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FACTORS AFFECTING

HE ACCUMULATION OF^NMATE

IN SOIL, WATER, AND PLANTS

Agriculture Handbook No. 413

Ü. S. DEPT. OF A^v

NATIONAL A'ir;!:!.'!T' '

FEL "

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Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE
FACTORS AFFECTING

THE ACCUMULATION OF NITRATE

IN SOIL, WATER, AND PLANTS

By FRANK G. VIETS, JR., and RICHARD H. HAGEMAN

Agriculture Handbook No. 413

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE

Washington, D.C. Issued November 1971

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402 — Price 65 cents
This publication reports research involving pesticides. It does not contain recommendations for their use, nor does it implj^ that the uses discussed here have been registered. All uses of pesticides must be registered by appropriate State and/or Federal

agencies before they can be recommended.

CAUTION: Pesticides can be injurious to humans, domestic animals, desirable

plants, and fish or other wildlife^—if they are not handled or applied properly. Use

all pesticides selectively and carefully. Follow recommended practices for the disposal

of surplus pesticides and pesticide containers.

«MMiiit or Actieuuuii

III
CONTENTS

Page

The Nitrate Problem __-. -. 1

Human and Animal Health Aspects - 1

Agricultural and Aquatic Aspects 3

In Ensilage and Food Preservation - 3

Nitrate is Not a New Problem 5

Nitrate and the Nitrogen and Carbon Cycles — 6

Natural Accumulations of Nitrate 7

Accumulations in Caves, Caliches, and Playas ^ 8

Agricultural Accumulations Before the Fertilizer Epoch ~ 10

The Sources of Nitrogen and Nitrate ^ 12

Geologic Deposits ^ 12

Soil Organic Matter, Peat and Muck 13

Livestock and Poultry Wastes ^ 16

Domestic Sevi^age - 17

Commercial Fertilizers ^ 18

Nonconcentrated Sources - - 21

Nitrate Transport and Accumulation in Relation to Hydrology and Water Movement 22

In the Root Zone „____ 22

In the Zone Below Roots and Above the Aquifer 24

In Aquifers 25

Recent Field Investigations on Nitrate Accumulation and its Sources 26

In Arid and Semiarid Regions 26

In Humid Regions 31

Nitrate Concentration from Water Passage Through Soil 35

Nitrate Absorption, Reduction, and Accumulation in Plants - - 35

Nitrate Absorption 35

Evidence for Active Uptake of Nitrate „ 36

Influence of Companion Ion on Nitrate Absorption 36

Effects of pH on Nitrate Absorption and Conversely 36

Nitrate Absorption as Related to Plant Families, Specie, and Genotype 38

Nitrate Reduction -_-_ 39

Characteristics of Nitrate Reductase 39

Genetic Variation _. 42

Intraplant Distribution of Nitrate Reductase 42

Accumulation of Nitrate and Factors That Affect Accumulation 43

Nitrate Availability „ , 48

Drought and Temperature 48

Light 48

Mineral Deficiencies . 48

Endogenous Metabolites . 49

Herbicides 49

Time Trends of Nitrate Accumulation „ 50

Foods and Feeds 50

Soil and Water „ . „ 51

An Appraisal 53

Literature Cited ^ 55

IV
Factors Affecting The Accumulation of Nitrate in

Soil, Water, and Plants

By PRANK G. VIETS, JR., Chief Soil Scientist, Soil and Water Conservation Research

Division, Agricultural Research Service, and RICHARD H. HAGEMAN, Professor of

Agronomy, University of Illinois, Urbana

THE NITRATE PROBLEM

Human and Animal Health Aspects

Some environmentalists and ecologists say we

are faced with a nitrogen (N) crisis resulting

from use of commercial nitrogen fertilizers and

the release of nitrogen oxides by high temperature combustion in power plants and internal

combustion engines. They point to the high nitrate content of many water sources and to high

nitrate in food, particularly some canned baby

foods, as causes for real alarm. Commoner^ in

his address to the American Association for the

Advancement of Science called for elimination

of fertilizer N use for 10 years. In his paper

'The Balance of Nature," Commoner (1968)^

has been one of the most vociferous critics of

present technology and practices.

Nitrate upon reduction yields nitrite

(N02~), which is commonly regarded as 10

times as toxic as nitrate in water, food, and

feeds for equal concentration. Nitrite also can

be produced as a lag phase in the oxidation of

ammonium to nitrate by microorganisms. Not

all of the biochemical effects of highly reactive

nitrite are known. Nitrite has deleterious effects on red blood cells, possibly on muscle tis-

^ Commoner, B. Threats to the integrity of the

nitrogen cycle: nitrogen compounds in soil, water and

atmosphere and precipitation. Presented at the Dallas,

Tex., meeting of the American Association for the Advancement of Science. December 1968. In GLOBAL EFFECTS

OF ENVIRONMENTAL POLLUTION. S. F. Singer, Ed.,

Springer-Verlag, New York, pp. 70-95. 1970.

^ The year in italics after the author's name is the key

to the reference in Literature Cited, p. 55.

sue, and has been implicated in cancer. Nitrite

can overoxidize the ferrous (Fe+ + ) iron of

hemoglobin to ferric (Fe+ + + ) iron. The resulting methemoglobin in red blood cells is incapable of carrying oxygen. Nitrite can also combine

irreversibly with hemoglobin to form nitrohemoglobin which, like carbonmonoxyhemoglobin,

cannot carry oxygen. Nitrite can also combine

with the myosin of muscle tissue. Gruener and

Shuval (1970) gave a good short review of the

relation of nitrate to acute and chronic subclinical methemoglobinemia and possible effects on

fetal development. Winton (1970) discussed nitrate in water in relation to methemoglobinemia

of infants and the complicating factors of bacterial infection, genetic imperfections, and formula preparation involved in setting standards

for water quality.

If forages like corn (Zea mays) are high in

nitrate and are put in a silo, denitrification can

lead to the production of NO (colorless), N2O

(yellow), and N2O4 gases that are heavier than

air. The gases accumulate in silo drains, chutes,

and buildings that are attached to the silo.

These gases can be lethal to man and animals

unless fencing and adequate ventilation are provided (Hanway et al., 1963; Crawford and Kennedy, I960).

Nitrite reacts with secondary amines in an

acidic environment to produce nitrosoamines.

These compounds have received recent attention

because they can be carcinogenic, teratogenic,

or mutagenic (Lijinsky and Epstein, 1970),

DuPlessis and coworkers (1969) reported that

the high incidence of esophageal cancer in
AGEICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

Transkeian Bantus is related to high nitrate

levels in the maize, pumpkins, and beans that

they eat and have identified dimethylnitrosoamine in the fruits of Solanum incannum.

The juice of this fruit is used to curdle milk to

produce the curds eaten by the head boys. The

authors claim the plants are high in nitrate because of molybdenum deficiency in the area.

However, Malins and coworkers {1970) could

not detect more than 10 p.p.b. of dimethylnitrosoamine in aqueous systems of secondary

amines and nitrite much more acidic than those

existing when smoked chub salmon is preserved

with sodium nitrite.

The reader is reminded that nitrate is reported in many different ways. Much confusion

results from lack of uniformity. Units used

have been NOs", NO3--N, KNO3, and NaNOg.

Forty-five p.p.m. NO3" equals 10 p.p.m.

N03~-N. One p.p.m. equals 1 mg. per liter.

Like standards for radiation exposure, DDT

in foods, and safe speed on the highway, standards for acceptable levels of nitrate in drinking

water, canned foods, and animal forages are

highly controversial. The U.S. Department of

Health, Education, and Welfare's Public Health

Service {1962) standards call for less than 10

p.p,m. of N03~--N (45 p.p.m. NOg") in drinking water, 50 p.p.m. nitrate (dry weight basis)

in asparagus, 3,000 p.p.m. nitrate in spinach

(dry weight basis), none in squash and tomatoes for baby foods, 83 p.p.m. nitrate in

strained spinach for babies, and 200 p.p.m. of

nitrate or nitrite in preserved meats, and fish.

The California State Department of Public

Health notifies public water districts and the

local health officer when a source of supply exceeds 45 p.p.m. NOs" and urges the utility to

reduce the level. When the water has more than

90 p.p.m. NOs" in any significant portion of the

community, local health officers are requested to

notify all local practicing physicians of the

risks and hazards involved, and the water purveyor is required to develop a special sampling

program to determine the extent and levels of

nitrate in the distribution system. The Policy

for Administrative Procedures further states

that if the water supply continues to exceed 90

p.p.m. N03~ that each case will be specifically

investigated to determine an appropriate course

of action (Ward, 1P70).

The U.S. Department of Interior (IP<?S)

Water Quality Criteria set 10 p.p.m. NOs'-N

plus N02~-N as permissible in drinking water

for humans but set the desirable level at "virtually absent." No NOs" standards for water

for animals were established. Keller and Smith

{1967) said that water with 50 p.p.m. NO3--N

(perhaps 10) should be avoided for livestock.

Case {1970) gave a good discussion of animal

health in relation to nitrate N. The permissible

levels of nitrate in animal forages are far from

established (Crawford and Kennedy, 1960;

Wright and Davison, 196Jf; Hanway et al.,

1968). What the standards ought to be is outside the scope of this review, but to argue that

the current standards are too low is of little

comfort to those interested in the hazards of nitrate accumulation in soils, waters, and foods.

However, the present standards for nitrate in

potable water should not be considered closed

against réévaluation on the basis of new information. Many of the cases of infant death attributed to high nitrate may be caused by a

combination of nitrate, enteric infection, and

debility that arose from wells with fecal contamination. Winton {1970) stated that present

U.S. standards for public water supplies are

under review, but he cautioned against lowering

the standards simply because there are methods

of preparing baby formulas with waters higher

in nitrate than the limit now recommended in

the standards.

Gruener and Shuval {1970) stated that vitamin C can prevent or cure methemoglobinemia, and that use of citrus juices for infants

in Israel may explain the scarcity of the disease

in areas with high nitrate in drinking water.

What the future permissible level of nitrate

in potable water ought to be is a very important

consideration in the event that a substantial

buildup of nitrate in underground water supplies cannot be reversed without drastic measures. Stout and Burau {1967) were rather pessimistic about the Grover City-Arroya Grande

Basin in California they studied. They suggested that eventually many areas may have to

have two water supplies—one for drinking and
ACCUMULATION OP NITRATE IN SOIL, WATER, AND PLANTS

one for other uses. In California, some water

districts mix water from different wells and

surface sources to stay under the recommended

standard (Ward^ 1970), Israel is trying to

maintain N08"-N below 10 p.p.m. in the entire

National Water System. This is being attempted by mixing water from ground-water

supplies with the low nitrate water from the

Jordan sources.

Agricultural and Aquatic Aspects

In the management of N in the environment

to prevent the undesirable accumulation of nitrate above tolerable levels, several other aspects of nitrate must not be forgotten. Growth

of all organisms requires nitrogen. The photosynthetic organisms, whether they be crops on

land or phytoplankton and algae in water, require nitrogen either as ammonium or nitrate,

although they may use some simple soluble organic nitrogen compounds. Hence, ammonium,

total soluble nitrogen, and nitrate cannot be

considered independently. Some species of bacteria and blue-green algae are capable of fixing

atmospheric N2 to meet their requirements.

Their inputs must be considered in both land

and water environments.

All crops, grasses, and forest lands must have

one of these forms of nitrogen available to the

plant roots during vigorous vegetative growth.

Most of the nitrogen absorbed is probably absorbed as nitrate because ammonium is so

quickly oxidized to nitrate by the nitrifiers in

soil. In the United States, we use about 7 million tons of nitrogen annually to maintain soil

productivity. The world agricultural consumption is about 25 million tons. Too much absorbable nitrogen in soil, whether it comes from too

rapid oxidation of soil organic matter, too much

animal manure, or too high application rates

of commercial fertilizers can cause lodging in

cereals, low sugar in sugar crops, and deterioration of quality of fruit and vegetable crops.

Further, excessive nitrogen can be a hazard to

quality of underground water because of the potential nitrate loss by leaching. Nitrate poses no

danger to the soil and its properties, but excessive accumulation is generally associated with a

salinity problem.

The terrestrial situation is paralleled by that

in lakes and streams. Too little nitrogen can result in poor growth of photosynthetic plankton

and algae—the basis of the food chain that supports fish. The water is oligotrophic. A "biological desert" results. On the other hand, too much

nitrogen, along with sufficient amounts of other

nutrients, can result in overfertilized or eutrophic water that supports nuisance blooms of

algae. Nitrate, as well as ammonium and simple

soluble organic nitrogen compounds, can be assimilated by aquatic plant life. In studies of

lake eutrophication, the standard is usually

total soluble nitrogen rather than nitrate. Big

mistakes can be made in surface water studies

by just considering nitrate or ammonium.

In Ensilage and Food Preservation

Preservation of vegetation, fodder, or foods

of high nitrate content pose potential problems.

These problems are caused by nitrate which is a

good oxidant and which may react with reductants in the stored material, environment, or

with the container. The partly reduced products

formed from the nitrate are far more toxic than

the nitrate itself. The products formed from the

reductant may also be undersirable.

Fodder or vegetation of high nitrate content

converted to silage loses variable amounts of its

nitrate content during fermentation. Kurtz and

Smith (1966) estimated that, in general, half of

the nitrate is lost from the silage ; however, the

loss is variable and depends upon several factors. For example, silage made from material of

55-percent moisture content lost only 20 percent

of its nitrate, while material ensiled at 80-percent moisture lost from 61 to 98 percent (Jacobson and Wiseman, 1963). The addition of sugar

to high-moisture silage or laceration of the material before storage decreases nitrate loss.

The fermentative processes that occur in the

ensilage including denitrification, in general,

have been assumed to result from microbial

contamination. The fact that many microorganisms, such as Escherichia coli and Pseudomonas

aeruginosa, have a dissimilatory nitrate reductase that uses nitrate as a substrate lends credence to this view (Hewitt and Nicholas, 196Í).

However, Wang and Burris (1960) found that
AGEICULTÜRE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

silage made from asceptically grown soybean

seedlings produced the same kinds of nitrogen

oxides as nonsterile silage.

Mulder et al. (1959) and Klepper and Hageman {1969) showed that under anaerobic conditions the endogenous nitrate reductase of leaf

tissue uses carbohydrate material to reduce nitrate only as far as nitrite. Thus, nitrate reductase endogenous in the silage material could explain : (a) The production of oxides of nitrogen

by asceptic material ; (b) the greater loss of nitrate from high-moisture silage (based on the

fact that the greener, less mature, tissue would

have higher levels of enzyme) ; and (c) the decrease in nitrate loss resulting from laceration

of the tissue before storage, as this would tend

to inactivate nitrate reductase.

It is conceivable that the addition of sugar

decreases loss of nitrate by enhancing the fermentative production of acids that inhibit nitrate reduction or reactions that generate energy for nitrate reduction. The intent of this

section is to suggest that if nitrate reductase is

present in the ensiled material, it may act alone

or in conjunction with the nitrate reductases of

contaminating organisms to convert nitrate to

nitrite. Nitrate can be subsequently converted

to nitric oxide, nitrous oxide (the toxic silo

gases), and ultimately nitrogen. Although the

initial conversion of nitrate to nitrite is thought

to be biological, some of the subsequent interconversions are chemical.

Forages, such as oat hay, beet tops, and rape,

having high-nitrate content, are also potential

sources of nitrite during the postharvest curing. If forages of high-moisture, -nitrate, and

-nitrate reductase are handled in a way to cause

anaerobic conditions, the endogenous enzyme

systems can convert nitrate to nitrite. In contrast, forages stored under aerobic conditions

are more likely to convert nitrate to ammonia

(nitrate assimilation). Under aerobic conditions that are conducive to drying-^—low humidity, or high (30° to 40"^ C.) temperature—the

endogenous nitrate reductase will denature in

most tissues within a few hours. Nitrate loss

from such tissue would then be minimal. Forages containing high amounts of nitrate, adequate carbohydrate, and moisture (initial or

from subsequent precipitation) can become contaminated with microorganisms that can utilize

the nitrate as an electron acceptor (dissimilatory enzyme) and produce nitrite. While forages of excessively high-nitrate content can

cause nitrate toxicity (especially with ruminants), proper postharvest management is required to prevent conversion of the nitrate to

nitrite by endogenous nitrate reductase or contaminating microorganisms.

Reviews of the denitrification process, general aspects of the production of toxic "silo

gases," and practical management practices to

minimize nitrate toxicity in ensilage are given

by Petersen et al. (1958), Wright and Davison

(196A), and Kurtz and Smith (1966), respectively.

Foods preserved for human consumption that

frequently contain high levels of nitrate are

leafy vegetables (such as spinach), beets, and

to a lesser extent, green beans, carrots, and

tomatoes. Three major problems that are associated with the preservation of foods of high nitrate content are: (1) The adverse effects of nitrate per se ; (2) the detinning effects of nitrate

when foods are preserved in tin-lined cans ; and

(3) the production of highly toxic nitrite from

the nitrate by contaminating microorganisms

once the cans or frozen foods are opened or

thawed. Nitrate content of preserved foods per

se is not considered a major problem to adults

on a balanced diet.

The presence of nitrate in foods preserved in

tin-lined cans has been associated with the '*detinning" of cans and shelf life. Since these

foods are heat-treated and sterilize^, the oxidation reduction reactions associated with detinning of the sealed cans are chemical in nature.

The reduction products of nitrate are primarily

ammonia, although nitrous oxide and nitrogen

have also been identified in the headspace of

canned foods.

In canned whole tomatoes or tomato juice, it

has been shown that tin removal continues until

either the nitrate or the tin coat is exhausted.

The presence of nitrate in canned carrots,

beans, and spinach has been associated with detinning. However, the initial concentration of

nitrate in these products is not so reliable an

index to the degree of detinning as with tomato.

In beans, carrots, spinach, and tomatoes, the
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS

detinning process is more complex and dependent upon more acidic pH and oxalate content

(at least for spinach) of the pack. The use of

higher levels of phosphate fertilizer in the production of green beans, carrots, and spinach appears to retard detinning. Currently it is not

known whether this effect is caused by increased levels of inorganic phosphate in the

pack, or metabolic alterations in pH and oxalate

content of the product to be processed.

These detinning problems can be avoided by

use of enamel-lined cans, glass jars, and freezing the product and processing in plastic or

lined cardboard containers. However, these

treatments do not reduce the nitrate content of

the produce. Farrow, Charbonneau, and Lao

(1969) gave a more comprehensive coverage of

the detinning process and associated factors.

The problem of conversion of nitrate to nitrite by microbial contamination arises when

canned or frozen food products are opened and

thawed and not maintained asceptically until

the food is consumed. Simon (as reported by

Lee et al., 1970) showed that high levels (200

mg. N02~/l.) can be produced from nitrate of

cooked spinach juice held under nonsterile conditions at 20 "^ C. for 24 hours. Nitrite can also

be formed in vivo (up to 320 mg. NO2"/100 g.

fresh tissue after 4 days of storage) in fresh

spinach during transport and storage. In processing high-nitrate foods for consumption, discarding of the cooking water is recommended

because both nitrate and nitrite are water soluble.

Nitrate is Not a New Problem

The deleterious effects of excess nitrate on

human and animal health is not a new discovery. Nitrates were used for a long time in internal medicine as depressants, but were phased

out because of their severe depression of the

heart. Sea captains were claimed to have used

saltpeter as an anaphrodisiac.

Many western U.S. creeks were given unflattering names by the miners because of the

pharmocological properties of the water. According to U.S. Public Health Service (1962),

nitrate in drinking water was first proved to

produce serious changes in the blood chemistry

of infants in 1945 by Comly (19U)^ From 1947

through 1950, 139 cases of methemoglobinemia,

including 14 deaths caused by nitrate farm

water supplies, were reported in Minnesota

(Bosch et al., 1950), Lenain (1967) stated: ''In

1949, a nine-state survey revealed 262 cases of

infant cyanosis. In all but nine, nitrates were in

excess of 50 p.p.m. Minnesota revealed 139

cases, including 14 deaths, associated with nitrate contaminated wells. These were rural,

none connected to a public system. Cases of infant cyanosis related to high nitrate wells have

been reported in many parts of the world, particularly in U.S. and the central prairies of

Canada."

Schwüle (1969) reported that the ground

waters of the Mosel River Valley have high nitrate levels, but that even adjacent wells have

big differences. He says no cases of methemoglobinemia have been reported from this area,

and only two cases have been reported from the

entire Rhineland area.

Keller and Smith (1967) stated: ''Ground

water containing nitrate (including nitrite) in

toxic concentrations, occasionally high enough

to be lethal to human beings and livestock when

the water is used regularly, has been observed

in Kansas, Minnesota, Wisconsin, Iowa, Illinois, Michigan, Montana, Pennsylvania, and

elsewhere as an awareness of [nitrate] NO3""

poisoning increases." Gruener and Shuval

(1970) stated that about 2,000 cases of infant

methemoglobinemia have been reported in various countries since its first clinical recognition

in 1945 and cited clinical studies on it in the

U.S.S.R. and Czechoslovakia.

California has never had a reported case of

infant cyanosis caused by drinking water. Ward

(1970) also reported that "In 1962, eighty-five

community water systems under the jurisdiction of the California State Board of Public

Health were serving water at some time during

the year which had nitrate concentrations exceeding 45 mg./l. in the whole or in part of a

distribution system." Eighteen communities

now have the problem. Their populations range

from 2,000 to 32,000.

In the United States, no deaths from nitrate

in canned baby foods have been recorded ; but, in

Europe several cases have occurred.
AGRICULTURE HANDBOOK 418, U.S. DEPT. OF AGRICULTURE

There are many recorded instances of animal

deaths resulting from ingestion of high nitrate

forages, often produced under drought conditions. Mayo (1895) reported cattle deaths from

pasturing drought-stricken corn in Kansas 76

years ago. Case {1970) discussed the subclinical

effects of nitrate in water and forages on different kinds of livestock and the dearth of adequate statistics on deaths and harmful effects.

We have not been able to locate maps or compilations of statistics of NO3" concentrations in

ground waters, cases of methemoglobinemia, or

of the incidence of livestock ailments so that we

could relate them to other factors even qualitatively, or to determine whether any longterm

trends of general nitrate accumulation have occurred in ground water in the United States or

Canada. The infant cyanosis problem appears

to have been more acute in the 1940's and the

early 1950's than it is today. ''Public Health Reports" published monthly by the U.S. Public

Health Service carried only one article on nitrate and methemoglobinemia in the period

1960-69, inclusive. That 1962 incident involved

cyanosis of a baby on a formula prepared with

a municipal water supply in Colorado. According to Winton {1970), the child's mother had

not heard the warning given to physicians

about use of the water for babies. When the boy

developed diarrhea, the mother boiled the water

for the formula longer and concentrated the nitrate. The child survived after intravenous administration of méthylène blue.

The apparent lower incidence in the past 15

years may be related to a number of factors:

(1) More people use water from safe public

supplies (2) more adequate testing and construction of rural wells to protect against fecal

contamination, and (3) more widespread use of

citrus juices in babies' diets and general improvement of their health. Thus, the apparent

clinical picture cannot be interpreted to mean

that underground water qualities, in general,

have improved. Another conclusion, which cannot be fully documented, is that the central and

upper Midwestern States have had high incidences of nitrate problems in comparison with

the rest of the Nation. As will be discussed

later, this incidence may be related to the natural high organic N content of the soils and to

the long residence time of ground waters resulting from the close balance of precipitation and

évapotranspiration. Another area with high

NOs" in ground water is the San Joaquin and

other valleys in California (Ward, 1970):

In Israel, Gruener and Shuval {1970) reported that a definite upward trend in nitrate in

wells has occurred in the central and southern

coastal plains and the problem has now reached

serious proportions. In this densely populated

area, about 180 water supply wells now have

NOg^-N concentrations equal to or in excess of

10 p.p.m. Another 50 wells, now below this

limit, are increasing in this concentration at the

rate of 0.44 p.p.m. or more per year.

NITRATE AND THE NITROGEN AND CARBON CYCLES

Nitrate accumulation or depletion from soil

and water cannot be understood without considering the pertinent parts of both the nitrogen and carbon cycles. Water is important too.

