to the Colorado Department of Public Health and Environment
Water Quality Control Division to follow-up with results from this study (Attachment 2,
www.bugsconsulting.com/clients/awp).
The AWP represents more than 6 years of effort in collaboration between Municipal, State,
Tribal, Federal, and local entities to achieve workable solutions to issues in the Animas River
watershed, primarily related to nutrient enrichment. Currently, the group is in the process of
completing a Watershed Management Plan, and has completed a Sampling and Analysis Plan, in
order to provide an agreed-upon framework for watershed management and data collection on
the Animas River and tributaries. In addition, the AWP has compiled a comprehensive database
of existing water quality samples and measurements and compiled this data in a Geographic
Information System (GIS) database that contains spatial datasets such as land cover, discharge
locations, irrigation systems, and land ownership.
11
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Appendix 1. Narrative Description and Interpretation of Chlorophyll-a
Data; Mountain Studies Institute
Methods
We followed the methods outlined in EPA - ESS Method 150.1: Chlorophyll –
Spectrophotometric which consist of a field and laboratory component. During the field
component, we gathered periphyton from sites (Table 3) along the Animas River mainstem. We
collected periphyton samples from three separate cobbles located in riffle environments. Cobbles
were placed in a plastic tub, where a 5 cm2 diameter circle of periphyton was removed by
scrubbing with a toothbrush. Scraped periphyton was collected and stored on ice in sealed
Whirlpaks© for later laboratory preparation and analysis. Above each collected cobble,
measurements of flow velocity and depth were observed and recorded, with replicate samples
taken approximately every 15 cobbles.
Laboratory preparation was done within 10 hours of collection, with algal cell concentration
completed by filtering periphyton and a known volume of water through a Whatman 47 mm
filter, then freezing for later laboratory analysis.
Laboratory processing for Chlorophyll-a was conducted at B.U.G.S. Consulting1, using a
spectrophotometer where pigments were extracted from the concentrated algal sample in an
aqueous solution of acetone. The chlorophyll-a concentration was determined
spectrophotometrically by measuring the absorbance (optical density - OD) of the extract at
various wavelengths. The resulting absorbance measurements were then applied to a standard
equation (Nusch et al., 1980):
Chl a (g/L) = (A665-A750b)-(A665-A750a)(R/R-1)(v/V*1)(103)/
Where, Chl a = chlorophyll a corrected for phaeophytin content
665-750 b = before acidification
665-750 a = after acidification
R/R-1 = 1.7/0.7 and is the acid ratio for pure chlorophyll a
 = specific absorption coefficient for chlorophyll a (g*L-1*cm-1)
V = volume of water filtered, in L
v = volume of solvent used to extract sample, in mls
l = pathlength of spectrophotometer cuvette, in cm
Equation simplifies to:
29.6 (665 b-665 a)( v / V * l)
Phaeophytin may then be calculated as:
(g/L)= 20.8 (665 a (v / V * l) - Chl a (g/L)
1
B.U.G.S. Consulting http://www.bugsconsulting.com B.U.G.S. (Bioassessment Underwater GIS Stats) specializes
in aquatic ecology, water quality monitoring, wetlands, watershed studies, and GIS. Our lab facility in Durango, CO
provides in-house capability to process macroinvertebrate, nutrient, and bacteria samples.
12
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
We divided the sites into different land-use class groups (Table XX, Figure XX – land-use at
sites) and examined the differences in mean chlorophyll a concentrations, by conducting a
Kruskal-Wallis one-way analysis of variance on ranks (ANOVA) with sites blocked by land-use.
Results
Chlorophyll-a varied along the reach (Table 3, Figure 2) with the lowest values (0.2-1.5)
recorded at the upstream most sites (KOA Outfall, Hot Springs, Trimble Land and Hermosa
Sanitation) and the two most downstream sites (Trumble and Animas above Florida). The
highest values were found at the High School inflow, South Durango waste water treatment plant
(WWTP), Basin Creek, and Weaselskin (8.6-11.2). Overall, the pattern was of pulses of
increased chlorophyll- a, which were followed directly downstream by lower values.
When the sites were examined by land-use type we observed that the Urban and Agricultural
(Ag) sites did not differ significantly in concentration of chlorophyll-a (Urban – 4.1, Ag – 6.8),
however the Forested/Ag sites found in the upper sections did significantly differ from the Ag
sites (Forested/Ag – 1.1). These differences were pronounced when considered in a downstream
spatial context where low values were recorded until a high value was encountered (e.g., City
Diversion #1 - 6.5, Lightner Creek – 8.2, and South Durango WWTP 9.4), with subsequent,
downstream measurements having lower values.
