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1

This is to certify that the

thesis entitled

The Effect of Nitrate Contained in
Drinking Water on the Productivity
and Health of Farrowing Swine

presented by

Colleen S. Bruning-Fann

has been accepted towards fulfillment
of the requirements for

 

 

MLSJ degree in Large Animal
Clinical Sciences, Emphasis in
Epidemiology

 

Major professor

Jo B. Kaneene, DVM MPH PhD

Date October 1151991;

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

LIBRARY
Mlchlgan State
University

 

 

 

PLACE DI RETURN BOXtomnmmboMokoutromyourm
TOAVOID FINES rotunonorbdondatoduo.

DATE DUE DATE DUE DATE DUE ll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU loAnN'flrmdlvo Won-l Opportunity Institution

1

 

 

THE EFFECT OF NITRATE CONTAINED IN DRINKING WATER
ON THE PRODUCTIVITY AND HEALTH OE PARROWING SWINE

BY

Colleen 8..Bruning-Fann

A THESIS

Submitted to_
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Large Animal Clinical Sciences

1994

ABSTRACT

THE EFFECT OF NITRATE CONTAINED IN DRINKING WATER
ON THE PRODUCTIVITY AND HEALTH OF FARROWING SWINE

BY

Colleen 8. Bruning-Fann

A cross-sectional study design was utilized to determine
the effects of nitrate contained in drinking water on
farrowing’swineihealth.and.productivityu Swine farms (571) in
the 0.8. were monitored for 3-months. Two water samples
collected 3-months apart, were tested for nitrate. Data was
gathered through a daily log recorded by the operator and
administered questionnaires.

The data were analyzed on a farm basis using stratified
analysis, and.mu1tip1e linear regression or multiple logistic
regression. No ' association was seen between the nitrate
concentration of drinking water and average litter size,
average percentage stillborn, or the risk of having an above
median percentage of mummies. No association was seen between
nitrate and the risk of having an above median (>0) percentage
of swine ill or dead due to farrowing problems, other
reproductive problems, other known health problems, or unknown
health problems. The statistical power of this study was .90

(at a=.05).

This thesis is dedicated to my
husband John for his unwavering
faith and support and to my children

Lyle and Eric.

iii

ACKNOWLEDGMENTS

I am deeply indebted to my major professor, Dr. John B.
Kaneene, whose extensive knowledge on a wide range of subjects
has guided both my personal and professional development. I
am extremely appreciative of my committee members, Dr. Aryeh
Stein, Dr. Jim Lloyd, and Dr. Brad Thacker for their
expertise, guidance, and constructive criticism throughout
this project. I thank Drs. Phyllis York and Nell Ahl for
providing the initial impetus and supporting me in this
endeavor. I am grateful to RoseAnn Miller whose computer
skills greatly aided this research project and my training.

I thank the United States Department of Agriculture for
financial support, allowing' time for this jproject. while
working as a Veterinary Medical Officer, and for use of the
National Swine Survey data. I wish to acknowledge the
contribution of the many swine farmers without whose
participation in the National Swine Survey, this study could

not have been done.

iv

ABSTRACT

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . .

CHAPTER 1

INTRODUCTION

CHAPTER 2

REFERENCES . . . . . . . . . . . . . . . . .

OVERALL GOALS AND HYPOTHESES

CHAPTER 3 O O O O O O O O I O O O O O O O I O O O

A REVIEW OF THE EFFECTS OF NITRATE, NITRITRE,
AND N-NITROSO COMPOUNDS ON ANIMAL HEALTH

ABSTRACT . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . .
Nitrate Measurement: . . . . . . .
Nitrate and Nitrite Metabolism: .

NITRATE, NITRITE, AND NFNITROSAMINES

RUfiINANTS O O O O O O O O O O O O

Methemoglobinemia in Ruminant Animals

Chronic Toxicity . . . . . . . . .
N-Nitroso Compounds In Ruminants .

NITRATE, NITRITE, AND NFNITROSAMINES

MONOGASTRI CS 0 O O O O O O O O O 0
Acute Nitrate Toxicity . . . . . .
Methemoglobinemia . . . . . . .

Chronic Nitrate and Nitrite Exposure

N-Nitroso Compounds . . . . . . .
DISCUSSION 0 O O O O O O O 0 0 O O O 0
REFERENCES . . . . . . . . . . . . . .

IN

IN

Page
ii

vii

10
10

12
12
28
39

4O
4O
43
53
7O
71
77

CHAPTER 4 .

THE EFFECTS OF NITRATE CONTAINED IN DRINKING WATER
ON THE PRODUCTIVITY AND HEALTH

ABSTRACT . .
INTRODUCTION
Hypotheses Tested:
MATERIALS AND METHODS

National Swine Survey

Sample Selection .
Data Collection
Water Sampling .
Laboratory Testing

Data Analysis

RESULTS . . .
DISCUSSION .
REFERENCES .

CHAPTER 5 .

DISCUSSION

BIBLIOGRAPHY

vi

OF FARROWING SWINE

of Water

99

100
102
104
105
108
106
110
112
112
113
118
121
166

171

176

LIST OF TABLES

Page

3.1: Conversion Factors . . . . . . . . . . . . . . . 76
4.1a: Dependent Variables Utilized In

Statistical Analysis . . . . . . . . . . . . . . 127
4.1b: Independent Variables Utilized In

Statistical Analysis . . . . . . . . . . . . . . 128
4.2: Results of water analysis on swine farms:

National Swine Survey, 1989 - 1991. . . . . . . 129
4.3a: Selected characteristics of swine farms:

National Swine Survey, 1989 - 1991. . . . . . . 130

4.3b: Prevalence of selected practices on
swine farms: National Swine Survey, 1989 - 1991. 131

4.3c: Measures of sow health and productivity on swine
farms: National Swine Survey, 1989 - 1991. . . 132

4.4a: Spearman correlations between nitrate, potential
confounders and measures of sow productivity on
swine farms: National Swine Survey, 1989-1991. 133

4.4b: Spearman correlations between nitrate, potential
confounders and measures of sow productivity on
swine farms: National Swine Survey, 1989-1991. 134

4.4c: Spearman correlations between independent
variables and measures of sow productivity:

National Swine Survey, 1989-1991. . . . . . . . 135
4.4d: Spearman correlations between independent

variables and measures of sow health: National

Swine Survey, 1989-1991. . . . . . . . . . . . . 136

4.4e: Spearman correlations between independent
variables and measures of sow health: National
Swine Survey, 1989-1991. . . . . . . . . . . . . 137

vii

4.4f: Spearman correlations between independent
variables and measures of sow health: National
Swine Survey, 1989-1991. . . . . . . . . . . . .

4.5a: Selected characteristics of swine farms stratified
according to the level of
nitrate in the drinking water:
National Swine Survey, 1989-1991. . . . . . . .

4.5b: Prevalence of selected practices on swine farms
stratified according to the level of
nitrate in the drinking water:
National Swine Survey, 1989 - 1991. . . . . . .

4.5c: Measures of sow productivity and health
on swine farms stratified according to
the level of nitrate in the drinking water:
National Swine Survey, 1989-1991. . . . . . . .

4.6: Unadjusted stratified analysis of detectable
levels of nitrate, nitrate 245 ppm and nitrate 2100
ppm on farrowing sow productivity and health:
National Swine Survey, 1989-1991. . . . . . . .

4.7a: Summary of adjusted stratified analyses for the
association between Nitrate 24 5 ppm and Average
Litter Size 0 O O O O O O O O O O O O O O O O O

4.7b: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and the
Percentage of Stillbirths . . . . . . . . . . .

4.7c: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and the
Percentage of Mummies . . . . . . . . . . . . .

4.7d: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Illness Due
to Farrowing Problems . . . . . . . . . . . . .

4.7e: Summary of adjusted stratified analyses for the

association between Nitrate 245 ppm and Mortality
Due to Farrowing Problems . . . . . . . . . . .

viii

138

139

140

141

142

143

144

145

146

147

4.7f: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Illness Due
to Reproductive Problems Other Than Farrowing .

4.7g: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Mortality
Due to Reproductive Problems Other Than Farrowing

4.7h: Summary of adjusted stratified analyses (adjusted)
for the association between Nitrate 245 ppm and
Illness Due to Other Known Causes . . . . . . .

4.71: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Mortality
Due to Other Known Causes . . . . . . . . . . .

4.7j: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Illness Due
to Unknown Causes . . . . . . . . . . . . . . .

4.7k: Summary of adjusted stratified analyses for the
association between Nitrate 245 ppm and Mortality
Due to Unknown Causes . . . . . . . . . . . . .

4.8a: Results of a multiple linear regression model for
the effect of Nitrate on the farm average litter
size: National Swine Survey, 1989-1991. . . . .

4.8b: Results of a multiple linear regression model for
the effect of Nitrate on the farm average
percentage stillborn: lNational Swine Survey, 1989-
1991. O O O O O O O O O O O O O O O O O O O O O

4.8c: Results of a multiple logistic regression model
for the effect of Nitrate on the farm average
percentage born mummified: National Swine Survey,
1989-1991. . . . . . . . . . . . . . . . . . . .

4.8d: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sows ill due to farrowing problems: National Swine
Survey, 1989-1991. . . . . . . . . . . . . . . .

ix

148

149

150

151

152

153

154

155

156

157

4.8e: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sow mortality due to farrowing problems: National
Swine Survey, 1989-1991. . . . . . . . . . . . .

4.8f: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sows with reproductive illness other than farrowing
problems: National Swine Survey,
1989-1991. . . . . . . . . . . . . . . . . . . .

4.8g: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sow mortality due to reproductive illness other
than farrowing problems: National Swine Survey,
1989-1991. . . . . . . . . . . . . . . . . . . .

4.8h: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sows ill with other known problems: National Swine
Survey, 1989-1991. . . . . . . . . . . . . . . .

4.8i: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sow mortality due to other known problems:
National Swine Survey, 1989-1991. . . . . . . .

4.8j: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sows ill with unknown problems: National Swine
Survey, 1989-1991. . . . . . . . . . . . . . .'.

4.8k: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of
sows mortality due to unknown problems: National
Swine Survey, 1989-1991. . . . . . . . . . . . .

4.9: The approximate minimum detectable odds ratio when
a=0.05 (one-tailed), for an overall event
proportion (Pr0p) with a sample size of 571. . .

158

159

160

161

162

163

164

165

CHAPTER 1

INTRODUCTION

2

It is estimated that groundwater is the primary source of
water for 85% of the rural population and 50% of the entire
population of the United States (Council on Agricultural
Science:and.Technology, 1985). Evidence suggests that.nitrate
levels in groundwater are increasing in much of the world
(Vigil et al., 1965; Fraser and Chilvers, 1981; Hollander and
Sander, 1987). This is believed to have resulted from human
intervention either through agricultural activities, waste
disposal, or industrial processes (Bouchard et al., 1992).

Nitrate is associated with a number of adverse health
effects both in animal and human health (Bruning-Fann and
Kaneene, 1993a; Bruning-Fann and Kaneene, 1993b). The
National Academy of Sciences has recommended that
concentrations of nitrate in the drinking water of animals not
exceed 455 ppm. This recommendation was based on the meager
information available at that time (1974). Since then,
relatively little additional research has been done concerning
the effect of nitrate in drinking water on livestock species.

With groundwater serving as the primary source of
drinking water for livestock and the levels of nitrate in this
resource increasing, it is timely and important to ascertain
the significance of nitrate contamination of groundwater on
animal health and productivity. To this end, this study was
undertaken‘toiexplore the effects of nitrate found in drinking

water on farrowing swine health and productivity.

3

REFERENCES:

Bouchard DC, Williams MK, and Surampalli RY: Nitrate
contamination of groundwater: Sources and potential health
effects. J Am Water Works Assoc, 85:85-90, 1992.

Bruning-Fann C and Kaneene JB: The effects of nitrate,
nitrite, and N-nitroso compounds on animal health. Vet Human
Toxicol, 35:237-253, 1993a.

Bruning-Fann C and Kaneene JB: The effects of nitrate,
nitrite, and N-nitroso compounds on human health: A review.
Vet Human Toxicol, 35:521-538, 1993b.

Council for Agricultural Science and Technology: {Agriculture
and groundwater quality. Council for Agricultural Science and
Technology Report No. 103, p. 62, May, 1985.

Fraser P and Chilvers C: Health aspects of nitrate in
drinking water. Sci Total Environ, 18:103-116, 1981.

Hollander R and Sander J: Nitratkonzentrationen von
trinkwassern in Swaest-Niedersachsen. Zentralbl Bakter
Mikrobiol Hyg, 184:287-296, 1987.

National Academy of Sciences--National Research Council,
Subcommittee on nutrient and toxic elements in water:
Nutrients and toxic substances in water for livestock and
poultry. National Academy of Sciences, Washington, D.C.,
1974.

National Academy of Sciences-~National Research Council,
Assembly of Life Sciences: The health effects of nitrate,
nitrite and N-nitroso compounds. National Academy of
Sciences, Washington, D.C., 1981.

Vigil J, Warburton S, Haynes WS and Kaiser LR: Nitrates in
municipal water supply cause methemoglobinemia in infant. Pub
Health Rep, 80:1119-1121, 1965.

CHAPTER 2

OVERALL GOALS AND HYPOTHESES

Goal:

General

Hypothesis:

Specific

Hypotheses:

To determine the effect of nitrate in drinking
water on the productivity and health of

farrowing swine.

That nitrate at levels found in the drinking
water from wells during the National Swine
Survey adversely affects farrowing swine

productivity and health.

That the nitrate level of drinking water is

associated with the farm

1. Average number of pigs born per litter.

2. Average percentage of the litter
stillborn.

3. Average percentage of the litter born
mummified.

4. Percentage of swine with farrowing
problems.

5. Percentage of swine with reproductive

problems other than farrowing problems.
6. Percentage of farrowing swine with other

health problems.

10.

11.

6

Percentage of farrowing swine with
unknown health problems.

Mortality rate due to farrowing problems.
Mortality rate due to reproductive
problems other than farrowing problems.
Mortality rate due to other known
problems.

Mortality rate due to unknown problems.

CHAPTER 3

A REVIEW OF THE EFFECTS OF NITRATE, NITRITE,

AND N-NITROSO COMPOUNDS ON ANIMAL HEALTH

ABSTRACT

The clinical signs of acute nitrate toxicity vary among
species. In general, ruminant animals develop
methemoglobinemia while monogastric animals exhibit severe
gastritis. Nitrate ingestion has also been linked to impaired
thyroid function, decreased feed consumption, and interference
with the metabolism of vitamin A and E. Hematologic changes
observed with chronic high nitrate exposure include both
compensatory increases in red blood cells and anemia, along
with increased neutrophils and eosinophils.

Unlike nitrate, nitrite is apparently capable of inducing
methemoglobinemia in a wide range of species .
Methemoglobinemia has been reported in cattle, sheep, swine,
dogs, guinea pigs, rats, chickens, and turkeys. Ina rats,
chronic nitrite. exposure, causes pathologic changes in. a
variety of tissues, alters motor and brain electrical
activity, and affects absorption of nutrients by the gastric
mucosa. Nitrite affects the metabolism of sulfonamide drugs
in animals such as the pig, guinea pig, and rat.

The N-nitroso compound dimethylnitrosamine causes toxic
hepatosis in cattle, sheep, mink, and fox. Nitrosamines have
been reported in cows’ milk and have been found to pass into

the milk of goats under experimental conditions.