Many misinterpretations of data on nitrate are

possible if concurrent changes in ammonium

concentration, oxidizable carbon compounds,

biological oxygen demand, and oxygen supply

are not simultaneously considered. The flux and

direction of water movement, and sometimes

the water suction, are important in transformations. The September 1970 issue of Scientific

American had excellent summaries of the nitrogen, carbon, oxygen, and water cycles.

Only the most pertinent aspects are briefly

stated here. Nitrate in either soil or water is

formed by the oxidation of ammonium ion to nitrite by Nitrosomonas spp. and of the nitrite to

nitrate by Nitrobacter spp. These bacteria derive energy from the oxidation. Oxygen and favorable moisture and temperatures are required. Nitrification cannot occur if the soil is

waterlogged so that oxygen becomes depleted. It

cannot occur in soil or water loaded with oxidiz-
ACCUMULATION OF NITKATE IN SOIL, WATER, AND PLANTS

able organic compounds, such as sewage or animal wastes, to the extent that molecular oxygen

is depleted. Nitrite can accumulate in soil as a

lag phase in nitrification under alkaline or other

conditions unfavorable to Nitrobacter spp.

Ammonia either in soil or water can come

from ammonium fertilization, air, or rain, but

one of the main sources is the oxidizable nitrogen-containing compounds contained in animal

and plant waste, sewage, and soil organic matter. Favorable temperatures and moisture are

required for the host of microorganisms that accomplish ammoniñcation, either with or without oxygen. Oxygen will be used as long as it is

available. Both the ammonium released and the

biodegradable organic matter are part of biological oxygen demand (BOD).

Denitrification is the reduction of nitrite or

nitrate chieñy to N2 gas, but some gaseous oxides of nitrogen may be formed. This process

requires favorable temperatures and absence of

molecular oxygen, the nitrate and nitrite giving

up their oxygen during nitrate respiration of

organisms.

Nitrate data obtained on open bodies of water

are difficult to interpret because nitrate concentration is affected by supplies of oxidizable

carbon and dissolved oxygen and by nitrate use

by green plants of all kinds. Although the NOa"

concentration in itself may be of significance in

water supply systems, inferences about sources

of pollution, total N load and time trends are almost useless without the associated data needed

for interpretation. Just as the nitrate in a soil

can be depleted by a plant, so can nitrate in a

lake, river, or an open agricultural drain be depleted by growth of algae and phytoplankton.

Just as nitrate in a soil can be depleted by mixing crop residues with high C :N ratios, such as

straw in the soil, so can nitrate in a water body

be depleted by "slugging" the water with material with high BOD demand. Both in water and

in soil, NO.s" will appear again under favorable

temperature conditions when the oxidizable carbon is depleted, oxygen returns to the system

from the air, and ammonification and nitrification can occur. Perhaps one of the best illustrations of the fluctuations in nitrate for a lake is

the data of Serruya and Berman (1970) on eutrophic, temperature and chemically stratified

Lake Kinaret (Sea of Galilee). They found a

range of N03~-N in this constant volume lake

of from zero to about 2,600 metric tons over a

4-year period with definite seasonal influences

apparent. Nitrate comes from the Jordan River,

mostly in the spring, and from the oxidation of

ammonia released from the hypolimnion on fall

turnover. Highest NOs"" occurs in the spring.

This nitrate is assimilated in the summer by

photosynthetic organisms or is denitrified in the

hypolimnion when the lake restratifies.

NATURAL ACCUMULATIONS OF NITRATE

The accumulations of nitrate in soil, water,

and geologic strata can be divided into three

time periods for convenience in assessing the

impacts of modern technology. We have chosen

to discuss the data in three time classifications :

(1) The accumulations related to ''geologic"

time before there was much impact of man ; (2)

the period before much commercial fertilizer

was used, which terminated after the end of

World War 11; and (3) the period of accelerating commercial fertilizer use since 1945. Only

the first two periods are discussed in this section. Section on Recent Field Investigation on

Nitrate Accumulation and its Sources (p. 26)

covers the third period.

These are arbitrary classifications, as nitrate

in the environment has multiple sources from

geologic deposits, decomposition of soil organic

matter and peat, animal and human waste, and

fertilizers. In a chronology, each new source of

input is merged into the previous sources. Imperfect and as arbitrary as it may be, this classification may give the reader some appreciation of the trends with time. An opinion about

trends can only be a subjective one, as there is

no continuous record of nitrate measurement in

any soil or water body.

To avoid the fragmentation of a particular

study by this classification, we have put it

where its dominant results apply.
AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

Accumulations in Caves, Caliches, and

Playas

Nitrate deposits can accumulate under

special conditions of nitrogen availability, movement by water, and concentration by evaporation of water at a surface. Mansfield and Boardman il9S2y reported nitrate deposits in 23

States and classified them into cave, caliche, and

playa deposits. This publication was preceded

by eight earlier ones of the U.S. Geological

Survey on the occurrence of nitrate deposits in

the United States,

Cave deposits occur on the walls and ceilings

of caves where animal excreta has the opportunity to oxidize in air and the water evaporate.

Oxidation of soil organic matter and plant detritus in the overburden may also contribute nitrate to the deposit. Such deposits occurred in

the caves of such States as Indiana, Kentucky,

and Virginia and were used by the American

Confederacy in gunpowder during the American Civil War. Smith {1967) discussed these

caves in the Missouri Ozarks and their contribution to nitrate in water supplies.

The best known caliche deposit is the northern Chilean deposit on the Tarabaca Plateau in

the Atacama desert that contained 250 million

tons of NaNOs. The most probable explanation

of this accumulation, like that on the offshore

islands of Peru, is the oxidation of guano and

subsequent accumulation of nitrate. However,

the theory that the Chilean deposits resulted

from electrical discharge in the cloudy, but

rainless, atmosphere has had its supporters.

A corollary of the caliche-forming process

was used for several centuries in the production

of nitrate. Nitriaries in Germany and France in

the 18th and 19th centuries produced nitrate by

the outdoor oxidation of animal waste. The

United States imported 2,500 tons of nitrate

for munitions in 1916 from India, where the nitrate was produced by composting of human

and animal waste.

The accumulation of nitrate in playas, along

with other salines, was one of the interesting

controversies of western U.S. agricultural development. Hilgard (1906) attributed the nitre

spots in western cultivated soils to the more

rapid nitrification of the organic matter in the

warm arid climate when they were irrigated.

Headden (1911, 1913)^ and in at least seven

other bulletins, and Sackett (1911) attributed

the nitrate accumulation in Colorado to the

stimulation of Azotobacter spp. by cultivation.

The story is best told by quotation from Stewart and Peterson (1916) of the Utah Station:

1. Certain soils in western arid America

contain spots which are rich in nitric nitrogen and contain in some instances several

hundred times as much nitric nitrogen as

occurs in normal, cultivated, productive irrigated soil.

2. These spots are locally known as 'nitre

spots.'

3. Three theories have been presented regarding the origin of these accumulations

(a) Hilgard's conception was that the accumulation was due to more rapid nitrification of the organic matter of the soil of

arid climates, (b) Headden mentions that

they are due to the fixation of the atmospheric nitrogen by the nonsymbiotic bacteria, (c) Stewart and Peterson believe

that they are the direct result of the leaching of nitrate out of the pre-existing deposits in the country rocks and locally concentrated by seepage.

Hi * *

6. The nitrate deposits are not limited to

the country rock of a definite geologic series. They have been found in large quantities in the Cretaceous, Tertiary, and Triassic with smaller quantities in the Jurassic.

¡^ ^: ^

8. The accumulation in the virgin soil possesses all the characteristics of the nitre

spots in cultivated soils, i.e. (1) a high nitrate content, (2) the presence of excessive

quantities of other soluble salts, (3)

brownish color, (4) thin crust over

surface. . .

* * *

10. The non-symbiotic bacteria are not essential to the production of the nitrate or

of the color of the nitre spots.

Tables 1 and 2 give some analyses of rocks reported by Stewart and Peterson (1914-, 1916).

Note that the nitrate is only a very small frac-
ACCUMULATION OP NITRATE IN SOIL, WATER, AND PLANTS

TABLE 1.-—Sodium nitrate concentrations in

western rocks^

Age Maximum Minimum Average

Percent Percent Percent

Cretaceous :

Sandstone 0.293

Shale 1.026

Clay . .115

Tertiary :

Sandstone .284

Shale .750

Clay .940

Jurassic :

Sandstone .039

Shale .019

0.005 0.055

.003 .104

.002 .074

0 .074

0 .147

0 .053

0 .008

0 .005

^ Data from Stewart and Peterson (1914),

TABLE 2,—Nitrate and total soluble salt content

of rocks'^

Location

Number of Soluble Nitrate

samples salts N

West of St. George,

Utah

East of St. George,

Utah 10

Northern Arizona 9

Mt. Carmel, Utah 14

Cedar City, Utah 11

P.p.m. P.p.m.

48,962 156.0

54,256 74.3

51,858 27.3

17,734 67.7

36,055 28,7

^ Data from Stewart and Peterson (1916),

tion of the total soluble salts. They stated that

all Cretaceous samples contained nitrate. Rankama and Sahama (1959) gave average values

for total N in igneous rocks as 46 g./ton and

sedimentary rocks, 510 g./ton.

The data on a drainage well constructed near

Grand Junction, Colo,, to relieve the artesian

pressure on an experimental area illustrate the

amounts of salt and nitrate that can come from

an aquifer in contact with the Mancos shale.

This well was pumped continuously at a rate of

about 260 gallons per minute. The water was

highly saline with a dissolved solids content of

9,570 and 35 p.p.m. of nitrate~N. In 24 hours,

the well pumped about 110 pounds of nitrate~N

or about 20 tons of nitrate-N per year into the

Colorado River.

The occurrence of natural nitrates, particularly in profiles and geological strata of semiarid and desert environments, is sufficient to

often complicate interpretation of data obtained

today to determine if fertilizer, sewage, and industrial waste disposal are enriching soils and

waters with nitrate. Dyer {1965) reported

nitrate-N concentrations that were in the range

of 1,400 to 4,200 p.p.m. of soil solution to a

depth of 50 feet for four sites located on Panoche soils, formed on alluvial mud flows on the

west side of the San Joaquin Valley in California, Concentrations of nitrate were low in the

upper 3 or 4 feet of the profiles caused by natural leaching. On adjacent, sprinkler-irrigated

sites, the upper 20 to 30 feet of profile contained

very little nitrate, but large accumulations occurred below these depths.

Smith (1967) gave an interesting account of

the nitrate outflow of springs in the Missouri

Ozarks : ". . . largely a forested area with little

use of fertilizers and a sparse population of

both humans and livestock." Smith wrote :

. , . Big Spring in Carter County has a

maximum daily flow of more than 800 million gallons of water. An average daily flow

of 253 million gallons has been found over

a 17-year period. Analysis of the water in

1964 showed a nitrate-nitrogen content of

2.4 to 3.0 ppm. Three parts per million in

252 million gallons of water is more than

6,000 pounds of nitrogen. The drainage

area of Big Spring has been estimated as

440 square miles. Assuming that onefourth of the 48 inches of annual rainfall

percolates through the soil, an average of

250 million gallons daily would be obtained.

A discharge of 6,000 pounds of nitrogen

daily would amount to less than 8 pounds

of nitrogen per acre per year, a realistic

leaching loss even on the low-fertility forest areas of the area. Five or more pounds

of nitrogen per acre is added to soils in

precipitation each year. This regular addition of nitrogen in rainfall could account

for most of the nitrate discharged by Big

Spring. It is also of interest that the use of
10 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

nitrogen fertilizer in the drainage area of reached on eight of the sites had nitrate-N eonBig Spring in 1963 was only about 65% of tents varying from 0.1 to 19 p.p.m., with an avthe nitrate discharged in the flow of this erage of 11.5 p.p.m.

single spring."

Agricultural Accumulations Before the

Smith further stated that about one-third of Fertilizer Euoch

the known caves in Missouri have been reported

to contain bat guano. Good correlations have ^^^^ ^^ ^^® available data on nitrate in

been obtained between the nitrate in spring wa- "^^^^^ ^^^ *^® overlying mantle collected before

ters from these caves and the presence of guano extensive fertilizer use or from areas with little

deposits. Obviously, the amount of nitrate and fertilizer use before sampling are pertinent Nithe concentration in water from Big Spring trogen fertilizer use before the end of 1945 did

hold much interest, but it is impossible to assign ^^* approach the scale of use now.

the sources. This example is typical of the mod- Fertilizer N consumption of 1945 amounted

ern problem. Nitrogen in rainfall is but one to only about 650,000 tons in the United States

input of nitrogen into a system that can yield compared with about 7 million tons in 1969. In

nitrate. Smith implied that part of the nitrate evaluating data of this "prefertilizer era," it is

could have come from rainfall, but recent stud- impossible to separate the nitrate that may

ies cloud the picture. Taylor et al. (1971) re- have existed in "geologic'' accumulations from

ported that the total N runoff from an Ohio those related to man's occupancy of the land,

wooded watershed was only 12 percent of that Whitehead and Moxon (1952) reported that

in the precipitation over a 3-year period, and eight wells in South Dakota, some of them causthat for farmland was 80 percent The research ing cattle losses, had N03~-N concentrations

by Borman and Likens (Deevey, iP70) on run- varying from 235 to 525 p.p.m. These wells

off from forest land at Hubbard Brook, N.H., were sampled in the period 1944-49. They do

showed that the runoff of nitrogen is less than not give the well depths. They reported that the

that in the precipitation. water in one stock tank was 240 p.p.m. higher

Stewart et al (1967a, h) reported that 17 in nitrate-N than the well due to concentration

cores taken from the soil surface to the water of the water by evaporation and ice formation

table or drilling impediment, in native grass- in the tank.

land of northeastern Colorado, averaged 90 The data in table 3 show appreciable amounts

pounds of NO3--N in 20 feet of profile. (See of nitrate-nitrogen in the water of the Arkantable 11.) Of the 124 analyses reported in pro- sas River, Colo., in 1940-41, and that nitrate

files taken to a depth of 12 meters, 62 samples did not become more concentrated downstream

contained no nitrate, 52 contained 1 to 3 p.p.m. as did the other dissolved salts. As in most irri-

(dry soil basis), nine had 4 to 10 p.p.m., and gated areas, seepage flows into the river and the

only one had more than 11 p.p.m. Water that water is reused. The data indicate that either

entered the drill holes when the water table was crops, plants in the stream channel, or release

TABLE 3.—Salt content of Arkansas River, water year WW-Ul

o i 4.. Flow Dissolved solids btation ^^Q^g .^-^^ -^i^^- Ca Mg Na HCO3 SO. Cl NO3--N

^^^^ /*• P.p.m. P.p.m. F.^m, P.p.m. F/p,m. P.p.m, P.p.m.

Pueblo 503 346 237,000 59 15 18 137 122 7 0.56

Nepasta 568 542 419,000 92 24 40 165 249 12 .95

La Junta 108 1,088 160,000 154 45 99 207 555 27 .56

Caddoa 310 1,376 580,000 170 61 158 195 775 36 AO

Holly. 169 2,034 468,000 217 88 241 243 1,050 81 .95

Source: U.S. Bureau of Eeclamation.
ACCUMULATION OP NITRATE IN SOIL, WATER, AND PLANTS 11

of untreated sewage by towns selectively removed nitrate.

Table 4 gives the water and nitrate budget

for the Sunnyside Valley Irrigation District. All

water diverted into irrigation canals or leaving

the district via drains was measured. Appreciable concentration of nitrate occurred in the return flow, even though little nitrogen fertilizer

TABLE 4.—Input and output of water and

nitrate N from Sunnyside (Wash,) Valley

Irrigation District

Item Unit 1943 1944

Water :

Input Acre-foot 167,931 164,060

Output do. 68,033 62,456

NO3-N

Concentration :

Input P.p.m. 0.27 (^)

Output do. 2.29 1.66

Total NO3---N:

Input Pounds 123,000 ^'0

Output do. 427,000 284,000

N Release:

NOs'-N - Pounds per acre 8.5 8.0

^ Trace.

^ Assuming that trace equals zero. Irrigated acreage

in the Division was 37,896 acres.

Source: Unpublished data of C. S. Scofield and L. V.

Wilcox, U.S. Salinity Laboratory.

was used in the district in those war years. The

principal sources of nitrogen were barnyard

manure and alfalfa. The contribution of an acre

of land in 1943 was about 8.5 pounds of

NO;r-N; in 1944 it was 8.0. This budget does

not consider organic nitrogen in the water

which was not measured. Crops grown in the

area were corn, alfalfa, hops, sugarbeets,

wheat, barley, asparagus, and mint.

Table 5 gives the input and output of mineral

salts of the Sutter Basin, Calif., in 5 different

years. In two of the 5 years, there was more nitrate in the irrigation water than in the drainage water. In 1947, when data were available on

total water flow, a N03~-N output of about 0.5

pound per acre was computed. Much of the land

of the Sutter Basin is used for rice production.

Rice, which is flooded from planting until

shortly before harvest, is conducive to denitrification of nitrate in the soil. This may explain

the much smaller outflow of nitrate from this

basin compared with the Sunnyside Valley project in Washington.

Table 6 gives the nitrate concentrations in

canal waters and some wells in the Columbia

Basin Project, Washington, in 1954 and 1955.

This is a former semidesert area where irrigation had just been started. Nitrate in the water

leaving a part of the project (sampling station

2) had less nitrate than water entering the project through sampling station 1. Of particular

TABLE 5,—Input and output of mineral salts—Sutter Basin, Calif.'

Input

Year Output Salts Ca Mg Na NCO3 SO, Cl NO3--N

P.p.w. P,p,m, P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. P.p.m.

1931 Input 140 17 10 11 107 8 9 0.027

1931 Output 1,008 77 59 150 268 30 364 .013

1932 Input 125 15 10 12 97 5 6 .027

1932 Output 1,294 96 78 193 283 36 518 .013

1933 Input 140 15 8 16 100 6 11 .069

1933 Output 950 67 57 152 276 31 335 .110

1946 Input 103 12 6 9 75 7 5 .069

1946 Output 603 52 41 105 277 36 184 .14

1947 Input 125 14 8 13 88 11 9 n

1947 Output 611 52 40 102 255 25 197 0.25

^Acreage irrigated: 45,028. 1947 water input—^299,300 acre feet; water output—152,110 acre feet.

^ Trace.

Source: U.S. Salinity Laboratory.
12 AGRICULTURE HANDBOOK 413, U.S. DEPT. OP AGRICULTURE

TABLE 6.—Salinity of water in the Columbia Basin Project

Sampling station EC 25' C= Ca Mg Na HCO3

Mfnhos/cm, . P,p,m.

1. Lake Roosevelt ^ 150 21 5 2 '76

2. Frenchman Wasteway ^ -.__-213 22 8 10 91

3. Moses Lake^^ ._.-.._. . 896 28 15 33 182

4. Wasteway 204 25 8 8 101

5. Potholes E. Canal Mile 0 345 24 13 28 163

6. Lind Coulee 801 32 10 15 113

7. Scooteney Wasteway 159 21 6 4 82

8. Potholes E., Canal Mile 26 335 24 13 26 159

9. Potholes E., Canal Mile 38 321 24 12 24 154

10. Potholes E,, Canal Mile 66 „ 318 24 12 24 146

A. Well _- 834 92 46 20 201

B. Do. ._ . .653 44 44 37 299

C. Do. . ...431 38 16 26 201

D. Do. 955 58 45 86 530

E. Do. 495 40 25 25 173

P. Do. 517 31 37 29 263

SO4 CI NO3--N

12 1 0.17

20 4 .11

33 11 .44

19 4 .44

27 10 .24

37 12 .55

13 1 ,15

24 11 .11

25 10 .04

26 10 .11

214 12 15.0

203 12 .40

52 10 .69

63 24 3.02

34 16 15.0

51 3 4.4

^ Water into project.

^ Water out of project.

Source : Bureau of Reclamation- -data for 1954 and 1955.

interest is the high nitrate in at least two of the

wells. This nitrate could not have come from

high rates of nitrogen use in irrigation farming

or from livestock.

THE SOURCES OF NITROGEN AND NITRATE

Nitrate unlike ammonia is not a constituent

of the igneous rocks of the earth. Only small

amounts come down in rain as the result of electrical discharge or some form of atmospheric

pollution. Except for factory-produced nitrate,

nitrate has to come from the oxidation of ammonia. There has to be some mechanisms of

concentration for it to be of much environmental concern. Hence, the processes of accumulation of organic nitrogenous compounds that can

be ammonified and nitrified are of paramount

importance in consideration of nitrate accumulation. The nitrate produced is then often further concentrated by evaporation of water.

Geologic Deposits

The accumulations of nitrates on cave walls

and ceilings^ the deposits of caliche containing

nitrates and other salts^ and the accumulation

of nitrates in playas and seeps are all the result

of evaporation in a dry atmosphere of nitratebearing solutions. In all instances, the source of

the ammonia to produce the nitrate appears to

have been accumulations of biological waste

such as dung, animal remains, and animal and

plant detritus deposited in caves, on rocks, or in

the bottom of a shallow sea (marine deposits)

to become consolidated into sandstones, siltstone, or shales. These processes have their

modern counterparts to be discussed. Although

geologic may liot be the best terminology for

these old deposits of nitrate, the outflow from

them and their occurrence in semidesert and desert soils, and in aquifers in humid regions, is a

problem in estimating the impact of modern

technology. In most instances, this "background'' is so variable and so little attention has

been paid to measuring it that we lack knowledge about what would happen if we could eliminate current sources of nitrogen input.
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 13

Soil Organic Matter, Peat and Muck

Cultivation of some soils has increased their

contents of humus and organic nitrogen. Substantial accumulations of these important soil

fractions have occurred in New Zealand and

Australia, where white or subterranean clovers

have been used in grass-legume pastures with

high rates of phosphate and sulfur fertilization

(Walker et al, 1959), Western U.S. soils put

under irrigation may maintain their organic

matter and nitrogen contents, but only when alfalfa or heavy applications of manure are used

(Viets, 1959), Information is lacking on the effects of the higher rates of commercial fertilizers now in general use on humus and organic

nitrogen contents of most soils. However, the

predominant effect of long-time cultivation has

been depletion of organic matter and nitrogen.

Few people realize the magnitude of these losses

of nitrogen that have occurred from grassland

and woodland soils put under cultivation in the

temperate zone, where nitrate problems are of

most concern to people. Also, peats and mucklands, when drained and cultivated, undergo

rapid subsidence and always show losses of organic matter and organic N. These losses of soil

N have been well documented at numerous locations, and estimates of their significance on a

regional scale have been made.

How much organic N was accumulated in our

soils before they were cultivated? A figure of 10

billion tons is frequently quoted for the 48

States. Table 7 adapted from Schreiner and

Brown (1988)^ who adapted their data from C.

F. Marbut's analysis of 250 to 300 carefully selected soil samples taken to a depth of 40 inches,

shows a total of 3,494 billion tons for 720 million acres. The latter is about a third of our

total land area, that not represented in table 7

being the podzol soils, organic soils, various

nonzonal soils, and all land west of the Rocky

Mountains.

Smith and Vandecaveye (19^6) reported that

soil N losses on Palouse silt loam at Pullman,

Wash., were greater in wheat-fallow rotation

than under a continuous wheat system for a

23-year period. In the wheat-fallow system, soil

N continued to decline even when N fertilizer

(as NaNOs) or crop residues, either alone or

TABLE 7.—Average total N content of virgin

soils in soil regions of the United States east

of the Rocky Mountains^

Ap- In 40 inches

prox. Surface Per- Regional

Soil region area 6 inches cent Tons total

Million Millón

acres Percent tons

Brown

forest 180 0.05—0.20 0.05 3.35 603

Bed and

yellow 150 .05— .15 .03 2.00 300

Prairie 113 .10— .25 .12 8.00 854

Chernozem

and

Chernozem like..- 123 .15— .30 .12 8.00 984

Chestnut -..„._ 102 .10— .20 .08 5.85 546

Brown 52 .10— .15 .06 4.00 208

Total 720 3,495

Total of 48

States 2,266

^ Adapted from Schreiner and Brown {1938),

combined, were used. Bracken and Greaves

(19^1) found that soils in wheat-fallow systems

in the Cache Valley of Utah had lost 15.9 percent of their organic N in the first foot compared with adjacent virgin grassland. Of the

2,560 pounds of N per acre lost from the upper

2 feet of soil, 735 pounds could be accounted for

by crop removal, leaving an unexplained loss of

1,825 pounds, or 71.3 percent of the total loss.