Overall, the chlorophyll a values recorded in periphyton were below the levels considered the
boundary between oligotrophic/mesotrophic (20 ug/cm-2) (Dodds et al, 1998). However, the
pattern of elevated chlorophyll a found at sites in the urban and agricultural areas suggest that
nutrient inputs to the system in these land-use areas were increasing primary productivity above
levels that may be considered background for the Animas River.
13
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Table 3. Chlorophyll a (ug/cm-2) for sample sites along the Animas River mainstem.
Latitude
Longitude
Kilometers
Downstream
Primary
Land-use
Chlorophyll-a
(ug/cm-2)
37.44917
107.8013
0
Forested/Ag
1.5
Animas Up Hot Springs 37.44758
107.8024
0.2
Forested/Ag
0.2
37.3856
107.8367
8.9
Forested/Ag
1.1
37.38519
107.8428
9.6
Forested/Ag
1.1
37.2962
107.8702
26.7
Urban
6.5
37.28715
107.8719
27.8
Urban
8.6
37.2856
107.8722
28.1
Urban
2.0
37.28116
107.8733
28.6
Urban
3.0
37.27768
107.8827
29.7
Urban
4.9
37.26874
107.8863
30.9
Urban
8.2
37.25908
107.8773
32.4
Urban
3.3
37.23419
107.8683
35.6
Urban
3.0
37.20361
107.8473
40.4
Ag
9.4
Animas at Basin Creek
37.18503
107.8792
44.6
Ag
11.2
Animas at Weaselskin
37.15221
107.8883
49
Ag
8.6
Powerline Return Flow
37.13327
107.8891
51.4
Ag
4.9
Trumble Inflow
37.10082
107.8878
56.4
Ag
1.1
Animas Up Florida
37.05109
107.8757
63
Ag
0.4
Site
Animas Up KOA
Effluent
Animas Up Trimble
Lane
Animas Up Hermosa
Sanitation Effluent
Animas Up City
Diversion Inflow
Animas Up High
School Inflow
Animas Up Junction
Creek
Animas Up Fish
Hatchery
Animas Up Skate Park
Animas Up Lightner
Creek
Animas Up Durango
Effluent
Animas Down High
Bridge
Animas Up S. Durango
WWTP
14
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 2. Chlorophyll a concentrations (ug/cm2) at study sites along the Animas River mainstem
from KOA Outfall (37.44917, 107.8013) to the Animas above Florida River (37.05109,
107.8757).
Figure 3. Chlorophyll a concentrations (ug/cm-2) arranged by adjacent land-use classification.
15
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 4. Chlorophyll a concentrations at study sites along the Animas River mainstem (samples
collected 8/2 – 8/5/2010).
16
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 5. Land-use patterns across study sites
17
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 6. Upper Chlorophyll a sites and adjacent land-use
18
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 7. Middle Chlorophyll a sites and adjacent land-use
19
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 8. Lower Chlorophyll a sites and adjacent land-use
20
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
References
Nusch, E. A. 1980. Comparison of different methods for chlorophyll and phaeopigment
determination. Arch. Hydrobiol. Beih. Ergebn. Limnol. 14: 14-36.
Dodds W. K., Jones, J. R., Welch, E. B. 1998. Suggested classification of stream trophic state:
Distribution of temperate stream types by chlorophyll, total nitrogen, and phosphorus. Water
Resources 32(5) 1455-1462.
21
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Appendix 2. Narrative Description and Interpretation of N15 Data;
Mountain Studies Institute
Nitrogen Pollution as Threat to Water Sources
Globally, nitrogen pollution to streams and lakes is a significant threat to water quality and use
of water supplies for humans, biota, and the natural systems they both depend upon (Smith,
2003). As summarized in Camarago and Alanso (2006), excess nitrogen in the aquatic
environment can lead to a number of deleterious effects, including: (1) an increase the in
concentration of hydrogen ions in low ANC freshwater ecosystems, resulting in acidification of
those systems; (2) an increase in the development, maintenance and proliferation of primary
producers, leading to eutrophication of aquatic ecosystems; (3) and, toxic concentrations of
nitrates, nitrites and ammonia, impairing the ability of aquatic animals to survive, grow and
reproduce. In addition to the impacts on natural systems, inorganic nitrogen pollution of ground
and surface waters can cause adverse effects on human health and economy. These effects can be
seen in increased risk for non-Hodgkin’s lymphoma (Ward et al., 1996), heart disease (Cerhan et
al., 2001), and may contribute to mutagenicity and other birth defects (Luca et al., 1987), and
though the evidence may be inconclusive (Fwetrell, 2004), the US EPA has set as its maximum
contaminant level goal (MCLG) for nitrate at 10ppm (Safe Drinking Water Act, 1988 & 1992).