INTRODUCTION

Nitrate containing compounds were first implicated in the
toxicosis of man in 1793 according to a review by Beck (1909).
The earliest recorded case of nitrate poisoning in animals was
noted in 1895 by Mayo. It was Comly in 1945, who first
associated methemoglobinemia in children with consumption of
water high in nitrate and advocated limiting the use of
drinking water which exceeded 45 ppm nitrate (10 ppm as
nitrate nitrogen).

Since Comly's work, it has become apparent that nitrate
is not stable in biological environments but can be converted
to nitrite and in the presence of other naturally occurring
nitrogen compounds, to N-nitroso compounds. The National
Academy of Science after reviewing the literature has
recommended that drinking water for humans not exceed 45 ppm
nitrate and that of animals not exceed 445 ppm (100 ppm as
nitrate nitrogen) (National Academy of Sciences, 1981;
National Academy of Sciences, 1974). While the toxicity of
nitrates, nitrites, and N-nitroso compounds in humans has been
reviewed relatively recently (Hartman, 1982; Green et al.,
1982; Magee, 1982; National Academy of Sciences, 1981), it has
been 20 years since the effect of nitrate and nitrite on
animal health has been reviewed (Singer, 1968; Singer, 1972;
wright and Davison, 1964; Ridder and Oehme, 1974a; National

Academy of Sciences, 1974). With the controversy surrounding

10
the effects of nitrate on health, a wide variety of views on
the potential of nitrate to cause adverse health effects has
developed. This review was undertaken in an attempt to
clarify the situation by considering the effects of nitrate,

nitrite and N-nitroso compounds in animals.

Nitrate Measurement:

There are several different 'ways to report. nitrate
content including the levels.of:nitrate ion, nitrate-nitrogen,
and potassium or sodium nitrate. Conversion factors may be
used to compare the different units of nitrate measurements in
various studies (see Table 3.1). Due to differences in
molecular weights, 1 ppm potassium or sodium nitrate is
equivalent to 0.61 ppm nitrate or 0.14 ppm nitrate-nitrogen.
Likewise, conversion from nitrate-nitrogen to nitrate ion can
be accomplished. by' multiplication by a factor of 4.45.
Multiplication by 6.1 will convert nitrate-nitrogen to

potassium nitrate or sodium nitrate.

Nitrate and Nitrite Metabolism:

When discussing the health effects of nitrate, it is
important to consider how nitrate is metabolized by different
species. Many of the toxic effects attributed to nitrate are
actually the effects of metabolites of nitrate. It is through

examining all the effects of the various products that one can

11
thoroughly understand the importance of nitrate exposure to
health.

There are considerable differences in the toxicity of
nitrate depending on the animal species involved. The anatomy
and microflora of ruminant animals such as cattle and sheep,
allows the in viva reduction of nitrate to nitrite to occur
rapidly, while the large cecumiand colon.of the horse provides
an area for microbial reduction of nitrate to occur which is
intermediate between that of monogastrics and ruminants. In
ruminants only a small proportion of nitrate is eliminated in
the urine (Kearley et al., 1962; Lewis, 1951a), while in
monogastrics such as the dog (Greene, 1954; Greene and Hiatt,
1955), most of the nitrate is eliminated in the urine. In an
experiment involving young calves, large quantities of
nitrates were excreted in the urine (Prewitt and Merilan,
1958). This finding reflects the fact that calves function
initially as monogastrics, becoming ruminants later as their
diet changes from milk to roughage.

Nitrate poisoning usually occurs subsequent to reduction
to nitrite. .Acute .nitrate ‘poisoning, though common in
ruminants, is rare in monogastric animals. Nitrate poisoning
usually occurs subsequent to reduction to nitrite. Nitrite is
estimated to be 2.5 times more toxic for ruminants and 10
times more toxic for monogastrics than nitrate (Emerick,
1974). Substantial species differences in the rate of

methemoglobin formation (Smith and Beutler, 1966; Spicer,

12

1950), methemoglobin reduction (Smith and Beutler, 1966), and
response to methylene blue are known to occur (Smith and
Beutler, 1966). In general, ruminants exhibit the most rapid
rates of methemoglobin formation and reduction and respond
most rapidly to the administration of methylene blue. In
monogastrics, the pig shows the least response to methylene
blue and the slowest rate of methemoglobin formation and
reduction. Humans tend to be intermediate in methemoglobin
formation and reduction but respond exceptionally well to
methylene blue.

There are substantial species differences in nitrate and
nitrite pharmacokinetics. The elimination half-life of
nitrate is 44.681 hours in dogs, 4.233 hours in sheep, and
4.821 hours in.ponies, while that of nitrite is 0.499 hours in
dogs, 0.475 hours in sheep, and 0.566 hours in ponies
(Schneider and Yeary, 1975). Little is known about the long

term effects of nitrate exposure in man or animals.

NITRATE, NITRITE, AND N-NITROSAMINES IN RUMINANTS

Methemoglobinemia in Ruminant Animals:

Poisoning in cattle due cornstalks containing high levels
of nitrate was first reported by Mayo in 1895. Illness and
death in cattle after consuming oat hay was later reported by
Newsom et al. (1937). Numerous other reports of "corn stalk"

(Case, 1954; Muhrer et al., 1955; Schwarte et al., 1939) and

13

"oat hay poisoning" soon followed (Bradley et al., 1939;
Davidson et al., 1941; Thorp, 1938) . Subsequently it was
shown that nitrates were the causative agent (Bradley et al.,
1939) . Since then, methemoglobinemia subsequent to
consumption of nitrate containing roughages has been commonly
reported in ruminant animals. Nitrate poisoning has resulted
from the consumption of green oats (Dodd and Coup, 1957) ,
sugar beet tops (Savage, 1949), variegated thistle (Silybum
marianum) (Kendrick et al., 1955), turnips (Anon., 1978;
Cawley and Collings, 1977), silage (Bjornson et al., 1961;
Cawley and Collings, 1977), kale (Cawley and Collings, 1977),
Sudan forage (Boermans, 1990; Vermunt.and Visser, 1987), Sudan
(Sorghum species) hay (Brown et al., 1990 ; Carrigan and
Gardner, 1982; Haliburton and Edwards, 1978; Hibbs et al.,
1978), ryegrass (Nicholls and Miles, 1980; O'Hara and Fraser,
1975), Italian ryegrass and white clover pasture (Vermunt and
Visser, 1987), hay (Brown et al., 1990; Smith and Suleiman,
1991) and pigweed or redroot (Amaranthus retroflexus) (Dodd
and Coup, 1957). I

Toxicity due to well water containing nitrate has also
been reported (Bjornson, 1961; Bjornson, 1964; Campbell et
al., 1954; Fincher, 1936; Osweiler et al., 1985). An unusual
source of nitrate poisoning in cattle involved water
consumption from blasted watering holes (dugouts) (Yoong et
al., 1990). Water nitrate levels were found to be 4.8 g/I.and

7.0 g/L. These extremely high nitrate levels were due to the

14
use of fertilizer containing nitrate as a portion of the
explosive charge.

Acute poisoning can occur when forages exceed 0.5%
nitrate (on a dry weight basis) or 500 ppm nitrate in drinking
water (Buck, 1970) . The finding that some feed samples
implicated in toxic episodes contained high levels of nitrite
in addition to nitrate suggests that both substances should be
measured in feed samples (Smith and Suleiman, 1991).

Nitrate can be absorbed across the ruminant epithelium
probably by the C1’/HCO3, exchange mechanism (Wiirmli et a1. ,
1987). Nitrate can also be microbially reduced in the rumen
to ammonia with nitrite as an intermediary step (Lewis, 1951a;
Lewis, 1951b; Wang et al., 1961). During this reduction
process, nitrite may be absorbed into the bloodstream (Wang et
al., 1961) where it reacts with hemoglobin to form
methemoglobin (Bodansky, 1951) . There is a linear
relationship between the amount of nitrate or nitrite
administered and the extent of methemoglobin formation within
a specific dosage range (Crawford et al. , 1966; Lewis, 1951a) .
At dosages above this level, the reduction of nitrite to
ammonia becomes a limiting factor allowing nitrite to
accumulate at a much higher concentration in the rumen. At
this point, rapid absorption of nitrite into the bloodstream
results in a much greater increase in methemoglobin (Crawford
et al., 1966; Lewis, 1951a). The administration of 2.5 grams

of potassium nitrite intravenously elicited a fatal

15
methemoglobinemia in sheep within two hours while the
intravenous administration of 2.5 grams of potassium nitrate
caused no serious problems demonstrating that nitrate must be
reduced to nitrite prior to entering the blood stream in order

for methemoglobinemia to occur (Pfander et al., 1957).

Clinical Signs of Methemoglobinemia:

Clinical signs of methemoglobinemia become apparent when
the percentage of hemoglobin converted to methemoglobin
reaches 30 to 40%, and death ensues when the conversion
exceeds 80% (Buck, 1969; Buck, 1970). Clinical signs of
methemoglobinemia include weakness, ataxia, trembling,
hypersensitivity, gasping for breath, and a rapid pulse rate
(Bradley et al., 1940a; Davidson et al., 1941; Dodd and Croup,
1957; Fincher, 1936; Kendrick et.a1., 1955; Mayo, 1895; Newsom
et al., 1937; Schwarte et al., 1939; Sloan, 1971; Thorp,
1938). Respiration and urination are frequently increased
though temperature remains normal. The most characteristic
sign of methemoglobinemia is the chocolate brown appearance of
the blood which causes a peculiar "muddy" cyanosis of the
mucus membranes and tongue. Symptoms are exacerbated by
stress and exertion. Petechial hemorrhages in the epicardium,
endocardium, peritoneum, mucous membranes of the respiratory
tract and rumen, and the serosal surface of the digestive

tract are frequently found on necropsy. Congestion of the

16
lungs and gastrointestinal tract are often seen, and the odor
of nitrogen oxides may be noticeable on necropsy.

Animals poisoned by forages usually die within 2 to 10
hours (Beath et al., 1953). It has been reported that signs
typical of nitrite poisoning are more obvious approximately 2
hours after peak blood nitrate and methemoglobin levels (Diven
et al., 1962a). The methemoglobin level peaks when the
nitrite level of the blood is at maximum (Pirincci and
Kelestimur, 1987). Maximum concentrations of rumenal nitrite
coincide with peak methemoglobin levels (Wang et al., 1961)
indicating the rapidity with which nitrite is absorbed from
the gastrointestinal tract and reacts with hemoglobin. In
cattle, maximum methemoglobin levels appeared 3 hours after
intrarumenal.dosing (Wang et al., 1961) or drenching (Crawford
et al., 1966) with nitrate. When nitrate was fed once daily,
the maximum methemoglobinemia occurred approximately 8 hours
after feeding, but when fed twice daily, peak levels occurred
4 to 5 hours after feeding (Crawford et al., 1966; Jainudeen
et al., 1964).

Blood levels of nitrate and methemoglobin vary directly
with the dose of nitrate (Diven et al., 1962a; van.’t.Klooser,
1982). However, large interanimal variation is seen in the
amount of methemoglobin induced in animals of the same species
when given the same dose of nitrate (Diven et al., 1962a;
Prewitt and Merilan, 1958; Simon et al., 1959a; Winter, 1962;

van ’t Klooser, 1982). In experimental trials using sublethal

17
doses in sheep, peak. methemoglobin, levels were achieved
approximately 4 to 8 hours after intrarumenal administration
of a single 20 gram dose of sodium nitrate in one study
(Emerick et al., 1965) ; another research team found that
maximum plasma nitrate and nitrite concentrations occurred 2
hours after drenching and peak methemoglobin formation
occurred approximately 4 hours after drenching (Setchell and
Williams, 1962). When sheep received a lethal dose of 20
grams of sodium nitrate as a drench, plasma nitrate, nitrite,
and methemoglobin levels continued to rise until the animals
died at 5 and 6.5 hours after treatment (Setchell and
Williams, 1962). These differences are probably related to
differing rates of nitrate intrarumenal metabolism because
when nitrite is administered directly to the blood, the extent
of methemoglobin formation is relatively constant (Winter,
1962). Blood levels of nitrate are also related to lactation
status with.higher nitrate levels seen in dry cows compared to

lactating cows (Hambitzer et al., 1987).

Abortion:

Abortion in ruminants has been reported as a sequela
following nitrate toxicosis (Bjornson, 1964; Bradley et al.,
1940a; Carrigan and Gardner, 1982; Case, 1957a; Case, 1957b;
Davidson et al., 1941; Garner, 1958; Haliburton and Edwards,
1978; Hibbs et al., 1978; Jainudeen et al., 1964; Nicholls and

Miles, 1980; Ruhr and Osweiler, 1981; Stuart and Oehme, 1982;

18
Thorp, 1938; Vermunt and Visser, 1987). Abortions occurred 3
to 7 days after the toxicity episode. Case (1957a), reported
high levels of methemoglobinemia in stillborn calves born to
cows receiving a high nitrate ration. Fetal death and
abortion was seen in pregnant cattle given large doses of
potassium nitrate (Simon et al., 1959a). Nitrates are the
suspected causal agent of "lowland abortion syndrome" in
cattle (Simon et al., 1958; Simon et al., 1959b; Sund et al.,
1957). Rations containing 3.4% nitrate may have caused
abortion in sheep (Davison et al., 1965). In aborted fetuses
and ill or dead calves, elevated ocular nitrate concentrations
suggest that excessive maternal nitrate ingestion may have
been a causative or contributing agent (Johnson et al. , 1983) .
Abortion may be due to methemoglobinemia in the dam, resulting
in fetal death from Ihypoxia or interference with other
essential iron containing proteins (Wood, 1980). An
experiment on pregnant cows documented low oxygen saturation
of umbilical venous blood following nitrite administration to
the cow (van.’t.Klooster et al., 1982) which strongly suggests
oxygen transfer to the fetus is decreased after nitrite

administration.

Diagnosis:
Methods to ascertain the level of methemoglobin in blood
have been described (Evelyn and Malloy, 1938; Hainline, 1965;

Schneider and Yeary, 1973) . Methemoglobin determination

19
should be carried out as soon as possible after blood
collection because spontaneous methemoglobin reduction
(Bodansky, 1951; Eppson et al., 1960; Watts et al., 1969;
Wendel, 1939) occurs. The addition of one part blood to 20
parts phosphate buffer (pH 6.6) or a 1:20 dilution with
distilled water followed by refrigeration or freezing has been
effective in preserving methemoglobin (Ruhr and Osweiler,
1986). Methods to determine serum. nitrate and. nitrite
concentration have been described (Diven et al., 1962b;
Schneider and Yeary, 1973). Nitrate levels as measured by
ocular fluid offer some advantages over blood determinations.
Ocular nitrate values correlate well with serum values and
clinical signs while remaining stable for over 24 hours and
diagnostically significant for 60 hours after death (Boermans,
1990). However, if death occurs very quickly, ocular nitrate
levels will be well below serum values presumably due to a
lack of time for equilibration. Urinary nitrite determination
may also be a useful diagnostic test (Watts et al., 1969).
Recently, utilization of ion-exchange liquid chromatographic
determination on nitrate and nitrite in serum, ocular fluid
and water has been described (Boermans, 1990; Lippsmeyer et

al., 1990).

Factors influencing Toxicity:
The extent of acute toxicity is dependent on a number of

factors. Of primary importance is whether nitrate or nitrite

20
is given, the route and rate of administration, and the total
dosage involved. For example, 60% of total hemoglobin was
converted to methemoglobin with 25 grams of sodium nitrate
intrarumenally, 10 grams of sodium nitrite intrarumenally, or
2 grams sodium nitrite intravenously (Lewis, 1951a).