In the Juab Valley of Utah, a loss of 1,186

pounds of N per acre had occurred in wheat-fallow systems, 402 pounds of which could be accounted for by crop removal, leaving 784

pounds or 66.1 percent of the loss unexplained.

Haas et al. (1957) concluded from a summary of the data from 11 dryland experiment

stations in the Great Plains that, in 33 years of

cultivation, the total N of the surface foot decreased 24 percent under continuous small

grain and 29 under an alternate fallow-small

grain system. Since most of these field experiments from which the data were derived were

located on relatively level sites with low water

erosion hazard, the average field losses of N
14 AGRICULTURE HANDBOOK 413, U.S. DEPT. OP AGRICULTURE

may have been underestimated considerably. Viets {1968) made some highly speculative

calculations about 1964 N losses in relation to

the 1964 needs of wheat in seven States of the

Great Plains based on the data of Haas et al.

{Î9S7). The speculation lies in the extrapolation to a Statewide basis of the annual N losses

and soil N contents at the experiment station

when the experiments were terminated. The

calculations show that for 1964 the sum of the

fertilizer N applied plus that lost from the soil

in 1964 about equalled that in the 1964 wheat

crop for five of the seven States for which the N

balances were made for annual cropping systems. However, for a wheat-fallow system, the

2-year N loss would be about twice that removed in the wheat crop.

Thompson et al. (1954) gave a list of 20

paired soil samples obtained from the surface 6

inches of cropland and adjacent virgin grassland in Iowa. The total N contents of the cultivated soils averaged only 70 percent of that in

the grassland soils. The average total N content

of the grassland soils was 0.3454, for the

cropped soils, 0.2422—a loss of 0.1032. Assuming an acre-6 inches of soil weighs 2 million

pounds, the N loss from just the surface soil averaged 2,060 pounds per acre.

Sufficient data exist to show conclusively that

cultivation has taken an enormous toll of our

soil organic N accumulated over centuries.

What the numbers mean on a national scale and

what they mean to nitrate accumulation in surface and ground waters are speculative. Although loss figures spanning a period of time

from a great number of locations are available,

enough measurements were not made and are

not being made no%v because of the experimental difficulties to know what fractions were lost

by crop removal, erosion, nitrate leaching, and

denitrification. In other words, we are far from

having a balance sheet in which everything was

determined even for one site.

Wadleigh {1968b) credits George Stanford

with the estimate that the cultivated soils in the

48 States have lost 1.75 billion tons of organic

N from the top 40 inches in the last 100 years.

Lipman and Conybeare {1936) estimated annual

crop removal for 1930 at 4.6 million tons, loss

by erosion—-5.0 million tons, and leaching of

soil N—4.0 million tons. For 1947, Mehring and

Parks (cited by Stanford et aL, JP70) made estimates of 6.5, 4.0, and 3.0 million tons for these

categories. Stanford et al. {1970) made new estimates for 1969 as 9.5, 3.0, and 2.0 for these

items (table 8). Note the question marks on estimates of leaching of fertilizer N and denitrification.

All of the estimates in table 8, except those

for fertilizer nitrogen, must be regarded as

highly speculative. We really know little about

the input and output accounts for nitrogen,

even for a selected farming system and soil association.

The effect of slow organic matter mineralization on the nitrate concentration of percolating

water can be computed if the amount of leachate is known or assumed. The assumption is

made that nitrate is uniformly distributed in

the percolate, that is, no negative or positive adsorption. Stout and Burau {1967) in their discussion of organic matter loss in relation to nitrate in percolating water, gave a number of

cases, one of which is summarized here: If a

rich prairie soil contained 0.1 percent of organic N initially in the top foot (a very conservative assumption), one-fourth of this N

was lost in 100 years of cultivation, and onehundredth of the loss leached as nitrate in 12.6

inches of percolate each year, then the percolate

would contain 28.6 p.p.m. of nitrate N.

Questions posed by such calculations using

the known facts on organic matter loss are

many. How have the known losses of soil N

been distributed among erosion, deep percolation of nitrate, crop removal and denitrification? Is their active denitrification below the

root zone? If the assumptions are correct, why

have all of the ground waters in the North Central Region not become loaded with nitrate long

ago? Are the rates of recharge and outflow

from the aquifers sufficient to keep the nitrate

from accumulating? Does denitrification take

place in the water table? Historically, there has

been a high incidence of nitrate phenomena,

such as livestock deaths and methemoglobinemia, where people depended on wells for

water supply, deaths in silos, silo explosions,

and toxic nitrate levels in animal forages in the

Midwestern States,
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 15

TABLES.—Balance sheet of nitrogen (N) in the United States: Estimated changes for 1930,

19Ji7, and 1969 on harvested cropland^

Item 1930^

Amount of nitrogen in—

1947« 1969

Inputs of nitrogen from :

Fertlilizer N

N ûxeà by legumes

N fixed (nons3rmbiotic)

Barnyard manure ..

Roots of unharvested portions of crops . Rainfall

Total

Million

tons

-- 0.3

-- 1.7

- 1.0

.- 1.9

- 1.1

-. .8

Removals of nitrogen by:

Harvested crops

Erosion

Leaching of soil N

Leaching of fertilizer N .

Denitrification

Total

6.8

4,6

5.0

4.0

0

(?)

13.6

Million

tons

0.7

1,7

1.0

1.3

1.5

1.0

Million

tons

6.8

2.0

1.0

1.0

2.5

1.5

7.2 14.8

6.5 9.5

4.0 3.0

3.0 2.0

0 (?)

(?) (?)

13.5 14.5

^Data of Mehring and Parks cited from Stanford, England, and Taylor {1970),

^ Lipman, J. G., and A. B. Conybeare. Preliminary note on the inventory and balance sheet of plant nutrients

in the United States. N. J. Agr. Expt. Sta. BuL 607. 1936.

'Mehring, A, L., and R. Q, Parks. Agricultural Chemicals, v., No. 10, pp. 36-50; No. 11, pp. 33-39 and

77-78, (See Stanford, England, and Parks 1970),

Peats and mucks represent great monuments

to a past or current eutrophication process that

have accumulated vast amounts of organic N.

When these peats and mucks are drained and

cropped, large amounts of nitrate can be released hy mineralization. Schreiner and Brown

(1938) indicated that there are 197,500 pounds

of organic N in an acre of peat 40 inches deep.

Wadleigh (1969) wrote that there are 7,500,000

acres of peat in Minnesota containing 6.4 billion

tons of peat. Assuming an average N content of

1.5 percent, he calculated this peat contained

102 million tons of stored N.

Many studies have been conducted on nitrogen mineralization in peat, but we cite here only

a few to indicate their potential as a nitrate

source. Neller (19H) reported the nitrate con»

tent with depth of Everglades peat in fallow

fields that had water tables at depths of 12 and

24 inches. The data shown in table 9 have been

recalculated to show the nitrate content in the

soil water of the samples assuming uniform distribution of nitrate in the water. Obviously,

sawgrass peat liberates high concentrations of

nitrate that can pollute drainage water unless

crop demands are sufficient to remove it. Deep

drainage to 24 inches allowed more nitrogen to

mineralize than shallow drainage.

TABLE 9.—Nitrate-nitrogen concentration on

March 31 in soil solution of fallow Everglades peat having drainage to two depths^

Drainage Drainage

Soil depth depth depth

(inches) 12 in. 24 in.

P,p,m. P.p.m*

0 to 6 84 182

6 to 12 58 100

12 to 18 84 59

18 to 24 15 54

^ After Neller, (19U).
16 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

The problem Israel is having with nitrate

coming from overdrained peat in the Hula Valley above the Sea of Galilee (now Lake Kinaret) illustrates the significance of peat as a nitrate source. In 1958, the Hula swamp and lake

that had been previously drained were prepared

for growing crops, mostly cotton. Soil of the

dried swamp is a low moor peat containing

from 30 to 80 percent organic matter. Soil of

the dried lake contains 60 to 80 percent lime

€5quivalent and 5 to 10 percent organic matter.

Water is supplied by subirrigation. Rainfall is

about 30 cm. and comes in the winter months.

Crops grow very poorly on patches comprising

about 10 percent of the total area (Levin,

1970),

Shoham and Levin {1968) showed that the

subsidence in the former swamp was about 10

cm, per year for 1958 to 1964 and about 9 cm.

for 1964-65. Table 10 shows the salt content as

measured by electrical conductivity and the nitrate concentrations in a lake bed area with

good crops (unaffected area) compared with

one with poor crops. The soluble salts are almost entirely gypsum and calcium nitrate. Soil

in the affected area had two to three times as

much nitrate as the unaffected area depending

upon the depth. Nitrate-N as high as 2,000

p.p.m. was measured in some affected areas.

This situation resembles that of the playa accumulation of nitrates noted in the Western

United States, except nitrate is the dominant

salt, and the source of the nitrate is not shales

and other rocks, but peat.

TABLE 1^,-—Electrical conductivity and nitrate

concentration in lake bed soil of Hula Valley,

Israel^

Depth, cm. Electrical conductivity NOa-

(dry soi

Poor

crops

^N

1 basis)

Poor

crops

Good

crops

Good

crops

0 to 80

30 to 60

60 to 90

90 to 120

Mm.hos/cm.

8,0

7.8

4.3

2.8

Mmhos/cm,

7.1

4.4

2.9

2.1

P.p.m,

1,550

1,480

1,020

P.p.m,

660

400

430

^Adapted from Levin, (1970),

According to Serruya and Berman (1970)^

largely because of the drainage of the Hula Valley peats, a large part of the total N (1,600 tons

in a normal year) entering Lake Kinaret from

the Jordan is nitrate. In 1969, the nitrate nitrogen inflow into Kinaret was 5,000 tons. Since

Israel is trying to maintain nitrate in all water

supplies at drinking standards (less than 10

p.p.m. nitrate-N), some Israelis are having second thoughts about the wisdom of draining the

Hula Valley. Much of the nitrate entering the

lake is metabolized or denitrified in the hypolimnion (Serruya and Berman, 1970).

Livestock and Poultry Wastes

Livestock, wild animals, and birds foraging

in their natural habitats do not ordinarily constitute a concentrated source of N supply that

can give rise to nitrate accumulations. The N is

recycled at or near the site it came from. However, bats and some bird species that nest

closely together accumulate deposits of guano

that are a potent nitrate source. But livestock

and poultry production has changed and is still

changing. Cattle are fattened in operations in

which as many as 200,000 head are crowded

onto 640 acres of land. Swine are fattened in

confinement. Poultry feeding is concentrated

into huge operations. The urine and manure

that were once scattered over the landscape are

now concentrated in a small area, the nitrogen

in the feeds coming from a large land area.

Wadleigh (1968a) stated that domestic animals in the United States produce over 1 billion tons of manure and 400 million tons of

urine a year. He estimated that as much as 50

percent of this waste may be produced in concentrated supply. To give some idea of the significance of concentrated supply, the following

calculations are given (Viets, 1970a). A typical

Colorado feedyard is used continuously and has

about 360 head of fattening steers and heifers

per acre. In one year these cattle will produce

4,200 tons of wet manure and urine containing

24.9 tons of nitrogen. The depth of water excreted annually in the urine and manure

amounts to almost 31 inches. For a feedlot with

a capacity of 200,000 head, being constructed

but not fully stocked now, the total N excretion

will be about 14,000 tons per year.
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 17

Mature healthy monogastric animals on good

diets can excrete nitrate in the urine ; cows and

sheep do not (Case, 1970), However, nitrifying

organisms can quickly oxidize the ammonia

originating from,hydrolysis of urea and uric

acid and from ammonification of the organic N

compounds of the feces to nitrate. Animal

wastes are usually high in salts so that the runoff or percolate from their oxidation on land

after evaporation results in a nitrate accumulation not unlike the playa deposits noted earlier.

Animal and poultry wastes volatilize ammonia

which can be absorbed by water as shown by

Hutchinson and Viets (1969) for lakes in the

vicinity of cattle feedyards in northern Colorado.

Eunoff from feedlots can contain nitrate and

always contains soluble nitrogen compounds

that can quickly nitrify. In the studies of runoff

from beef cattle feedlots at Mead, Nebr., Gilbertson et al {1970) reported that NOs^-N in

winter runoff ranged from 0 to 80 p.p.m. and in

rainstorm runoff from 0 to 17 p.p.m. Heavy

rates of land disposal of animal wastes can be a

source of nitrogen for nitrates in percolating

water. Shallow wells can become polluted with

nitrate. In Michigan, Deutsch {1968) reported

concentrations of N03~-N ranging from 54 to

216 p.p.m. in shallow dug wells in several counties. The source was human and animal excreta.

Several cases of infant methemoglobinemia

were observed. Near Guelph, Ontario, Canada,

Gillham and Weber {1969) reported that nitrate contamination of water in shallow wells is

a serious problem in the area because rural

wells are frequently located close to barnyards.

In a study of the contaminated zone resulting

from a barnyard located on glacial till, they

found that the barnyard contributed 4.4 pounds

of N03"-~N to the water table in a 5-month period. The lowest concentration of NO3 in the

ground water was found in the summer.

Smith {1967) contended that the major

source of nitrate contamination of shallow

ground water in Missouri is livestock feedlots

and barnyards. Frink {1969a) estimated the N

input budget for highly specialized dairy farms

in the Northeastern States. He concluded that

under certain conditions dairy farms could contribute significant amounts of nitrate to ground

water. He believed the loss of nutrients can be

minimized by providing more cropland per cow,

by selecting crops and livestock that are

efficient users of N, and by applying both manure and commercial fertilizers during the

growing season. For Connecticut, Frink {1969b)

estimated that animal wastes contributed 10

percent of the total N input budget, whereas

agricultural fertilizer N contributed 3.5 percent,

and nonagricultural fertilizer N use contributed 2.2 percent. Much of the N in animal

wastes comes from imported feeds.

Although some waste from hog and poultry

feeding operations is being put through sewage

treatment plants not unlike those used by cities,

the magnitude of the physical plant required

(one cow equals the sewage equivalent of 10 to

12 people), the high water requirements for

transport and digestion, and the costs all make

conventional sewage treatment largely out of

the question. Needless to say, the animal waste

problem has created problems for pollution control agencies, all of which are not solved.

Animal wastes, unlike the other sources of

potential nitrates discussed in previous sections,

have enough oxidizable carbon along with the

nitrogen so that riddance of the nitrogen by

managed denitrification is an alternative to letting nitrate accumulate where it is not wanted.

Domestic Sewage

People are not included in the section with

animals and fowl because some readers might

object, but people are also a concentrated source

of N. Because of the high dilution of city sewage, the concentration of effluent from a sewage plant is not the problem that the runoff

from a feedlot may be. The usual figure for N in

city sewage is about 4.4 kg. of N per year per

capita. For a city of 7 million people that

amounts to about 34,000 tons of N per year.

City sewage plants, depending upon their design and operation, remove part of this nitrogen in sludge and by denitrification. Although

all of the organic N is not removed by treatment, most of the N in the effluent is ammonia

and nitrate. The proportion between the latter

depends on the aeration and holding time in the

aeration process (trickling filter or activated
18 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

sludge). The ammonia in the effluent can be a

significant part of the total BOD released, since

the ammonia will be nitrified by the nitrifiers if

molecular oxygen is available.

Stratton (1968, 1969) showed that substantial amounts of ammonia can be volatilized

from the effluent of secondary sewage treatment

plants when that effluent is retained in a eutrophic pond or lake. The combination of high

concentration of ammonium ions and high pH

resulting from CO2 assimilation by algae are

conducive to ammonia escape.

The transition from the period when we

dumped raw sewage into lakes and streams to

the present period of requiring at least secondary treatment greatly complicates the interpretation of water analysis for nitrate only in

water bodies. Nitrate in the water body can

come from agricultural areas, a sewage treatment plant, or from nitrification of sludge previously accumulated in the water body but not

oxidized because of the continual pollution with

wastes with high BOD.

Nitrate in rural wells can come directly from

improperly located cesspools and septic tanks.

This is not desirable recycling.

Commercial Fertilizers

No concentrated source of nitrogen has attracted as much attention from the ecologists

and environmentalists concerned about the nitrate problem as has the spiraling increase in

the use of commercial fertilizers. This is in part

probably due to the availability of statistics on

fertilizer use and the cases where misuse or

overuse of nitrogen fertilizers have created a

problem in ground water. Outright bans on N

fertilizer use have been suggested and legislation has been introduced in some States to regulate N use. Viets {1970,1971a, h) discussed this

problem in its many ramifications. Suffice to say

the controversy over use of commercial fertilizers is far from over. Dodge (1970) claims that

the NH4+ containing fire retardant chemicals,

often used to fight forest and range fires, contains insufficient N to contribute to NOg' in

runoff water, the amount being much less than

is used in a range or pasture fertilization program.

Saltpeter has been used as a nitrogen fertilizer since the time of Virgil. Up until the invention and development of the Haber process

(1904-08) for fixation of atmospheric N and

the first full-scale plant put on stream in 1913,

the use of commercial N fertilizers grew at a

very slow rate. Agriculture had to depend on

guano, the Chilean saltpeter deposits, which

were mined from 1880, the ammonia from coke

ovens, human and animal wastes, and nitrogen

fixation by legumes as sources of nitrogen. Indeed, before the invention of the Haber process

and some other processes, such as the electric

are and cyanamide, which turned out to be too

expensive to use, Malthusian doctrine of 1798

appeared to have come true. World population

would be limited by availability of N for plant

protein synthesis. Crop yields in Europe had remained stationary for centuries. Yields were

comparatively high in the United States because we had a rich supply of N accumulated in

grasslands to exploit. But the Haber process

and its commercialization into enormous N fixation plants have changed the outlook completely

in all countries where N is available, and there

is a market for crops in excess of family needs.

In the United States, N consumption on

farms has increased from about a third of a

million tons in 1930 to almost 7 million tons

today. The world consumption in 1965 was

about 16 million metric tons. According to

**Chemical Information Services" (1969), the

world synthetic ammonia capacity in 1969 was

48.7 million metric tons or 14 kg. per capita.

Capacity by 1973 will be 69.7 million metric

tons or 18 kg. per capita. Of course, only part of

this ammonia is used for fertilizer.

The use of commercial N fertilizers is expanding at a rapid rate. For the World, the rate

of increase since 1950 has been about 11 percent

a year. For the United States from 1940

through 1966, it was 1,290 percent or about 40

percent a year. Ibach (1966) estimated that by

1980 U.S. consumption of N will have to be

12.2 million tons to meet our requirements for

agricultural production. This estimate of need

compares with the 6.8 million tons we used in

1969.

In 1968, about 90 percent of the commercial

N fertilizer used on farms was ammonia, am-
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 19

monium salts, and urea, which hydrolyzes

quickly to ammonium ions. Although ammonium is retained against leaching by all soils except sands, it is quickly nitrified to nitrate if

soil is moist and the temperature above 45^ F.

Some attention is being given to the inhibition

of ammonium nitrification by the use of chemicals such as azide and picolines. Applying ammonia when soils are cold or the using of nitrification inhibitors offers some control of nitrate

leaching, but the long-term effect on loss of nitrate from cropland by such practices is unknown. Other developments are coating fertilizer pellets with resins and sulfur to reduce the

rates of solution and nitrification.

One of the problems confronting the commercial N fertilizer enthusiast, and the organic N

enthusiasts too, in these days of concern about

eutrophication and possible nitrate accumulation, is to explain why the recovery of supplemental N by crops is so low. What happens to

the rest of it? How can recovery be improved?

The same problem is presented in explaining

where all of the N went that has been lost from

our former grassland and forested soils placed

under cultivation.

Commoner (1968) answered the question on

fertilizer N this way: *^ . . we must look for

some of the 6 million tons of nitrogen added to

U.S. soils during the last year not in the soil,

or in the harvested crops, but in the Nation's

lakes and rivers, and in the rain and snow that

falls on the land." He estimated that 15 percent

of the fertilizer nitrogen is lost by leaching and

to surface waters, and that an equal fraction is

lost to the air.

Allison (1955, 1965, 1966) made three excellent reviews on the enigma of soil nitrogen balance sheets and the recovery of N from inorganic fertilizers and manures. He stated that N

in harvested crops will more often be less than

50 percent instead of greater than 50 percent of

the N applied or mineralized from organic matter. Recoveries slightly higher than 80 percent

have been achieved in the field on soils with low

N and organic matter levels, and no leaching,

but such high recovery is rare. Even when

leaching losses are prevented or accounted for

as in lysimeters, Allison's (1955) summary for

51 uncropped lysimeters and 106 cropped to

nonlegumes showed that 15 percent of the fertilizer N added or mineralized from soil organic

matter decomposition could not be found. The

presumption is that this N is lost by denitrification , mostly to N2, but this has not been proved

in the field with N^^ and the gaseous products

identified. Allison concluded that the main mode

of nongaseous loss in the field is through the

leaching of nitrate in the spring or fall when

there is apt to be a surplus of water, because of

low évapotranspiration demands.

Agronomists and soil scientists have been interested in response of crops to various rates of

N application with the objective of attaining

good yields and good recovery of the fertilizer.

They have not, however, had much concern for

nitrogen that was lost in runoff or leached

below the root zone, except that it was not

available for plant use. So in spite of the perhaps hundred thousand experiments that have

been conducted, we have little information on

how much and how fast nitrate percolates, and

whether it reaches the water table. Whether a

near maximum yield of any crop can be produced with natural rainfall, or even under carefully controlled irrigation without having some

nitrate percolate below the root zone, is an unanswered question. Although a plant will extract all of the NOs" from a culture solution or

a soil if the plant is starved for N, can roots remove all of the NOs" from a porous media such

as soil and still have sufficient N for its maximum growth ?

There are many examples of overuse of N

fertilizers under conditions of slope, excessive

rainfall, or method of application that are not

recommended. These examples often give fertilizer use a bad name and are sometimes generalized to be the condition that exists in agriculture. The reports of Moe et al (1967, 1968)

showed what can happen. They applied 200

pounds of N per acre as pelletized ammonium

nitrate to fescue sod plots (clipped at 2 inches)

and to bare fallow plots on Zanesville silt loam

on a 13-percent slope in Ohio. The soil had a

fragipan at 24 to 36 inches. They then applied 5

inches of artificial rain in 24 hours. Fallow

plots with a surface crust lost 8.2 percent of the

applied N in runoff. Fallow plots plowed in the

spring lost only 3.1 percent of the N. Loss of or-
20 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

ganic N by erosion exceeded the loss of fertilizer N. On sod plots, loss of fertilizer N depended on the initial soil moisture content, but

was 14.0 percent when the soil moisture was

high (25.8 percent).

On the same plots, Moe et al. {1968) applied

400 pounds of N per acre as pelletized urea or

ammonium nitrate: The fescue had been clipped

at 3 to 4 inches. The second year fallow plots

had been plowed in mid-May and disked twice

in mid-July before the fertilizers were applied.

Two and one-half inches of artificial rain were

then applied. Total loss of fertilizer N from the

plots ranged from 2.4 to 12 percent of the N applied. Urea was found in the runoff only from

the sod plots. These losses of N are, of course,

economically significant to the farmer who

would apply N in this way under these conditions. Viets (1970a) pointed out that even very

small losses of fertilizer N that would be of no

economic significance to a farmer can contribute sufficient N to surface waters to give them

concentrations frequently associated with eutrophy.

There are many examples of overuse of nitrogen fertilizers at rates that appear to be far in

excess of crop needs. Wüvd (1970) cited a report of the California Department of Water Resources stating that the average annual application of N for strawberries was 145 pounds per

acre, and for celery was 290 pounds per acre, in

the Arroya-Grande—Grover City area. This

area has high concentration of nitrate in the

shallow water table. In some areas in California, so much residual nitrogen is left in the soil

after heavy fertilization of vegetable crops that

sugarbeets with high sugar content and purity

cannot be produced. Bingham et al. (1971) reported that the average nitrogen fertilizer rate

on a 960-acre watershed in citrus in Southern

California was 130 pounds per acre per year.

Nitrate in the percolate was equal to about 45

percent of the nitrogen applied.