Excess nitrogen in river systems can fundamentally alter food webs, as nitrogen is typically the
element most limiting to primary production and most responsible for eutrophication (Nixon et
al., 1996; Dodds and Welch, 2000), which when it occurs, often leads to increased treatment
costs for waters destined for agricultural or domestic uses.
Reasoning and Utility of Using 15N to Determine Anthropogenic Sources
of Nitrogen
The nitrogen 15 (15N) isotopic signature, is a measure of the amount of the heavier nitrogen
isotope when compared to the more common and lighter, nitrogen 14 isotope (14N). Studies
examining stable isotopes at or near natural abundance levels are usually reported as delta (δ), a
value given in parts per thousand or per mil (‰). For example, the ratio of the natural
concentration of heavier 15N relative to the lighter 14N is >> 99 to 1 (14N – 99.64%, 15N – 0.36%;
Hoefs, 1980). Delta values are not absolute isotope abundances but differences between sample
readings of the natural abundance standards, which are considered delta, or zero (Ehleringer and
Rundel, 1989). Absolute isotope ratios (R) are measured for a sample and a standard, and the
relative measure delta is calculated as:
[
]
[
]
Where:
[
]
15
N values have been used to detect anthropogenic connections to aquatic food webs for over 40
years (Kohl et al., 1971; Peterson and Fry, 1987) as the fractionation of the lighter 14N from the
15
N happens preferentially as energy (i.e., food) moves upward through tropic levels, enriching
individuals with greater amounts of 15N. Human and animal-derived wastewaters typically have
22
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
higher 15N values than waters not affected by these discharges because of the preferential
volatilization of lighter 14N with ammonia during the hydrolysis of urea (Peterson and Fry, 1987;
Wayland and Hobson, 2001; Silva et al., 2002; Ulseth and Hershey, 2005). Different sources of
nitrogen often have distinctive isotope signatures which can provide a better understanding of the
system than simply measuring concentrations of nitrogen. An increase in 15N usually signifies an
increasing anthropogenic source. Nitrate from human and animal wastes generally has greater
15
N enrichment (typically +7-20‰) than nitrate derived soil nitrogen or from synthetic fertilizers
(Wassenarr, 1995), which generally have low 15N values, generally ranging from -3 and 2‰
(Macko and Ostrom, 1994) as most synthetic fertilizers fix atmospheric nitrogen through the
Haber-Bosch process, whose 15N value is ~0‰ (Heaton, 1986; Mayer et al., 2002). Typically,
natural soil organic 15N has values that range from –3 to +5‰ and is driven primarily by
microbial nitrification process occurring in the organic horizons of the soil profile (Kendall,
1998).
In aquatic systems, 15N has been used to determine the sources of nitrate pollution from urban
sites, (Aravena and Robertson, 1998), Confined Animal Feeding Operations (CAFO’s) (Komor
and Anderson, 1993), and from synthetic agricultural fertilizer (Mayer et al., 2002) as well as to
determine the influence of different land-use complexes on aquatic wood webs. For example, in
Toda et al. (2002) 15N in periphyton (surface algae, bacteria, and fungi) increased with increasing
distance from headwaters. The increase in periphyton 15N also correlated with increasing relative
contribution of nitrate (NO−3) from sewage and animal waste. A similar relationship was found
between increasing agricultural use of watersheds and increasing 15N by Luecke and Messner
(2005). Additionally, in the 2006 nutrient source identification study conducted on the New
Mexico portion of the Animas River, an increase in 15N was observed downstream of the Aztec
Wastewater Treatment Plant and continued for several miles through the more urbanized lower
portion of the river (B.U.G.S. Consulting, 2008).
