Please Participate with Team #20 in
Nitrate Testing
in
Urban and Rural Areas
 
                by: Wayne Cowell, Nancy Gettman, Mark Little, Elmer Romero
All from the great state of Colorado
 
ABSTRACT:
    Although New Jersey and Colorado do not have similarities in weather, both have diverse environments that influence the nitrate concentrations in surface and ground water.  These environments are agriculture, industry, municipalities, and households that continually contribute to the loading of nitrates in the water.  The introduction and background of this research project describes the impact of high concentrations of nitrates on living systems, the nitrogen cycle, and how nitrates enter ecosystems.  Data collection at 6 different New Jersey sites was done to answer our problem and support our hypothesis. The equipment and supplies were funded by the NSF Woodrow Wilson National Fellowship Foundation Environmental Institute and the protocol was done according to Hoyle....Oops, we mean Hach.  The graph from our data is summarized in our results and conclusion, which did not support our hypothesis.  For more information about the application of this project within your school, standards, resources,  extensions and a clean student data lab sheet, please proceed at your own rate.  We are here to assist you as a reference. Thank you to the people from the Woodrow Wilson Environmental Science Institute at Princeton that were instrumental in helping with this project.
 
INTRODUCTION:
    New Jersey has diverse environments that influence the nitrate concentrations in surface and ground water.  These environments are Agricultural practices that include cropland and livestock, Industrial operations such as mining and construction, Municipal,- landfills and waste water treatment plants, and Households with septic tanks and the improper disposal and use of cleaners, solvents, automobile and lawn maintenance products.
    High nitrate concentrations lead to a loss of other nutrients from soils, increased acidity in surface and ground waters, blooms of algae along coast lines, and an excess of  nitrogen (N) compounds entering the air as pollutants.  The impact of high nitrate concentrations in living systems has been linked to lymphomas caused by N-nitroso compounds, "blue baby syndrome" or methenoglobinemia where a blue tinge on the nose and ear tips appear along with other symptoms such as diarrhea, lethargy, and coma, and a higher incident of spontaneous abortions.  While this nitrogen provides many benefits for society in the form of higher yields from crops, nitrate levels in drinking water in agricultural areas of the United States carried out by the United States Geological Survey (USGS) found nitrates often exceeding safety standards and is increasing steadily.
 
PROBLEM:
    Is there a higher nitrate concentration in water from rural areas when compared to urban areas?
 
HYPOTHESIS:
    Surface water obtained from agricultural practices will contain higher levels of nitrate concentrations when compared to water obtained from urban/suburban areas.
 
BACKGROUND:
    Nitrates is the form of nitrogen that is an essential nutrient for plants and animals as the building block to make proteins.  Making up almost 80 percent of the atmosphere, most nitrogen in the environment exists in the form of gas (N2).  Most plants cannot use nitrogen in this form, but can be transformed into several other compounds that plants can be use which includes nitrates (NO3-), nitrites (NO2- ), and ammonium (NH4+).   Of these, the ammonium and nitrate ions are the most common forms taken in through plants roots.
    Nitrogen is recycled continually by plants and animals called the nitrogen cycle. Nitrogen gas must be fixed before plants and animals can utilize it; therefore, the first step in the nitrogen cycle is nitrogen fixation.  Nitrogen fixation is the conversion of gaseous nitrogen into ammonia.  This crucial step is carried out by bacteria.  Some of the more common Genera are Rhizobium, Anabaena, Clostridium, and Anxobacter.  The second step involves the conversion of ammonia to nitrate.  This process is carried out by bacteria such  Nitrosomonas and Nitrobacter.  Once the plant dies, decomposing bacteria change the nitrogen in the plant back to nitrate and ammonia .  The last phase of the nitrogen cycle is called denitrification, that converts nitrates back to nitrogen gas and returns the nitrogen back to the atmosphere.
    Nitrates enter the water system through natural and human sources.  The natural sources include animal wastes and decomposing plant material.  Human sources of nitrates are sewage (septic tanks,  municipality discharge, and storm overflow), fertilizers from lawns and golf courses, and agricultural runoff from feedlots and cropland. When high concentrations of nitrates accumulate in the water systems, eutrophication of ponds and lakes occur by increasing organic material.  This in turn affects the oxygen production and ultimately the overall growth of that ecosystem.  Excess amounts of nitrates can cause low levels of dissolved oxygen (hypoxia) in animals, brown blood disease in fish,  blood poisoning in infants, hypertension in children, gastric cancers in adults, fetal abnormalities and spontaneous abortions.  In addition the nitrates react directly with the hemoglobin in the red blood cells to produce methemoglobinemia, which destroy the ability to transport the maximum amount of oxygen.  This potentially fatal condition in infants,  before the age of  6 months, results when their digestive system has a diminished capability to secrete  gastric acid, thus the pH level can rise to 5-7.  This causes a proliferation of bacteria in the stomach which then in turn increases the rate of transformation of nitrates to nitrites.  Due to the fact that nitrates are reduced to nitrites in the intestine, a minimum standard of 10 mg/L nitrates as the maximum concentration allowed in drinking water is set by the US Public Health Service.  Check out the River Watch Network.
 