The best estimates of how much nitrogen is

used on various crops and in various subregions

of the United States are the estimates of Ibach

and Adams (1967) for 1964. Some examples of

high average use of nitrogen fertilizers by

States expressed in pounds of N per acre are:

fruits and nuts in Arizona—149, vegetables in

California—136, potatoes in California—174,

potatoes in Delaware—130, fruits and nuts in

Florida—142, and potatoes in North Carolina

—154. Since practically all growers in these

States used nitrogen fertilizers, and the rates of

application quoted are averages, some growers

must use very high amounts of N in relation to

those who use only minimal amounts. Practically no information is published on how the

rate of use is distributed around the average.

To put the problem another way : In a given region is 25 percent of the fertilizer N put on 10

percent of the acreage in a given crop?

The potential for pollution of ground water

with high rates of nitrogen fertilizer is greater

than for low rates, but it is not necessarily true

that the higher rate will cause nitrate leakage

into the aquifer. The kind of crop, soil, amount

and distribution of rain, and many other management variables are important. Two hundred

pounds of fertilizer N per acre in one circumstance may not be as hazardous as 50 in another. Therefore, statistics on fertilizer use

must be interpreted with understanding.

Although a nonfertilizer use in the conventional sense, another interesting but insignificant use of nitrate salts has been their addition

to water as an oxidant to decrease the biological

oxygen demand (BOD) of polluted water. Sodium nitrate has been added to sewage effluent

being infiltrated into sand dunes in Holland to

accomplish more rapid oxidation of pollutants.

Nitrate is denitrified to yield oxygen to the denitrifying bacteria while they are digesting the

organic pollutants for energy.

The following are interesting remarks made

by Sawyer (1970) at the Twelfth Sanitary Engineering Conference at the University of Illinois :

Now I don't want to be criticized after

Tm through telling you about these things,

because in the light of the gospel that is

being preached today this is strict heresy.

But when you get into a hole, a real hole,

with regard to an odor problem, and the

paint is turning black on the buildings, and
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 21

the people are up in the arms, you're going

to do something about it and you turn to

nitrates. This is the quickest way. Today,

why sure you'd use a portable aerator and

you'd do a lot of things, but in years gone

by you didn't have as many facilities, and

nitrates were turned to because nitrates,

next to dissolved oxygen are the easiest

source for organisms to utilize oxygen. As

long as nitrates are present you never have

an anaerobic condition, but you do have anoxie conditions. . , Now for specific examples: nitrates were used for many, many

years on the Androscoggin River in Maine,

because there were suinte pulp mills on the

headwaters that couldn't see their way

clear to removing the B.O.D. that was necessary. Through court action they set a

precedent on the Androscoggin. Dr. Lawrence of Bates College and his crews sample

the water routinely, and when the dissolved

oxygen would go down to a certain level at

a certain point, nitrates would be added to

the Androscoggin River . . .

Nonconcentrated Sources

There are many sources that contribute N to

the environment and are important in consideration of input-output balances, the behaviour of

ecosystems, and enter significantly into crop

and water management practices, but in themselves may not be sources of hazardous concentrations of nitrate. Nitrogen content of precipitation has interested many for a long time.

Feth (1966) reviewed the recent literature

for the United States and northern Europe.

There are some indications that the amounts

may be increasing due to air pollution with ammonia and the nitrogen oxides from high temperature combustion. There may be direct absorption of NH3 from the air by plants, soil,

and water (Hutchinson and Viets, 1969) that is

not included in precipitation figures, Biggar

and Cory (1969) stated: "Nitrogen derived

from rainfall may range from 1 to 19 pounds

per acre per year at various locations, averaging 7 or 8 pounds. This is not enough to produce

marked increases in crop yield, but in runoff or

percolative waters, it becomes a steady and significant supply."

Campbell and Webber (1969) stated that

precipitation contributes more N to 2,100-acre

Canal Lake in Ontario, Canada, than does the

30,000 acres of unfertilized pasture land. They

estimated the total stream input to be 4,845

pounds of N03"-N, of which 480 pounds was

from nonagricultural sources (summer cottages). The agricultural land contributed 0.15

pound per acre. Feth (i^^^) said: "Data on the

nitrogen load added to surface water and ground

water by precipitation are hard to evaluate. Precipitation might be the most important single

source, for surface water at least. Inevitably,

then it must be an important source of nitrogen

in ground water also."

Nonsymbiotic bacteria fix nitrogen from the

air, but the magnitude of fixation is generally

small and low per unit of land area. In table 8,

nonsymbiotic fixation was estimated at 1 million tons annually. The blue-green algae contribute significant amounts of N to rice paddies

and sometimes to lakes. The symbiotic Rhizobia

in legume root nodules and root organisms on

many nonleguminous species fix large amounts

of N when grown under favorable circumstances. Alfalfa can fix as much as 600 pounds

of N per acre per year, but most of the N is removed in the hay. Note in table 8 that the estimates of N fixed by legumes were 1.7 million

tons in 1930 and 1947, and 2.0 million tons in

1969.

Stout and Burau (1967) suggested that nodulated legumes upon decomposition can contribute nitrate to percolating water. However,

Stewart et al. (1967a, h) found low concentrations of nitrate under alfalfa fields to a depth of

at least 20 feet. The nitrate concentrations were

about the same as those under virgin grassland.

Alfalfa either absorbed the nitrate that accumulated in the deep profile, or possibly denitrified it at the root surface. Legume nodules are

not efficient in fixing atmospheric N2 if they

have fixed nitrogen available. Hence, legumes

are probably not an effective factor in contributing nitrate to percolating water, unless large

amounts of leguminous residues are available

for decomposition.
22 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

NITRATE TRANSPORT AND ACCUMULATION IN

RELATION TO HYDROLOGY AND WATER MOVEMENT

Water is the solvent, the transporter, and a

storage reservoir for nitrate. Microbial mineralization of soil organic nitrogen and organic

wastes requires water at least in amounts sufficient to exceed the approximate wilting point of

the soil. Nitrate diffusion occurs very slowly in

soils or geological materials without appreciable

water movement, so that significant nitrate

transfer has to be in moving water. Since nitrate is not adsorbed or negatively adsorbed by

soil particles, water in soil is a storage media

for nitrate just as in a lake. Hence, an understanding of the physics of soil water and its

movement, of hydrology, and of climatology is

essential for a thorough understanding of nitrate. All we can do in this review is to point

out some of the more important aspects. Some

aspects of nitrate pollution in relation to hydrology have been reviewed by Goldberg

(1970), he Grand (1970), and Feth (1966),

In the Root Zone

We use the term **root zone" in a general

sense since its thickness depends on the kind of

plant, soil, and climate. The Monograph "Soil

Nitrogen" edited by Bartholomew and Clark

(1965) covered the behavior of nitrogen and nitrate in the root zone in detail. It contains a

chapter by Gardner (1965) giving a mathematical treatment of nitrate movement in the soil

profile. Biggar and Cory (1969) discussed the

roles of diffusion and tortuosity of the migration path in porous media like soil in spreading

out the advancing front resulting from a pulse

of nitrate solution followed by water lacking nitrate. The further the advancing front of nitrate moves, the more diffuse or spread out it

becomes. Nitrate may move faster, slower, or at

the same rate as the average total water in the

soil. A general rule, applicable to soluble salts

too, is the depth of maximum nitrate concentration resulting from nitrate application at the

surface will advance about 1 foot for each foot

of water infiltrated.

If the direction of water movement is upward

i-esulting from absence of rain and high evaporation, then nitrate can accumulate at the soil

surface as a crust along with other salts as an

evaporite. As noted previously, this is probably

the explanation of the pla,ya accumulations.

Malpeaux and Lefort (1912) found that sodium

nitrate placed at a 40-inch depth in soil reached

the surface 6 inches of soil in less than 6 weeks.

This work was of significance when W. P.

Headden in Colorado was trying to explain the

nitre spots as accumulations resulting from

nonsymbiotic N fixation by Azotohacter. Accumulations of nitrate in the soil surface in welldrained soils in the dry season has been measured in Uganda (Stephens, 1962), in Kenya

(Robinson and Gagoka, 1962)^ and in northern

Australia (Wetselaar, 1961), to mention only a

few reports.

Lateral movement and accumulation of nitrate in ridges of crops grown under irrigation

are of great significance to agriculture. Perhaps

the first instance noted of such accumulation

was by McBeth (1917) j who reported accumulations of nitrate in nitre spots that contained

as much as 1 percent N as nitrate between furrows in California citrus groves.

Harding (195Í) reported that as much as 3

tons of salts containing 800 pounds of nitrate N

per acre collected in the interfurrow ridges of

irrigated oranges in southern California, Nielsen and Banks (1960) pointed out that nitrate

accumulation in ridges (where the plant

grows) between irrigation furrows may be a

problem in getting good N recovery from N fertilizer. Stout (1961) indicated that such nitrate

accumulation in ridges in the intermountain

area of the United States can be very detrimental to sugarbeet quality if fall rains wash

this nitrate into the root zone and the beets resume growth. Regulation of nitrate nutrition of

sugarbeets is difficult in such situations. Nelson

(195S) studied nitrogen placement in relation

to the position of the irrigation furrow and

found that side placed fertilizer N must be

placed below the water level in the furrow to
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 23

avoid nitrate concentration in the ridge and immobilization there by evaporation.

Two conditions are essential for nitrate to

move below the root zone—there must be nitrate present and downward water movement.

Except in regions with excessive precipitation

or irrigation requiring tile drainage, there is

seldom any direct estimate of the amount of

water percolating below the root zone. In arid

and semiarid regions, the estimates of ground

water recharge are at best only estimates not

based on direct measurements. Therefore, the

alternative approach of measuring precipitation

and subtracting estimated évapotranspiration

and runoff to get the deep percolation by difference must be used.

Nelson and Uhland (1955) were perhaps the

first to use this approach in relation to possible

nitrate loss from the root zone. They constructed a map of the eastern half of the

United States showing the hazard in four

zones of applying fertilizers in the fall. The parameters were fall and winter precipitation and

time when an average minimum air temperature of 40"^ F. was reached, the latter being significant since the nitrification of injected NH^

and ammonium salts depends on temperature.

Allison (1965) stressed the importance of

curves of precipitation and évapotranspiration

plotted on a monthly basis and the soil storage

capacity for water in evaluating the potential

for nitrate leaching. Presumably, runoff would

also have to be considered. Allison stated:

'Trobably the first fact that needs to be emphasized is that leaching of available nitrogen beyond the plant root zone usually does not occur

to any marked extent in cultivated, medium-textured humid soils in the United States during

the main growing season, unless the annual

rainfall is above about 50 inches. Most of the

leaching occurs in the late fall, in the winter if

the soil is not frozen, and in the early spring.''

In using the précipitation-évapotranspiration

concept, it is important to have current data

rather than long-term averages. The latter may

be of value for broad generalizations spanning

both area and time, but there is no way to explain the nitrate accumulation below the root

zone in wheat-fallow systems observed in northeastern Colorado by Stewart et al. (1967a, &),

where the average rainfall is only 14 to 17

inches and the root zone seldom becomes filled

with water. It is possible to explain the downward movement by a sequence of several years

with precipitation above normal. These do

occur. A series of wet years could result in a

''pulse'^ of nitrate escaping below the root zone.

One further point that needs consideration is

the depth of nitrate in the root zone. The nitrate in the upper part of the soil is generally

more available for plant root absorption than

that which is deeper, simply because the root

system of most plants has decreasing density

with increasing depth. Gass (1969)^ placed

K^^NO.s at depths from 0.3 to 6 feet in an eastern Nebraska silty clay loam in plots, each of

which had accumulated 364, 417, and 520

pounds of residual soluble nitrogen per acre in 6

feet of profile resulting from differential N fertilization the preceding 3 years. Corn absorbed

N^^ at progressively deeper placement depths

with continued growth, but more N^^ was present in the mature stover from the 0.3-foot

placement than from deeper ones. More isotope

in the grain, however, was absorbed from the 2-

and 4-foot depths. Recovery of N^^ from the 6-

foot placement decreased with increase in the

amount of residual nitrogen.

As noted in another section, alfalfa has the

apparent capacity to remove nitrate from the

profile to at least a depth of 20 feet in alluvial

soils and so is a promising crop for ''decontaminating" soil profiles of their nitrate.

Nitrate distribution in the root zone, particularly when a growing crop or furrow irrigation

is involved, has shown so much heterogeneity

that researchers interested in studying its uptake by plants or movement below the root zone

have been completely frustrated by soil sampling procedures.

In irrigation projects and in irrigated fields,

the soluble salts applied in the irrigation water

must be balanced over a period of time by the

salt draining from the root zone. The leaching

requirement can be calculated and water in excess of évapotranspiration applied to remove

^ Gass, W. B. Recovery of residual nitrogen by corn

plants from various soil depths as measured by ^^N tracer

techniques. Thesis, Univ. Nebr., Lincoln, Nebr. 1969.
24 AGRICULTURE HANDBOOK 413/U.S. DEPT. OF AGRICULTURE

salts from the profile. In most situations when

adequate water is available, excess water is applied in most irrigations during the season because of the operational difficulties of matching

amount of irrigation to the amount of water

needed to bring the root zone back to field capacity. Obviously, the best time to accomplish

leaching is late in the season after the crop has

removed most of the nitrate from the root zone.

Research agronomists and soil scientists are

now giving increasing attention to the effect of

fertilization with commercial fertilizers, animal

wastes, sewage sludge, and effluents on nitrogen

recovery by crops, and to subsequent availability and distribution of the nitrogen unused by

the crop. A review of all the literature, most of

which cannot be related directly to the question

of nitrate accumulation in ground waters because of lack of direct measurement of nitrate

in percolate below the root zone, is beyond the

scope of this paper. However, such a review

needs to be made.

Although an unsurfaced cattle feedlot or a

dairy cow loafing pen does not have a root zone,

it can soon have one if the livestock are removed. Weeds grow promptly and crops usually

can be grown without difficulty. The soil under

an animal enclosure has many forces operating

that are at least more intense, if not different,

than in a cropped field. All of the forces and the

results are not understood, but this is an active

area of research.

Viets {1971c) indicated some of the effects on

nitrogen transformations derived from field and

laboratory studies. In a cattle feedyard stocked

at high density and continuously, the ammonia

comming from hydrolysis of urea in urine

makes the surface extremely alkaline so that

the ammoniu is volatilized rather than converted to nitrate by the nitrifiers. In lower

density yards where there may be some nitrification, the nitrate may be leached to a lower

depth along with soluble carbon compounds

where the combination of high BOD along with

high water content results in denitrification. In

a feedyard that is operated only part of the

year, opportunities for nitrification and leaching of the nitrate toward the water table are

undoubtedly much greater than in a yard used

continuously. These differences in possible behavior of the N wastes under different conditions of management are undoubtedly the explanation for the results of Stewart et al.

{1967a, ly)\ who found that the NO3--N in 20

feet of profile varied from none to over 5,000

pounds per acre under cattle feedyards in

northeastern Colorado. Each profile had differ^

ent distribution of nitrate with depth. These

studies suggested the possibility of choosing

sites for feedlots and operating them so that

there will be a zone of nitrification near the surface and an anaerobic zone below where the nitrate is denitrified. Studies of a feedlot with the

desired site conditions are now underway at

Central City, Nebr., by the U.S. Department of

Agriculture in cooperation with the Nebraska

Agricultural Experiment Station and the Water

Quality Office of the Environmental Protection

Agency (Mielke et al., i5 70 ).

In the Zone Below Roots and Above

the Aquifer

The presumption is that nitrate below the

root zone and above the water table is stable,

there being" an insufficient supply of oxidizable

carbon for the denitrifiers. There is no significant source of ammonium for the nitrifiers, the

ammonia being adsorbed and held against

leaching by the clays and humus of the root

zone. F. E. Clark found low populations of bacteria in this zone compared with surface soil,

but there was some increase in population in

the capillary fringe above the water table under

irrigated fields and cattle feedlots (Stewart

et al, Ï967a).

One peculiarity observed by investigators of

nitrate concentrations in cores is the marked

variation with depth (Stout and Bnrau, 1967;

Stewart et al., 1967a). Nitrate expressed on the

basis of dry weight of soil is expected to vary

widely because the soil texture and water content may vary widely; for example, a layer of

clay impedes water movement into a lower layer

of loamy sand. However, large variations hav»

ing a threefold or fourfold range are still apparent when the nitrate is expressed on the basis

of the water in the strata as it existed in the field.

The variations ax-e too large to be caused by
ACCUMULATION OP NITRATE IN SOIL, WATER, AND PLANTS 25

negative adsorption of nitrate. Biggar and

Corey (1969) discussed these variations. The

best explanation may be that they are caused by

*'pulses'* of solution of high nitrate concentration coming from the surface which are not

obliterated by diffusion as water moves dowm

through the profile. The possibility that denitrification is occurring in these zones of low concentration has not been excluded, however. At

present, the presumption that nitrate below the

root zone will eventually reach the water table

appears to be correct, but it cannot be accepted

as proven.

In Aquifers

Biggar and Corey (1969) discussed the mixing, stratification, and differences in flow velocities in aquifers in relation to nitrate accumulation and lateral movement. Stratification and

incomplete mixing are common in ground water

bodies. Rates of lateral movement may vary

from 1 foot a year to several miles per month.

The size and rates of turnover of the water in

the basin or aquifer must also be considered.

Carlson (196Ji) found that the mean residence

time of recharged ground water in a Wisconsin

drainage basin as 45 days and in a New Jersey

basin as 30 days.

Contrast this humid region situation with one

existing in the semiarid region of the Great

Plains using the estimates cited by Duke

(1967) for the Ogallala aquifer under Colorado's northern high plains before irrigation

wells were drilled. Duke estimated that this

aquifer contained 80 million acre-feet, the recharge rate is 430,000 acre-feet per year, and

that 390,000 acre-feet underflow eastward into

Kansas and Nebraska at the rate of about 2 miles

per year. By dividing the aquifer storage (80

million acre-feet) by the recharge rate (430,000

acre-feet per annum), one gets a mean residence

time of 186 years.

The Ogallala contains very "old" water. Obviously, on a time-scale nitrate pollution and depollution of the New Jersey basin can occur

2,265 times as fast (186 years/30 days) as the

Ogallala formation. Admittedly, application of

simple dilution chemistry to an aquifer is extremely simplistic, but it does illustrate the importance of considering the hydrology of the

region. We should take particular note of Carlson's (196i) statement that the water in 90 to

95 percent of United States' available ground

water reserves is probably over 50 years old,

which makes their tritium content useless for

dating them.

Some concept of the slow movement in the

Ogallala may be gained from the experiments of

Scalf et al. (1967) near Amarillo, Tex., who injected water containing DDT, tritium, and nitrate into a recharge well for 10 days and then

pumped the well, after 3 hours of equilibration,

for another 12 days to recover the tracers.

Water containing an average of 5.435 p.p.m. of

N03~-N was injected at a rate of 348 g.p.m.

The injected nitrate came from NaNOs added

to the recharge water that contained initially

1.72 p.p.m. of N03"-N. Water from the supply

well contained 72 pounds of N03~-N and that

from NaNOa was 156 pounds, for a total of 228

pounds. After 12 days of pumping at 504 g.p.m,,

all of the tritium and 94 percent of the nitrate

were recovered, but most of the DDT remained

in the aquifer. In accounting for the lower recovery of NO;i than of tritium, Scalf and coworkers stated that no denitriñcation occurred.

They attributed the lower recovery to difficulties in estimating the nitrate concentration in

the aquifer initially but do indicate the possibility that nitrate diffused further than the tritium. Such a possibility is questionable. They

stated that nitrate pollution of the aquifer by

nitrate is of more concern than DDT pollution

because of the ability of nitrate to move

through the aquifer. However, their own data

indicate that such movement must be very slow.

Nitrate composition of well waters fluctuates

seasonally and often erratically. Even adjacent

wells will differ when sampled at the same time

because of the differences in depth and position

on the aquifer. Feth (1966) reviewed some of

the data on fluctuations in well water composition. These fluctuations make it difficult to determine if trends in composition are occurring

unless a near continuous record is kept of composition and flow.

Very little is known about the stability of nitrate in ground water. The fluctuations in nitrate content of well water discussed by Feth
26 AGRI€ULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

(1966) ave probably mostly dilution and concentration phenomena related to the source, either from infiltration from above or lateral displacement. Stratification also occurs, and so

changing composition of well flow can be related to depth of drawdown. However, reduced

water tables do occur containing Fe++, SÖ3",

HoS, NH3 and even methane. Under such conditions, any infiltrating nitrate should be reduced, but Stewart et al (1967a) found nitrate

in water samples with redoxpotentials of —300

mv. Samples from cores of soil or earth had insignificant nitrate when the E^ was below^

-l-3ö0mv.

Ammonia occurs in high concentrations in

spring waters from Lake and Colusa Counties

in California (Feth, 1966). Its source is unknown. Undoubtedly, some reduction of nitrate

occurs when septic tank or percolate from feedlots reaches the water table. F. E. Clark (in

Stewart et al., 1967a) obtained much higher

counts of total bacteria in the capillary fringe

above the water table than in the soil above it,

both under feedlots and irrigated fields. Since

from 0.9 to 1.3 g. of biodegradable carbon is

needed to denitrify a gram of NOs^-N, it appears unlikely that much microbial denitrification can occur in the water table. The safest assumption then is that once nitrate reaches the

water table it will remain until flushed through

orjumped out.

RECENT FIELD INVESTIGATIONS' O^ NIHRATE

ACCUMULATION AND ITS SOURCES

In Arid and Semîarîd Regions

In California, Johnston et al. (1965) in a

study of the N inputs in fertilizer and irrigation water, and losses in the tile effluent and

runoff from four fields in the San Joaquín Valley, found that the losses as percentages of

inputs varied from 9 percent for cotton to TX)

percent for a field 70 percent in cotton and 30

percent in rice. Application rates of fertilizer

were 19r5 pounds per acre for cotton and 66 for

rice. The nitrate in the irrigation water applied

ranged from 4.8 (cotton) to 31 (rice) pounds

of N per acre for the four tiled fields. The

nitrate-N concentrations in the tile effluent varied from 1.8 to 62A p.p.m. The mean concentration was 25*1 p.p.m. No unfertilized controls

were included, and so the amount of nitrate lost

assignable to fertilizer application cannot be determined. The drainage from a recently instalkd line under a field that had never been

farmed or irrigated contained 1 p.p.m. of nitrate N. The tile effluent from a field in firstyear alfalfa, planted on land that had not been

N fertilized for 2 years, ranged from 2.0 to 14.3

p.p.m. Peak discharge of nitrate N was 4.7

pounds per day for 90 acres in alfalfa. Although

the alfalfa field received 1,717 pounds of N as

nitrate in the irrigation water, only 282 pounds

of nitrate N appeared in the drainage.

The nitrate-N losses in runoff from a natural

'l,0Ö0-ai3relysimeter," located on granitic soils

over granite or gabbro near Riverside, Calif.,

were investigated by Davis et al. (1969) ana

compared with the 135 pounds of N as urea per

acre applied annually in split applications to the

citrus grove. The losses in 1966 and 1967,

respectively, were 57 and 72 pounds per acre,

amounting to 42 and 53 percent of the N applied. Nitrate-N concentrations of the drainage

were generally above 10 p.p.m. Both the amount

of N and irrigation applied were excessive

according to 0avis and coworkers. The experiment lacked an unfertilized control. In a subsequent paper, Bingham et al. (1971) gave additional data for 1968 and 1969. Nitrogen losses

in runoff and percolate for 1967 were 45.6

pounds per acre, for 1968—55.1 pounds, and for

1969—69.6 pounds. They stated that nitrogen in

drainage averages about 45 percent of the nitrogen appliexi. Although the irrigation water is

relatively saline, significant salinization of the

soil is not developing because 40 to 50 percent

of the rain and irrigation water percolates. Average NOs" concentration of percolate for any
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 27

2-month period in the 3 years ranged from 37 to

81 p.p.m.

Doñeen (1966) measured the nitrate in the

tile effluents from three irrigated fields in the

San Joaquin Valley and compared the results

with the nitrate in the saturation extracts of

two adjacent virgin land sites that had never

been irrigated, and from which no drain effluent

was available. One field had grown cotton, rice,

barley, and lettuce in the 1962-66 period. The

average annual application of N fertilizer was

167 pounds per acre. The irrigation water contained 1.7 p.p.m. of nitrate N. Water applied

ranged from 7.4 to 12.1 feet per year. The nitrate N in the tile effluent averaged 44.5 p.p.m.,

which was two to four times the concentration

of the saturation extract of the profile samples.