Methods
We followed methods similar to those outlined in Toda et al. (2002), for collection of periphyton
for later analysis of the stable isotope 15N. Specifically, between August 2nd and 4th 2010,
periphyton samples were collected at seventeen locations along the Animas River (Figure 1)
between the Hot Springs (37.44758, 107.8024) and the Powerline Return (37.13327, 107.8891)
sites (Table 1, Table 2). The sites are orientated along an axis of upstream to down and reflect
three general types of land use in the watershed. The upper sites (KOA Effluent, Hot Springs
Inflow, Animas Up Trimble Lane, and Animas below Hermosa Sanitation) (Figure 2) are within
land use complexes with low housing densities, agriculture, and grazing. The middle sites, (City
Diversion Inflow, High School Inflow, Junction Creek Inflow, Fish Hatchery Inflow, Skate Park
Inflow, Animas Up Lightner Creek, Durango Effluent, and Animas Down High Bridge) have
land use patterns reflecting the urbanized Durango corridor (Figure 3). The lower sites, (Animas
Up South Durango Effluent, Animas at Basin Creek, Animas at Weaselskin, Powerline Return
Flow, Trumble Inflow, and Animas Up Florida) are located in an area of low housing densities,
with agriculture and oil production being the other primary land use. Given the different land-use
patterns our analysis groups our sites by upper (forested/Ag – low density housing), middle
(Urban – high density housing), and lower (Ag/Oil and Gas – low density housing) (Figure 4).
Periphyton sampling
At each site periphyton were collected by scraping an area of 10 cm2. The dislodged periphyton
was placed into a labeled container and stored on ice. Following the periphyton collection, the
23
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
longest axis of each rock was measured and recorded. Within 10 hours of collection, samples
were filtered on ashed glass fiber filters and stored frozen. Periphyton samples which consisted
of clumps or filaments were blended prior to filtration. Within 3 days, the frozen filter samples
were dried at 60°C, and then encapsulated in tin capsules. Samples were shipped to the Colorado
State University Stable Isotope Laboratory for analysis by mass spectrometry
(https://nrel.colostate.edu/isotope-laboratory.html).
Colorado State University Laboratory/Isotope Methods
CSU Isotope laboratory methods for solid sample 15N analysis utilizes a Carlo Erba NA 1500
(Milano, IT) elemental analyzer coupled to a VG Isochrom continuous flow isotope ratio mass
spectrometer (Isoprime Inc., Manchester, UK). An integrated thermal conductivity detector
allows the determination of carbon and nitrogen concentrations simultaneously with an isotopic
determination. Ratios of 15N/14N are expressed relative to known standards (VPDB2 and
atmospheric N, respectively) in per mil (‰) notation. The reference laboratory standard is
Glycine, and was run three times prior to the first sample and then after every 7 – 8 sample runs.
The laboratory reported measurement errors (1SD) for the standard was ~0.22‰ (δ15N).
Statistical Analyses
All statistical analyses were run Sigma Plot 11 (Systat Software, San Jose, CA), with datasets
analyzed for normality and transformed as needed using either log10(x) or x2 transforms.
Statistical significance of all tests was judged at p < 0.05. Analysis of variance (ANOVA) was
used to evaluate the effect of different land uses and geographic location on the enrichment or
depletion of 15N signatures on collected periphyton. Land use classes were used as replicates
for 1-way ANOVA’s to evaluate the effect of land use. Two sets of analyses were run, the first
set including the Hot Springs, Trumble, and Animas above Florida sites, the second set excluded
these sites. Reasoning for this is due to the confounding factors of the geothermal waters at Hot
Springs enriching the 15N values, and the depleting effect of the agricultural return flows on the
values at Trumble and Animas above Florida. Comparisons used the Fishers least significant
difference (LSD) method. The power of the Fishers LSD test is that it computes the pooled
standard deviation (SD) from all the groups, which, if groups are sampled from populations with
the same SD, using all the data to compute the pooled SD gives a more accurate value for the SD
and provides more degrees of freedom, hence more robust statistical significance (Fisher, 1949;
Ott and Longnecker, 2001).
Results 15N
The general pattern of 15N enrichment goes from upstream to down, with the exception of the
Hot Springs site where δ15N value was in the top 33% of all values. The lowest values were
recorded at Trimble Lane (1.8‰) and the highest values were found at Weasel skin (7.4‰). δ15N
values in the sections upstream of Durango were generally lower (mean - 2.8‰) than sites in or
downstream from Durango, even when the Animas Up Hot Springs site was included (Figure 5,
Table 2). When the Hot Springs site is excluded, the upper three sites had a mean of 1.9‰. This
is contrasted with the section beginning at City Diversion Inflow and ending below Durango at
Powerline Return Flow, where the mean δ15N value is 5.1‰. Overall, the downstream pattern is
positive with 15N increasing ~0.06‰ for every kilometer below the KOA Effluent site (adjusted
R2 0.351, p <0.004) (Figure 6).