EQUIPMENT & SUPPLIES:
SUMMARY OF PROTOCOL
    Nitrates are measured using the cadmium reduction method with a colorimeter or spectrophotometer.  CAUTION:  This test generates cadmium waste which is toxic and should be discarded properly.
PROTOCOLS

Measuring Nitrates Samples:
1.     Label specimen bottles.  Collect 100 mL samples in labeled bottles from appropriate
        sites.
2.     Refrigerate samples unless test results are done immediately, within 4 hours.
3.     Using the Hach DR/20 spectrophotometer, dial the wavelength to 500nm for the Nitrate
        Program 355. (Fig. A)
4.     Pour 25 mL of the first water sample into two sample cells.  Wearing gloves, add the
        NitraVer
        5 Powder Pillow to one sample cell. (Fig. B) With the stopper in place, shake vigorously
        for one minute. (Fig. C) Then allow to set for five minutes.
5.     Place the blank cell with sample water  (the one without NitraVer 5) into the
        spectrophotometer and allow to "zero".
6.     Remove the blank.  Place the water sample (the one with NitraVer5) into the
        spectrophotometer and press "read". (Fig. D)
7.     Repeat the reading using the other collected water samples.
 
 

Fig. A 
Fig. B
Fig. C
Fig. D
                                                                                                                                    

Preparing the Standard Nitrate Concentrations using a B&L Spectrophotometer.
    To prepare a 50 mg/L stock of nitrate nitrogen standard solution:
1.  Pour 10 mL of the Nitrate Standard Solution (500 mg/L) into a 100 mL volumetric flask.
     Add distilled water until the final volume reads 100 mL resulting in a final concentration of
     50 mg/L nitrate.  Swirl to mix
2.  Label six 50 mL Erlenmeyer flasks or test tubes at the 25 mL level, one for each standard.
     Label the flasks or tubes 0.00, 2.0, 4.0, 6.0, 8.0,10.0.
3.  Use volumetric pipettes to transfer corresponding volumes of the nitrate standard solution to
     each flask or test tube to prepare standard concentrations:
        Standard Concentrations                                            Volume (mL)
            (mg/L NO3)                                                Nitrate Standard Solution
                    0.0                                                                    0.0
                    2.0                                                                    1.0
                    4.0                                                                    2.0
                    6.0                                                                    3.0
                    8.0                                                                    4.0
                   10.0                                                                   5.0

    Before using each pipet the first time, clear by filling once with the standard solution and
    forcing out.
4. Fill the remainder of each tube with distilled water to the 25 mL line.  If using Erlenmeyer
    flasks, measure the water using a graduated cylinder to create a volume of 25 mL.  Swirl to
    mix.
5. Wearing gloves, add the contents of on NitraVer5 Powder Pillow to each standard.  Cover
    with parafilm and swirl for one minute.  Wait 5 minutes.
6. Turn the B&L spectrophotometer on and allow to warm up for 10 minutes.  Set at 400 nm and
    adjust the spectrophotometer.
7. Pour distilled water into a curvette to use for the blank.  Wipe with a Kimwipe and adjust.
8. Pour each of the sample into a curvette.  Wipe with a Kimwipe.  Record the absorbance fo
    each standard.
9. Graph an absorbance versus concentration graph.
 

 SITES:
    Data for this research was collected at 6 different sites in Mercer County, New Jersey.  A variety of environments was incorporated in the sites with a selection from an urban area and several selections from rural cropland and livestock runoff area.