Another field had been cropped to tomatoes,

cotton, wheat, safflower, barley, and lettuce. Annual applications of fertilizer N had been about

100 pounds per acre. Water applications ranged

from 2.5 to 6.1 feet per year and contained 2.1

p.p.m, of nitrate-N. The average nitrate~N concentration of the drain water was 23 p.p.m.

Another field cropped to safflower and cotton

received fertilizer N applications of 75 to 131

pounds per acre per year. The average

nitrate-N concentration of the drainage was 37

p.p.m. On the basis of the saturation extracts

and ground water composition, one of the virgin sites contained more nitrate and one contained less than that of the cropped, fertilized,

irrigated sites. The data illustrate the problem

of assigning source among fertilizer, irrigation

water, organic matter decomposition, lateral

movement, and earlier nitrate accumulations in

evaluating a nitrate problem.

Nightingale (1970) made a statistical study

of 18 years' records of the nitrate composition

of the Fresno-Clovis urban area well water and

the surrounding irrigated, highly N-fertilized,

agricultural area. The urban area now contains

two sewage treatment facilities. The data were

broken into three 6-year periods. Well waters

were a third more saline under the agricultural

area than under the urban area. The mean

nitrate-N content of the well water for the

1950-67 period for the agricultural area was

2.29 and 3.20 p.p.m., or 40 percent higher for

the urban zone. In the urban zone, the

nitrate-N increased from 2.31 p.p.m. for the

first 6-year period to 3.64 p.p.m. for the last.

Under the agricultural zone, the nitrate-N

increased from 1.22 to 3.51 p.p.m. For the

last 6 years nitrate increased faster under the

agricultural area than under the urban area.

The declining rate of nitrate increase under the

urban area in the last 6 years was attributed to

decreasing use of septic tanks and more connections to sanitary sewers.

Behnke and Haskell (1968) showed that the

ground water contained from 5.5 to almost 8

p.p.m. of nitrate-N down gradient from the

Clovis sewage plant and leaching ponds. Similar

values were noted in areas of septic tank use.

Stout and Burau (1967) conducted one of the

best studies to date in trying to understand the

N balance of a water basin in relation to nitrate

accumulation in underground water in the

Grover City-Arroya Grande Basin near San

Luis Obispo, Calif. They quote State of California estimates that 450,000 pounds of N are imported and 150,000 pounds are exported in farm

products annually from 2,700 acres used for

crop production. The net agricultural N remaining in the Basin is 74 pounds per acre per year.

On the assumption of 2.5 feet of percolate annually from over-irrigation, Stout and Burau

(1967) calculated that the nitrate-N in the

leachate would be increased 10.9 p.p.m. each

year. They calculated that the protein intake by

the 13,500 inhabitants would add 16.4 p.p.m. of

N to the water pumped for domestic use, ignoring evaporation and the input of N from lawn

fertilization. They could not make estimates of

other N inputs such as N-fixation by legumes

and denitrification losses from the system.

Cores taken under different kinds of land use

were analyzed for nitrate. In an irrigated

strawberry field, located on a sandy soil and irrigated with water containing 26.6 p.p.m. of

nitrate-N, the water in the profile from 4 to 39

feet had an average concentration of 21 p.p.m.

Water in the saturated zone below 36 feet contained 16.6 p.p.m. Water percolating downward

had only 79 percent the nitrate concentration of

the irrigation water. The percolating water contained 90 percent of the N in the irrigation

water and fertilizer applied less that removed

in the crop. Under a heavily fertilized crop of
28 AGRICULTURE HANDBOOK 413, U.S. DEPT. OP AGRICULTURE

celery located on a clay soil, they found nitrate

concentrations varying from 14 to 122 p.p.m. in

the soil water from depths of 10 to 43 feet.

Since these depths were sealed off from surface

percolation by an impermeable clay layer, they

concluded that the nitrate deep in the profile

was from pre-irrigation sources. For a permanent pasture they found an average soil solution

concentration of 2.78 p.p.m. and estimated that

only 4.4 pounds of nitrate N per acre is leached

out of this pasture annually.

In sewage effluent lagoons used for ground

water recharge and operated on an intermittent

drying and filling basis, Stout and Burau

(1967) found the nitrate concentrations in the

profile were high in some layers and low in others. They attributed this stratification to

^^slugs" of nitrate coming from the ponds. When

the ponds were dried, nitrification of the

sludge produced high concentrations of nitrate

that was leached when the ponds were filled.

After this slug of nitrate was leached, the low

nitrate water of the effluent moved down. Stout

and Burau emphasized that any fertile soil will

produce nitrate for leaching, and that this release of nitrate is impossible to separate from

that coming from fertilizers and decomposition

of crop and legume residues. For a basin with

high nitrate waters at shallow depths, being

continually recycled by pumping, they recommend drawing water from deep wells uncontaminated by nitrate for domestic use, and recycling of the shallow nitrate-laden water for

agricultural use.

Dyer (1965) showed graphs of nitrate concentration with soil depth for four nonirrigated

and four adjacent sprinkler-irrigated profiles of

the Panoche series in the San Joaquin Valley.

The unirrigated sites had from 1,400 to 2,800

p.p.m. of nitrate N in the soil water to the depth

of drilling, 50 feet on three sites, and 14 on the

other. Soil material in these sites was dry

throughout the profile. Irrigation and cropping

had removed most of the nitrate from the upper

20 feet of the irrigated profiles, but accumulations were noted at depths of 20 to 30 feet. The

NO3" concentration-depth curves in these zones

of accumulation were sigmoidal in shape, characteristic of a wetting front. Nitrate concentrations as high as 4,200 p.p.m. of soil water in

these zones of accumulation were noted. Since

soil water data are not given and these irrigated fields had been in alfalfa about half the

time since inception of irrigation, no further

analysis is possible. Stewart et al. (1967h)

found little nitrate under an alfalfa crop to a

depth of at least 20 feet and concluded that alfalfa has the ability to remove nitrate from the

profile to an appreciable depth.

Meek et al. (1969) showed that in the Imperial Valley of California only 1.5 percent of the

250 pounds of N per acre applied to cotton on a

silty clay loam reached the drainage tile as nitrate. They attributed the loss to denitrification

in the poorly drained soil above the tile, and

showed that the redox potential dropped below

300 mv. below 152 cm. of depth. The tile lines

were 183 and 244 cm. in the two parts of the

field. Tile lines in the Imperial Valley often become clogged with MnO^ and Fe precipitates,

indicating that there is a zone of intense reduction in the water above the tile lines.

In the Coachela Valley of California, Bower

et al. (1969) found little nitrate in the drainage. Lack of nitrate in the drainage may be related to limited use of water for leaching of

salts. On a chemical equivalent basis, nitrate

amounted to about 0.20 percent of the salts in

the irrigation water from the All American

Canal. In the valley main drain, nitrates constituted about 0.77 percent of the soluble salts.

For the San Joaquin Valley of California,

Glandon and Beck (1969) reported the results

of an investigation on tile drain flows and the

nitrate concentrations in them. In the 42 tile

systems monitored, the annual discharge

ranged from 0.3 to 17.0 acre-feet per acre per

year. The average nitrate N concentrations varied from 2 to 400 p.p.m. Flows were highest

during April through September and decreased

when irrigation was stopped. Nitrate concentrations in tile drainage from several rice fields

declined rapidly during flooding and increased

when the fields were drained. In the northern

part of the valley where an average of 42

pounds of N fertilizer had been used per acre

per year during the 1957-67 period, the tiles delivered 35 pounds of nitrate N per acre per year

for the period 1966-67, inclusive, and the average concentration was 9 p.p.m. Corresponding
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 29

numbers for the central San Joaquín Valley

were 88 and 83 pounds per acre and 33 p.p.m.,

and for the southern part of the valley the values were 74, 2, and 9. They reported that many

of the soils contained residual (geologic) nitrate, and that one experimental tile system located in a basin with an accumulation of salt

delivered water ranging from 370 to 2,000 p.p.m.

of nitrate N. No conclusions about the impact of

fertilizer use on nitrate in drainage can be

drawn from these studies. However, the data

do show that the San Joaquin Valley had a

vast accumulation of nitrate in the subsoils before the beginning of agriculture.

In his summary of the California situation,

Ward {1969) pointed out that there were five

areas in California where nitrates in ground

waters frequently exceed the standards. For the

Verdugo Basin, he stated the California Department of Public Health had concluded that the

major contributions to the high nitrates in

ground water being pumped for domestic purposes were from domestic sewage discharge

from individual household systems. In the Arroya Grande-Grover City Basin, high nitrates

were found in wells 70 feet or less in depth or in

deeper wells having gravel packing, extending

to shallow formations; or deep wells having

some of their perforations at shallow depths.

For the Delano area, Ward summarized: "Nitrates were found to have highest concentration

in areas where the ground water has risen

markedly during recent years, where soils of

high permeability require greater use of irrigation water, or where irrigation has been practiced for longer periods of time . . . domestic

sewage was excluded as being a major source of

nitrate. . ."

The problem of high nitrate in the drainage

waters of the San Joaquin Valley induced two

Federal agencies and the California Department of Water Resources to collaborate on a research program on their sources and methods of

removal so that nitrates would not contribute to

the eutrophication of San Francisco Bay. Some

of the recent developments are reported in "Collected Papers Regarding Nitrates in Agricultural Waste Water" (noted in the literature citations to Price, 1969) and Glendon and Beck,

1969),

In the Hawaiian Islands, Mink (1962) found

that the average NO^'-N concentration of

wells in the nonirrigated sections of southern

Oahu was 1.0 úz 0.22 p.p.m., but in the irrigated

regions was 8.2 ± 2.4 p.p.m. He attributed the

difference to use of commercial fertilizers.

In Idaho, Carter et al. (1971) made a balance

study of the N and P entering and leaving a

202,702-acre tract near Twin Falls, irrigated

from the Snake River, for the water year beginning October 1, 1968. They estimated that 36

percent of the total input of water from irrigation, precipitation, and runoff onto the tract

was lost by évapotranspiration, 14 percent was

lost by surface runoff, and 50 percent was lost

by subsurface drainage into drainage tunnels

carved out of the underlying basalt and into

special drainage wells. Little opportunity existed for contamination of the drains by sewage

or nitrate use by photosynthetic organisms.

Two hundred and ten tons of N03~-N entered

the tract from irrigation water, 32 tons left it

in surface drainage, and 3,194 tons were in the

subsurface drainage. The net output of

nitrate~N was 3,016 tons, or 30 pounds per

acre. Average N03~-N in the irrigation water

was 0.12 p.p.m. and in the subsurface drainage

water w^as 3.24 p.p.m. Carter and coworkers estimated that the N fertilizer use for the entire

area was 53 pounds per acre per year. Salt loss

from the tract was high because of excessive

rate of irrigation. Nitrate loss was also high,

but the relative contributions of various sources

are unknown.

In Washington State, Sylvester and Seabloom

(1963) reported on the nitrate balance of the

Yakima River Basin for 1959 and 1960. They

found that the nitrate-N in the Yakima River

water become concentrated from 0.25 p.p.m, at

the point of diversion to 2.5 p.p.m. in the subsurface agricultural drains and to 0.8 p.p.m. in

the surface drains. On a per-acre basis of agricultural land, they give the nitrate-N inflow as

4.2 pounds and the total outflow as 19.3. They

reported that nitrate concentration in the open

surface drains was reduced two-thirds during

the growing season by surface inflow and

aquatic vegetation.

Near Spokane, Wash., Crosby et al. (1971)

reported that profiles under dairy cow loafing
30 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

areas with high cow density ranged in nitrate

content from 10 to 85 p.p.m. of dry soil (2 to 16

p.p.m. N03~-N) from the 8-foot depth to 50 or

70 feet for the two cores taken. A field control

area was very low in nitrate. Since the cores

taken under the loafing areas were very dry,

Crosby and coworkers postulated that the profiles were now sealed against water infiltration

and that the nitrate must have leached down

soon after the loafing pens were established.

The association of the current dry profile in

which little water is moving and the low relative nitrate content indicate no hazard to water

quality. A danger could arise when the land is

returned to crop production, but the effects

would be very localized from the lot little more

than one-fourth acre in size.

In Colorado, Stewart et al. {1967a, h) coredrilled 129 sites from the surface to the water

table, or to consolidated rock, in the South

Platte Valley and analyzed the cores for nitrate.

The five kinds of land use ranged from virgin

grassland to cattle feedlots, some of which had

been used for over 50 years. Average amounts

of nitrate in the top 20 feet of profile and the nitrate in the water table that entered the drill

hole are shown in table 11. The nitrate in the

grassland and alfalfa land profiles was low and

was uniformly distributed with depth. The land

in alfalfa previously had grown fertilized crops

as part of crop rotation used in the area. Alfalfa removes nitrate from the profile to its

depth of rooting by some mechanism, possibly

simple absorption. The dryland fields usually

grow unfertilized wheat after fallow in a 2-year

sequence to store moisture. These profiles were

much higher in nitrate and showed definite accumulations at depths of 8 to 12 feet in most

profiles. The irrigated land, which was mostly

in corn and sugarbeets, had larger accumulations of nitrate but it was more diffuse in the

profile. On a soil-water basis, NOg^-N usually

ranged from 20 to 40 p.p.m. For 1964, the estimated fertilizer N use for this irrigated area

was 83 pounds per acre for corn, 100 for sugarbeets, 60 for potatoes, and 40 each for wheat

and barley. These rates of fertilization are regarded as essential for good production. Cattle

feedlots showed highly variable amounts of nitrate ranging from none to over 5,000 pounds in

20 acre-feet of profile. Water from shallow

wells located in or near feedlots often had offensive odor and the wells were not used. The

nitrate concentration in the water at the top of

the water table (table 11) was highly variable

for each kind of land use, making it impossible

to draw the conclusion that the surface-land use

was affecting directly the composition of the

water in the surface of the water table. Nitrate

was not found in profile samples that had fieldmeasured redox potentials less than 300 mv.

In Nebraska, Knudsen and others (1965)

found that nitrate was higher in shallow water

tables than in deep ones. Water samples containing more than 10 p.p.m. of nitrate-N were

collected from wells where the average depth to

water was SO feet. Samples containing less than

2 p.p.m. came from wells that had an average

depth of 61 feet Herron et al. {1968) could not

find appreciable leaching of nitrate below 6 feet

in irrigated cornfields located on deep loess in

eastern Nebraska, unless the soils were very

high in N or had been highly fertilized. Under

average conditions of fertilization, they found

nitrate below 6 feet but ''not appreciably in excess of amounts found in nonfertilized soils.'*

Their experiments were located on Prairie and

Chernozenic soils that have high natural organic N content.

In New Mexico, Bower and Wilcox (1969)

analyzed the data on nitrate accumulated over

the period 1943-63 on the salt balance in the

upper Rio Grande. This was a period in which

fertilizer N use on the irrigated crops in the

valley had increased tremendously. The total nitrate load in the river decreased during the

study period. They attribute this decline to decreased river flow. Comparison of 1934-43

means with 1954-63 means showed the following annual N03~-N loads of the river in metric

tons : Percha Dam—161 to 73; Leasburg Dam

—298 to 140; American Dam—183 to 38; and

El Paso-Hudspeth County line—151 to 51. They

also mention that a substantial fraction of the

NOg in the river at the latter location comes

from the El Paso sewage treatment plant For

the four sampling stations and the three 10-

year periods, the average NOs-^-N concentration in the river varied from 0.11 to 0.68 p.p.m.

The latter value was for the period 1954-63 on
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 31

TABLE 11.—Average nitrate in 20'foot profiles and water at surface of

water table^

Land use

Number

of

profiles

NO3--N

in

20 feet

Number

of water_

tables

NOa--N

Mean Rang^e

Virgin grassland 17

Dryland . 21

Irrigated land (except

alfalfa) 28

Irrigated land (alfalfa) 13

Feedlots ^^ . 47

^ Adapted from Stewart et al. {1967a).

samples taken below the El Paso sewage plant.

The agricultural drainage water contained from

a trace to 2.84 p.p.m. for the three valleys and

the three decades. The volume of drainage

water in relation to the irrigation water diverted varied from 0.20 to 0.61 for the three

valleys and the three decades.

Power {1970) at Mandan, N. Dak., found

that nitrate moved below the root zone of corn

on sandy loam that was fertilized annually with

a high rate of N. Fertilization of continuous

wheat and grassland gave little evidence of nitrate moving below the root zone, although he

does admit to the possibility of some nitrate

leaching below the root zone under systems

with fallow or on other kinds of land use when

a series of years with precipitation above normal occurs.

In Humid Regions

In Missouri, Smith {1967) and Keller and

Smith {1967) reported extensive studies of nitrate distribution and its sources in rural wells.

Smith concluded that most of the nitrate pollution comes from animal waste and septic tank

drainage into improperly constructed, shallow

wells and not from fertilizers, except for some

heavy applications on the sandier soils of southern Missouri. He reported that cattle and hog

numbers by counties when correlated with N

use by counties gave a correlation coefficient

(r) of 0.75. In the northwestern part of the

State where the deeper waters are saline, 50 to

75 percent of the shallow wells contain sufficient

nitrate to be of concern in livestock production.

Lh.¡acre P.p.m. P.p.m.

90 8 11.5 0.1-19

261 4 7.4 5-9.5

506 19 11,1 0-36

79 11 9.5 1-44

1,436 33 13.4 0-41

He also reported that few farm ponds contained more than 0.5 to 0.75 p.p.m. of NOs'-N,

even when they received drainage from livestock areas. Table 12 shows the amounts of nitrate in the soil profile he found at varying distances from old cattle feedlots. Table 13 gives

similar data where the source was a septic tank.

He also reported that a Putnam silt loam profile

that had received 120 pounds of N annually on

corn for 17 years contained 360 pounds of

NO3--N per acre 10 feet, but a nonfertilized

cornfield contained only 148 pounds.

Keller and Smith {1967) collected more than

5,000 samples of well water and found that

about 42 percent, ranging from 12 to 75 percent

of the samples for individual counties, contained over 5 p.p.m. of N03~-N. The maximum

concentration observed was over 300 p.p.m. Nitrite, which they stated is 10 times as toxic as

nitrate, was detected in only 1 to 2 percent of

the wells in winter months and 3 to 4 percent of

them in warm weather. They stated that

N03"-N higher than 50 p.p.m. should be

avoided for livestock. Correlation of the N use

by counties with the percentage of wells containing undesirable water for livestock yielded

a nonsignificant r of -0.0295. When livestock

numbers by counties were correlated with

water supply nitrate data, the r was 0.58. It

should be noted that in 1964, which was about

the time these data were obtained, Missouri was

not a heavy user of fertilizer N.

Ibach and Adams {1967) showed that only

38.7 percent of the total cropland in Missouri

received commercial fertilizer N and that the
32 AGRICULTURE HANDBOOK 413, U.S. DEPT. OP AGRICULTURE

TABLE 12.—Nitrate nitrogen content of two soils at varying distances

from old cattle feedlots^

Farm A^

re, :NO:r-N

Farm B'

Distance from Pounds N/ac- Pounds N/acre,

contaminated oto 18 ft. groundwater, 0 to 13 ft.

^ area, ft. p.p.m.

0 2,425 73 1,375

150 1,475 48 357

300 1,014 13 317

600 780 (*) 275

5,280 958 227

^ Adapted from Smith (1967).

^ Loess soil, level topography, approximately 80 percent silt.

^ Silt loam, but with 25 to 40 percent clay below 24 in.

* Trace.

rate of application was only 52.2 pounds per

acre on the land fertilized.

In Illinois, Dawes et al. (1969) analyzed the

NOs" data for Illinois rivers dating back to

1945. The median concentrations of some selected rivers are shown in table 14, They stated

that since 1945 the NOs" concentration has almost doubled. They blame the 129-fold increase

in use of commercial N fertilizers. Their data

also showed, in general, that chloride, total dissolved minerals, and hardness of the water have

also increased. Although they showed the sewered population for the watershed contributing

water to the sampling stations, no information

is given on other stream parameters such as

BOD, NH4^-N, or total N in solution needed to

determine the extent that installation of sewage

treatment facilities, during the period of study,

had on nitrate concentrations or loads in the

TABLE IB.-—Nitrate nitrogen content of soil and

groundwater at varying distances from

septic-tank drainage field. Saline County^

Mo.-'

Distance from

septic tankj

ft.

Pounds N/acre

0 to 13 ft. soil

N groundwater,

p.p.m.

60

86

112

474

375

308

22.0

8.8

7.4

^ Adapted from Smith (1967).

rivers. Viets {1970b) has indicated that the increases in NOs" concentration observed in Illinois rivers "may be the first symptoms of

stream recovery from organic pollution by raw

sewage."

Harmeson and Larson (1970) of the Illinois

State Water Survey summarized the nitrate

measurements on Illinois streams and in ground

waters. The following are some of their conclusions. The list of potential surface water supply

sources in which the nitrate concentration exceeds the Public Health Standards during some

part of the year has increased from zero to nine

in the past 14 years. The highest concentration

of nitrate in surface waters is most often found

in the spring and early summer when the water

flow is also the highest. The highest nitrate concentrations in surface waters are more consistently found in areas of intensive agricultural

production on fertile, well-drained soils which

are naturally rich in organic nitrogen. For the

Kaskaskia watershed above Shelbyville they

made these estimates of inputs of total nitrogen: Soils—61.5 percent; commercial fertilizers

—-26.8; animal wastes—7.6; atmospheric

sources—8.8; and domestic wastes—0.3 percent. They concluded that each watershed seems

to possess unique characteristics as to sources

of nitrogen, making it impossible to reach generalized conclusions about the entire State.

Ground water contamination by nitrate from

fertilizers is not now a problem in Illinois, at

least not of the magnitude of contamination of
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 38

surface waters according to Harmeson and Larson.

In Illinois, Walker (1969) reported that 25

percent of the wells 50 feet deep or less contained water with 10 p.p.m. or more of

NO3--N.

Alldrich {1970), aware of nitrate and eutrophication problems in Illinois, strongly defended the recommended use of N fertilizers in

the State. He endorsed fall application of ammonium fertilizers after the soil reaches 50° F.

at the 4-inch depth. Curtailing fertilizer use

would require a great expansion of crop acreage

onto land with high erosion and percolation

hazards. He points out that a typical deep, dark

Illinois soil releases about 125 pounds of N per

acre per year, which is not enough for economic

production. The 1969 corn crop in Illinois absorbed over a million tons of nitrogen from the

soil-water system. Just for corn, the N absorbed

was more than twice the fertilizer N applied to

all crops, pastures, golf courses, home lawns,

and all other areas in the State, according to

Alldrich.

In Michigan, Erickson and Ellis (1971)

measured the composition and total flow of

drainage from tile lines and in creeks in an intensively farmed area in 1969, Some of the data

for nitrate are shown in table 15. The Muck

Farm is an experimental farm located on deep

peat. They concluded that the nitrate in the

drainage is only a small fraction of the fertilizer N applied and discussed the difficulties of

assigning sources among fertilizer, rainfall, and

organic matter decomposition.

In West Germany, Kick and Kretzschmar

(1968) investigated the sources of the increasing NO3"" in the ground water on the lower terrace of the Rhine north of Bonn. They took

cores to a depth of 4 meters under different

kinds of land use. They found layers of accumulation of NO3", Ch, and SOr, which were especially pronounced when concentrations were

expressed on the basis of the soil water present.

The nitrate concentrations were higher in intensively cropped soils than in deciduous forest

and wasteland soils. Differences in Cl^ and

SOr were smaller. Since the concentration of

ions within the undisturbed profiles also varied

horizontally, they stated it was difficult to trace

their changes and migrations in the field. They

said: "The increase of the nitrate load to

ground water cannot exclusively be attributed

to fertilizing, which in the area is done in relatively high applications. The sewage from

urban waste waters and water courses must

also be considered,'*

In The Netherlands, Kolenbrander (1969)

found that the nitrate in drain water from

cropped land and from pastures fertilized with

liquid or solid manure, and liquid ammonia in

the fall or winter, showed a distinct maximum

in the spring (March through May) and a minimum in November. [Some of the Illinois rivers

TABLE 14.—Nitrate concentrations in Illinois rivers not exceeded 50

percent of the time^

River 1945-51 1951-56 1956-61 1961-66 1966-69

P.p.m. -_

10.4

4.8

8.2

8.2

2.5

3.8

8.7

minois at Peoria 9.5

3.8

18.8

5.0

12.9

4.3

17.7

16.5

Kaskaskia at—

New Athens 5.0

Vandalia

Shelbyville

Skillet Fork at—

Wayne City 1.8

15.5

24.3

Ohio at Cairo -

Sangamon at Mahomet 32.6

Embarass at Camargo 28.0

^ Adapted from Dawes and others (1969)
34 AGKICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

TABLE 15.—Nitrate N in the drainage of three

farms and in water of two creeks in 1969 in

Michigan^

Drainage composition Fer-

- tilizer N

Location Maximum Minimum Total applied

P,p.m. P.p>m, Lh./acre LbJacre

Perden Farm.___. 8.1 0.9 10.8 80

Davis Farm _-.„ 7.2 1.8 7.4 35

Muck Farm -—-__ 2.8 0.2 16.7 50

Dear Creek. _- .„. 4.4 0.4 ^3.2

Sloan Creek —_ .... 3.7 0.3 ^3.1

^ From Erickson and Ellis (1971),

^ 8 months.

also show this pattern of concentration (Dawes

et al, 1969; Harmeson and Larson, 1970).]