The rapidity of administration is also very important.
Hay that was consumed without problem when fed free-choice,
produced intoxication when force fed within 4 hours (Dollahite
and Holt, 1969). A single intrarumenal dose of 20 grams of
potassium nitrate was fatal to sheep while the same dosage
incorporated into the feed caused no ill-effects (Sinclair and
Jones, 1967). The LD,o for cattle when nitrate is given by
drenching is estimated to be 330 mg of nitrate ion per kg of
body weight (Bradley et al., 1940a) but increases to 990 mg/kg
when nitrate is consumed with forage (Crawford et al., 1966).
There is no difference between the toxicity of naturally
accumulated nitrate and nitrate added to the forage (Davison
et al., 1965).

There is evidence that ruminants adapt to the ingestion
of nitrate. Several days of dietary nitrate administration
were necessary before nitrites appeared in substantial amounts
in rumenal fluid (Nakamura et al., 1976; Tillman et al.,
1965b) and blood during two studies (Tillman et al., 1965b).
Another team of researchers found that rumen flora were
capable of nitrate reduction immediately but that

methemoglobin did not appear for several days (Wang et al.,

21

1961) . One week after the initiation of daily nitrate
treatment, blood nitrate and methemoglobin levels were at
their peak (Sinclair and Jones, 1964). Subsequently, blood
nitrate decreased and methemoglobin levels returned to normal.
In calves given nitrate or nitrite for a period of several
weeks, a gradual decline was seen in the induced methemoglobin
levels though the dosage of nitrate or nitrite remained the
same (McIlwain and Schipper, 1963). Experimental evidence
suggests that nitrate intoxication in the ruminant is more
likely to occur after a several days of high nitrate ingestion
(van ’t Klooster et al., 1982). Field reports of toxicity
episodes support the premise of nitrate adaptation. During an
acute toxicity episode, 4 of the 7 deaths in a group of 90
cattle were from a group of 6 cattle recently introduced into
the herd (Slenning et al., 1991).

Under conditions of chronic nitrate ingestion, there are
changes in the rumen flora which accounts for adaptation. ‘The
ability of rumenal flora to transform nitrate and nitrite is
enhanced after nitrate exposure (Cheng et al., 1985; Jamison,
1959; Nakamura et al., 1976). This enhanced ability of rumen
microbes to degrade nitrate and nitrite was found to transfer
to control animals housed in adjacent pens but not to widely
separated control animals (Cheng et al., 1985). Changes in
the molecular percentage structure of volatile fatty acids in
the rumen is seen in sheep treated with nitrate, nitrite and

hydroxylamine suggesting other alterations occur in the

22
rumenal flora (Jamison, 1958). It is possible that treatment
with antibiotics could alter the microbial population in the
rumen thereby altering the response to nitrate ingestion
(Smith, 1965).

Case, stated that any amount of nitrate over 0.5% of the
total ration was a potential cause of trouble (Case, 1957a);
however, several studies have shown that under certain
conditions, much higher levels of nitrate can be tolerated.
Studies have shown that animals on an adequate ration can
handle up to 1.2% nitrate without a significant increase in
methemoglobinemia (Wallace et al., 1964; Weichenthal et al.,
1963). Animals on high quality rations can withstand much
higher levels of nitrate than those on deficient rations
(Case, 1957a; Case, 1957b; Holtenius, 1957; Pfander et al.,
1957; Sapiro et al., 1949; Sokolowski et al., 1960) because of
an increased ability to break down nitrite in the rumen
(Holtenius, 1957).

Cattle receiving urea in the ration had lower blood urea,
nitrate and methemoglobin levels than cattle receiving soybean
meal (Clark et al., 1970). Nitrate added to the ration
decreased weight gains in sheep fed soybean meal but increased
weight gains in sheep fed urea (Hatfield and Smith, 1963).
This finding suggests that either soybean meal inhibits the
reduction of nitrate, or urea stimulates the reduction of

nitrate in the rumen.

23

The addition of glucose to the ration increases the in
vitro reduction of nitrate and nitrite (Pfander et al., 1957;
Sapiro et al., 1949). With corn or sucrose added to the
ration, the average peak methemoglobin level was significantly
decreased with intrarumenal nitrate administration, but not
with intravenous nitrate administration (Emerick et al.,
1965). Up to 4% sodium nitrate in the ration, in conjunction
with an active rumenal flora and adequate sugar, produced no
harmful effects (Clark and Quin, 1951). This evidence shows
that carbohydrates act to prevent the accumulation of nitrite
in the rumen by increasing the rate of nitrite reduction.

There. is evidence that. other' ration. components are
important in the utilization of nitrate. The addition of
inorganic sulfur to the ration appears to increase. the
utilization of nitrate in growing lambs (Sokolowski et al.,
1961). .Adding butylated.hydroxy anisole to:rations containing
up to 2% potassium nitrate increased digestion, absorption,
and retention of ration nitrogen but blood nitrate levels were
lowered (Ketchum et al., 1957). Digestion and/or absorption
of ration nitrogen was also increased when sodium meta
bisulfite was added to the ration (Ketchum et al., 1957).
Molybdenum is necessary for in vivo nitrate reduction while
iron and copper may be important (Tillman et al., 1965b). ‘The
addition of L-cysteine with administered nitrate suppressed
nitrite production and.methemoglobin formation (Takahashi and

Young, 1991).

24
Non-Protein Nitrogen:

In low to moderate levels, ruminant animals can utilize
nitrate as a source of non-protein nitrogen (Clark and Quin,
1951; Osweiler et al., 1976; Sapiro et al., 1949; Sokolowski
et al., 1961; Tollett et al., 1961). The addition of sodium
nitrate and a source of sugar to poor quality hay results in
accelerated cellulose digestion (Clark and Quin, 1951). At
high levels, nitrite can accumulate resulting in
methemoglobinemia. The potential for synergy between nitrate
and non-protein nitrogen feed supplements has been considered
by a number of authors. Non-protein nitrogen has been linked
to an episode of acute nitrate toxicity although other factors
(use of monensin and improper feeding practices) may have
contributed to the deaths (Slenning et al., 1991). In sheep,
nitrate.decreases the.in vitro and.in vivo utilization of urea
(Bloomfield et al., 1961a). It appears that nitrate is
utilized by microorganisms before urea, thereby decreasing the
rate of urea hydrolysis (Carver and Pfander, 1974). Soybean
meal, urea or sodium nitrate all appeared to be utilized
equally well as sources of N2 when used to increase the
protein content of a ration fed to sheep (Hoar et al., 1968).
Carver and Pfander (1973) found urea and nitrate could be fed
in a properly supplemented, high concentrate ration as long as
an adaptation period was followed. Following an adaptation
period, as much as 5.0% potassium nitrate can be given with

urea without problems when fed to low producing dairy cows

25
whether the ration was high or low energy (Sebaugh et al.,
1970).

The use of monensin in conjunction with high-nitrate
rations may act to precipitate nitrate toxicity in animals.
For example, cattle that had been grazing turnips for 6 weeks
without incident, developed methemoglobinemia less than 1 day
after monensin was introduced. Following recovery, the cattle
were once more allowed to graze the turnips. Again,
methemoglobinemia developed when monensin was added to the
ration (Malone, 1978). The use of monensin has been
associated with forage-related nitrate toxicosis in dairy
heifers (Slenning et al., 1991). These animals had excessive
nitrate exposure as measured by ocular nitrate values but the
clinical signs departed somewhat from the classical syndrome
as not all animals displayed cyanosis and chocolate-colored
blood. One author has attributed the ability of monensin to
precipitate nitrate toxicity t0» a :rapid shift in. rumen
microbial population in favor of the nitrite producers
(Malone, 1978).

There are numerous reports of nitrate poisoning occurring
after forage was wetted (Davidson et al., 1941; Smith et al.,
1959; Thorp, 1938). These accounts suggests the
transformation of nitrate to nitrite in the wet forage prior
to consumption (Olson and.Moxon, 1942). Since nitrite is much
more toxic than nitrate, clinical signs would occur at a much

lower dose. Conversion of a small amount of nitrate to

26
nitrite in drinking cups of pigs was noted in one study
(Seerley et al., 1965). Also, the conversion of nitrate to
nitrite is known to occur in 'vegetables during storage
(Walker, 1975). Adternatively, nitrate concentrations are
known to vary considerable even in the same species of plant
grown in the same location. Assuming nitrate levels in the
feed were extremely variable, clinical signs would become
apparent only when the portions of the roughage containing

high levels of nitrate were consumed.

Prevention:

There is considerable variation in the nitrate
concentration of plants due to differences in the plant
species, stage of plant maturity, and growing conditions
(Kretschmer, 1958; Ruhr and Osweiler, 1986; Walker, 1975;
Wilson, 1943; Wright and Davison, 1964). A wide variety of
plants including many of the common forages fed to livestock
may accumulate high levels of nitrate (Bradley et al.,1939;
Bradley et al., 1940a; Knight, 1985; Ruhr and Osweiler, 1986;
Whitehead and Moxson, 1952; Wilson, 1943). The level of
nitrate varies considerably even among plants of the same
species and is influenced by a number of factors such as
available nitrogen (Williams, 1989; Wilson, 1949; Wright and
Davison, 1964) and the stage of plant growth (Garner, 1958;
Knight, 1985). Also, different parts of the same plant will

contain different amounts (Knight, 1985; Whitehead and Moxson,

27

1952). Growing conditions such as drought have been found to
foster nitrate accumulation (Whitehead and Moxson, 1952) . The
use of herbicides increases both the nitrate content of plants
(Ruhr and.Osweiller, 1986) and.the palatability of some plants
thereby increasing the likelihood of a toxic episode. In
general, plants in rapid vegetative growth with heavy
fertilization, drought, and high or low environmental
temperatures are likely to accumulate excessive nitrate.

To prevent excessive nitrate accumulation in plants, care
should be exercised in fertilizing crops. Sufficient time
must elapse between applications of fertilizer and harvesting.
Because drought can cause nitrate accumulation in plants,
irrigation during dry periods can prevent excessive nitrate
levels in plants. Since stems accumulate more nitrate than
leaves, raising the height of the cutter bar to lessen the
amount of stem cut will help to lower the nitrate content of
the hay (Ruhr and.Osweiller, 1986). Ensiling often lowers the
nitrate content of forages (Ruhr and Osweiller, 1986).

If high nitrate forages must be used, an adaptation
period will allow animals to adapt, thereby preventing
problems. Also, dietary supplementation with high quality
forages or a source of readily utilized carbohydrate will aid
in preventing methemoglobinemia by increasing the rate of
nitrite reduction. For each animals ration, the addition of
1 kg grain/200 kg body weight has been suggested (Ruhr and

Osweiler, 1986) . The addition of L-cysteine to the ration may

28
be effective in preventing nitrite accumulation and subsequent
methemoglobinemia (Takahashi and Young, 1991). Maintaining
adequate feed and water is important as several outbreaks of
nitrate toxicity occurred after a period of time without feed,
probably either due to rapid feed consumption and/or loss of
nitrate adaptation (Brown.et al., 1990; Slenning et al., 1991;

Haliburton et al., 1978).

Treatment:

In mild cases, removal of the source of nitrate and
keeping the animal quiet is usually sufficient. More severe
cases can be treated with intravenous injections of methylene
blue at a rate of 4 of 15 mg/kg of body weight or more
(Burrows, 1980). In cattle, 8 mg/kg of body weight is usually
sufficient (Beath et al., 1953; Bradley et al., 1940a; Bradley
et al., 1940b). Although ascorbic acid reduces methemoglobin,

the mode of action is much slower (Pirincci et al., 1988).

Chronic Toxicity:

Hematologic Effects:

Studies in which animals developed significant
methemoglobinemia for an extended period of time as a result
of the ingestion of high levels of nitrate, suggest that
compensatory increases in hemoglobin concentration, packed red

cell volume, and blood volume occur in both cattle and sheep

29
(Davison et al., 1965; Jainudeen et al., 1964; Winter and
Hokanson, 1964). One study found that methemoglobin and total
hemoglobin increased proportionally and therefore the
percentage of methemoglobin was unchanged (Crawford et al.,
1966). It has been reported that sodium nitrate administered
intrarumenally in sheep for 5 weeks at 0.2 g/kg increased the
erythrocyte count whereas at 0.3 g/kg, a hypochromic anemia
was seen (Kelestimur et al., 1988). Other blood changes such
as higher leukocyte counts, increased neutrophil and
eosinophil percentages, and lower lymphocyte percentages were
seen in treated sheep whether 0.2 or 0.3 g/kg of sodium
nitrate was given, compared to controls (Kelestimur et al.,

1988).

Transplacental Transfer:

The finding of nitrate in amniotic fluid of cows given
sodium nitrate orally suggests nitrates can pass the bovine
placental barrier (Slanina et al., 1990). Pregnant heifers
given nitrate in the ration at levels sufficient to maintain
a daily peak methemoglobin level at 40 to 50% of total
hemoglobin, had calves with elevated red blood cell and
hemoglobin concentrations (Winter and Hokanson, 1964). This
transplacental effect may be due to fetal anoxia subsequent to
maternal methemoglobinemia or from the passage of nitrate or

nitrite across the placenta.

30
Transmammary Transfer:

The composition of milk from animals receiving high
levels of nitrate has been studied by several researchers. In
one study, nitrate but not nitrite was found in the milk of
ewes and a cow given sodium nitrate orally (Slanina et al.,
1990). The methemoglobin levels of lambs increased after
consumption of this high nitrate milk (Slanina et al., 1990).
However, other studies found either a small increase in the
nitrate content of milk from cows consuming high nitrate fed
(Davison et al., 1964) or no significant difference in milk
nitrate levels (Kammerer et al., 1992; Sebaugh et al., 1970).
The differences between the studies are likely due to the
large dosage in the first experiment relative to the other
studies.

It has been demonstrated experimentally that ingested
potentially carcinogenic nitrosamines could appear in the
blood and milk of lactating animals (Juszkiewicz and Kowalski,
1974). The formation of nitrosamines in the rumen occurred
only under acidic conditions, which do not normally exist in
the healthy animal. However, nitrosamines have been found in
some plants and fish meals which may be used in animal feeds

(Juszkiewicz and Kowalski, 1974).

Thyroid:
The goitrogenic effect of nitrate discovered in rats by

Wyngaarden et al. (1952), stimulated the study of this effect

31
in ruminants. The concentration of serum I131 as protein-bound
iodine in sheep receiving nitrate in the diet was
significantly less six days after injection of I131 (Bloomfield
et al., 1961b; Bloomfield et al., 1962) but higher at 27 days
compared to controls (Bloomfield et al., 1962). A tendency
for dietary nitrate administration to decrease thyroid
activity as measured by triiodothyronineifl“ uptake was noted
initially (Carver and Pfander, 1973) but further study found
no significant effect (Carver and Pfander, 1974). No effect
from the ingestion of nitrate was seen on thyroid weights or
thyroid acinar heights in dairy cattle (Jainudeen et al.,
1965). These data do not support a goitrogenic effect of
nitrate in ruminant animals except perhaps a transitory effect

prior to nitrate adaptation.

Pituitary:

The only study to monitor the pituitary gland reported
pituitaries from cattle fed up to 660 mg/kg body weight were
heavier than those from cattle not receiving initrate
(Jainudeen et al., 1965). It is unknown.if pituitary function

was affected.

Vitamin A:
Vitamin A deficiency in cattle was first attributed to
dietary nitrate by Case (1957a) . Since then, several theories

have been examined concerning the effect of nitrate and

32
nitrite on vitamin A status. One theory proposes that nitrite
oxidizes iron-containing enzymes, such as dioxygenase enzymes,
thereby decreasing the conversion of beta-carotene to vitamin
A (Wood, 1980).