SITE 1 was collected from the Hobler Park bridge over Beedens Brook where the Stoney Brook Millstone Watershed Association is renovating a park by reforestation.  The brook is between a grassland (poa and wheat grass) and a cornfield.

 

SITE 2 was collected from the Rock Brook inlet on Route 601 bridge that flows into Sullivan Lake.  The brook is in a small meadow of sedge grass where 1000s of geese have become established off of Route 601.  South of the area is a suburban environment with a golf course.

 

SITE 3 was collected from Rock Brook at Skillman Dairy Farm, Dairy Farm Drive.  The Skillman Dairy Farm has about 150 head and milks 60 Jersey cows year-round on a 1000 acre farm run by state prison inmates.  Almost 90 percent of the cows are bred via artificial insemination (AI) resulting in a need for only a few "clean up" bulls.  The bull calves are sold and the heifer calves are used in the replacement herd.
 

 

SITE 4 was collected from Rock Brook effluent less than 2 km from the North Princeton Water Treatment Plant. Renovated in the early 1990s, the small treatment plant is a primary, secondary, and tertiary plant established down stream from the dairy.  At the tertiary level, alum is added to neutralize the phosphates and chlorine, to kill bacteria.
 

 

SITE 5 was collected from the headwaters of  Rock Brook that begins in the Sourland Mountain Range in Mercer Co. New Jersey and directly comes in contact with other ecosystems .

 

SITE 6 was collected from the Delaware Canal at the crossing of Alexander Street on the Princeton University campus.

 
  Delaware Canal
 
DATA:
Nitrate Lab Sheet

Name:  Sample Data                                    Date:                                                     

School:                                                        Class:                                                    
 

 
Measurement of Standards
Concentration mg/L as N03-
  
 Absorbance        
 
  Comments 
0.00
 
 
2.00
 
 
4.00
 
 
6.00
 
 
8.00
 
 
10.00
 
 
From these numbers, prepare a standard curve on graph paper and attach this sheet.  Use this curve to find concentration of samples (see directions "Preparing a Calibration Curve and Converting to mg/L")
 

Measurement of Samples
   Bottle #     Site #       Absorbance Conc. mg/L  
(use std. curve)		                   Comments
1   1  N/A  2.4,2.5,2.8 Hobler Park
1   1  N/A 2.7,2.4,2.6  Hobler Park
 2  2 N/A  .8,.7,.6  Rock Brook Inlet 
 2 2  N/A  .5,.7,.6  Rock Brook Inlet 
 3 3  N/A  .4,.6,.5  Skillman State Dairy Farm 
 3 3  N/A  .7,.8,.6  Skillman State Dairy Farm 
 4 4  N/A  4.5,5.0,4.9  Effluent Rock Brook Tx Plant 
 4 4  N/A  5.0,5.2,5.2  Effluent Rock Brook Tx Plant 
 5 5  N/A  .7,.8,.8  Headwaters of Rock Brook
5 5 N/A .8,.9,.9 Headwaters of Rock Brook
6 6 N/A .8,1.1,1.1 Delaware Canal
6 6 N/A 1.0,1.2,1.2 Delaware Canal
 
To get a clean Student Nitrate Lab Sheet: click here
 

 GRAPH:


 

