Qualitative Nitrate Detection for Toxicity Potential
George B. Garner
Department of Biochemistry
This test is designed for survey use and for advising clients of the need
for quantitative nitrate analysis when the level of nitrate detected could possibly
cause nitrate/nitrite poisoning. The test can be used on water supplies, hay,
silage, fodder, pasture samples and mixed ground feeds.
Preparing the reagent
The reagent is prepared by placing diphenylamine (0.5 grams) in a graduated
cylinder or flask containing 20 milliliters of distilled water. While stirring, slowly add
88 milliliters of concentrated sulfuric acid (H2SO4).
This will dissolve the diphenylamine and generate heat.
Allow the reagent to cool, then place it in a clean dropper bottle. Since
the reagent contains sulfuric acid, it must be handled with caution. Burning
of skin, charring of clothing, etc., can best be stopped by washing with water
or, even better, with baking soda (sodium bicarbonate).
If, over time, the reagent becomes highly discolored, brown or blue, discard
it. The brown color is caused by foreign matter and blue is probably due to
touching the sample with the dropper tip. Don't allow the dropper tip to contact
Diphenylamine crystals in 0.5 grams per packet are available upon request
from the MU Department of Biochemistry. Sulfuric acid solutions are not mailable.
Distilled water and sulfuric acid are available at area soil testing laboratories.
Testing solid substances
To acquaint yourself with the test, split a normal appealing corn stalk and
test each node from the ground up with a few drops of reagent. The concentration
should be highest at the lower end and become less toward the top of the stalk.
If ample nitrogen has been available, a dark blue spot would be expected to
develop almost instantaneously at the first node. Each node toward the tassel
should show a light blue spot or take longer to become dark blue.
When an ear has formed and proper nitrogen fertility is present, the node
above the ear should be very light blue and a brown color may begin to appear.
The brown coloration indicates carbohydrates and may occur anywhere in the plant
if nitrates are absent. Only a blue, blue-black color is a measure of nitrate
All gradations of blue color from light blue to blue-black are possible.
A rating system of 1+, 2+, 3+ may be useful to correlate with quantitative data
on samples tested.
For testing silage, hay and pasture samples, two procedures may be used.
The simplest is to drop the diphenylamine reagent on the cut cross section of
the stem or pith and observe the color. The second method is to squeeze out
liquid from silage or pasture samples onto a white spot plate and add a few
drops of the reagent. With dry silage and hay, enough water should be added
to moisten the sample throughout and then express the liquid and test as described
Pour a few milliliters of water into a clean test tube and carefully pour
1 to 3 milliliters of the diphenylamine reagent down the side of the slanted
test tube. If a blue color does not appear at the interface of the water and
sulfuric acid within 30 seconds, hold the test tube between the thumb and index
finger and tap gently to start a swirling action. Approximately 1/2 of the reagent
layer should be left. Samples that do not show blue coloration at the interface
within 30 seconds are quite low in nitrate (less than 20 ppm). Expressed liquids
from silage, hay and pasture samples can also be handled this way, but they
take more time than the spot plate.
Interpreting the test
The moisture content of material to be tested markedly affects the interpretation.
Wet silage may show less blueness than dryer silage but still have equal nitrate
For silages, since the nitrate content is higher in the stem or stalk
at time of harvest, sort out several stems or pieces of pith for testing. Drop
reagent on the material and observe.
If the silage is moist enough, express a few drops on a spot plate to test.
A light blue color indicates some nitrate present. A rapidly developing dark
blue color that appears nearly black indicates the silage should be analyzed
quantitatively for nitrate. To further test, take the leafy portion of the silage
and express a drop of liquid from it. Add reagent. If it rapidly develops a
dark blue-black color, feed the silage cautiously until a quantitative test
For green chop, a few drops of liquid usually can be expressed and
tested. Rapidly developing dark blue and black color indicates a level of nitrate
that requires a quantitative analysis. For corn that has a small to large normal
ear, split the stalk and test by nodes. If only a weak test is observed above
the ear node, there will be a dilution of at least half of the nitrate in the
base nodes nitrate when chopped. No problems should be anticipated in direct
feeding or ensiling.