Another theory states that vitamin A is readily destroyed
by oxidation and since nitrates are oxidizing agents, nitrates
could destroy vitamin A (Mitchell et al., 1967). In vivo
experiments indicate nitrite is capable of destroying carotene
(Keating et al., 1964; Olson et al., 1963; Pugh et al., 1962)
and vitamin A (Keating et al., 1964; Pugh et al., 1962;
Roberts and Sell, 1963). However, nitrate seems to affect
carotene and vitamin A in some circumstances but not in
others. The conversion of carotene to vitamin A in vitro was
reduced by nitrate (Reddy and Thomas, 1962) but in vitro
carotene destruction was not increased (Davison and Sen,
1963). The addition of high levels of nitrate to the rumen
liquor of steers in vitro increased the destruction of vitamin
A only in steers fed high concentrate diets (Keating et al.,
1964). Experiments in which nitrate was added to the ration
have failed to find any effect on the in vitro (Keating et
al., 1964) or in vivo destruction of vitamin A (Mitchell et
al., 1965; Mitchell et al., 1967) or in vitro destruction of
carotene (Davison and Sen, 1963) . Likewise, feeding 2%
potassium. nitrate to sheep had no effect on vitamin. A
concentrations in abomasum fluids, indicating little

destruction of vitamin A in vivo (Roberts and Sell, 1963).

33
Dietary administration of nitrite decreased liver vitamin A
levels in sheep (Holst et al., 1961). These results indicate
that only with relatively large doses of nitrate and nitrite
(greater than is seen with the dietary addition of nitrate)
are carotene and vitamin A metabolism noticeably affected.

The thyroid is important.in the conversion of carotene to
vitamin A in rats (Johnson and Baumann, 1947) . Research
indicates that this is also true in ruminants. Treatment of
steers with the thyroid hormone triiodothyronine resulted in
increased plasma carotene and liver vitamin .A in ‘those
supplemented with carotene or vitamin A and increased plasma
carotene and vitamin A in steers without supplementation
(Jordan et al., 1963). In addition, the in vitro conversion
of carotene to vitamin A was reduced with hypothyroidism and
also by the presence of nitrate in vitro (Reddy and Thomas,
1962). Since the thyroid is important in. transforming
carotene to vitamin A, anything that alters thyroid activity
should also affect vitamin A status. Though many studies have
been undertaken with the assumption that nitrate affects
thyroid function, that premise has not been proven.

The last proposed mechanism of vitamin A impairment
involves hydroxylamine. Hydroxylamine has been shown to be an
intermediate product in the reduCtion.of nitrate to ammonia.in
vitro (Lindsey and Rhines, 1932). It has been proposed that
hydroxylamine is also formed in the ruminant and that it is

hydroxylamine which adversely affects vitamin A status

34

(Tillman et al., 1965a). Though administration of
hydroxylamine did decrease plasma vitamin A levels in sheep,
there‘was no effect on liver vitamin A levels (Tillman et al.,
1965a). The authors of that article suggest that not enough
time had elapsed during the experiment to allow changes in
liver vitamin A to become apparent. However, since only very
low levels of serum hydroxylamine have been found in cattle
receiving nitrate (Winter, 1962), it is unlikely that this
mode of action is biologically important.

Regardless of the hypothesized mode of nitrate action, a
number of studies have measured serum carotene, serum vitamin
A, liver carotene, and liver vitamin A in relation to various
levels of nitrate or nitrite. Although there are significant
relationships between serum carotene, serum vitamin A, liver
carotene, liver vitamin A, these items are not directly
correlated in cattle (Diven et al., 1960).

Most studies in ruminants, found no significant effect
due to dietary nitrate on either plasma carotene (Davison et
al., 1965; Goodrich et al., 1964; Miller et al., 1965; Wallace
et al., 1964; Weichenthal et al., 1963), plasma vitamin A
(Davison et al., 1965; Miller et al., 1965; Smith et al.,
1962; Wallace et al., 1964; Weichenthal et al., 1963), liver
carotene (Wallace et al., 1964; Weichenthal et al., 1963), or
liver vitamin A (Cline et al., 1963; Davison et al., 1965;
Hale et al., 1961; Smith et al., 1962; Wallace et al., 1964;

Weichenthal et al., 1963). Although a few studies found

35
nitrate to decrease plasma vitamin A (Hatfield et al., 1961)
and liver vitamin A (Goodrich et al., 1962; Goodrich et al.,
1964; Hatfield et al., 1961) and some studies suggest nitrate
may inhibit the conversion of carotene to vitamin A (McIlwain
and Schipper, 1963) or increase the depletion of liver vitamin
A (Jordan et al., 1963). The weight of evidence supports the
view that nitrate has relatively little effect on in vivo

vitamin A status in ruminants.

Vitamin E:

In. sheep receiving' dietary' nitrite, no {evidence of
vitamin E deficiency (as measured by hemolysis time) was noted
(Holst et al., 1961). The addition of vitamin E to sheep
receiving nitrate did not have any beneficial effect on
vitamin A storage or the rate of gain (Cline et al., 1963).
Apparently, nitrate has no effect on vitamin E in the

ruminant.

Weight Gain and Feed Efficiency:

In most studies, no significant difference was seen in
weight gain in cattle (Davison et al., 1963; Smith et al.,
1962; Wallace et al., 1964) or sheep (Cline et al., 1963;
Davison et al., 1965; Eppson et al., 1960) receiving nitrate
in the ration. Either no difference in weight gain was seen
in steers fed high-nitrate silage as compared to those fed

low-nitrate corn silage (Jordan et al., 1961; Jordan et al.,

36
1963) or average daily weight gains were increased for the
higher-nitrate silage group (Zimmerman et al., 1962). These
results are consistent with the finding that nitrate can be
utilized as a source of non-protein nitrogen in ruminants
(Clark and Quin, 1951; Osweiler et al., 1976; Sapiro et al.,
1949; Sokolowski et al., 1961; Tollett et al., 1961).

Nitrate in the ration at 1% or more can decrease feed
intake in cattle (Hale et al., 1961, Hale et al., 1962; Jones
et al., 1966; Weichenthal et al., 1963) while the same result
occurs in sheep at 3% or greater (Carver and Pfander, 1974;
Davison et al., 1965; Goodrich et al., 1964). The addition of
vitamin A lessened the decrease in feed consumption seen with
sheep (Goodrich.et.al., 1964) but.not with cattle (Weichenthal
et al., 1963). When the amount of feed was limited, adding
2.5% nitrate (34 grams/head daily) did not affect rate of
weight gain, feed efficiency or carcass grade (Goodrich et
al., 1964). This evidence strongly supports the premise that
nitrate does not decrease feed efficiency. Rather, any
decrease in weight gain is brought about by decreased feed
consumption possibly related to poor palatability.

Reduction in daily weight gain in steers was seen when 1%
nitrate was fed ‘with. a ration low in total digestible
nutrients (low-concentrate). But when the steers were fed a
ration high in total digestible nutrients (high-concentrate),
an increase in weight gain was seen (Hale et al., 1961, Hale

et al., 1962). These findings are consistent with evidence

37
that high nitrate levels reduce feed consumption and
carbohydrates in a high-concentrate ration increase the

utilization of nitrate as a source of non-protein nitrogen.

Reproduction:

Research in sheep and cattle has shown nitrate to have
little effect on reproduction. No detrimental effect on the
maintenance of pregnancy was observed in heifers fed nitrate
at levels sufficient to maintain a daily peak methemoglobin
level of up to 50% of total hemoglobin (Winter and Hokanson,
1964), nor were significant differences found in length of
gestation, birth weight of calves, average length of estrous
cycle or the number of services per conception (Davison et
al., 1963). In sheep, no adverse effects were seen from
nitrate ingestion on the pregnancy (Eppson et al., 1960), the
birth weights of the lambs born (Setchell and Williams, 1962;
Sinclair and Jones, 1964 ; Davison et al., 1965), or growth of
the lambs (Eppson et al., 1960).

The only effect seen from nitrate administration relates
to progesterone levels. While serum progesterone
concentrations were not statistically different in early
pregnancy of mid-pregnancy cows fed the same high and low
nitrate rations (Page et al. , 1990) , serum progesterone
concentrations were decreased in open, luteal phase cows fed

a high nitrate ration (1,600 ppm nitrate) compared to open,

38
luteal phase cows fed a low nitrate ration (<400 ppm nitrate).

The mechanism of action is unknown.

Milk Production:

Since Case first reported lower milk production in dairy
cows receiving high nitrate rations, several studies have
explored this area (Case, 1957a). A study utilizing only two
cows also reported declines in milk production (Muhrer et al.,
1956) as.did an epidemiological study involving nitrate in the
drinking water (Olson et al., 1972). However, the small
sample size in the first study and the uncertainty that
confounding factors (such as feed, housing, and management
factors) were controlled in the second study make these
results uninterpretable. Most studies have found no
significant difference in the rate of milk production of cows
receiving high levels of nitrate compared to cows receiving
low levels of nitrate (Davison et al., 1963; Jones et al.,
1966; Kahler et al., 1975; Morris et al., 1958; Murdock and
Hodgson, 1972; Page et al., 1990; Sebaugh et al., 1970). A
decrease in milk production was reported in cattle given
nitrate at levels that produced 24 to 35.5% methemoglobinemia
(Stewart and Merilan, 1958). High producing cows and those
cows not previously treated with nitrate, were the most
affected by nitrate administration. Clearly, nitrate has no
effect on milk production unless given at doses sufficient to

cause significant methemoglobinemia.

39

N-Nitroso Compounds In Ruminants

Outbreaks of toxic hepatosis occurred in Norway in 1961
and 1962 affecting a substantial number of cattle and sheep
(Hansen, 1964; Koppang, 1964). Ruminants of all ages and.both
sexes were stricken although pregnant ewes and dairy cows were
most susceptible. Initially inappetence, decreased milk
production, decreased rumenal movements, and lethargy were
seen. As the disease progressed, apathy, anorexia, abdominal
pain, and ataxia of the hind legs occurred. The animals
became unresponsive to stimuli and exhibited an abnormal
posture of the head and extremities. A peculiar
characteristic odor was often perceptible from the breath,
skin, and milk of these cattle. Morbidity and mortality
varied considerable from herd to herd. The most striking post
mortem findings were grossly enlarged livers with variations
in coloring such as light yellowish-brown areas alternating
with darker, hyperemic areas. The causative agent was a toxic
substance contained in nitrite preserved herring meal fed to
these animals as a protein supplement (Koppang, 1964) . It was
determined that sodium nitrite used to preserve the herring
had reacted with amines present in stale fish to produce
highly toxic dimethylnitrosamine (Sakshaug et al., 1965).

Nitrosamines (N-nitroso compounds) have been found in
certain plants and fish meals used as animal feeds
(Juszkiewicz and Kowalski, 1976) and it has been demonstrated

experimentally that ingested volatile nitrosamines can pass

40
into the milk of goats (Juszkiewicz and Kowalski, 1974). The
possibility that toxic N-nitrosamines may pass into the milk
of cattle is suggested by the unusual odor associated‘with.the
milk of cattle poisoned with dimethylnitrosamine (Hansen,
1964). N-nitrosodimethylamine has been reported in milk from
cows which the authors attribute to nitrate ingestion
(Rubenchik et al., 1988). The importance of these findings to

humans remains to be determined.

NITRATE, NITRITE, AND N-NITROSAMINES IN MONOGASTRICS

Acute Nitrate Toxicity:

Acute nitrate intoxication has been experimentally
induced in pigs by the administration of large doses of
potassium nitrate. On necropsy, the pigs which died within 24
hours of dosing with nitrate had a severe gastritis with
blood. The submucosa of the stomach was swollen and
edematous. No changes were seen in blood color, heart,
mesenteric lymph nodes, spleen, kidneys, bladder, respiratory
tract, and large or small intestines. Necropsy lesions in a
pig which received multiple sublethal doses of sodium nitrate
consisted. of“ hyperemic liver, kidneys, and stomach, and
edematous stomach walls (Wanntorp and Swahn, 1953). A pig
that lived 9 days after nitrate administration exhibited

similar pathology (Gwatkin and Plummer, 1946).

41
Histopathologic examination revealed extensive liver and
kidney lesions. ‘The severe gastritis noted in the acute cases
of nitrate toxicosis resembles that seen with salt poisoning
(Ellis, 1942; Blood et al., 1983).

There are only a few cases of acute nitrate toxicity
reported in swine subsequent to consumption of high-nitrate
feed (Case, 1957c; Smith et al., 1959) or water (Bjornson et
al., 1961). Smith et al., (1959) noted illness and death in
pigs which had consumed wet oat and wheat straw. Clinical
signs included labored breathing, inappetence, and increased
water consumption. One animal had a convulsive seizure prior
to death. Findings at postmortem examination were mild
congestion of the lungs and acute gastritis. Samples of the
wet straw were positive for both nitrate and nitrite, though
the specific levels were not mentioned. It is not known if
the nitrate, nitrite, or both.compounds‘were the causal agent,
however the necropsy lesions more closely resembled those seen
with nitrate intoxication as described by Gwatkin and Plummer
(1946). Serum nitrate levels increased in pigs as the amount
of nitrate in the ration increased. In pigs fed 2% potassium
nitrate in the ration, serum nitrate was 16 mg 100 ml compared
to control pigs at 1-3 mg 100 ml (Garner et al., 1958).

Acute nitrate toxicosis has been reported in a dog
receiving large daily doses of potassium nitrate from the
owner (Whitehead, 1953). The dog exhibited behavioral

changes, vomiting, diarrhea, polyuria, polydipsia, depression,

42

inappetence, and dehydration. It recovered completely
following cessation of nitrate administration. A rabbit given
5 g. of potassium nitrate orally developed a rapid, weak
pulse, muscular trembling and died within one hour. The
necropsy revealed congestion of the lungs, the cardiac portion
of the stomach and a portion of the small intestines (Mayo,
1895).

Water containing nitrate has been reported to cause
toxicity in.chinchillas (Bjornson, 1964) and.turkeys (Adams et
al., 1969). Clinical signs of nitrate toxicity in turkeys
include slow growth, excessive salivation, incoordination, and
anorexia. Nitrate at levels of 3,990 ppm and above caused
increased mortality. Necropsy lesions were suggestive of salt
toxicity (Adams et al., 1969).

Experiments using rats have shown that the LD,o to be 5 g
of sodium nitrate per kg of body weight (Wanntorp and Swahn,
1953). Clinical signs appeared soon after administration of
sodium nitrate by intragastric tube. Initially, listlessness
was noted, followed by apathy. In 2-3 hours, coma developed,
lasting until death occurred at 6-24 hours after
administration. Experiments with Japanese quail (Coturnix
coturnix japonica) show mortality is significantly increased
when the birds are given drinking water containing nitrate at
3960 ppm or more (Adams, 1974).

Since pigs, dogs, rabbits, chinchillas, turkeys, quail,

rats, and man (Phillips, 1968b; Carlson and Shapiro, 1970;

43
Christian, 1928; Eusterman and Keith, 1929) can all develop
acute nitrate toxicosis, it seems likely that most if not all
mammalian species are susceptible to nitrate given a

sufficiently large dose.

Methemoglobinemia:

Methemoglobinemia has been induced in horses by dosing
with 50 g of nitrate per 100 lb of body weight. Clinical
signs appeared 11 hours after administration in one animal and
21 hours in the other. The animals became weak, ataxic,
restless, and perspired profusely. The administration of
nitrate at 100 grams/cwt. resulted in death with 70% of the
hemoglobin converted to methemoglobin (Bradley et al. , 1940a) .