2
VPDB - Vienna Pee Dee Belemnite
24
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
The abundance of 15N in waters in the upper sites generally ranged from 1.8‰ (Trimble Inflow
and Hermosa Station Effluent) to 5.5‰ (Hot Springs), (ῡ - 2.8‰, sd – 1.8‰) (Figure 7, Figure
8) . When the Hot Springs values are excluded the mean for the upper sites is 1.9‰; exclusion of
the Hot Spring site was considered reasonable, as the 15N in the effluent waters from the Hot
Springs sites could be largely driven by geologic processes and not anthropogenic causes. In the
middle sites the 15N values were generally higher (ῡ - 4.7‰, sd – 0.8‰), and in the lower sites
the 15N values were, on average the highest (ῡ - 5.8‰, sd -1.4‰). As with the upper sites, there
were two sites which had values that were ~1.5‰ lower than the other sites (Trumble Inflow
4.6‰ and Animas Up Florida 4.4‰). When these sites are excluded the mean for the lower sites
is 6.7‰. The rationale for potentially excluding these sites in the overall analysis is that these
sites may potentially be integrating the effects of the agricultural return flows, which would add
depleted 15N water if the nitrogen found in those waters was derived from synthetic fertilizer. If
the three values that had relatively high (Hot Springs) and low values (Trumble Inflow, Animas
Up Florida) are excluded then the downstream relationship becomes much more robust, with
15
N‰ increasing ~0.2‰ for every kilometer below the KOA Effluent (adjusted R2 0.872, p
<0.001) (Figure 7).
Analysis of variance for all sites (Table 3) indicates that the difference between sites is
significant (F 8.78, p – 0.003) however, the difference is small and the pairwise comparisons
suggest that the Lower and Middle sites are not significantly different (p – 0.28). This is
contrasted with the analysis done excluding the Hot Springs, Trumble and Animas above Florida
sites (Table XX – all sites excluding HS, TRU, AaF), where the overall effect of geographic
location is much stronger (F 53.61, p <0.001), additionally the pairwise comparisons show a
larger difference in 15N values between the different geographic regions (Table 4).
Conclusions 15N
The general pattern of increasing 15N from upstream to down is evident in both the geographic
analysis as well as when individual regions are compared on a pairwise basis (Figure 9, Table 4).
15
N is generally lowest in the reaches above Durango, with the exception of the Animas Up Hot
Springs site. The more enriched values identified at the Hot Springs site may be due to higher
temperatures of the waters preferentially volatizing the lighter 14N, or may be due to a point
source of enriched 15N directly upstream, regardless the more enriched values of 15N observed at
Animas Up Hot Springs do not seem to influence the sites below and appear anomalous for the
upper sites. Moving downstream into the more urbanized central Durango reaches of the study
we observed increasing enrichment of 15N as distance downstream increased. This pattern
continues until the Powerline Return Flow site, where the 15N values at Trumble Inflow and
Animas Up Florida are much lower than those upstream. One potential reason for this is that
return flows are washing in nitrogen from synthetic fertilizers applied to the agricultural lands in
the southern part of the watershed. Further understanding of these lower values in the southern
reaches of the study may require that agricultural return flows are sampled for 15N and a two part
mixing model applied to the different source waters.