As long as the crop is green and has not been severely drought damaged (whitish-green
to gray leaves), normal ensiling will further reduce the nitrate. There will
be some reduction of nitrate between the time of cutting and feeding. Overnight
storage on wagons is not recommended because nitrite could build up. Chop and
feed as needed. There is one exception: where cyanide (prussic acid) is a problem
in some sorghums, overnight storage on wagons or trucks would result in loss
Dried fodders and hays
Test stems and stalks. If dark blue-black color rapidly develops, check
the leaves. If leaves are also very positive, make a quantitative analysis before
feeding the forage or feed it cautiously.
Test stems of plants. If stems test positive, then test leaves. If
both are positive, excessive nitrates are present and quantitative analysis is
needed along with cautious feeding. Sudan, Sudex, etc., are more likely to be
a problem than cool season grasses. You may occasionally find that grasses in
old feedlots or well fertilized pastures are a problem
Mixed ground feed
Grains would be free of nitrates as measured by this reagent. Contamination,
accidental addition of nitrate salts to rations, and the use of beet pulp or
molasses with high nitrate content would be the most likely cause for a positive
test. Moisten the feed to paste consistency on a white spot plate and drop reagent
on the edge of the paste. Observe the color of the reagent. Only a blue color
is positive because the high sugar-starch content of the feed will react with
the sulfuric acid to form a charred mass that is black and impart a brown color
to the excess reagent.
Water should be tested by using a test tube and layering of the reagent.
A small light blue ring at the interface indicates between 20 and 30 ppm of
nitrate (NO3-). A dark blue ring indicates at least 50
ppm. If the entire reagent layer becomes dark blue, the nitrate level is in
excess of 100 ppm nitrate. U.S. Public Health recommended safe limit is 45 ppm
nitrate. Pond water should be negative by this test. Often a green chlorophyll
color will be seen if there is algae in the water. Whenever nitrate is detected
in pond water, there is a source of contamination (be careful not to test suspended
soil particles), and that source should be determined. If the source is removed.
the organisms in the pond will soon use up the nitrate.
Water testing warning
Nitrite at a few parts per million causes the reagent to become a violet-blackish
color, usually seen as violet to greenish above the dark black ring. If reagents
are available, make a test for nitrite. Pond water and water from livestock
tanks or other non-sterile holding basins (cisterns) often contain water with
nitrite present if nitrate contamination is also present.
Sampling should be done carefully. The analysis can be no better than the
Collect water in a clean, sterile bottle (2 ounce minimum). When collecting
from a water system, allow enough flow to replace all the water in the lines
and dilute out the pressure tank. Then take the sample.
Ponds may be sampled from the drain pipe. In unimproved ponds, sample so
as to avoid mud contamination.
Whenever possible, send in a core sample from 15 to 20 bales. (Use
Penn State Sampler or equivalent). For loose hay or without bale sampler, open
10 to 15 bales and take a grab sample of approximately one handful from each.
Cut the forage into 2- to 4-inch pieces, being careful to preserve all leaf material.
Pack tightly in a container.
Silage and fresh grass
Pack the sample (approximately 5 pounds) in a plastic bag. This sample
should be taken from a composite sample of at least four areas in the silo or
Samples should be taken or sent immediately to the laboratory. If transit
time is more than a few hours, freeze the samples of water, silage, fresh grass
or green chop to minimize bacterial action. Otherwise, test results will be
lower than in the water or feed offered to livestock.
Chemical preservatives such as mineral acids, chloroform, toluene, etc.,
should not be added unless they have been cleared by the laboratory doing the
G9811, reviewed October 1993