Experimentally, sodium nitrate has caused
methemoglobinemia in rabbits, with maximum :methemoglobin
readings seen between 4 and 7 hours after ingestion (Kilgore
et al., 1959). Furthermore, methemoglobin levels increased as
the level of nitrate in the diet increased whether the source
of nitrate was naturally contained in the feed or added to the
diet as sodium nitrate (Kilgore et al., 1959). The results of
balance studies show that some conversion of nitrate to
nitrite does occur in vivo in the rabbit (Kilgore et al.,
1959).

Cyanosis was seen in a rat (2.49 g methemoglobin per 100
ml of blood) which had consumed nitrate at a rate of 10 g/kg

of body weight (Kilgore et al., 1959). This suggests that in

44
some monogastrics given large doses of nitrate, some
conversion to nitrite may occur.

Toxicity in turkeys grazing on plants high in nitrate has
been reported (Riggs, 1945) . Though the turkeys had been
grazing this field for some time without problems, toxicity
was seen after a rain. The turkeys developed anorexia,
cyanosis, and diarrhea. Pathologic changes included dark
blood, dehydration, mucoid enteritis, hyperemia of the
intestinal mucosa, and petechial hemorrhages in the
epicardium. Experimentally, the author was able to reproduce
the syndrome in a turkey with potassium nitrite but not with
sodium nitrate. In this case, the weight of evidence points
to the reduction of nitrate to nitrite in the plants prior to
consumption by the turkeys.

In order for methemoglobin to result from nitrate
ingestion, the reduction of nitrate to nitrite must occur in
vivo. Microbial reduction in known to occur in human infants
and ruminant animals where conditions suitable for nitrate
reducing bacteria exist. That nitrate ingestion can result in
methemoglobinemia in the horse and rabbit suggests the
enlarged cecum of these species may provide a suitable
environment for nitrate reduction.

Studies in other monogastrics indicate that exposure to
nitrite but only rarely nitrate, can result in
methemoglobinemia. One researcher observed methemoglobinemia

in dogs after oral administration of high levels of sodium

45

nitrate (Hiatt, 1940). 131a.later'experiment, this researcher
was unable to experimentally induce methemoglobinemia in dogs
with nitrate even after nitrate reducing bacteria were
introduced into the gut (Greene and Hiatt, 1954) . Other
studies on nitrate metabolism provide no evidence that nitrate
is reduced to nitrite in the dog, nor that methemoglobinemia
may be a sequela to nitrate administration (Greene and Hiatt,
1954; Greene and Hiatt, 1955; Keith et al., 1930). These
studies indicate that for nitrate to induce methemoglobinemia
in the dog and probably most other monogastrics, nitrate must
be converted to nitrite prior to ingestion.

There are several reports of methemoglobinemia and
elevated methemoglobin levels in pigs due to nitrite (London
et al., 1967; Sleight et al., 1972; Wood et al., 1967; Emerick
et al., 1965) in the literature. Unusual cases include
poisoning from the consumption of whey containing nitrite
(Wanntorp and Swahn, 1953) and a case where nitrate contained
in well water was converted to nitrite during the cooking of
soup (Winks et.al., 1950). Elevated methemoglobin levels.have
been reported in pigs given sodium nitrite in the ration at a
rate of 0.5% (Koch et al., 1963). One study reported the
methemoglobin levels in pigs increased as the amount of
nitrate increased in drinking water (Anderson and Stothers,
1978) while another study found no significant effect (Woods
et al., 1967). The.difference in the study results may be due

to the high levels of sulfate in the water in one study which

46
by itself also seemed to increase the methemoglobin levels.
Experimentally, pigs given up to 100 ppm nitrite in the
drinking water had small but significant increases in
methemoglobin levels compared to control groups (Seerley et
al., 1965).

Oral administration of sodium nitrite to pigs resulted in
methemoglobinemia with clinical signs appearing 10-15 minutes
after intake. Fasted pigs reached maximum methemoglobin
levels at 1.5 to 2 hours after oral dosing, with normal
methemoglobin levels seen in 8-10 hours (Wanntorp and Swahn,
1953). Sows given subcutaneous injections of sodium nitrite
developed maximum methemoglobin levels approximately 2 hours
after administration (Sleight et al., 1972). The severity of
clinical signs varied.directly with the.dose of nitrite as did
the extent of methemoglobinemia (London et al., 1967; Sleight
et al., 1972).

Clinical signs are apparent. when 20% of the total
hemoglobin is present as methemoglobin, and consist of
restlessness, frequent urination, and detectable dyspnea
(London et al., 1967). As methemoglobinemia worsens, ataxia,
weakness, dyspnea, and cyanosis develop (Wanntorp and Swahn,
1953; Winks et al., 1950; London et al., 1967; Sleight et al.,
1972). Finally, the most severely affected.animals are unable
to rise, vomiting occurs along' with severe dyspnea and
cyanosis. Death results when more than 75% of the total

hemoglobin is present as methemoglobin and is often preceded

47
by severe convulsions. No lesions are present on necropsy
though the blood is the characteristic dark brown (chocolate)
color characteristic of methemoglobinemia.

Sodium nitrite given intravenously to dogs at a rate of
30 mg/kg of body caused methemoglobinemia (Wendel, 1939).
Methemoglobin reached maximum concentrations of 60 to 70 mg
100 ml in 60 to 100 minutes. Reconversion to hemoglobin
occurred at a rate of 0.03 mg 100 ml per minute with no
methemoglobin present after 8 to 9 hours. Another study
reported nitrite levels peaked in the blood, saliva, and bile
of dogs in approximately 30 minutes following intravenous
administration of sodium nitrite (Fritsch et al., 1985). The
time differences between the studies may be due to the
different doses on sodium nitrite administered, or there may
be a lag period between nitrite entering the blood and the
development of methemoglobin.

Experimentally, the methemoglobin level of rats given
sodium nitrite peaked at 1-1.5 hours after oral administration
(Wanntorp and Swahn, 1953). Methemoglobin levels are reduced
by 50% every 90 minutes (Shuval and Gruener, 1972) until no
methemoglobin remains after 4-6 hours (Wanntorp and Swahn,
1953) . Increases in methemoglobin accompany increases in the
level of nitrate in the diet of rats whether the source of
nitrate is naturally contained in the feed or added to the

diet as sodium nitrate (Kilgore et al., 1959).

48

Chicks fed a ration containing 0.4% nitrite had greater
mortality than controls. Chicks receiving nitrite exhibited
anorexia, mild ataxia, dyspnea, paleness, and tired quickly
when excited (Sell and.Roberts, 1963). Similar clinical signs
along with poor growth, muscle tremors and frothing around.the
mouth were reported for nitrate and nitrite toxicity in
turkeys and chickens (Marrett and Sunde, 1968). With nitrite
at 400 ppm in the ration, turkeys exhibit reduced growth, a
lack of balance, rapid breathing, frothing at the mouth,

dehydration and high mortality (Sunde, 1964).

Factors Influencing Methemoglobinemia:

The presence of food in the gastrointestinal tract
apparently has a protective effect towards nitrite toxicity.
Fatal methemoglobinemia was seen in pigs (20 to 45 lbs) given
5 grams of potassium nitrite when fasted, but when given the
same dosage on a full stomach, mild methemoglobinemia
developed (Gwatkin and Plummer, 1946). It is likely the food
slowed the rate of absorption of nitrite to a level the pigs
could tolerate. Pigs given sodium nitrite intravenously
developed less methemoglobinemia when treated at 1 week of age
than when treated at 3 months or 5 1/2 months of age (Emerick
et al., 1965). This may be related to the slower rate of in
vitro methemoglobin reduction seen for older pigs compared to

those 1 week of age (Emerick et al., 1965).

49

One study found that ascorbic acid deficient guinea pigs
developed significantly higher levels of methemoglobin from
nitrite administration than ascorbic acid supplemented
controls (Kociba and Sleight, 1970). However, another study
reported ascorbic acid had no effect on the susceptibility of
guinea.pigs to nitrite toxicity (Kilgore et al., 1964). Large
interanimal variation in response to nitrite is often seen.
It is likely that the small sample size (4 guinea pigs) in the
latter study contributed to the study’s failure to detect a
difference in nitrite response due to ascorbic acid
administration. Diets containing 1% ascorbic acid slightly
reduced nitrite-induced methemoglobin blood levels in guinea
pigs compared to diets with 0.02% ascorbic acid. The finding
that ascorbic acid protects against methemoglobinemia agrees
with human studies (Armstrong et al., 1958; Bodansky, 1951;
Deeny et al., 1943; Shuval and Gruener, 1972; Super et al.,
1981) and what is known about the mode of action of ascorbic
acid (Bodansky and Gutmann, 1947; Bodansky, 1951).

The addition of 1% methionine to the guinea pig ration in
conjunction with 1% or 2% ascorbic acid resulted in a 50%
reduction in methemoglobin levels (Stoewsand et al., 1973).
High nitrate-containing beets or high-nitrate diets enhanced
nitrite induced methemoglobinemia in guinea pigs (Stoewsand et
al., 1973). The mechanism of action may be related to the
ability of guinea pigs to reduce nitrate to nitrite in the

oral cavity (Woolley and Sigel, 1982). The sulfhydryl

50
compounds glutathione and ergothioneine are protective against
nitrite induced methemoglobinemia in rats though the mode of
action is unknown (Mortensen, 1953).

Experiments showed that pregnant rats had higher
methemoglobin levels than non-pregnant rats receiving the same
doses of nitrite (Gruener and Shuval, 1973; Shuval and
Gruener, 1972) . This finding agrees with previous work
showing the increased sensitivity of pregnant rats to nitrite
(Metcalf, 1962). Starvation. was found 'to increase: the
sensitivity of rats to nitrite while riboflavine increased the
resistance of hemoglobin to oxidation with nitrites (Metcalf,
1939).

Young turkeys were more sensitive to the effects of
nitrate and nitrite than young chickens (Sunde, 1964). The
addition of vitamin A to the ration mitigated the toxicity of
nitrite in studies involving chickens.and.turkeys.(Marrett.and
Sunde, 1968; Sell and Roberts, 1963; Sunde, 1964) . These
studies point to interference with vitamin A utilization as a

mode of action for nitrites in chickens and turkeys.

Abortion and Placental Transfer:

Case (1957c) first reported abortion 2 weeks prior to
full term along with stillbirths and.deformities in pigs which
had been pastured on oats and rape containing high levels of
nitrate. Testing showed a low level of plasma vitamin A and

excessive nitrate in the pigs. Since then, several

51
researchers'have explored the effect of nitrate and nitrite on
the fetus.

Elevations in serum nitrate were found in dead pigs born
to sows fed potassium nitrate compared to control pigs, with
the increases in serum nitrate corresponding to the levels of
nitrate fed in the dams’ ration (Garner et al., 1958). Though
litter size was unchanged, liveability and the number of
strong pigs appeared to decrease with higher levels of nitrate
(Garner et al., 1958). These results suggest nitrate
ingestion by sows may have some affect on their fetuses.

In a group of sows given sodium nitrite at high doses,
only 1 of the 8 sows which survived the nitrite treatment, had
fetal deaths associated with the toxic episode (Sleight et
al., 1972). This sowrwas given nitrite for 2 consecutive days
and farrowed 4 days after the second dose. .At farrowing, 2 of
the 9 pigs appeared to have died during the period of nitrite
toxicosis. This study found a narrow range between the dose
that killed sows and the dose that allowed sows to survive,
suggesting that toxicity episodes severe enough to cause death
of the fetuses without causing maternal death would be rare.

No methemoglobin was found in the fetuses of sows which
died within 2 hours of nitrite administration (Sleight et al. ,
1972). However, oxygen saturation of fetal blood decreased
significantly (P <0.001) after nitrite was administered to the
dam (Sleight et al., 1972). The oxygen saturation of fetal

blood declined as time elapsed between administration of

52

nitrite to the.dam.and blood collection from the fetus, though
the correlation was not linear. These findings suggest that
fetal death may be due to hypoxia in the fetuses as a result
of maternal methemoglobinemia. Sleight et al. suggest that
little of the nitrite crossed the placental barrier because
the methemoglobin levels of the fetuses did not appear to be
affected by nitrite administration. However, the study did
not measure nitrite levels in fetal blood. It is also
possible that not enough time had elapsed between dosing the
sow and testing the fetuses to allow for a full transfer of
nitrite and methemoglobinemia to occur.

Abortions as a sequelae to nitrite toxicosis, occurred in
a high percentage of ascorbic acid deficient guinea pigs while
no abortions occurred. in ‘the control group (Kociba and
Sleight, 1970) . However, fetal methemoglobin levels were
similar in both ascorbic acid deficient and control guinea
pigs following nitrite administration (Kociba and Sleight,
1970). This result suggests that the nitrite-induced
abortions are not related to methemoglobinemia in the fetuses.

In a series of experiments with rats, Gruener et al.
(1973) , demonstrated the transfer of nitrite through the
placenta and production of methemoglobinemia in the fetuses.
The transplacental transfer effect was discovered to have a
threshold dose of 2.5 mg/kg of sodium nitrite. Nitrite levels
rose in fetal blood approximately 20 minutes after the dam's

level rose and the increase in fetal nitrite was followed by

53
a rise in fetal methemoglobin. These studies indicate that
nitrite can cross the placenta, at least in one species.

Rat fetuses were found to have approximately ten times
more methemoglobin reductase than adult pregnant rats (Gruener
et al. , 1973) . This suggests that rat fetuses are less
sensitive to the oxidation properties of nitrites than adults,
in contrast to humans where fetuses have less methemoglobin

reductase than adults.

Maternal Milk:

Dogs given intravenous nitrate had increased milk nitrate
but the levels remained at or below plasma levels (Green et
al., 1982). In rats, although the dams showed high
methemoglobin levels due to ingestion of nitrites in the
drinking water (after giving birth), no rise in methemoglobin
levels were seen in suckling rats (Shuval and Gruener, 1972).
These findings suggest that.the passage.of nitrate and nitrite

into maternal milk may be species specific.

Chronic Nitrate and Nitrite Exposure:

Tissue pathology:

In rats given massive doses of sodium nitrite for up to
18 weeks, degenerative vascular and parenchymatous lesions
were found in the heart, lung, brain, kidney and testes

(Hueper and lendsberg, 1940). Pathological examination of

54

tissues from rats receiving drinking water that contained up
to 3000 mg of nitrite per liter for 24 months showed no
changes in the pancreas, adrenal, or brain though necropsy did
reveal lesions in the lungs and heart, especially with higher
doses of nitrite (Shuval and Gruener, 1972). After 24 months
of study, most control rats had some degree of thickening of
the intramural coronary arteries, many with marked hypertrophy
and narrowing. However, the rats receiving water containing
nitrate (especially at 3000 mg/l) had coronary arteries that
were thin and dilated (Shuval and Gruener, 1972). No
explanation is known for these changes.

Elevated serum urea, creatinine, and glutamic-pyruvic
transaminase concentrations provide evidence of kidney and
liver damage.in.chickens fed.nitrate and nitrite in the.ration
(Atef et al., 1991). Lower bursa of Fabricius weights,
decreased hemagglutination response, and reduced delayed
hypersensitivity reaction in chickens fed nitrate and nitrite
(Atef et al., 1991) suggests interference with the immune
system, although further work is required to confirm this

inference.