Drainage from grassland was more constant in

composition. Because of the fluctuations in nitrate content, Kolenbrander stressed the need

for obtaining samples throughout the year. He

found an average N03~"N concentration of 1 to

2 p.p.m. from grassland and from 4 to 10 p.p.m.

for cropped arable land, values being higher

for sandy soils than for heavy-textured ones.

From lysimeters filled with 120 to 125 cm. of a

soil profile containing more than 35 percent

clay, little nitrate was lost in the 35 cm. of

leachate either from mineralization or fertilizer

application. With sandy soils containing zero to

10 percent clay, he found that nitrate loss

amounted to 60 kg. per ha., and that 20 percent

of the N fertilizer was lost from the profile.

In southwestern Ontario, Canada, Bolton

etal. {1970} measured the quantity and composition of tile drainage from plots in continuous

corn or bluegrass and from the crops in a 4-

year rotation in which each crop was represented each year in the rotation. Half of the

plots were fertilized with N, P, and K, l)ut only

the annual rates of N application are shown in

table 16. Both the N concentration and the total

N loss, practically all nitrate, were higher from

the fertilized plots than from the unfertilized

ones. They stated that the nitrogen application

rate for corn in the rotation with alfalfa was

much higher than that usually recommended. In

spite of the high N rates and the presence of alfalfa in the rotation, the N losses appear to be

small in relation to the N available. Both the

concentrations and losses of nitrate under the

bluegrass sod were very low. Since these experiments were conducted on a Brookston clay with

tile at about 70 cm., the low losses of nitrate

under all systems may be caused by denitrification in the lower root zone. They found that the

nitrate loss was predominantly affected by the

amount of drainage. For continuous corn the

drainage was 15,5 cm. per year and for the

bluegrass, 6.45 cm. This difference probably accounte for most of the difference in nitrate loss

from continuous corn vs. bluegrass.

TABLE 16,—Average annual nitrate-N composition of tile drain effluent

from nonfertilized (NF) a^nd fertilized (F) continuous corn, continuous

bluegrass, and a 4-year rotation on Brookston clay for 1961-67, inclusive,

in southwestern Ontario, Canada^

Nloss

System N applied

Kgjha,

Kotation :

Corn ( 1 ).__.__, „.__„. 128.8

Oats & alfalfa (2) 16.8

Alfalfa (3) 0

Alfalfa (4) 0

Continuous corn 128.8

Continuous bluegrass 16.8

' Adapted from Bolton and others (1970),

NF

N03--N

NF

Kg./ha. Kg./ha. P.p.m. P.p.m.

5,6 15.1 8.5 14,0

4.3 5.7 6.4 8.5

4.8 3.9 6.3 5.8

4.7 8.6 9.3 10,1

Q.ß 14.0 4.4 8.9

0.3 0.7 3.5 1,1
ACCUMULATION OP NITRATE IN SOIL, WATER, AND PLANTS 35

Nitrate Concentration by Water

Passage Through Soil

Table 17 is a compilation of the nitrate concentrations in the input water, irrigation, or

rain (R), and the subsurface or tile drainage

water for some investigations cited in this

paper. In all cases with complete data, the

drainage is much higher in nitrate than the

input water. In many instances nitrate exceeds

the standards for potable drinking water. Table

5 for the Sutter Basin in California showed that

drainage often had lower nitrate concentration

than the irrigation water. Much of this drainage was surface drainage from ricefields. In all

cases, these measurements were made on soils

that had good to excellent fertility as judged by

the areas, fertilizer programs, and kinds of

crops grown. Reduction in nitrate concentration

might have been observed if the soils had had a

very low state of fertility and were cropped.

These increases in nitrate in the drainage were

observed, even though the system as a whole

was conserving nitrogen and the total outflow

of nitrate was less than the total input.

This behavior of a fertile soil in enriching the

drainage with nitrate is somewhat akin to the

degradation of w^ater with soluble salts as water

passes through the profile and most of the

water is lost by évapotranspiration. However,

the resemblance in behavior of nitrate and salt

is only superficial Salt is affected less by reactions with soil and crops as it moves in water,

whereas nitrate is subject to a myriad of reactions that affect its concentration.

TABLE 11,-—Nitrate enrichment of subsurface drainage compared with water source

Location Reference Input

N03--N inDrainage

California - Johnston et al. (1965)

Do Davis et al. {1969)

Do Doñeen (1966)

Do . Stout and Burau {1967)

Do Binghani et aL (1971)

Idaho ^- Carter et al. (1971)

Washington ^ Sylvester and Seabloom (1963)

Michigan Erickson and Ellis (1971)

Netherlands Kolenbrander (JPöö), grassland

Do, . Kolenbrander (1969), cropped land

Washington, 1943 Table 4, unpub. dataWashington, 1944 do,

Ontario, Canada Bolton et al. (1970) ,hluegT&Bñ

Do. Bolton et al. (1970), unfertilized crops

Do Bolton et al. (1970), fertilized crops

P.p.m. P.p,m.

(?) 25.1

(?) >10

1.7 44.5

2.1 23

26.6 ^21

0.28 8.4 to 18.2

0.12 3.24

0.25 2.5

rain 0.2 to 8.1

do. 1 to 2

do. 4 to 10

0.27 2.29

trace 1.66

rain LI to 3.5

do. 4.4 to 9.3

do. 5.8 to 14.0

^ Average in soil water, 4 to 39 feet.

^ Unpublished data of C.S. Schofield and L. V. Wilson, U.S, Salinity Laboratory.

NITRATE ABSORPTION, REDUCTION, AND

ACCUMULATION IN PLANTS

Nitrate Absorption

Although the literature on ion and salt absorption and absorption mechanisms is voluminous, a definite and detailed mechanism for ion

absorption has not been described. It is generally concluded that entry into and accumulation

of inorganic ions by plant cells is via a carrier

(the carrier hypothesis) and is energy dependent. The location of adenosine triphosphatase
36 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

(ATPase) on the plasmalemma (Lott and

Hodges, 1970) has led these workers to postulate that this enzyme in conjunction with adenosine triphosphate (ATP) may be a part of a

metabolic system involved in active transport,

at least with certain ions (Fisher et al., 1970).

Whether the energy (ATP) is used to synthesize or activate the carrier or in the binding of

the ion is not known. In comparison with energy dependent (active) uptake, nonenergy dependent (passive) uptake by diffusion, mass

flow, etc, is currently relegated to a minor role

in actual ion uptake. More complete discussions

of ion absorption including the many unresolved problems are detailed in various reviews

(Brouwer, 1965; Laties, i^^^; Leggett, 1968;

Russell and Barber, 1960).

Relatively few absorption studies with nitrate are recorded in the literature in comparison with the amount of work done with other

ions, especially cations. Two reasons for the

limited use of nitrate in absorption studies are

the lack of a convenient radioisotope, and the

fact that nitrate may be reduced and assimilated once absorbed. There is enough data available to conclude that the absorption process for

nitrate is not qualitatively different than for

other ions.

Evidence for Active Uptake of Nitrate

Historically, the concept of active anion uptake (anion respiration) is based on the observations of Lundegardh and Burstrom 1933).

Althoug:h this work shows a relationship between respiration and salt uptake, it does not

provide precise evidence for the coupling of energy to anion uptake.

Active uptake is suggested by Langmuir adsorption isotherms (evidence of binding) obtained when rates of nitrate absorption by

maize roots were plotted against external nitrate concentrations (van den Honert and

Hooymans, i^55). The half-values of nitrate

absorption were 0.023 and 0.032 meq./l. at pH

6.0 and 7.4, respectively. It was concluded that

neither pH nor temperature markedlj^ altered

the uptake (binding sites). From this data, a

Km value of 2.0 x 10"^M was estimated for the

hypothetical nitrate carrier. Similar adsorption

isotherms and half-value (0.033 meq./l) were

found with perennial ryegrass roots (Lycklama,

1963), Lycklama (1963) also noted that increases in temperature (5° to 35"^ C.) caused

an increase in rate of nitrate absorption over

the entire range ; however, the greatest increase

in absorption rate occurred between 15° and

25^ C.

Additional evidence for an active nitrate uptake is provided by transmembrane eleetropotential measurements of pea and oat seedling

tissue (Higinbotham et al., 1967). With anions

including nitrate, an electrochemical gradient

was established from the tissue to the solution ;

hence, an active influx system for anions was

suggested.

Indirect evidence for a nitrate carrier or a requirement for energy for nitrate uptake is suggested by the work of Minotti et al. (1968).

Nitrogen deficient wheat seedlings when transferred to a solution of Ca(NO,02 exhibited a

lag period (up to 240 min.) before initiation of

a steady increase in nitrate absorption. Also of

interest is the report of Greidanus et al (í^fO)

that cranberry cuttings were unable to absorb

nitrate from nutrient solutions as indicated by

the lack of disappearance of nitrate from the

culture medium and plant growth. This would

indicate the lack of a nitrate carrier in the roots

of this species. This leads one to speculate that

the amount of carrier could vary among varieties within a species and for a given genotype

depending onphysiological age and status.

It is of interest to speculate about the nature

of the binding site for the nitrate ion on the

proposed nitrate carrier, especially in view of

the highly soluble (relatively nonbinding) characteristics of the nitrate ion. In this vein, the

observation of Lycklama (1963) seems pertinent. Ryegrass seedlings absorbed more (twofold) nitrate from a complete nutrient solution

that contained 0.2 mg./l ammonium molybdate

than from a similar solution devoid of molybdenum. Lycklama (1963) concluded ^ . . nitrate absorption is hampered by molybdenum

deficiency,'' although the seedlings were grown

and cultured in nonpuriñed solutions. Alternatively, it is conceivable that the enhanced

effect of molybdenum could be the formation at

the root cell surface of a molybdo-protein carrier for nitrate. This is based on the concept
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 37

that during the reduction of nitrate by the

molybdocontaining nitrate reducíase, nitrate is

bound near or by the molybdenum site.

One of the many complicating factors in ion

absorption (influx) studies is the efRux of ions

from the adsorbing tissue or cells. Recently, evidence for an appreciable efflux of nitrate that

occurs simultaneously with net nitrate uptake

has been reported by Morgan (1970). Ryegrass

grown for 28 days in ^^NOs" solutions lost significant amounts of ^^NOs" to the external Ca

(^^N03)2 solution over a 6-hour period. Moreover, net nitrate uptake from Ca (^^N09)2 exceeded that from Ca (^^N03)2 solutions. The

latter observation was interpreted as evidence

of recycling of endogenous ^^NOs" that reached

the absorbing site by efflux from the roots. The

results suggest that net nitrate uptake is the

resultant of the influx and efflux processes. As

previously mentioned, nitrate absorption studies

are further complicated by the simultaneous assimilation of the absorbed nitrate.

Influence of Companion Ion on Nitrate

Absorption

Wright and Davison (196^) indicated that

plants absorb more nitrate from solutions made

with potassium than from calcium or sodium

salts. It has also been observed that increases in

nitrate absorption are associated with increases

in the potassium content of the nutrient media.

However, increasing the amounts of potassium

supplied to field-grown plants has not consistently increased nitrate accumulation. In a similar vein, it has been noted that increased levels

of phosphorus have caused a decrease in nitrate

absorption by soybeans grown in nutrient cultures. Again, similar treatments with field

grown plants have given variable results

(Wright and Davison, 196A).

In solution cultures, the presence of ammonium ions interferes with nitrate absorption.

Wallace and Mueller (1957) reported the ratio

of NH4"^ : NOs" absorbed by lemon cuttings was

approximately 1.84, even though the ratio of

NH4^ :N03" in the solution varied from 2:8 to

8:2. Lycklama (1963) measured simultaneously

the absorption of NH4+ and NOs" by ryegrass

when nitrate content of the media was held constant (1.0 meq./l.) but ammonium ion levels

were increased from 0 to 0.9 meq./l. As the

NH4^ levels were increased there was a decrease in NO3" absorption from 55 to 1.8

meq./l. x 10"^ In the reverse experiment

(NH4'^ supplied at a constant rate of 0.2

meq./l. and NOs"" varied from 0 to 3.0 meq./l.)

NH4^ absorption was decreased but slightly.

While it is established that NH4+ interferes

with the absorption of NOs", the mechanism of

this interference is not clear. Since direct interference at the carrier site seems unlikely, the

interference is most probably indirect and attributable to assimilation. Wallace and Mueller

(1957) reported that pretreatment of lemon

cuttings with I'^NOs" subsequently resulted in

increased absorption of i^NH4'^ but had no effect on i^NOs" absorption. In contrast, pretreatment with ^^NH4'^ subsequently resulted

in decreased absorption of both i^NH4"^ and

^^NOg". Since pretreatment with NH4"^ could

have lowered the level of organic acids (Kirkby,

1968) and nitrate reductase (Hewitt and Nicholas, 196Jf)^ there seems to be some supporting evidence for Lycklama's conclusion (1968)

that NH4+ interferes with NOs" absorption at

the assimilation stage. A decrease in assimilation of NO.s" could in turn decrease net uptake

by the influx-efflux concept (Morgan, 1970).

Effects of pH on Nitrate Absorption and

Conversely

Most data indicate that maximum nitrate absorption occurs when the external media are

slightly acidic (pH of 4.5 to 6.0), although some

workers have indicated that nitrate absorption

is not markedly affected by pH. These views

may be reconciled by the findings of Wallace

and Mueller (1957) that, when nitrate concentrations of the medium were low, (2 meq./l.)

nitrate absorption was unaffected by pH. In

contrast, when nitrate was supplied at higher

concentrations (8 meq./l), nitrate absorption

was decreased at higher pH values. Nutrient solutions containing nitrate as the sole source of

nitrogen frequently became more alkaline because of the differential absorption (nitrate is

usually absorbed at a faster rate than its accompanying cation) by plants.

To maintain electrical neutrality, it is suggested that hydrogen ions are absorbed and me-
38 AGEICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

tabolized rapidly by the plant (Dijkshoorrij

1962) or that a basic ion (bicarbonate or hydroxyl) is excreted (leaked) by the cells (Jackson and Adams, JPÖf ; Dijkshoorn, 1962). The

bicarbonate (or hydroxyl) ion could have its origin in decarboxylation reactions in the cells, as

oxidation or synthesis of organic acids is the

major mechanism by which plants maintain cytoplasmic pH balance. As a consequence of unequal absorption of nitrate cellular levels of organic acid should be depleted. However, in

general this is not supported by experimental

results (Ulrich, 1941). In fact, unequal absorption of nitrate may be associated with an increased level of cellular organic acid (Hoagland, 19U iBijkshoorn, 1962),

These experimental observations are explainable by the assimilation of the absorbed nitrate.

If nitrate remained unassimilated, the expected

depletion of organic acids could be anticipated

(Dijkshoorn, 1962), Since nitrate is assimilated, metabolic changes are associated with

this process. The reduction of nitrate to nitrite

could enhance the production of organic acids

(Klepper, 1969), and the conversion of nitrite

to amino-form would alter the cation-anion bal=

anee, thus leading to the requirement for synthesis of organic acids to maintain electrical

neutrality and pH balance (Dijkshoorn, 1962).

Consistent with this view are Kirkby's findings

(1968) that nitrate grown plants had 30 percent less reducing sugars and 30 percent more

organic acids than comparable plants supplied

with ammonium salt.

From a practical view, the alkalinizátion of

the nutrient medium from more rapid removal

of nitrate by the plants could tend to retard nitrate uptake. However, physiological iron deficiency and related problems caused by the alkaline medium (pH 7.5 to 8.5) are more likely to

cause poor growth of plants cultured in nutrient media (Hewitt, 1966), These problems

can be avoided or minimized by flowing cultures, large volumes of nutrient medium, or recirculation of medium with frequent adjustments of pH, the use of a mixture of ammonium

and nitrate ions as the nitrogen source, e.g.

Hoagland's solution No. 2 (Hoagland and

Arnon, 1950), or by the use of carboxy resins in

static cultures (Hageman et al, 1961b).

Nitrate Absorption as Related to Plant

Families, Specie, and Genotype

In their review^ Wright and Dmimn (1964)

listed Amaranthaceae, Chenopodiaceae, \Cruciferae, Compositae, Grwmineae, and Solanaceae

as plant families that have been shown to accumulate high levels of nitrate. Because nitrate

content varies with: (a) Rate of absorption,

rate of assimilation, and rate of translocation,

including efflux (each of these processes are dependent on metabolic status of the plant and

probably vary with plant age) ; (b) environment, including nitrate and molybdenum supply; and (c) tissue sampled, the available data

do not permit a precise ranking of plant families even with respect to nitrate accumulation

let alone absorption.

Variations in nitrate accumulation have been

recorded for species within a family and for

genotypes within a species (Wright and DaviBon, 1964), In a cooperative study involving

seven universities, it was noted that varietal

characteristics for each of three vegetables, was

one of the major factors influencing nitrate accumulation (Farrow et al., 1969), Few reports

were found of experiments specifically designed

to measure nitrate variation as a function of

genotype. For example, Hoener and DeTurk

(19S8) concluded that "high protein" corn

plants absorbed and assimilated more nitrate

when cultured on a medium_ containing 100

p.p.m. nitrate than did the related '*low protein" strain. This is supported by the observation that the "low protein'' strain contained 49,

48, and 71 percent as much total nitrogen, nitrate, and dry weight, respectively, as the ''high

protein'' strain. However, when the plants were

supplied with 25 p.p.m. nitrate in the nutrient

media, the ^^low protein" strain had 3, 25, and

15 percent more total nitrogen, nitrate, and dry

weight, respectively, than the *-'high protein"

strain. From this it can be argued that the "low

protein" strain had a more efficient absorption

system—lower Km for niti^ate by the carrier

than the "high protein" strain. In this study,

root weight, type of root system, and assimilation rate factors that could influence absorption

were not measured. Thus, there seems to be lit»

tie or no specific data concerned with differ-
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 39

enees in nitrate absorption as a function of genotype in the literature.

Nitrate Reduction

This section will be limited to recent developments and reemphasis of certain aspects pertinent to nitrate reduction. The reader is referred

to the recent review of Beevers and Hageman

{1969) for a more detailed coverage

In the review article, it was estimated that on

a worldwide basis, 10 billion tons of nitrogen

are assimilated annually. In the United States

alone, 7 million tons of nitrogen fertilizer are

applied annually. Since much of the nitrogen is

taken up as nitrate and the reduction of nitrate

to the usable form (ammoniacal) requires eight

electrons, the reduction of nitrate to ammonia

utilizes a massive amount of solar energy, either directly or indirectly.

It is generally accepted that in both chlorophyllus and nonchlorophyllus tissue that the reduction of nitrate to the ammoniacal form proceeds as follows :

nitrate nitrite

N03~ + 2e——> N02" + 6e -> NH*"^

reductase reductase

The first and rate limiting step is catalyzed by

reäuced nicotinamide adenine dinucleotide

(NADH) : nitrate oxidoreductase (nitrate reductase). This reaction, which occurs in the cytoplasm, is a typical transfer of two electrons

from NADH. The NADH would be generated

via carbohydrate oxidation and would be considered an indirect utilization of solar energy.

The second step is catalyzed by nitrite reductase, presumably ferredoxin (Fd) : nitrite

oxido-reductase, in leaf tissue.

It has been shown in vitro that reduced ferredoxin can provide the electrons for the reduc=

tion of nitrite. Localization studies have indicated that both ferredoxin and nitrite reductase

(but not nitrate reductase) are in the chloroplasts. Based upon these observations, but with

no direct proof of in vivo function, the reduction of nitrite in green tissue is considered to be

closely linked to solar energy. In nonchlorophyllus tissue (for example, roots and corn scutellum), the immediate electron donor for nitrite reduction has not been identified. It does

not, however, appear to be ferredoxin. In nonchlorophyllus tissue, the source of energy for

reduction of both nitrate and nitrite would be

via carbohydrate oxidation and, thus, indirect

utilization of solar energy. Miflin {1970) reported that part of the nitrate and nitrite reductase in barley roots is associated in a single

particle. This particulate preparation when supplied with organic acids and ATP can convert

nitrate to ammonia (Bourne and Miflin, 1970).

Nitrite reductase from leaf tissue behaves as

a single protein that converts nitrite to ammonia without the release of free intermediates,

such as hyponitrite and hydroxyl amine. The

current data indicate that nitrite reductase affects the transfer of six electrons, a baffling

thermodynamic and kinetic phenomenon. However, it is conceivable that there could be more

than one active site on the enzyme surface. The

mechanism of this complex reduction is unknown. Two hydroxylamine reductases have

been isolated from leaf tissue (Hucklesby,

1968). One of these enzymes also utilized ferredoxin as an electron donor (Fd: hydroxylamine

oxido-reductase). The role of these enzymes in

vivo is not known and it should be emphasized

that the reduction of nitrite to ammonia by nitrite reductase in vitro (illuminated chloroplasts and exogenous Fd as the electron donor

system) is not dependent on either of the two

hydroxylamine reductases.

Characteristics of Nitrate Reductase

Some of the more pertinent characteristics of

the enyzme are: (a) Nitrate reductase is

NADH-specific ; (b) the enzyme is a metaloprotein and contains both iron and molybdenum;

(c) maximum activities of 600 to 700 nmoles of

NO2" produced per minute per mg. of protein

have been reported for the enzyme; (d) the enzyme is adaptive, that is it is inducible by nitrate; (e) the enzyme is most unstable both in

vivo and in vitro (the enyzme exhibits a 4-hour

half life in detached corn leaves at 35'' C.) ; (f)

plants subjected to mild moisture and temperature stress exhibit reduced levels of enzyme activity; and (g) the enzyme exhibits both a seasonal and diurnal variation.

Because of recent developments, a few com-
40 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

ments on these characteristics are required.

With respect to the electron donor for nitrate

reductase, it now appears that the enzyme has a

specific requirement for NADH, even for the

soybean leaf enzyme. Evans and Nason (1953)

originally reported that the soybean leaf enzyme could utilize either NADH or reduced nlcotlnamide adenine dinuoleotide phosphate

(NADPH) as electron donor. This finding was

widely accepted and, in fact, many texts indicate NADPH to be the preferred cofactor.

Beevers et al. (19 6i) confirmed the fact that

the soybean enzyme could utilize both cofactors ;

however, the enzyme from other plant species

had a specific requirement for NADH. Wells

and Hageman (1970) have now shown that the

NADPH-activity of the soybean enzyme is an

artifact, and this activity is due to the presence

of contaminating levels of a phosphatase that

converts NADPH to NADH. The latter compound is the electron donor for nitrate reductase.

While it is true that nitrate reductase can utilize reduced flavins as well as NADH for electron donors, it does not seem likely that the

reduced flavins are operative in vivo. This is because (a) the amounts of flavin required for optimum activity in vitro are in excess of the estimated in vivo levels ; and (b) reduced flavins

are auto-oxidizable, and there is no precedent

for them functioning as independent (not associated with protein) electron carriers in metabolic systems. Current work would indicate that

reduced nitrate reductase is composed of two

tightly coupled protein subunits, (a) NADHspecific diaphorase and (b) a flavo-metalo-protein, that are not readily separated by normal

purification procedures (Panaque and Losada,

1966; Schrsíder et aL, 1968; Oji and Izawa,

1969), Thus, in vitro electrons can be accepted

by the diaphorase from NADH and transferred

to the attached flavo-protein or the flavo-protein

can also accept electrons from reduced flavins.