RESULTS & CONCLUSION:
    The Environmental Protection Agency (EPA) has set a maximum contaminant level of 10 mg/mL for nitrate-nitrogen in public water supplies.  This level provides a margin of safety regarding a significant risk for human health.  Nitrates is a relatively non-toxic substance that occurs naturally as part of the nitrogen cycle.  The EPA believes that water containing nitrate-nitrogen at or below the 10 mg/mL level is acceptable for drinking every day over the coarse of one's lifetime and does not pose any health concerns.
    However, nitrates can be converted readily by bacteria into nitrite.  This occurs in the environment, in foods, and in the human mouth and gastrointestinal tract.  Once nitrogen is converted into nitrates, it can have harmful health effects.  For example, as stated in the background, high nitrates in drinking water can cause methemoglobinenia resulting in the reaction of nitrites with hemoglobin in the red blood cells affecting the ability of the blood to carry sufficient oxygen to individual cells of the body.
    Nitrate contaminants in water sources can occur via point or non point discharge.  Water from point sources include industrial and municipal discharge pipes, municipal and private landfills, and chemical storage facilities.  Non point nitrate contaminants are transported in the water by direct runoff from broad portions of the landscape such as urban storm water runoff from buildings and streets and agricultural runoff from rains washing over fields containing farm waste from livestock and fertilizers from fields.  This research project is testing primarily the nitrate levels of non point sources.
    The 6 sites selected for this research project involves a variety of non point nitrate source environments.  From previous research, water collected from rural sites have a higher level of nitrates than urban sites which would support our hypothesis. Therefore, most of the water collected were from rural non point sites.
    Data was collected two times and none of the sites indicated a high nitrate level.  The headwaters of the Rock Brook (site 5), tested with an average nitrate levels of 0.8, was done to compare to the other sites.  The average range of nitrates in the urban area (site 6) was 1.0 mg/mL, which is low according to EPA.  While the average nitrates level of rural areas from cropland runoff was 2.5 mg/mL (site 1), the livestock (site 3) nitrate level was only 0.6 mg/mL which was lower than the headwaters.  The geese inhabited area (site 2) resulted in a level of 0.6 mg/mL.  Surprisingly, the highest level of nitrates in the water was obtained from the effluent of the wastewater treatment plant (site 4) which resulted in an average of 5.1 mg/mL.  According to Heathwaite (1993), nitrates in an effluent are an indication of complete oxidation and stability, being the end product of oxidased ammonical nitrogen.  Further increase in nitrate concentration downstream may be due to biodegradation of ammonia .  This being an unstable form of nitrogen, results in a high potential for further nitrification downstream, whereby biodegradation of ammonia releases more nitrate into the stream.  This could explain the rise in nitrate concentration within the first 2 km downstream that our data shows.  Consequently, a monitoring of nitrite and ammonia concentrations, not just nitrates, should be done since incomplete bioxidation of organic nitrogen is likely to release unstable ammonia with a high potential for further decay.
    According to EPA, the nitrate concentrations from all our sites do not pose any danger or health hazards. Our recommendations are: Soil Nitrate Tests and Water Quality:
APPLICATION OF STANDARDS: click here
 
RESOURCES:
Behar, Sharon.  Testing the Waters- Chemical and Physical Vital Signs of a River.  Dubuque:
   Kendall/Hunt Publishing Co., River Watch Network, 1997.
Havel, John, Christopher Barnhart, and Janice Greene.  The American Biology Teacher.
   "Experimental Investigations of Water Quality: The Bioassay".  Vol. 59; June, 1997.  (Bioassay).
Mitchell, Mark, and William B. Stapp.  Field Manual for Water Quality Monitoring.  Dubuque:
   Kendall/Hunt Publishing Co., 1996.
National Science Education Standards.
Standard Methods for the Examination of Water and Wastewater.  US Government, 1992.
Strategic Diagnostics Inc., 128 Sandy Dr., Newark, DE 17713.  800-544-8881. (Omhicrom).
See other interesting  projects about nitrates from the Woodrow Wilson Environmental Science Summer Institute:  Team #3:  Nitrates and golf courses: a comparison between agricultural and suburban areas; Team #11:  Nitrate levels in agricultural and suburban areas; Team #25:  Nitrates in agricultural, suburban and recreational areas; Team #38:  Wetlands and Nitrate biofilters; Team #50:  Nitrates and soil effects on water down stream; and Team #55:  Fertilizers and levels of nitrates in streams.
 
ACKNOWLEDGMENTS:
Team #20 wishes to acknowledge those people who were instrumental in the compilation and completion of our research based project. The time and effort to put a research based project of this nature take many hours of frustration and learning of new techniques to complete.   Thanks to:
REFERENCES:

Wayne Cowell                                                       Nancy Gettman
Biology Teacher                                                      Science Educator (all disciplines)
Standley Lake High School                                     Woodlin School District R-104
Westminster, CO  80021                                        Woodrow, CO  80757
(303) 982-3311                                                      (970) 386-2223
(303) 982-3312 FAX                                              (970)386-2241FAX
wcowell@jeffco.k12.co.us                                       gettman@iguana.ria.net
 

Mark Little                                                            Elmer Romero
Biology/Anatomy Teacher                                       Life Science/Physical Science Teacher
Broomfield High School                                          Bilingual Education
Broomfield, CO  80020                                           Hill Middle School
(303)466-7344                                                        Denver, CO  80220
(303)447-5390 FAX                                               (303)399-0254
little@bvsd.k12.co.us                                              (303)764-6844 FAX