Central Nervous System:

Motor activity was significantly decreased in rats given
nitrites in the drinking water at doses which produced up to
15% methemoglobinemia (Shuval and Gruener, 1972). Vitamin C

given concurrently with drinking water containing 2000 mg/l of

55
nitrites prevented. methemoglobinemia, but motor activity
remained decreased (Shuval and Gruener, 1972). Three-month-
old.rats exposed to nitrite in the drinking water for'2 months
had irreversible changes in brain electrical activity even at
levels of 100 mg/l (Shuval and Gruener, 1972). It appears
nitrite exerts an effect on the central nervous system other

than that due to methemoglobinemia.

Hematological Effects:

Sows given 2% nitrate in the ration had serum nitrate
levels up to 16 mg 100 ml compared to 1-3 mg 100 ml for
controls (Garner et al., 1958). These figures indicate that
some of the absorbed nitrate circulates in the bloodstream.
No difference in hemoglobin or methemoglobin levels were seen
in pigs given drinking water that contained nitrate at levels
up to 500 ppm nitrate for 79 days (Bouwkamp and Counotte,
1988). However, when.pigs were given sodium nitrite at levels
sufficient to cause a prolonged increase in methemoglobin,
hematologic changes included increased hemoglobin, packed cell
volume, and lymphocytosis (London et al. , 1967) . Nitrate
administered in the drinking water of turkey poults at levels
of 675 ppm (Arends et al., 1967) or up to 1,485 ppm (Adams et
al., 1969) for 24 weeks had no effect on blood hematocrit
values. iHowever, sodium nitrate (4.2 g/kg of diet) and sodium

nitrite (1.7 g/kg of diet) added to the ration for 4 weeks

56

resulted in increased methemoglobin levels from 1.1% of total
pigment to 8% and 25.6%, respectively (Atef et al., 1991).

Pregnant rats given 2,000 mg/l of sodium nitrite in the
drinking water for an extended period developed anemia while
non-pregnant rats exposed to the same treatment did not
(Gruener et al., 1973). In chickens fed nitrate and nitrite,
erythrocyte counts increased initially (at 7 days into the
trial), but then decreased in comparison to control birds
(Atef et al., 1991). It appears compensatory increases in
hematocrit are seen at low nitrite levels but with prolonged
or high-dose administration, anemia results. The cause of the

anemia is unknown.

Immunosuppression:

Rats given 400 ppm sodium nitrite or 400 ppm sodium
nitrate in the drinking water for 6 months developed much
lower antibody titers than control rats when injected with
Newcastle virus (224) . This suggests nitrate and nitrite

exert an immunosuppressive effect in rats.

Thyroid Gland:

The goitrogenic action of nitrate and possibly nitrite
was originally discovered in:rats by”Wyngaarden et al. (1952).
After seventeen days of nitrate treatment, the thyroid glands
showed minimal hypertrophy, hyperplasia and a slight reduction

in iodine concentrations. Since then a number of researchers

57

have repeated these findings in rats. Dietary nitrate was
found to significantly increase the‘weight (Bloomfield et al.,
1961b; Welsch et al., 1961) and cause hyperplasia and
hypertrophy (Welsch et al., 1962) of thyroid glands in rats.
The mode of action of nitrate on the rat thyroid is through
inhibition of both the collection (Bloomfield et al., 1961b;
Wyngaarden et al., 1952) and retention of the iodide ion in
the thyroid gland (Wyngaarden et al., 1952).

A goitrogenic effect due to nitrite has also seen in
chickens. Nitrite in the feed of chicks at a rate of 0.4%
increased thyroid gland weights. Massive doses of vitamin A
partially overcame the thyroid hypertrophy caused by nitrite
(Sell and Roberts, 1963). No effect was seen from the
administration of 675 ppm nitrate in the drinking water of
turkeys on thyroid activity or the size of the thyroid,
spleen, or adrenal gland (Arends et al., 1967). It is not
known if the differences between the results seen with turkeys
and chickens reflect species differences or differences in
study protocol.

Dogs given sodium nitrate at levels up to 1,000 ppm in
drinking water for 1 year showed no evidence of thyroid
dysfunction (Kelley et al., 1974). The finding that nitrate
ingestion interferes with rat and chicken thyroid function but
has no effect on dogs indicates substantial species
differences in the effect of nitrate on this organ.

Vitamin A:

58

Several researchers have explored the effect of nitrate
and nitrite on vitamin A, with conflicting results. In pigs,
one study found no difference due to nitrate in the vitamin A
levels of the liver (Anderson and Stothers, 1978). While
several authors found 0.3% nitrate decreased liver vitamin A
levels even when beta-carotene 3,520 IU/kg or vitamin A 1,172
IU/kg were provided (Garrison et al., 1966; Wood et al.,
1967). Conflicting findings were also seen with nitrite in
pigs as one study noted nitrite at levels up to 100 ppm in the
drinking water for 105 days had no effect on liver vitamin A
values (Seerley et al., 1965). Other researchers reported
liver vitamin A levels decreased with 0.04% nitrite even
though beta-carotene 3,520 IU/kg or vitamin A 1,172 IU/kg were
provided (Garrison et al. , 1966; Wood et al. , 1967) . The only
study to measure serum vitamin A levels in pigs found that in
general serum vitamin A varied inversely with the level of
nitrite (London et al., 1967). With such disparity in
results, no conclusions can be drawn as to the effect of
nitrate or nitrite on vitamin A metabolism in the pig.

Nitrate was found to decrease vitamin A stores in the
liver (Yadav et al., 1962) and to be associated with more
rapid depletion of vitamin A stores in the liver of rats
(Garner et al. , 1958) . In rats with adequate vitamin A stores
fed a high-nitrate silage diet, liver stores of vitamin A were
maintained by the addition of vitamin A but declined when

carotene was given (Smith et al., 1961) . One study found

59
liver storage of vitamin A was not effected in rats fed
nitrate when vitamin A was administered regardless of whether
vitamin A was given orally as an oil solution, orally as a
water solution, or injected in an oil solution (Emerick and
Olson, 1962) . However, nitrate did significantly reduce liver
storage of vitamin A with intragastric administration of
carotene (Emerick and Olson, 1962). These results indicate
nitrate acts to either destroy carotene, inhibit the
conversion of carotene to vitamin A, or to prevent the
absorption of vitamin A in the rat. It appears that vitamin
A supplementation may overcome this effect of nitrate. A
possible mechanism of action may be through the goitrogenic
effect nitrate has on the thyroid in rats. The thyroid is
known to be important in the conversion of carotene to vitamin
A (Johnson and Baumann, 1947) and rats given adequate iodine
stored more vitamin A than rats on a thyroid deficient diet
(Yadav et al., 1962) Nitrite administered to vitamin A
deficient rats caused a more rapid depletion of liver vitamin
A (O'Dell et al., 1960). Also, nitrite given to vitamin A
depleted rats reduced the liver storage of vitamin A whether
given orally in an oil—based solution, orally as a‘water based
solution, or with intragastric administration of carotene
(Emerick.and.Olson, 1962). However, when.an oil-based vitamin
A solution was injected in vitamin A deficient rats receiving
nitrite, storage was increased. The reason for this increase

in storage is unexplained. Apparently, nitrite interferes

60
with liver storage of vitamin A either through inhibited
storage, accelerated depletion, or both mechanisms.

The addition of nitrite at 200 ppm to the drinking'water,
reduced vitamin A levels in the liver of chickens and both the
vitamin A and beta-carotene levels in the liver of turkeys
(Adams et al., 1966). Chickens receiving 0.4% nitrite in the
feed had significantly reduced liver vitamin A levels compared
to control chickens except when high levels of vitamin A were
injected. Adding vitamin A or carotene to the ration did not
increase liver stores to the same extent (Sell and Roberts,
1963). This finding suggests that nitrite acts toidecrease to
absorption of vitamin A and carotene, to increase the
destruction of carotene and vitamin A, or both. Chickens
receiving 0.74% potassium nitrite had lower concentrations of
vitamin A than control birds in both the ventriculus and
intestinal ingesta suggesting that nitrite acts to destroy

vitamin A in these organs (Roberts and Sell, 1963).

Vitamin E:

Relatively little research.has been.done on the effect of
nitrite on vitamin E. Vitamin E levels varied inversely with
the level of nitrite administered to pigs though signs of
vitamin E deficiency did not develop (London et al., 1967).
The addition of 0.3% nitrite to a ration considered to be
adequate in vitamin E, precipitated a vitamin E deficiency in

rats on that diet (O’Dell et al., 1960). Research thus far

61
indicates nitrite has a detrimental effect on vitamin E though

the mode of action remains unknown.

Reproduction:

No teratogenic effects were noted in pig fetuses 10 days
after treatment, when sows were given doses of sodium nitrite
at 30 mg/kg of body weight on days 15, 31, 32 and 33 of
gestation (Sleight et al., 1972). A review of other animal
studies found no evidence for teratogenic effects attributable
to nitrate or nitrite (Fan et al., 1987). Although human
studies have attempted to link nitrate consumption to
congenital malformations (Scragg et al., 1982; Dorsch et al.,
1984), the weight of evidence does not support this
conclusion.

In pigs, no effect on corpora lutea numbers (Tollett et
al., 1960) or percent implantation (Tollett et al., 1960) was
seen from dietary nitrate. Litter size was found to be
unaffected by the addition of up to 2% nitrate in the ration
of pigs though liveability and number of strong pigs appeared
to decrease at higher nitrate levels (Garner et al., 1958).
Birth weight, litter size at.weaning, and.daily weight gain in
pigs whose dams were consuming drinking water containing
nitrate at 300 ppm were found to be unaffected (Seerley et
al., 1965). There is very little evidence that nitrate exerts

a detrimental effect on reproductive performance in pigs.

62

Administration of nitrate in the drinking water of
rabbits at concentrations of up to 500 ppm for 2 successive
gestations had no detrimental effects on conception, litter
size, or the weights of the offspring at birth or weaning
(Kammerer and Siliart, 1993). Nor were the plasma levels of
estradiol or progesterone affected.

Reproduction in guinea pigs was not affected by potassium
nitrate in the drinking water at levels up to 10,000 ppm
(Sleight and Atallah, 1968). But at 30,000 ppm, reproductive
performance was 8% of the untreated control group (Sleight and
Atallah, 1968). Nitrite caused massive fetal death at much
lower dosages than nitrate. Fetal losses in pregnant guinea
pigs given drinking water containing nitrite at 5,000 or
10,000 ppm were 100% (Sleight and Atallah, 1968). Nitrite
administered at a rate of 60 mg/kg subcutaneously to guinea
pigs during the last 15 days of pregnancy caused fetal death
approximately 1 hour after administration (Sinha and Sleight,
1971). Nitrite was found in the fetuses, though at lower
levels than the dams, indicating at least partial passage of
the nitrite ion.across the placenta (Sinha and Sleight, 1971).
A relatively narrow range was found in the doses of nitrite
which either had no effect on reproduction, caused fetal
death, or caused maternal death (Sinha and Sleight, 1971).
Methemoglobin concentrations were significantly lower in
fetuses than guinea pig sows, and no changes occurred in the

placenta prior to fetal death (Sinha and Sleight, 1971).

63
These data suggest that fetal death occurred due to hypoxia
subsequent to maternal methemoglobinemia. Conception appeared
unaffected by nitrate or nitrite.

Drinking water with nitrite levels of 2,000 mg/l and
3,000 mg/l given to pregnant rats had a pronounced effect on
offspring mortality, particularly in the 3-week period prior
to weaning (Shuval and Gruener, 1972). Though birth weights
were unaffected, growth rates were slower in the groups
receiving high-nitrite water. Dullness and thinning of the
fur was observed in pups whose groups received high-nitrite
water (Shuval and Gruener, 1972). The mechanism of action is
unknown.

In chickens, no consistent relationship was seen in the
rate of egg production when consuming nitrate at levels up to
300 ppm or nitrite at levels up to 200 ppm nor were egg weight
or shell thickness affected (Adams et al., 1966). Chickens
consuming drinking water with nitrate at 300 ppm exhibited a
lowered peak of early egg production though later egg
production (during lower environmental temperatures)
approached that of control groups (Bentley et al. , 1965) .
These results may indicate that more water was consumed during
the warmer periods resulting in higher nitrate dosages in the
chickens, or that early production is more sensitive to the
effect of nitrates than later production, or that the effect
of nitrite is greater during periods of warmer environmental

temperatures.

64
Diarrhea:
Laying hens consuming 150 or 300 ppm nitrate in the
drinking water had fluid feces for the first three weeks of
the trial, after which the hens apparently adjusted and no

further problems were noted (Adams et al., 1966).

Growth:

Feed efficiency in swine was not adversely affected by
nitrate (Koch et al., 1963; Seerley et al., 1965) or nitrite
(Koch et al., 1963). One study found slightly better feed
efficiency with high nitrite administration which the
investigators attribute to decreased activity levels because
of dyspnea caused by nitrite-induced methemoglobinemia (London
et al., 1967).

No significant differences in average daily weight gain
(Anderson and Stothers, 1978; Seerley et al., 1965; Wood et
al., 1967), feed.consumption.(Andersonland Stothers, 1978), or
feed-to-gain ratio (Anderson and Stothers, 1978) were seen in
pigs consuming up to 300 ppm nitrate in the drinking water.
Nitrate at levels of 200 ppm in weaned pigs and 500 ppm in
finishing pigs had no effect on rate of weight gain, feed
efficiency, or feed consumption (Bouwkamp and Counotte, 1988) .
Tollett et al. (1960) reported significantly depressed weight
gains in pigs fed over 1.84% nitrate, though the amount of
feed consumed was not monitored. Studies with ruminant

animals show decreased weight gains to be strongly associated

65

with decreased feed consumption. The evidence points to
nitrate having no effect on feed efficiency or weight gain,
except perhaps in regards to decreased feed consumption.

Giving guinea pigs drinking water that contained nitrate
at levels of up to 10,000 ppm nitrate or nitrite at up to
5,000 ppm had no effect on weight gain, or the consumption of
feed or water (Sleight and.Atallah, 1968). ‘Vitamin Aidepleted
rats fed 3% nitrate or 0.5% nitrite weighed significantly less
than controls at the end of the 6-day trials (Emerick and
Olson, 1962). No significant differences were seen in growth
or development of 3-month-old rats (non-vitamin A depleted)
given up to 3000 mg/l of nitrite in the drinking water (Shuval
and Gruener, 1972). This points to interference with vitamin
A metabolism as the cause of decreased weight gain in rats.

A reduction in growth was seen in chicks given drinking
water containing 200 ppm. nitrite (Adams et al., 1966).
Decreased weight gain was also noted in Balady chickens but
because of this breed's foraging habits, feed consumption
could not accurately be measured (Atef et al., 1991). Chicks
consuming 0.4% sodium nitrite had significantly decreased feed
consumption and rate of weight gain (Sell and Roberts, 1963).
When daily feed consumption was equalized between chicks
receiving nitrite and controls, no difference in weight gain
was seen if chicks were supplemented with vitamin A. However,
chicks on a nitrite-containing, vitamin-A-free ration gained

far less than all other treatment groups while consuming, on

66
average the same amount of feed (Sell and Roberts, 1963) .
Clearly nitrate acts to decrease feed consumption in chickens.
It appears that decreased feed efficiency when seen, is
related to interference with vitamin A metabolism.