An in vivo nitrate reductase assay has been

used by Klepper (1969) to determine the metabolic system that generates NADH for nitrate

reduction in leaf tissue. He concluded that the

cytoplasmically located 3-phosphoglyceraldehyde dehydrogenase was a major source of the

NADH used for nitrate reduction. This interrelationship between photosynthate, glycolytic

metabolism, and nitrate reduction in vivo is

shown in figure 1.

The chloroplasts when illuminated and supplied with GOo produce the gamut of phosphorylated glycerate, trioles, and hexoses, and sugars and starches. Certain of these intermediates

termed "transport metabolites'' by Heber and

Willenbrincks (196Í) move into the cytoplasm

where they are metabolized by the glycolytic enzymes. Since the pyridine nucleotides do not

move readily from the chloroplasts, the oxidation of 3-phosphoglyceraldehyde provides a

source of NADH. Since nitrate reductase is also

localized in the cytoplasm, it has access to this

NADH and uses it as a source of energy for the

reduction of nitrate. Although it can be argued

that the oxidation of pyruvate (a major end

product of the aerobic glycolysis) by the mitochondria could be a source of NADH for nitrate

reduction, the data obtained by Klepper (1969)

indicated that exogenous hexoses and trioses

were much more effective in stimulating nitrate

reduction than organic acids of the Krebs cycle.

This scheme (fig. 1) provides an explanation

for the marked stimulation of light on nitrate

reduction dependent upon CO2 observed by

Burstrom in 1943 and more recently by Kannangara and Woolhouse (1967). The data obtained are also consistent with Kirkby's (¿^^5)

observation that plants grown on ammonium

salts contain 30 percent more reducing sugars

and 30 percent less organic acids than comparable plants grown on a nitrate medium. In addition, figure 1 emphasizes the interrelationship

of light (photosynthate), carbohydrate, and nitrogen metabolism.

It has been shown that nitrate content varies

inversely with the level of nitrate reductase activity in the same tissue (Hageman et al,

1961a), Experiments with wheat (Croy and

Hageman, 1970; Eilrich, 1968) reveal that additions of nitrogen fertilizer over and above

that usually considered optimal for cultural

practices are correlated with increases in nitrate reductase level and protein content of the

plant or grain. Based on these findings, it is

concluded that the level of nitrate reductase

plays a major role in controlling the accumulation of nitrate.
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 41

Metabolic factors that regulate the level of

nitrate reductase then should be directly related

to the accumulation of nitrate. The rapid fluctuation of nitrate reductase that occurs daily

(Hageman et al., 1961a), seasonably (Zieserl et

al, 196S), when plants are placed in the dark

(Candela et al., 1951 ; Hageman and Flesher,

1960), or exposed to heat or drought (Younis et

al., 1965), indicate an effective in vivo regulatory mechanism. Evidence has been obtained

that suggests that the rate of synthesis versus

the rate of degradation, inactivation, or inhibition is one mechanism for controlling the enzyme level (Warner et al., 1969] Kannangara

and Woolhouse, i5><?7).

The rate of synthesis in vivo is dependent

upon the complex and poorly understood process of protein synthesis. Obvious requirements

are energy, the metabolic system involved such

as ribosomes, messenger and transfer-nucleic

acids; activating enzymes; and enzyme components, such as the amino acids, iron, and molybdenum. Travis and Key (1970) reported that

synthesis of nitrate reductase by etiolated leaf

tissue is light dependent. They suggest that the

light requirement is related to the formation

and maintenance of polyribosomes. Earlier

work (Beevers et al, 1965) had shown that

light was also indirectly involved in induction

by increasing the amount of nitrate in the inducing tissue. Although nitrate reductase is induced by substrate (nitrate), the process by

which it induces synthesis of the enzyme is not

well understood. It is also known that as nitrate

Light

CO

2 ^-*.

ADP ATP

1-3 PGA

^NADH,

^NADADP

ATP

3 PGA

)i

3 PGAId

:::::::::- /

^ADP

^ATP

pyruvate

Chloroplast

Cytoplasmic glycolytic

enzymes

n ATP

(energy)

CO,

Mitochondria

FIGURE 1.—-A simplified metabolic scheme illustrating the interrelationship between photosynthate, glycolytic metabolism, and nitrate reduction. (Abbreviations are: RuDP, ribulose diphosphate; 3PGA, 3-phosphoglyceric acid;

3 PGAld, 3-phosphoglyceraldehyde, DHAP, dihydroxyacetone phosphate; FDP, fructose diphosphate; F6p',

fructose-6-phosphate; NAD and NADH, oxidized and reduced nicotinamide adenine dinucleotides, respectively; and ADP and ATP, adenosine diphosphate and adenosine tri-phosphates, respectively.)
42 AGRICULTURE HANDBOOK 413, U,S. DEPT. OF AGRICULTURE

disappears or is used by the tissue, nitrate reductase disappears, thus synthesis must be

maintained to balance against continual degradation.

Although Filner {1965) has reported that

certain amino acids will repress nitrate reductase synthesis in cultured tobacco cells, attempts to repeat this work with corn leaf tissue

were unsuccessful (Schrader and Hageman,

1967).

Coumarin, trans-cinnamic, and trans-o-hydroxy-cinnamic acids will inhibit the induction

of nitrate reductase ; however, it is believed that

this is caused by the inhibition of protein synthesis in general and is not specific for nitrate

reductase (Schrader and Hageman, 1967).

With respect to rate of degradation or inactivation of nitrate reductase, it has been suggested

that this may be caused by an endogenous proteinaceous inhibitor or protease. This is based

on the observation of Travis et al. (1969) that

the rate of disappearance of nitrate reductase

activity in the dark was decreased upon the addition of protein synthesis inhibitors. In support of this view, Purvis (unpublished work at

the University of Illinois) found that nitrate

reductase extracted from young wheat seedlings is stable for 2 to 3 weeks at 3° C, after a

single ammonium sulfate fractionation. Since

the enzyme in the original extract was unstable,

it is suggested that the fractionation removed

an inhibitor or protease. The enzyme from

young corn seedlings cannot be stabilized by

similar or even more complex purification procedures.

Nitrate reductase can be inhibited in vitro by

cyanide and cyanate, two naturally occurring

m.etabolites (Schrader and Hageman, 1967).

The effectiveness of these two compounds as in

vivo inhibitors of nitrate reductase is not

known.

High temperature and moisture stress have

been associated with decreases in nitrate reductase activity in vivo. Younis et al. (1965) reported that increases in temperature (20'' day

—15^^ night, to 35° day~30° night) were associated with significant decreases in nitrate

reductase activity of corn seedlings. Plants supplied with ''optimum" levels of water had significantly higher levels of enzyme activity than

plants with '^restricted" water supply at all

temperatures tested. The measurement of enzyme activity indicated no significant interaction of temperature and moisture stress, Huffaker et ah (1970) demonstrated that barley

seedlings subjected to 4 days of ''mild" moisture

stress (water potential of control was —7 atmosphere versus —11 to —12 atmosphere for

the stressed plants) had 58 percent less extractable nitrate reductase than the controls. The

moisture stress decreased nitrate reductase activity to a greater extent (58 percent) than

three carboxylative photosynthetic enzymes (11

to 28 percent).

Genetic Variation

Corn inbreds and hybrids vary (up to fivefold) in level of nitrate reductase activity

(Zieserl and Hageman, 1962). The level of nitrate reductase activity is highly heritable

(Schrader et aL, 1966). It has been shown for

B14 and Oh43 and their progeny that the two

loci control the level of activity (Warner et al,

1969). Marked differences in the level of nitrate

reductase activity among wheat genotypes has

also been demonstrated (Croy and Hageman,

1970] Eilrich, 1968). Higher levels of nitrate

reductase activity are associated with higher

rates of reduction of nitrate (Hageman et al.,

1961a ; Schrader and Hageman, iP<?5).

Intraplant Distribution of Nitrate Reductase

Because in some plant species, there is evidence that roots may play an important if not a

major role in reducing nitrate (Bollard, 1960),

it is worthwhile to consider the relative abundance of nitrate reductase in roots and shoots

(leaves). In corn seedlings the amount of nitrate reductase in the leaves exceeds that in the

roots by threefold to fivefold when expressed on

a per-gram fresh weight or per-part basis

(Hageman, unpublished work; and Hageman

and Flesher, iP^O).

Miflin (1967) reported that the activity in

roots is greater than in leaves of barley seedlings if expressed on a per unit of extractable

protein; however, if expressed on a per-gram

fresh weight or per-plant-part basis, the shoot

has much more total activity than the root. Wallace and Pate (1965) found that the shoots had

as much activity as the roots when field peas
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 43

were grown in a media that contained less than

10 p.p.m. nitrate. In contrast, when the plants

were cultured on low (500 p.p.m.) levels of nitrate, the shoots had from 3 to 12 times more

enzyme than the roots. In other studies with

field peas, the capacity for nitrate reduction appears to be confined to the elongation zone ; that

is, the region of most active respiration (Carr

and Pate, 1967; Pate, 1968).

Although Bollard's data {I960) conclusively

showed that little or no nitrate exists in the exuded xylem sap in the many woody plant species

sampled, it should be pointd out that no mention

was made of the soil nitrogen status at time of

sampling.

In this vein, Klepper and Hageman (1969)

found that roots, stems, and petioles, and leaves

from apple seedlings cultured on a medium containing 0.015 M nitrate had nitrate reductase

activities of 351, 809, and 1,312 ^moles NOa"

produced g. fresh wt."^ hr.~i and nitrate contents of 298, 21, and 45 ¡ng. NO3--N, respectively. The higher level of enzyme activity in

the leaves and petioles and stems could account

for the lower levels of nitrate, at least in part.

Nitrate and nitrate reductase activity were also

found in leaves taken from a mature apple tree

well supplied with nitrogen throughout the

growing season (unpublished). Comparison

under defined levels of nitrate nutrition of the

nitrate reducing system of roots from plants

that reduce nitrate in the roots (field pea and

woody plant species) with those that do not

(cocklebur), as reported by Wallace and Page

(1967), would do much to clarify this problem.

Highest concentrations of nitrate reductase

activity have been found in the leaf blades of

the crop plants tested (corn, wheat, oats, soybeans, and sorghum). In general, maximum activity (20 to 30 /xmoles NO2" produced g. fresh

wt. ~i hr.~i) occurs in young (8-day-old corn

and 12- to 14-day-old wheat). In older plants,

maximum activity occurs in leaves before or

with the attainment of maximum leaf area. Nitrate reductase activity declines in the lower

(shaded) leaves of the canopy in normal field

stands of corn. In contrast, all leaves maintain

approximately equal and high activity, except

for the extreme top and bottom leaves, when intra-plant competition is minimal (Zieserl et

al., 1963). Measurements of nitrate reductase

activity made on fully expanded, unshaded, corn

leaves throughout the growing season show

cyclic variations with maxima in the seedling

stage just before tassel emergence and during

the ear filling period (Warner et al., 1969),

Leaf sheathes and ear husks contain small

amounts of the enzyme. Nonchlorophyllus tissue

from the above ground part of the plant (stalk,

ear shank, cob, and developing grain) is devoid

of extractable activity.

It is interesting to note that nitrate reductase

can be induced in both barley aleurone layers

(Ferrari and Varner, 1970) and in corn scutellum (Eisner, 1968) by incubating the tissue in

a medium containing nitrate. In intact seedlings

cultured on nitrate, the corn scutellum also develops appreciable levels of nitrate reductase

(Eisner, iP(55).

From an agronomic standpoint and in view

of the current use of high amounts of nitrogenous fertilizers, work done in our laboratory

(Eilrich, 1968, Hageman and Flesher, 1960 \

Schrader and Hageman, 1965; Zieserl et al,

1963) and that of Wallace and Pate (1965,

1967) suggested that, except for the early seedling stages, the bulk of nitrate is reduced in the

leaf blades of crop plants, excluding legumes

and paddy rice.

Accumulation of Nitrate and Factors

That Affect Accumulation

Nitrate is not accumulated uniformly by the

various tissues and organs of plants, and even

within a tissue there is variation depending on

location (e.g. tip versus base of the leaf) and

age. Nitrate accumulation in a tissue or organ

results from the rate of absorption and translocation, the rate of reduction (assimilation) and

the ability of the tissue to store the unassimilated nitrate. Because there are so many environmental and metabolic factors that influence

nitrate accumulation, extreme variations are

encountered in nitrate content even in the same

tissue and from plant to plant and species to

species.

In general terms, the stems (stalks) and petioles (mid-rib) of corn contain higher levels of

nitrate than the leaf blade. Work of Deckard
44 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

(1970) indicates that the nitrate content of the

basal part of the leaf mid-rib of corn is comparable with that of the stalk adjacent to its point

of attachment. Although there is little data

available as to the nitrate content of roots,

Wright and Davison (196^) stated that the

roots contain more nitrate than leaves but less

than stems. Limited data suggest that very little nitrate is found in the reproductive parts.

Inselberg (1956) noted that the third and nondeveloping corn ear initially accumulated nitrate in a linear manner (up to 420 fjLg.

NOH~-N/g. dry wt. over an 11-day period). In

contrast, the first and second ear initially exhibited a loss of nitrate with development. Although these nitrate levels are relatively low in

comparison with leaf tissue, the data show that

some nitrate is transported to the reproductive

tissue and suggest that these small amounts can

be assimilated in situ.

The data of table 18 (unpublished work, G.

Wells^) showed variation in nitrate content of

leaves of several plant species throughout the

1969 growing season. Similar results were observed in 1968. These plants were grown on the

^ Copy on file at Urbana, 111., Agronomy Dept.

University of Illinois farm on soil of normal

fertility but without preplant or supplemental

fertilizer or irrigation. Although all six species

exhibited a decrease in leaf nitrate content

throughout the season, the two monocotyledonous species, especially corn, showed the

earliest and greatest depletion of nitrate in the

leaf blades.

Other works with wheat (Croy and Hageman, 1970; Eilrich, 1968) and corn (Zieserl et

al., 1963) showed similar patterns [high levels

of nitrate (1,000 fig. NOg-^N/g. fresh wt.) in

young leaf tissue but rapid depletion with

development]. High initial levels of nitrogen

fertility and supplemental nitrogen applications

will tend to increase and maintain a higher level

of nitrate in the leaf blade than is shown by the

corn data of table 19. However, after the early

vegetative stage and with normal development,

the cereal crops usually do not accumulate nitrate in the leaf blades to the extent exhibited

by other plant species (table 18). One factor

for the decrease in nitrate content in the leaf

blades of corn may be due to its apparent inability to absorb nitrate in the later stages of development (Sayre, 1948; Deckard, 1970).

Sayre found that nitrate absorption by the

TABLE 18,—Nitrate content of leaf tissue of several plant species throughout the 1969 growing

season ^

Plant family Nitrate content, (ßg. NO,-r-N per g. fresh wt. ) -

and

species June

24

July

1

July

8

July

15

July

22

July

29

Aug.

5

Aug.

12

Aug.

19

Amaranthus hybridus 423 212 258 96 106 102 60 43 23

(pigweed)

Setaria Faberii 249 81 112 51 34 257 10 11 7

(foxtail)

Abutilón theophrasti 263 409 449 230 278 102 46 100

(velvetleaf)

Glycine max 386 434 280 69 172 40 19 13

(soybean)

Zea mays (corn)^ 322 41 44 9 -= 3 9 2 4

^ Unpublished work of G. Wells, Urbana, 111.

^Each value is the average of three replicate samples taken from a complete random block design. Plantine:

rates were such that there was essentially no intraplant shading.

^ The midrib was removed from the corn leaves, which in part accounts for their lower level of nitrate.
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 45

corn plant started to decrease 45 days after

planting and essentially ceased 15 to 20 days

later, A second factor is that corn leaf blade has

a relatively active reductive and assimilatory

system for nitrate. A possible third factor could

be an alteration in leaf anatomy during development that precludes accumulation.

The data in table 19 show that the last supplemental application of nitrogen (applications

made 44, 59, and 72 days after planting) was

least effective in increasing nitrate content of

stalks and leaves. It was estimated that only 10

percent of this last application was absorbed by

the plant. At maturity, the amount of nitrate in

the stalks (average of the six hybrids) was

33.5, 228.7, 195.1, and 145.9 mg. of NO3--N per

plant for the control and the three treatments

(44, 59, and 72 days), respectively. These values correspond to 97.3, 99.4, 99.4, and 99.2

percent of the total nitrate content in the aboveground part of the plant. Earlier studies established that the bulk of this nitrate is confined to

the lower third of the stalk (Schrader and

Hageman, 1965). Since the level of nitrate reductase in the stalk is extremely low, it is assumed that the stalk has a minimal assimilatory

function but does serve as a storage site for nitrate. Although there is little specific and direct

evidence on this point, it is presumed that the

nitrate is transported from the stalk to the

leaves for reduction and assimilation as the

plant develops.

Experiments with winter wheat, grown

under nitrogen regimes that minimize lodging,

show that the relatively high levels of nitrate

(200 to 500 fxg. N03"-N g. fresh weight) in

the leaf blade observed in March are rapidly depleted to negligible levels during the 6-week

period starting in mid-March (Croy and Hageman, 1970; Eilrich, 1968), Under certain environmental conditions, the level of nitrate in the

leaf blade can be depleted to nondetectible levels

by the first part of April (Stage B-4 to B-5 on

the Feekes scale). Although the stems (stalks)

of wheat, like corn, are probably not major sites

of assimilation (lacking enzymes of nitrate assimilation) , they are not good storage tissue for

nitrate. The level of nitrate in the stalks is

higher than that of the leaf blades and the depletion pattern parallels that of the leaf with a

lag of 1 to 2 weeks. In contrast to corn, the

stalks of wheat and the rest of the vegetation

and grain contain negligible levels of nitrate at

maturity.

Wheat also appears to differ from corn in

that it can continue to absorb nitrate late into

the reproductive stage of development (Barat

et al., 1969; and unpublished work of

Hucklesby^). It has been shown that wheat

plants effectively absorb and rapidly reduce and

assimilate nitrate applied during April and

early May (Eilrich, 1968; Hucklesby et al.,

1971), Eilrich applied 68 kg. N ha.-^ on April 8

to Ottawa and Gage wheats at a time when the

leaf nitrate was approximately 225 and 210 /¿g.

N03~--N g. fresh wt., respectively. By May 10,

the leaf nitrate was approximately 30 and 10 ¡xg.

N03~~N g. fresh wt. for the treated plants, and

10 to 5 fig. N03~-N g. fresh wt. for the control

plants of Ottawa and Gage, respectively.

Hucklesby et al. (1971) found that increase

in nitrogen of the grain and straw of the

treated plants over that of the controls was

more than adequate to account for the uptake

and assimilation of the 112 kg. N ha.~^ applied

May 9 (booting stage for all three varieties

used). Since isotopic nitrogen was not used, this

result is based upon depletion of an equivalent

amount of nitrogen in excess of the applied fertilizer nitrogen. No residual nitrate was detectable in the aboveground vegetation or grain at

maturity in any of the material of these two

wheat experiments.

In contrast, the mature vegetation of soybean

and three weed species contained appreciable

levels of nitrate (table 20). The bulk of the nitrate for these four plant species was found primarily in the nonleafy tissue. The higher level

of nitrate in the leaf tissue found in these species than in corn is partly because the midribs

and veins were not removed.

It is fortunate that the cereal grains used so

widely for food do not accumulate appreciable

^ Copy on file at Agronomy Dept., University of

Illinois, Urbana.
TABLE 19.—Nitrate content of stalk and leaf material of corn at different stages of development as affected by supplemental

nitrogen fertilization^

Plant 58 days when

sampled Plant 82 days when sampled Plant 120 days (matnrity) when sampled

Nitrogen applied at—

0 day 44 day 0 day 44 day 59 day 72 day 0 day 44 day 59 day 72 day

Hybrid

No. Stalk Leaf Stalk Leaf Stalk Leaf Stalk Leaf Stalk Leaf Stalk Leaf Stalk T,paf Stalk Leaf Stalk Leaf Stalk Leaf

.ßg, NOîr-N/ß*. drv weîffht"^__ _

1 8,410 259 10,670 432 1,010 14 2,570 44 1,420 14 1,670 24 234 27 3,452 27 2,141 28 1,755 38

2 7,240 235 8,310 317 1,320 16 2,810 60 2,270 44 1,940 13 316 26 2,644 25 2,106 29 2,106 22

3 10,200 255 14,050 403 1,260 14 3,390 30 2,650 40 1,980 21 1,158 51 3,054 57 2,808 45 1,977 42

4 10,040 274 15,020 610 1,090 15 4,580 154 3,380 99 1,650 35 281 33 3,276 48 2,925 52 2,223 43

5 8,810 212 13,440 526 870 5 3,040 36 1,930 31 1,450 5 199 56 3,288 35 2,141 40 1,778 37

6 9,940 230 11,810 302 850 19 3,530 36 3,510 79 1,550 31 410 43 2,656 44 3,498 42 1,977 38

' Adapted from Deckard, E. L. ('l5^70;.

* All plots received 90 kg, N har^ before planting and 337 kg. N ha. "^ were applied to the treatment plots, 44, 59, and 72 days after planting. These

dates correspond to 14 days after tassel initiation, tassel emergence, and initiation of silking. All plots were irrigated (2 inches of water, overhead

sprinkler) each time supplemental nitrogen was applied. Plant population was 59,000 plants ha.""^.

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ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 47

TABLE 20,—Nitrate content of the total aboveground portion of soybean and three weed

species at maturity and the distribution of

nitrate between leafy and nonleafy tissue

Nitrate content and

distribution

Plant family Mg. NO3---N/ Percent

and species ^ plant in leaves

Setaria faberii

(foxtail) 78 9

Glycine max

(soybean) 88 15

Amaranthus hybridus

(pigweed) 117 18

Abutilón theophrasti

(velvetleaf) 203 9

^ Same experimental material as used for the data of

table 18.

levels of nitrate. McNamara et al. (1967) used a

modification of a sensitive and specific enzymic

assay for nitrate developed by Lowe and Hamilton (1967) to survey 113 seed samples representing 37 different plant species. Seeds of 28

wheat samples and 20 oat samples had low

(maximum 1.9 /xg. N03~-N g. dry wt. ~~^) or

negligible levels of nitrate. The nitrate content

of 11 corn grain samples ranged from 1.1 to 3.8

/xg. NOa~-N g. dry wt,~\ Many of these samples of wheat, oats, and corn were known to

have been produced on highly fertile soils, and

some of the wheat and corn plants were given

large amounts of supplemental nitrogen. Nitrate was not detected in polished rice or grain

sorghum (1 sample each). Barley, navy, and

lima bean seeds (1 sample each) had 4.6, 3.4,

and 1.3 ¡xg, NOs^-N g. dry wt.~^, respectively.

Soybean seeds (10 samples) ranged from 8.6 to

23.8 jttg. N03~-N g. dry wt.~^ (these seeds were

obtained from the U.S. Regional Soybean Laboratory, Northern Marketing and Nutrition Research Division, Urbana, 111.). While cultural

conditions used in the production of soybean

seeds were not ascertained, it is presumed that

they were produced without supplemental nitrogen. These data suggest that investigation of

the effect of cultural conditions and varietal differences on nitrate content of soybean seed is

warranted. Of all samples examined, the weed

seeds had the highest nitrate content (195 samples with values ranging from 8.9 to 122.3 fig,

N03"-N g. dry wt.~^) than seed from other

plant species.

In summary, experiments with several cereal

plant species under ''normal" conditions indicate that in the early vegetative stages, the leaf

tissue can accumulate nitrate in relatively high

concentrations (400 to 1,500 fxg, N03~-N g.

fresh weight"^). This initial accumulation, under normal conditions, is attributable to available water and nitrate in the soil that favors

high rates of absorption. Thus, in the early

stages of development, absorption and translocation exceeds the capacity for nitrate reduction and assimilation. As the plant develops the

concentration of nitrate decreases in both leafy

and nonleafy tissue, with the more rapid decrease occurring in the leaf tissue. The decrease

in nitrate content is attributable to the following several factors: (a) Rapid growth tends to

dilute the nitrate concentration ; (b) the capacity of the leaf tissue to reduce and assimilate nitrate is increased not only from having a

greater amount of the proper enzymes but having an adequate supply of energy for the reductive steps; and (c) a reduction in available

water and nitrate in the soil. In mature plants,

nitrate accumulates primarily in tissue that exhibits a low capacity for reduction and assimilation (nonchlorophyllus stem and stalk structures). From studies with weeds and soybeans

(Wells^, unpublished work), it appears that

other plants exhibit a similar pattern of nitrate

accumulation.