25
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Table 1. δ15N summary statistics
N15
Site
Mean
4.6
Max
7.4
Animas Up
Weaselskin
Min
1.8
Trimble Inflow
Median 4.6
Table 2. Locations and Delta 15N values for periphyton samples along the Animas River
δ15N
Kilometers
Primary
Site
(‰)
Latitude
Longitude Downstream
Land Use
Forested/Ag
Animas Up KOA Effluent
2.1
37.44917
107.8013
0
Forested/Ag
Animas Up Hot Springs
5.5
37.44758
107.8024
0.2
Forested/Ag
Animas Up Trimble Lane
1.8
37.3856
107.8367
8.9
Animas Up Hermosa
Forested/Ag
Sanitation Effluent
1.8
37.38519
107.8428
9.6
Animas Up City Diversion
Urban
Inflow
3.8
37.2962
107.8702
26.7
Animas Up High School
Urban
Inflow
5.0
37.28715
107.8719
27.8
Animas Up Junction
Urban
Creek
4.0
37.2856
107.8722
28.1
Urban
Animas Up Fish Hatchery
5.5
37.28116
107.8733
28.6
Urban
Animas Up Skate Park
4.1
37.27768
107.8827
29.7
Animas Up Lightner
Urban
Creek
5.9
37.26874
107.8863
30.9
Animas Up Durango
Urban
Effluent
3.9
37.25908
107.8773
32.4
Animas Down High
Urban
Bridge
5.5
37.23419
107.8683
35.6
Ag
Animas at Basin Creek
5.7
37.18503
107.8792
44.6
Ag
Animas at Weaselskin
7.4
37.15221
107.8883
49
Ag
Powerline Return Flow
7.0
37.13327
107.8891
51.4
Ag
Trumble Inflow
4.6
37.10082
107.8878
56.4
Ag
Animas Up Florida
4.4
37.05109
107.8757
63
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Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Table 3. ANOVA results table for Log2 15N, for all sites grouped by geographic location
Location
N
Mean
SD
SEM
Upper
4
0.90
0.54
0.27
Middle
8
1.54
0.18
0.06
Lower
5
1.74
0.24
0.11
Source of Variation
Between Groups
Residual
Total
DF
SS
MS
F
P
2
1.67
0.84
8.78
0.003
14
1.33
0.10
16
3.01
Diff of
LSD
Pairwise Comparison
Means (alpha=0.05)
P
Lower vs. Upper
0.83
0.44
0.001**
Lower vs. Middle
0.20
0.38
0.28
Middle vs. Upper
0.64
0.41
0.005*
* indicates significant difference at <0.01, ** indicates significance at 0.001
Table 4. ANOVA results table for Log2 15N, for all sites grouped by geographic location,
excluding Hot Springs (Upper) and Trumble and Animas above Florida (Lower)
Location
N
Mean
SD
SEM
Upper
3
0.63
0.10
0.06
Middle
8
1.54
0.18
0.06
Lower
3
1.90
0.13
0.08
Source of Variation
Between Groups
Residual
Total
DF
SS
MS
F
P
2
2.65
1.33
53.61
<0.001
11
0.27
0.025
13
2.92
Diff of
LSD
Pairwise Comparison
Means (alpha=0.05)
P
Lower vs. Upper
1.26
0.28
<0.001***
Lower vs. Middle
0.36
0.23
0.006**
Middle vs. Upper
0.91
0.23
<0.001***
* indicates significant difference at <0.01, ** indicates significance at 0.001, ***is significant at
<0.001
27
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 1. Animas River Basin δ15N sampling sites.
28
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 2. Animas River N15 Upper Sites
29
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 3. Animas N15 Lower Sites
30
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 4. Land use map for the middle portion of the Animas River basin.
31
32
Figure 5. Delta (δ) 15N for seventeen sites located along the lower Animas River. Sites are arranged upstream to downstream (left to
right).
*
32
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 6. Relationship between delta 15N and distance downstream from the KOA
campground outfall site (37.44917, 107.8013). Solid line indicates least squares fit of
data (adjusted R2 0.351, p <0.004), dashed lines indicate 95% confidence interval of the
mean. Red markers indicate sites with abnormally high or low values – see text for
explanation. 15N is predicted by the equation 2.98 + (0.0557 * Kilometers Downstream
from KOA).
33
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 7. Delta 15N ranges for all the sites in the Upper, Middle, and Lower reach of the
study area. Vertical bars represent +/-1 SD, horizontal bars indicate mean.
Figure 8. Delta 15N ranges for sites in the Upper, Middle, and Lower reach of the study
area excluding the Hot Springs site in the Upper reach and the Trumble Inflow and
Animas Up Florida in the Lower reach. Vertical bars represent +/-1 SD, horizontal bars
indicate mean.
34
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
Figure 9. Relationship between delta 15N and distance downstream from the KOA
Effluent site (37.44917, 107.8013). Solid line indicates least squares fit of data (adjusted
R2 0.872, p <0.001), dashed lines indicate 95% confidence interval of the mean. 15N is
predicted by the equation: 1.057 + (0.118 * Kilometers Downstream from KOA). Green
circle indicates the upper sites, yellow indicates the sites located in the urban portion of
the watershed, and the red circle indicates those sites downstream from Durango.
35
Narrative Description and Interpretation of N15 Data; Mountain Studies Institute
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