Nitrate administered at levels up to 1,485 ppm
continuously in the drinking water of turkey poults from 1 day
of age to 24 weeks had no significant effect on growth rate or
feed conversion (Adams et al., 1969) . One study reported
water containing 675 ppm nitrate given to turkeys for the
first 4 weeks of life, resulted in turkeys which were heavier
than controls at 16 weeks of age (Kienholz et al., 1965).
Subsequent work mentioned that males were more affected than
females, and that testes weights were far smaller in the
nitrate treated groups (Arends et al., 1967) thus suggesting
that nitrate has a "caponizing" effect. Nitrate in the feed
at 1000 ppm for the first 4 weeks did not promote growth at
later ages (Arends et al., 1967). The finding that nitrate in
the feed did not have a similar effect and the disagreement
with the study done by Adams et al. (1969) casts doubt on the
existence of 'the "caponizing" effect of nitrate. .Feed
consumption and growth decreased in turkeys as the amount of
nitrite (50 to 200 ppm) in the drinking water increased (Adams
et 1., 1966). It is likely that the decrease in growth is
related to a decrease in food consumption.

Weight. gain and feed. consumption in .Japanese quail

(Coturnix coturnix japonica) decrease with nitrate in the

67
drinking water at 1,320 ppm and 3,960 ppm but not at 2,640
ppm, suggesting that the decreased feed consumption and weight
gain may have been due to some other factor rather than

nitrate (Adams, 1974).

Sulfonamide Drug Metabolism:

Nitrites have been shown to affect the metabolism of
sulfonamide drugs in animals. The deamination of
sulfathiazole, sulfamethazine, and sulfadiazine in the rat
(Nelson et al., 1987; Paulson, 1986; Paulson, 1987; Woolley
and Sigel, 1982), sulfadiazine in the guinea pig and rat
(Woolley and Sigel, 1982), and sulfamethazine in swine
(Paulson and Feil, 1987; Struble and. Paulson, 1988) is
accelerated by high dietary nitrite.

Since nitrite is required for the deamination reaction,
dietary nitrate must be reduced to nitrite in vivo (e.g. in
the oral cavity or stomach) if nitrate is to affect
sulfonamide drug metabolism. Studies have shown that rats and
most calves do not reduce nitrate, while guinea pigs can
readily convert nitrate to nitrite, and swine are intermediate
in.their ability (Paulson, 1987; Wooley and Sigel, 1982). The
concentrations of nitrite in the oral cavity of swine
increased when fed 500 and 1000 ppm nitrate though the amount
of nitrate reduced was small (Paulson and Aschbacher, 1990).
Bacterial reduction of nitrate is suspected as nitrate was

converted to nitrite in regular pigs but was not converted in

68

germ-free pigs (Struble and Paulson, 1988). Likewise in
guinea. pigs, orally administered. nitrate ‘was. reduced. to
nitrite but not when administered intramuscularly or
intraperitoneally (Wooley and Sigel, 1982). These results are
in agreement with human studies which show nitrate to be
reduced to nitrite in the oral cavity by bacterial activity
(Tannenbaum et al., 1974; Tannenbaum et al., 1976).

Experimental evidence in swine shows that at 1000 ppm
nitrate in the ration the amount of desaminosulfamethazine
increased from 1.1 ppm to 6.3 ppm (Paulson and Aschbacher,
1990). Because production of this metabolite of
sulfamethazine is enhanced by nitrite, this finding indicates
the deamination of sulfamethazine can occur in swine
subsequent to the reduction of nitrate to nitrite in vivo.
The effect of nitrite on sulfonamide drug metabolism has been
thoroughly reviewed elsewhere (Paulson, 1987). In general,
high levels of ingested nitrite act to increase excretion of
drug-related compounds in the feces and decrease
concentrations of the parent drug in the blood and other
tissues (Paulson, 1987). Animals that receive high levels of
nitrite or that reduce ingested nitrate to nitrite in the oral
cavity or gastrointestinal tract, may require a different dose
of the sulfonamide drug to provide the same level of
prophylactic or therapeutic action (Paulson, 1987). Ascorbic

acid counteracts the effect of nitrite ingestion on

69
sulfamethazine disposition in rats (Paulson, 1987) and may

exert a similar effect in other animals and humans.

Intestinal Absorption:

In sedentary rats, orally administered potassium nitrate
whether given acutely (one dose at 800 mg/kg of body weight)
or chronically (100 mg/kg of body weight for 90 days), did not
affect intestinal absorption nor alter the metabolic
parameters of the small intestinal mucosa (Grudzinski and
Szymanski, 1991b and 1991a) . However when the rats were
exercised, potassium nitrate caused reduced gastric mucosal
absorption as measured by D-xylose absorption in both the
acutely and chronically dosed rats (Grudzinski and Szymanski,
19910 and l991d). In the chronically dosed rats, decreased
alkaline phosphatase and Na+/K*-ATPase activity, and atrophy
of intestinal villi was seen with exercise (Grudzinski and
Szymanski, 1991c).

Sodium nitrite given orally to rats at a dose of 80 mg/kg
of body weight, increased gastric mucosal absorption as
measured by D-xylose absorption and increased the uptake of
oxygen in the small intestine (Grudzinski and Szymanski,
1991b) . However with chronic administration of sodium nitrite
at 5 and 10 mg/kg of body weight for 90 days, the absorption
of D-xylose was decreased by the rat gastric mucosa while
oxygen uptake was unchanged (Grudzinski and Szymanski, 1991a) .

Alkaline phosphatase and Na+/K+-ATPase were unaffected by

70

sodium nitrite at a dose of 80 mg/kg of body weight but were
significantly reduced with daily administration of doses of 10
mg/kg (Grudzinski and Szymanski, 1991a) . Exercise exacerbated
the effects of both acute and chronic nitrite administration
(Grudzinski and Szymanski, 1991c and 1991d). Further study
revealed that while nitrite reduces the activity of alkaline
phosphatase and Na+/K+-ATPase in intestinal mucosa, it does
not increase Na+/K+-ATPase inhibition produced by ouabain and
inhibition of alkaline phosphatase produced by L-phenylalanine
(Grudzinski and Szymanski, 1991e).

On histopathologic examination, the rats that received
sodium nitrite at a dose of 80 mg/kg body weight had a
reduction in the amount of mucus on the gastric surface, small
erosions, slight swelling of the villi, and a decrease in
lactate dehydrogenase activity in the superficial layer of
glands (Grudzinski and Szymanski, 1991b). Chronic daily
dosing with sodium nitrite at 10 mg/kg of body weight caused
considerable swelling and atrophy of villi within 21 days
(Grudzinski and Szymanski, 1991a). Further research is needed

to explain the mechanism and importance of these findings.

N-Nitroso Compounds:

Outbreaks of toxic hepatosis occurred in Norway in 1961

and 1962 affecting a substantial number of cattle and sheep

(Hansen, 1964; Koppang, 1964). The toxic substance was found

71
to have been contained in nitrite-preserved herring meal fed
to the animals as a protein supplement (Koppang et al. , 1964) .
It was determined that sodium nitrite used to preserve the
herring had reacted with amines present in stale fish to
produce highly toxic dimethylnitrosamine (Sakshug et al.,
1965). As research progressed, toxic herring meal was found
to be the cause of hepatosis seen in mink and foxes since 1957
(Koppang, 1966; Koppang and Helgebostad, 1966) . Further
research has shown mink to be exceptionally susceptible to the
effects of dimethylnitrosamine (Carter et al., 1969). In fur
animals, this disease is a slowly progressive disease with
restlessness, fur biting and dry, dull, fur observed
initially. As the disease advances, inappetence develops
along with ascites, anemia, and thin tarry feces. The
mortality rate can be quite high. On necropsy, the liver is
markedly affected. Both the gross and histologic appearance
of the liver varies with the acuteness of the disease

(Koppang, 1966; Koppang and Helgebostad, 1964).

DISCUSSION

Methemoglobinemia in ruminant animals subsequent to
consumption of high-nitrate forages will continue to be a
problem especially with erratic rainfall patterns. Monitoring
of forage nitrate levels and judicious use of fertilizers,

herbicides, and irrigation are useful in limiting nitrate

72

accumulation in plant material. Methemoglobinemia in animals
due to nitrate-contaminated water is rarely reported, though
a human fatality was described in the United States as
recently as 1986 (Johnson et al., 1987). In regard to nitrate
toxicity in ruminants, there remain a number of unanswered
questions. Many substances such as soybean meal, urea,
antibiotics, and monensin affect nitrate metabolism. It would
be beneficial to explore the nature and extent of these
interactions in order to better understand and prevent
toxicity episodes.

Large doses of nitrate are toxic to monogastric animals
without the occurrence of 'methemoglobinemia.
Methemoglobinemia has been reported in monogastrics from the
consumption of high-nitrate forage under conditions suitable
for nitrate reduction to nitrite in the forage prior to
consumption.

Ingested nitrites or nitrates in species with nitrate
reductase activity, may be of regulatory interest as
deaminated sulfonamide drugs in swine and calves have much
longer half-lives but are not detected by the Bratton Marshall
test (commonly used to measure sulfonamide drug residues in
animal tissues) (Paulson, 1986). In this case, sulfonamide
drug residues would be increased in food animals while the
efficacy of the analysis for the total tissue residue burden

was decreased.

73

A variety of other effects have been attributed to
nitrate. A goitrogenic effect on thyroid function by nitrate
has been demonstrated to occur in some species but not in
others. This aspect of nitrate may be a species specific
response, though the data remain incomplete. Little work has
been done on pigs, an animal that may serve as an appropriate
model for the response of nitrate in humans. Hematological
changes noted in cattle, sheep, pigs, rats and chickens
suggest that humans may respond in a similar manner to chronic
nitrate exposure. The finding of nitrite-induced tissue
pathology and irreversible changes. in brain electrical
activity of rats is of considerable concern. The question of
whether such devastating effects also occur in humans remains
unanswered.

Vitamin A deficiency has been associated as a risk factor
for cancer both epidemiologically and experimentally (Rose,
1983). The work done in animals such as the rat, chicken and
turkey showed nitrate to have a detrimental affect on vitamin
A metabolism in some species. This suggests the possibility
that nitrate may exert an effect on cancer through this
mechanism.

A hypothesis for the role of nitrate, nitrite and N-
nitroso compounds in the etiology of gastric and other human
cancers has been proposed (Correa, 1987; Correa et al., 1975).
A number of researchers have examined the effect of these

compounds on the rate of gastric cancer (Cuello et al., 1976;

74

Buitti et al., 1990; Gilli et al., 1984; Takacs, 1987; Zemla,
1980) . Epidemiologic studies using humans have been burdened
by a variety of difficulties, and the importance of nitrate,
nitrite and N-nitroso compounds in the etiology of gastric
cancer remains uncertain. To determine the nature and extent
of exposure to these compounds, one must determine the
sources, amount, and time period involved, all of which are
extremely difficult with human subjects. Because of the
difficulties encountered when using humans in studies, a
monogastric animal might serve as a suitable research subject.

Experiments in cattle, guinea pigs and rats suggest the
ability of nitrate to cross the placenta. Animal studies
suggest nitrite may also traverse the placenta (Gruener et
al., 1973; Globus and Samuel, 1978) or exert a transplacental
effect (Inui et al., 1979; Inui et al., 1979) . Investigations
of nitrate poisoning indicates abortion may occur in ruminants
subsequent to acute toxicity. The possibility of nitrate
traversing the human placenta (Gelperin et al., 1971) or
having a role in infant death or malformation has been
suggested, but remains unproven (Scragg et al., 1982; Dorsch
et al., 1984; Schmitz, 1961). A review of animal data found
no evidence for teratogenic effects attributable to nitrate or
nitrite ingestion (Fan, 1987). The finding of nitrates in the
milk from cattle and dogs and N-nitroso compounds in the milk
from goats is worrisome. At this time, it is not known

whether nitrate, nitrite or N-nitroso compounds cross the

75

placenta or can pass to children through their mothers’ milk.
Until further research clarifies the situation, pregnant women
and nursing mothers would be wise to avoid ingestion of
nitrate, nitrite, and N-nitroso compounds in food or water.

Currently, little.is known about the long term.effects of
nitrate, nitrite and N-nitroso compounds on animal health.
With the number of adverse health effects known or suspected
to be associated with nitrate, nitrite, and N-nitroso
compounds in human health, further animal research is needed

to answer the remaining questions.

76

Table 3.1: Conversion Factors

 

Nitrate in PPM = Nitrate in mg/kg = Nitrate in mg/L
PPM X 0.0001 = % Nitrate

% Nitrate X 10,000 = PPM

Potassium or sodium nitrate X 0.61 Nitrate

Potassium or sodium nitrate X 0.14 Nitrate-nitrogen
Nitrate-nitrogen (NO -N) X 4.45 = Nitrate

Nitrate-nitrogen (NO -N) X 3.29 Nitrite

Nitrate-nitrogen (NO -N) X 6.1 = Potassium or Sodium Nitrate

77

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CHAPTER 4

THE EFFECTS OF NITRATE CONTAINED IN DRINKING WATER

ON THE PRODUCTIVITY AND HEALTH OF FARROWING SWINE

99

100

ABSTRACT

A cross-sectional study design was used to determine the
effects of nitrate contained in drinking water on farrowing
swine health and productivity. The study was conducted from
November 1989 through February 1991.on randomly selected.swine
farms in the United States. The study consisted of 571 farms
containing 27,207 farrowing swine. At the beginning and end
of each farm’s 3-month monitoring period, the drinking water
provided to the farrowing swine was tested for the levels of
nitrate and 15 other elements and compounds. Nitrate was
detected in 53.2% (304/571) of the well-water samples with a
mean concentration of 17.9 ppm and a median of 2.1 ppm. Pre-
tested questionnaires administered to farm operators by
trained personnel, were used to collect data on management,
housing, and environmental parameters. During the farm's
monitoring period, data on farrowing swine health and
productivity were observed and recorded daily by the animal
caretaker.

The data were analyzed on a farm basis using stratified
analysis, and.multiple linear regression or multiple logistic
regression. No association was seen between the nitrate
concentration of drinking water and farrowing swine
productivity as measured by average litter size, average

percentage of the litter stillborn, or the risk of having an

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above median percentage of the litter born mummified. No
association was seen between nitrate and the health of
farrowing swine as measured by the risk of having an above
median (>0) percentage of swine ill or dead due to farrowing
problems, other reproductive problems, other known health
problems, or unknown health problems. The statitistical power
of this study was .90 (at a=.05) to detect if nitrate in the
drinking water explained 3% (partial R2=.03) of the variation
in either farm average litter size or percent stillborn.
Statistical power was sufficient to have detected an odds
ratio (at a=0.05, B=.20) as low as 1.3 for farrowing illness;
1.4 for illness due to reproductive, known, or unknown
problems; 1.5 for death due to farrowing or known problems;
1.8 due to unknown problems; and 2.5 for death due to

reproductive problems.

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INTRODUCTION

Nitrate affects both animal (Bruning-Fann and Kaneene,
1993a) and human (Bruning-Fann and Kaneene, 1993b) health in
a variety of ways. Methemoglobinemia from the consumption of
nitrate-contaminated water was first reported in humans in
1945 (Comly, 1945). Cases continue to occur with the latest
U.S. fatality reported in 1987 (Johnson et al., 1987). Many
episodes of methemoglobinemia and abortion in animals have
been attributed to acute nitrate toxicosis (Carrigan and
Gardner, 1982; Haliburton and Edwards, 1978; Hibbs et al.,
1978; Nicholls and Miles, 1980; Ruhr and Osweiler, 1986;
Vermunt and Visser, 1987). Nitrates are suspected of causing
"lowland abortion syndrome" in cattle (Simon et al., 1959a;
Sund et al., 1957). When pregnant dairy cattle were given
large doses of potassium nitrate, fetal death occurred
followed by abortion (Simon et al., 1959b).