There are many metabolic factors that are associated with the accumulation of nitrate in

plant tissue. Since nitrogen and carbohydrate

metabolism are closely interlinked through the

need for carbohydrate to provide the skeletons

for formation of nucleotides and amino acids,

and the energy for reduction of nitrate (Klepper, 1969) and synthesis in general, factors

that are unfavorable for carbohydrate metabolism will also adversely affect nitrogen metabo-

^ See footnote 4, page 44.
48 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

lism. If the specific factors affect reduction to a

greater extent than absorption and translocation, nitrate can be expected to accumulate.

Some factors are known to specifically decrease

nitrate reductase activity, while other factors

are more indirectly related to the accumulation

of nitrate. In the following section this will be

discussed in some detail.

Nitrate Availability

While listing soil nitrate as a factor may

seem obvious, the availability of soil nitrate

available for absorption by the plant is most

complex. Soil nitrate levels are extremely variable as nitrate can be lost by leaching, volitalization, microbial utilization, or simply unavailable

to the plant due to location or lack of water. Nitrate is considered to be contained entirely in

the soil solution; hence, the rate of replenishment (from organic or ammoniacal forms) may

be more important than the amount of nitrate

present at a given time (Bould and Hewitt,

Î96S),

Another truism is that nitrate must be present and available to the roots that are actively

absorbing. Specific and direct data bearing on

this point especially for field-grown plants are

minimal, Barat et al. (JP^P) indicated that

wheat plants in full head continue to absorb nitrate. The data obtained by Hucklesby et al.

{1971) showed that wheat plants in the booting

stage can effectively absorb soil applied nitrate.

In contrast, the results of Sayre (194-8) and the

data in table 19 suggested that corn does not absorb appreciable amounts of nitrate after tassel

emergence.

Drought and Temperature

Generally, when nitrate accumulates in

plants grown under droughty conditions, high

temperatures are usually an associated factor.

Both high temperature and moisture stress

have been shown to drastically reduce or inactivate nitrate reductase (Younis et al., 1965),

Huffaker et al. {1970) demonstrated that nitrate reductase activity was much more adversely affected by very slight moisture stress

than were certain enzymes of the photosynthetic system. These results suggest that either

high temperature or moisture stress has a more

adverse effect on nitrate reduction than the absorption and translocation of nitrate ; hence, the

accumulation of nitrate by the plant. Obviously,

for nitrate to accumulate under conditions of

drought, sufficient nitrate and water must be

present in the soil to permit absorption and

translocation by the plant.

Light

Light has long been known to enhance the reduction and assimilation of nitrate, although

the effects are complex. The level of nitrate reduction in leaf tissue varies diurnally and is influenced by the intensity of illumination (see

the review by Beevers and Hageman, 1969, for

full details). There is good evidence that light

influences the level of the enzyme per se.

Kannangara and Woolhouse {1967) reported

that there is a direct and absolute requirement

for photosynthetic CO2 fixation for the induction of nitrate reductase in leaves of Perilla.

This requirement does not appear to hold true

for all tissue, as the enzyme can be induced in

the dark in various tissues. Thus, the effect of

light would appear to be indirect, either by providing energy for synthesis of the enzyme or in

some way increasing and stabilizing polyribosome formation (Travis and Key, 1970), or altering membrane permeability so that transport

of nitrate is enhanced. Adequate light would

also insure photosynthate for reductive energy

via carbohydrates (Klepper, 1969) and carbohydrate skeletons for amino acid and nucleotide

formation. Shade or inadequate light can lead to

the accumulation of high levels of nitrate in

plant tissue (Knipmeyer et al., 1962).

Mineral Deficiencies

Molybdenum is a constitutive part of nitrate

reductase (Evans and Hall, 1955; Anacker and

Stoy, 1958), In most plants, molybdenum is

thought to have minimal additional metabolic

functions ; therefore, its deficiency leads to the

accumulation of very high concentrations of nitrate in leaf tissue (Hewitt and Jones, 19Í7;

Mulder, 19^8; and Wilson, 1948). There is some

evidence that iron is a constituent of the enzyme of higher plants (Anacker and Stoy,
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 49

1958) and conclusive proof that it is a part of

the enzyme of Neurospora (Garrett and Nason,

1967), Several workers have also shown that

phosphate stimulates nitrate reductase activity

(Hewitt and Nicholas, 196i). However, deficiencies of iron or phosphate do not lead to the

excessive accumulation of nitrate noted for

molybdenum deficient plants. This is probably

due to the deficiencies of iron and phosphate that

would affect metabolic energy generation systems and metabolism in general as well as affecting nitrate reductase activity per se; thus

absorption, transport and reduction of nitrate

would all be decreased in varying amounts depending on conditions and species. Manganese

deficiency has also been associated with the accumulation of both nitrate and amino acids.

These excesses are probably indirect deficiency

effects, as it seems logical that the main effects

of manganese deficiencies are on photosynthesis

and respiration. These effects would be complex

and diverse. Deficiencies of other elements, such

as potassium, could conceivably cause similar

complex alterations of metabolism.

Endogenous Metabolites

The inhibition of nitrate reductase or repression of its synthesis by endogenous components

could lead to the accumulation of nitrate in the

plant. However, it is not known whether the inhibitors or repressers of nitrate reductase that

are effective in vitro are responsible for accumulation of excessive amounts of nitrate in vivo.

Both cyanide (Hewitt and Nicholas, 19^^; Maranville, 1970) and cyanate, a derivative of carbamyl phosphate (Schrader and Hageman,

1967) will inhibit nitrate reductase, in vitro,

Filner (1965) reported that certain amino acids

will repress the synthesis of nitrate reductase

in cultured tobacco pith cells. Smith (1969)

showed that amnxonia and certain amino acids

will repress the enzyme in Chlorella and barley

roots but not in leaf tissue of higher plants.

With both Chlorella and barley roots, Smith expressed the opinion that ammonia per se, or

derived from the various amino acids, or a metabolite of ammonia (glutamate) may be the specific represser in all cases. It does not seem logical that these metabolites and their regulatory

effects would lead to excessive accumulation of

nitrate in "normal" plants.

Herbicides

Sugarbeets (Stahler and Whitehead, 1950)

and broadleaf weed species (Willard, 1960)

treated with herbicidal levels of 2,4-dichlorophenoxy-acetate (2,4-D) were shown to accumulate nitrate. No record of nitrate accumulation has been reported for members of the

Graminae family, similarly treated. An explanation for this difference in response was given

by Beevers et al. (196S). They found that nitrate reductase activity was decreased in cucumber plants sprayed with herbicidal levels of

2,4-D solutions, while the opposite effect was

noted with corn seedlings. With both species, nitrate content varied inversely with enzyme activity. Reproducible results were obtained with

growth chamber plants, while much greater

variability was encountered with field-grown

plants.

In their review, Beevers and Hageman

(1969) reported that several workers have

found that treatment of plants with sub-herbicidal levels of 2,4-D alone, or fortified with

micronutrients, increases crop yields and grain

protein for barley. However, effects of treatments on nitrate accumulation were not given.

Subherbicidal levels of simazine have been reported to increase the growth rate and nitrogen

content of plants cultured on suboptimal levels

of nitrate or when grown at low temperatures

(Tweedy et al, 1967; Ries et al, 1967), Field

trials where six different plant species were

treated with subherbicidal levels of simazine

were reported to show that appropriate treatments increased crude protein production, per

acre, from 10 to 52 percent (Ries et al, 1968). It

was stated that simazine treatment enhanced

nitrate uptake from the media. The data indicated that in some instances the nitrate content

of the tissue was higher. The rationale of the

simazine effect is that simazine increases nitrate uptake by the plant and that the higher

level of nitrate in the tissue induces higher levels of nitrate reductase. Again, as with the

2,4-D treatments, the effects on growth, yield,

nitrate and protein content for a given crop
50 AGRICULTURE HANDBOOK 413, U.S. DEPT. OP AGRICULTURE

varied with simazine level and environmental

conditions.

Workers at the University of Auburn have

shown that plants treated with atrazine (a related triazine) have a larger shoot-to-root ratio

of nitrogen and dryweight than comparable

controls. It has been proposed that the subherbicidal levels of triazine are inhibiting the photosynthetic process, thereby reducing the supply of carbohydrate available to the root and

thus altering the shoot to root ratios.^

In summary: (a) The increase in yields and

protein content achieved with the sublethal levels of herbicides emphasizes the importance of

nitrate reductase in increasing grain and grain

protein production ; (b) nitrate reductase is interwoven with metabolism as a whole ; (c) herbicida! effects on nitrate reduction are probably

indirect as observed through changes in overall

metabolism and, hence^ are complex and variable.

TIME TRENDS OF NITRATE ACCUMULATION

Foods and Feeds

In the past decade^ public concern has been

aroused over the possible health hazards of

foods and feeds of high nitrate content, to both

humans and animals. This concern has basis in

the following series of well-publicized events

that occurred in the mid-1950s to mid-1960s:

(a) Several individuals died from breathing

silo gases (NO, NOo, N2O4) produced from ensilage of high nitrate content (Wright and Davison, 196Jf) ; (b) loss of livestock was associated

with feeding forages or ensilage of high nitrate

content (Wright and Davison, 196i^\ Hanway et

al, 1963; Wright, 1968; Case, 1970) ; and (c)

infant mortality resulting from the ingestion of

spinach of high nitrate content was publicized

in both scientific and popular press (Simon as

reported by Lee et al., 1970), In this latter case,

however,.it was shown that death was caused

by nitrite produced by bacterial action after the

canned spinach was opened. Although nitrate

per se can be toxic to animals, the major hazard

of high nitrate feeds normally results from the

conversion to nitrite by microñora of the digestive tract. This is especially true for ruminants.

In a comprehensive survey of the toxicity of nitrates of foods, Phillips {1968) concluded that

the nitrate content per se of foods currently

consumed by humans older than 3 months poses

no major health hazard.

Articles appearing in the popular press,

health culture magazines, and a few poorly researched scientific articles have led part of the

' Personal communication from Mason Carter, {1970),

general public to believe that high nitrate content of foods and feeds is a new phenomenon

and directly related to the use of inorganic nitrogen fertilizers. These views can be refuted

by a review of the literature. Schimper {1888)

reported that shaded leaves contained a much

higher content of nitrate than comparable

leaves grown in the sun. Mayo {1895) reported

the loss of livestock fed cornstalks of high (2.6

to 3.5 percent NOg^-N g. dry wt.~^) nitrate

content. Producers of truck crops have always

used highly fertile soils in which organic matter

was the major source of nitrogen until the development of the nitrogen fertilizer industry

after World War I. Nitrates will be produced in

high amounts by microbial action in soils supplied with amounts of organic nitrogenous matter adequate for profitable truck crop production. Thus, it is not surprising that Richardson

{1907) found high levels of nitrates in various

market vegetables, both fresh and canned.

With respect to human foods, two recent surveys (Jackson et al., 1967; Lee et al., 1970) indicate that there appears to be no evidence that

there has been any appreciable change in nitrate content of vegetables from 1907 to the

present date. Two specific examples taken from

the review of Lee et al. {1970) illustrate this

point (table 21). As indicated by these two reviews, there is a paucity of data on the nitrate

content of human foods.

The nitrate content of cornstalks reported by

Mayo {1895) is far in excess (threefold) of the

highest level of nitrate observed in cornstalks

by Deckard {1970). Deckard's plants were
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS

TABLE 21.—Nitrate content of two vegetables

51

Vegetable NO3' content

and

source High Mean Low Analysis by

P.p.m, P.p.m. P.p.m.

Beets :

Market 8,062 2,552 930 Richardson (1907)

Grown 1,333 Wilson (ÎH9)

Grown 4,500 600 Smith (1966)

Fresh and canned 1,654 1,197 671 Jackson et aL (1967)

Spinach :

Market 3,809 1,892 310 Richardson (1907)

Canned 1,949 1,100 266 Do.

Market 2,353 1,600 Wilson (19^9)

Frozen 3,636 Do.

Fresh and frozen 739 524 238 Jackson et al. (1967)

Canned 550 473 396 Do.

Canned 1,435 886 505 Phillips (1968)

Frozen 2,463 429 Do.

Fresh 2,330 1,501 Do.

grown in fertile soil that were given supplemental inorganic nitrate fertilizers (427 kg.-N

ha."i) and irrigation. These data strongly

argue that the current use of inorganic nitrogen

fertilizers has not led to marked increases in nitrate content of feeds.

As pointed out by Jackson et al. {1967) and

as shown in the section on Nitrate Absorption,

Reduction, and Accumulation in Plants, page

35, there are many variables that contribute to

the accumulation of nitrate by plants. However,

in spite of these variables, the available data do

not indicate that the use of inorganic nitrogen

fertilizers has increased the nitrate content of

foods or feeds. In contrast, as stated by Kurtz

and Smith (1966), the extensive use of inorganic fertilizers has caused a marked increase

in both yield and quality of feeds and foods.

Soil and Water

Nitrate in the root zone is extremely variable

depending on the timing of sampling in relation

to recency of fertilization, crop growth, and

rainfall or irrigation. It may vary from almost

none to 200 or more pounds of N per acre during the season. Such fluctuations are needed for

good crop growth and for prevention of leaching of nitrate. With the increased use of commercial fertilizers, there has been a growing

tendency for nitrate to accumulate deep in the

root zone. More soil testers are now recognizing

the importance of subsoil nitrate and are insisting on deeper sampling to improve their predictions of the amount of fertilizer N to use on

crops. This awareness may be evidence that nitrate is accumulating in areas with intensive

agriculture.

We have also noted the large decreases of soil

organic matter associated with cultivation.

These losses mean that one source of nitrate is

becoming of less importance and that the deficit

needed for good crop yields must be made up

with fertilizer N and increased cycling of N in

crop residues and animal wastes.

Whether there is a trend toward higher nitrate concentration in surface waters is impossible to evaluate because of the biological reactions in which it is involved. Increases in nitrate

in lakes and streams may simply be a manifestation of better supplies of dissolved oxygen because of progressively lower inflows of

raw or partly treated sewage. For these reasons, the authors of this paper cannot conclude

that there is any trend in nitrate in surface waters, although they would not deny that the

total N and total soluble N in many lakes and

rivers may have increased significantly. It

should be noted that the 10 p.p.m. NOs'-N re-
52 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

lated to Public Health standards for water is

far in excess of the 0.3 p.p.m. total soluble N

sometimes regarded as sufficient for eutrophication.

Nitrate in ground water appears to be much

more stable biologically than that in surface

waters, but its concentration is subject to wide

fluctuations caused by dilution, mixing, and

stratification. To have evidence of a trend, we

must have sampling of sufficient frequency and

extend over enough time to warrant a conclusion. The best evidence of increasing nitrate

concentration is the statistical study of Nightingale (1970) who showed that nitrate was increasing under both the agricultural and urban

areas of the Fresno-Clovis area in California.

The calculations and core analyses of Stout and

Burau (1967) for the Grover Gity-Arroya

Grande Basin in California would also point to

a trend of increasing nitrate in ground water.

The data of Gruener and Shuval (1970) also

indicate increasing nitrification of the water on

the central and southern Israeli coastal plain.

Much of the data available indicate that drainage and percolating waters are higher in nitrate

than the infiltrating water, but such enrichment

does not necessarily mean that the water table

is changing concentration because of dilution by

water lower in nitrate coming from other

sources. Although the evidence indicates that

nitrate concentration may be increasing in some

urbanizing areas with highly intensive agriculture, the conclusion that nitrate in ground waters is increasing generally cannot be justified.

The question about the time trends of nitrate

accumulation in ground water probably could

not be answered now, even if we had started intensive sampling back in 1945 when Comly

showed that nitrate caused methemoglobinemia

in infants. In humid regions, such as our Eastern and Southeastern States, where leaching of

nitrate can be great if there is a nitrate source,

the residence time of the ground water is probably too short to allow nitrate to accumulate

(Carlson, 196-Í). In the writers' opinion, the hazard of nitrate accumulation in leached environments appears to be very low, a statement supported by the lack of a significant number of

cases of infant methemoglobinemia and livestock disorders in high rainfall areas. In contrast, the Midwestern and Plains States present

an entirely different picture. The rate of water

recharge from deep percolation is so slow that

the possible nitrate pollution of aquifers from

our modern technology will take decades. However, once nitrate gets into the aquifer, decades

will be required to replace the water with low

nitrate water. Fifty to 100 years mighc be required to establish a time trend, considering the

heterogeneity of aquifers. By the time the trend

was established, a dangerous situation could be

in the making that could not be corrected in a

time shorter than it took to create it.

A third situation can be identified, and that is

the "closed" water basin where the rate of recirculation of the water between the aquifer

and the surface is rapid because of pumping for

both domestic and agricultural supply, and the

rate of recharge in part of the basin is high because of intentional recharge with waste water

and leaching of agricultural soils to maintain a

favorable salt balance in the root zone. These

basins occur in parts of the West, particularly

in California, and in other places in the World

that are shart of water and ground water is

used extensively.

The time trends of nitrate enrichment of

ground water that have been noted in areas

such as the Fresno-Clovis area in California

(Nightingale, 1970) and in Israel (Gruener and

Shuval, ÍP70) are areas where a rapid circulation of water between the aquifers and the surface occurs on account of pumping. At least in

California, the impact of man on nitrate accumulation is complicated by the laï'ge amount of

nitrate of *%eologic" origin that occurred in the

profiles before irrigation or recharge was

started. Glandon and Beck (1969)^ in summarizing their studies of nitrate in California

drainage, stated that "High residual nitrates

found in soils account for greater quantities of

nitrogen in tile drainage than that contributed

by fertilizers." It is of interest to note the

projections of salts and nitrates in the San Luis
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 53

Drain being constructed as a master drain for

the San Joaquin Valley waste waters to convey

them to San Francisco Bay. The projection for

1970 was that the drainage would have 7,000

p.p.m. dissolved salts an(î would fall to 3,000 by

the year 2020. However, nitrate nitrogen in the

drainage is expected to remain at 20 p.p.m.

(Price, 1969).

AN APPRAISAL

Our evaluation of the available information

on nitrate in soil, water, foods and feeds is that

the potential for nitrate accumulation does not

pose a threat of an environmental crisis. There

is no indication of widespread upward trends of

nitrate concentrations in foods, feeds, surface

or ground water.

Based primarily on a survey published by the

National Institute of Environmental Health

Sciences (Lee et al., 1970) and an authoritative

article in the Canadian Institute of Food Technology (Phillips, 1968), the levels of nitrate

currently found in foods pose no major health

hazard to human adults. In isolated cases, nitrate content of water and foods (e.g. spinach)

is toxic to infants (under 3 to 6 months). In

many of these cases, the toxicity resulted from

microbial contamination that converted nitrate

to nitrite. However, even these limited number

of cases can be eliminated by more extensive

testing for nitrate in water and foods consumed

by humans. This can be achieved by increased

surveillance of nitrate levels of water and foods

and by educational programs sponsored by National and local health agencies.

Contrary to opinions expressed by environmental alarmists and lay opinion, the enhanced

use of inorganic nitrogenous fertilizers has not

lead to increases in nitrate of foods and feeds.

The reasons for this are that the cereal grains

used so extensively for food and feeds inherently do not accumulate nitrate, and truck crops

have also been produced on fertile soils. In general, soil organisms are equally adept at producing nitrate from organic or inorganic forms of

reduced nitrogen. Moreover, the accumulation

of nitrate in foods and feed crops depends more

on the species and environmental adversities

prior to harvest, such as drought, poor light,

and high temperature, than they do on the

available N supply of the soil. Knowledge of

these factors related to the accumulation of nitrate by food and feed plants, and the ease of

assaying for nitrate, should minimize the use of

foods or feeds possessing excessive levels of nitrate.

There is no indication of widespread upward

trends of nitrate concentrations in surface or

ground water. Absence of general trends, however, does not mean that nitrate is not accumulating in the shallow ground waters of some

areas with highly intensive agriculture and an

expanding suburban population lacking sanitary sewer systems. Areas of nitrate accumulation occur in the San Joaquin Valley and some

coastal valleys of California, the North Central

States of the United States, the coastal areas

of Israel, and in northern Europe. The nitrate

has multiple sources, some ancient and some

new. Their relative contributions are largely

unknown.

Since nitrate in ground water has many

sources and its movement to and residence time

in the ground water depends on the hydrology

of the area, each basin or watershed must be examined as an entity. Nitrate in ground water is

only one of the aspects of sanitary engineering

and water-related pollution control measures

that must be considered on a district basis. Certainly, Statewide or Nationwide controls on use

of fertilizer are not justified.

Computer models are being developed to better understand the dynamics and the equilibria

of nitrate in water basins, but the hydrological

and chemical data needed for use in models are

difficult to get. A simple input-output balance of

N for a basin can be calculated from quantities

that can be measured, but lack of data on denitriñcation in soils, profiles above water tables,

and in water, plus the lack of quantitative in-
54 AGRICULTURE HANDBOOK 413, U.S. DEPT. OF AGRICULTURE

formation on inputs by N-fixing organisms

makes the balance sheet approach entirely speculative for predicting the potential accumulation of nitrate.

Nitrate concentration in streams and lakes is

highly erratic because of absorption by photosynthetic organism.s, N mineralization in the

bottom sediments, N fixation by algae, and denitrification reactions resulting from pollution

with organic waste. With the cleanup of the Nation's waters by microbial oxidation of municipal wastes in treatment plants rather than in

waterways, increases in nitrate in surface waters are possible as streams or lakes recover

from organic pollution. Whether this will happen will depend on whether organic production

in the water body resulting from eutrophy can

be reversed. Such an increase in nitrate should

be regarded as evidence of improving health of

the water body rather than further deterioration.

Nearly all studies show that fertile soils produce subsurface drainage or water in transit to

the water table that is much richer in nitrate

than the rainfall or the irrigation water used.

Similar studies are lacking on soils of low N

fertility. Some nitrate leaching may be a necessity for a highly productive agriculture whether

it is in a humid region where some percolation

is unavoidable or in an arid, irrigated region

where some leaching is needed to avoid salt accumulation. This nitrate may come from nitrate

accumulated in the profile as a consequence of

its past history, from the oxidation of high levels of organic matter or animal waste additions,

and from fertilizers. Data do not exist that

allow a separation of the relative inputs of

these sources. A logical question is why does it

matter if there must be some leaching to have a

highly productive soil.

The significance of this downward movement

of nitrate to underground water quality depends on the characteristics of the aquifer, the

relative rates of recharge with water of low and

high nitrate concentration, and the residence

time of the water in the aquifer. Strong arguments can be made for having each intensively

cropped area surrounded by sufficient pasture

or forest land (green belts) that will yield percolate with a low concentration of nitrate. Their

spatial relationships depend on the hydrology of

the region. It is indeed possible that even higher

rates of N fertilization should be used on land

with low percolation and erosion hazard so that

more land can be returned to grass and forest

for overall environmental improvement. Since

much of our farmland has already lost much of

its native organic N, thereby impairing its capacity to produce unless fertilized, we must use

increasing amounts of fertilizer to utilize the

full potential of our better varieties and water

resources. The **balance of nature" can be better restored by using higher rates of N fertilizer than by using less.

We can probably accomplish the most in preventing nitrate accumulation in water by directing more attention to the concentrated

sources of N supply. These include sewage plant

effluents, concentrated livestock and poultry

feeding operations, and nitrate-rich drainage,

such as that of the San Luis drain in the San

Joaquin Valley. Possibilities for eliminating

much unwanted nitrate are the culture and harvest of algae in drainage and effluents, and managed denitrification in treatment plants, and on

land that receives large amounts of animal

waste.

Finally, almost every facet of the nitrate

problem needs further investigation from the

nitrate standards in water that will avoid methemoglobinemia to determination if there is any

form of fertilizer N that can be used that will

not yield some nitrate to the water table, and

yet be economic and permit near maximum

yields. Much greater team work is needed

among scientists and engineers of all specialties, but in particular, hydrologists and agronomists need to work together in reducing the potential hazards of a productive agriculture.
ACCUMULATION OF NITRATE IN SOIL, WATER, AND PLANTS 55

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