While the effects of acute nitrate toxicosis have been
well described, the effects of chronic, low-dose exposure in
humans and animals are unclear. Two epidemiological studies
in humans found the consumption of drinking water from wells
to be associated with congenital malformations and suggested
nitrate contained in the water may have been responsible
(Scragg et al., 1982; Dorsch et al., 1984). However, a review
of nitrate in regard to teratogenic effects concluded that no

such association has been shown (Fan et al., 1987). Some

103

animal studies found decreased conception rates (Davison et
al., 1964) and increased stillbirths (Sleight and Atallah,
1968) attributable to nitrate. However, other studies
reported no effect from nitrate exposure, on the maintenance
of pregnancy (Davison et al., 1965; Eppson et al., 1960;
Winter and Hokanson, 1964) or birth weights (Davison et al.,
1965; Eppson et al., 1960). High levels of nitrate (3000
mg/L) in drinking water have been associated with thinning and
dilation of coronary arteries in rats (Shuval and Gruener,
1972). In addition, nitrate has been linked to decreased
immune response in.rats (Kahraman, 1988; De Saint Blanquat, et
al., 1983) and chickens (Atef et al., 1991).

Relatively little is known about the effects of nitrate
in swine» Gastritis (Gwatkin and Plummer, 1946; Smith et al.,
1959), abortion, stillbirths and deformities have been
associated with the consumption of high levels of nitrate
(Case, 1957). Nitrate ingestion has been linked to reduced
weight.gain.(Tollett.et.al., 1960) and.decreased liver storage
of vitamin A in some studies (Koch et al., 1963; Garrison et
al., 1966; Wood et al., 1967) but not in others (Anderson and
Stothers, 1978; Seerley et al., 1965). Conflicting results
were also reported on the effects of nitrate on farrowing
swine productivity. One study reported an apparent decrease
in the number of strong pigs born and.their ability to survive
when sows were given 2% potassium nitrate in their rations

compared to control sows (Garner et al. , 1958) . However,

104

another study found.no effect on.corpora lutea number, percent
implantation, ovary weight, embryo weight or placenta weight
when up to 3.17% nitrate was added to the ration of gilts
(Tollett et al., 1960). A third study reported no difference
in litter size or average birth weight of pigs when sows were
given drinking water containing up to 300 ppm (parts per
million) nitrate (Seerley et al., 1965).

Although.the toxic effects of large.doses of nitrate have
been clearly established in humans' and animals, much
controversy remains as to the effects of chronic low-level
nitrate exposure. With nitrate levels increasing in
groundwater (Fraser and Chilvers, 1981; Shuval and Gruener,
1972; Vigil et al., 1965; Hollander and Sander, 1987; Moller
et al., 1989), it is important to establish the significance
of chronic low-level nitrate exposure to health. The
objective of this study was toidetermine if nitrate, at levels
currently found on swine farms, adversely affects the

productivity or health of farrowing swine.

Hypotheses Tested:

The nitrate level of drinking water is associated with the
farm:

1) average number of pigs born per litter,

2) average percentage of the litter stillborn,

The nitrate level of drinking water is associated with the

risk of a farm experiencing:

105

3) above median percentage of the litter born mummified,

4) above median percentage of swine with farrowing problems,

5) above median percentage of swine with reproductive
problems other than farrowing problems,

6) above median percentage of farrowing swine with other
health problems,

7) above median percentage of farrowing swine with unknown
health problems,

8) above median mortality rate due to farrowing problems,

9) above median mortality rate due to reproductive problems
other than farrowing problems,

10) above median mortality rate due to other known problems,
and

11) above median mortality rate due to unknown problems.

MATERIALS AND METHODS

National Swine Survey

The data used in this report are a portion of the data
collected during the National Swine Survey (NSS) conducted by
the United States Department of Agriculture (USDA), Animal and
Plant Health Inspection Service's, National Animal Health
Monitoring System (NAHMS). The NSS was conducted on swine
farms located in 18 states (Alabama, California, Colorado,

Georgia, Iowa, Illinois, Indiana, Maryland, Michigan,

106
Minnesota, Nebraska, North Carolina, Ohio, Oregon,
Pennsylvania, Tennessee, Virginia and Wisconsin) from November
1989 through February 1991. The NSS was a hybrid
epidemiologic study utilizing retrospective, prospective and
cross-sectional study designs. The data used in this analysis
were collected in a cross-sectional manner. Further
information on ‘the entire NSS is contained in the NSS

technical report (USDA, 1992).

Sample Selection
The sample size was determined primarily by the desire to

estimate preweaning mortality within 1% of true mortality

using the following formula:

1': = 523.2

d2
where:
n = the sample size
s2 = the variance of the proportion
z2== z value for the appropriate confidence level

c? = the acceptable bound on the proportion estimate

Preweaning mortality was thought to be approximately 13%
with a usual range of 0% to 35%. The variance for the
proportion of preweaning mortality was estimated using three
different methods (sz=pq; sz=AB2 [using the Gamma distribution

where A identifies the shape of the distribution and B is the

107

mean]; s’=(2p bar)2 [assumes the relationship of the standard
deviation to the mean is 2]). Ultimately, it was decided.that
1,400 farms would be sufficient to estimate the true
preweaning pig mortality rate with a confidence level of 99%.

The sampling process began with the selection of States
to be included in the NSS. Thirteen States were preselected
for inclusion in the study because of their prior involvement
in NAHMS projects. These States are Alabama, California,
Colorado, Georgia, Iowa, Illinois, Maryland, Michigan, Ohio,
Oregon, Tennessee, Virginia, and Wisconsin. Indiana and North
Carolina were also preselected because of their large swine
populations and strong interest in jparticipating in 'the
project. All remaining States which contained 2% or more of
the nation’s swine population were eligible to be included in
the NSS. Remaining States under consideration were placed in

1 of 2 strata by average herd size.

Stratum 1: Kansas, Minnesota, Missouri, Nebraska, and
South Dakota (states with larger average herd
sizes; criteria for farm eligibility was 10 or
more sows expected to farrow).

Stratum 2: Kentucky, Pennsylvania, South Carolina, and
Texas (states with smaller average herd sizes;
criteria for farm eligibility was 1 or more

sows expected to farrow).

108

A proportionate sample was selected based on the number
of hogs in the state. This resulted in the selection of
Minnesota, Nebraska, and Pennsylvania for participation in the
NSS. The 15 preselected States plus the 3 probability
selected States accounted for 62% of the swine operations and
81% of the hogs in the nation. Because the probability
selected States are representative of the States in their
respective strata, Minnesota and Nebraska represent the 3
remaining Stratum 1 States and Pennsylvania represents the 3
remaining Stratum 2 States. Therefore, the 18 selected States
represent a total of 24 States which contain 84% of the U.S.
swine operations and 95% of the swine population.

Potential study participants were identified by the
USDA’s National Agricultural Statistics Service (NASS).
Criteria for farm eligibility in the study were designed to
account for differences in average herd sizes among the
States. In States with a small average herd size (Alabama,
California, Colorado, Maryland, North Carolina, Oregon,
Pennsylvania and Virginia), farms with one or more sows
expected to farrow during the study period were eligible to
participate in the NSS. In the remaining 10 States (Georgia,
Illinois, Indiana, Iowa, Michigan, Minnesota, Nebraska, Ohio,
Tennessee and Wisconsin), farms were eligible if 10 or more
sows were expected to farrow during the study period.

Swine farms to be included in the NSS were selected by

NASS using a multiple frame sampling technique. This consists

109
of using both list (a roster of all eligible units) and area
frames (the actual land area is divided into sections, all
swine farms in chosen sections are sampled) to select farms

for participation in the NSS.

Farm population = farms from list estimate + farms from area
estimate that are not included on list

estimate

NASS personnel contacted randomly selected farms and
explained the NSS. During this visit, the General Swine
Survey was completed. Out of 3,184 farms initially identified
by NASS, 2,965 were located and met the criteria for
eligibility in the NSS. Of the 2,965 eligible farms, 1,320
refused to participate in the NSS and 3 farms were mislaid
prior to arrival at the NAHMS office. Of the 1,642 farm
contacts received by NAHMS from NASS, 746 operators refused to
participate in the study, 65 were ineligible, 16 were never
visited, and 815 agreed to participate (52.2% [815/1,561]).
Of the 815 farm operators who started the NSS, 103 left the
study prior to completion. The response rate for completion
of the NSS was 45.6% (712/1561). Although a total of 712
farms completed the study, data were excluded from 141 farms
because a water source other than a well was used (n = 106) or

because 2 well-water samples were not obtained (n = 35).

110
Ultimately, data from 571 swine farms were utilized in this

analysis.

Data Collection

Individual swine farms were monitored for 3 months with
the period of surveillance staggered throughout the year. In
this way, approximately equal numbers of farms were monitored
during each month of the study year. To ensure comparability
of data, training of NASS and NAHMS personnel was accomplished
prior to the first farm visit.

NASS personnel completed a General Swine Farm Report
during the initial visit to the swine producer. This
questionnaire emphasized current management practices and
other descriptive farm information. Farms whose operators
agreed to participate in the NSS were assigned to federal and
state veterinarians associated with NAHMS. These
veterinarians visited the farms on a monthly basis to collect
the farrowing diary cards and administer three more
questionnaires. Examples of the questionnaires and the
farrowing diary cards can be found in the NSS technical report
(USDA, 1992).

Completion of a Swine Health Report by a NAHMS
veterinarian during the second visit to each farm initiated
the start of the farm's monitoring period. This questionnaire

dealt with current vaccination and other preventive practices,

111
biosecurity and health problems experienced on the farm during
the past year.

A NAHMS veterinarian completed a Swine Facilities and
Feed Report on the third farm visit. Housing during all
phases of swine production was delineated with emphasis on the
monitored farrowing facilities. In addition, the composition
of all the feeds used on the swine farm during all phases of
production was described in detail.

A Swine Ending Inventory and Economics Report was
completed on the last farm visit by a NAHMS veterinarian,
which occurred at the end of each farm's monitoring period.
Information was gathered concerning purchases and sales of
swine, operating expenses, the amount of labor used in the
operation and the current swine inventory.

On farms expecting less than 100 farrowings during the 3
month monitoring period, all farrowing sows were monitored; on
farms expecting 100 or more farrowings, only sows entering
selected farrowing units were monitored. The NAHMS staff
selected farrowing units to be monitored using simple random
sampling. Data from a total of 27,207 sows on 571 farms in 18
states were used in this analysis.

A Farrowing Diary Card was completed for each monitored
sow and her litter. The animal caretaker recorded on the
diary cards all birth events, health events and preventive
practices as they occurred to the sow and her litter. Cases

of illness or death were classified into one of eight broad

112
categories of disease. Cards were initiated for each sow in
the monitored farrowing facility at the start of the farm's
monitoring period and for each sow that entered the monitored
facility during that period. Only sows that entered the
farrowing facility, farrowed and weaned their litters during

the farm’s monitoring period are included in this analysis.

Water Sampling

Water samples were collected from participating farms at
the first and last interviews (3 months between samples).
Samples from the water supply that served the farrowing unit
were collected as close to the point of water consumption as
possible. To ensure that the water sample was representative
of the well, the pipes were flushed by running the water for
at least one minute prior to sampling. All samples were
shipped by priority mail (delivery by the second day) to the
USDA's National Veterinary Services Laboratories (NVSL) for
analysisw Instructions for 'water sample collection .and

shipment were provided to the veterinarians.
Laboratory Testing of Water
NVSL analyzed the water samples for a number of elements

and. compounds using' an inductively' coupled. argon. plasma

emission spectrophotometer and an ion chromatograph.

113
Calibration standards were included at the beginning and end
of every batch, and control samples with known ion
concentrations were analyzed after every 10 samples. In cases
where the values were extremely high or the results were in

question, the analyses were repeated.

Data Analysis

Measures of Health and Productivity

Using the farm as the unit of comparison, various
measures were calculated in order to assess sow productivity
and health. Sow productivity was measured by determining the
average litter size (Avg Litter Size), the average percentage
of pigs stillborn (Avg % stillborn), and the average
percentage of pigs born mummified (Avg % Mummified) . The
average litter size was calculated by dividing the total
number of pigs born by the number of swine which farrowed.
Similarly, the average percentage of pigs stillbOrn (or born
mummified) was calculated by dividing the total number of pigs
stillborn (or born mummified) by the total number of pigs
born. The percentage of sows on each farm ill or dead were
calculated for each of the following disease categories:
farrowing problems (% Ill-Farrowing), reproductive problems
other than farrowing (% Ill-Other Repro. Prob.), other known

health problems (problems recognized by the animal caretaker

114
that are not included in other disease categories [% Other
Known Illness]), and unknown. health. problems (% ‘Unknown
Illness). The percentage of sows ill for each disease
category' was calculated by' dividing the number of sows
experiencing each disease problem by the number of sows at
risk for experiencing that particular disease problem during
the 3-month monitoring period. Similarly, the percentage of
sows dead in each disease problem category was determined by
dividing the number of sows dead in each disease category by
the number at risk. For use in stratified analysis and
logistic regression, the farm percentage of mummies and sows
ill or dead for each disease category were dichotomized as
above the median or not. These variables and their

descriptions are provided in Tables 4.1a.

Variables

To quantify the impact of nitrate (the independent
variable of interest) on the various measures of sow health
and productivity (the dependent variables), other factors
presumed likely to influence the dependent variables were
controlled in the analyses. A review of the literature
suggested that in addition to nitrate, a number of other
factors might affect farrowing sow productivity or health
(Leman et al., 1988; National Academy of Sciences, 1974;

Fraser et al., 1990). These potentially confounding and/or

115
effect modifying variables are the levels of ammonia, barium
and nitrite in the drinking water, the total number of swine
on the farm (TtlSwn) , the years of continuous swine farm
experience (YrsSwn) , and the farm average sow parity (Angar) .
Additional variables include Ihow' the farrowing’ unit. was
managed (all-in all-out or continuous farrowing [TypeFar]),
vaccination against Actinobacillus pleuropneumonia (Actino),
atrophic rhinitis (Rhinitis), parvovirus (Parvo),
leptospirosis (Lepto), pseudorabies (Pseudo), Escherichia
coli, rota virus (Rota V), Clostridium perfringens (Clos),
erysipelas (Erysip), transmissible gastroenteritis (TGE),
other diseases (Other V), the use of antibiotics in the feed
(Antibio F), and whether the farm manager administers
anthelmintics (Anthel). These variables and their

descriptions are provided in Tables 4.1b.

Statistical Analysis

The data were examined using descriptive statistics,
Spearman correlations, univariate logistic regression,
stratified analysis, multiple linear regression, and.multiple
logistic regression (SAS Institute Inc., 1988; SAS Institute
Inc., 1990).

Descriptive statistics allow comparison of the
composition of water and selected characteristics of the swine

population with other studies. Generation and examination of

j 116
these statistics afforded familiarity with the data, suggested
appropriate points at which to stratify the continuous
variables, and allowed determination of whether the data met
certain assumptions. Because most of the data were not
normally distributed, nonparametric statistics were employed.

Spearman correlations were used to examine the
relationships between the individual independent and dependent
variables and between nitrate and other independent variables.
Those variables associated with both nitrate and the dependent
variable(s) had the potential to confound the relationships
between nitrate and the dependent variables. To control for
confounding, those correlations with a statistical
significance of P50.2 were deemed portentous (Maldonado and
Greenland, 1993) and were included in stratified analysis and
multiple logistic regression.

It is recommended that univariate logistic regression be
accomplished prior to multiple logistic regression (Hosmer and
Lemeshow, 1989) . Univariate logistic regression was performed
on all independent variables hypothesized to be associated
with the dependent variables. This procedure aided in the
determination of whether any important predictor variables had
been missed with correlation analysis.

Stratified analysis was performed to assess effect
modification and to control confounding. Unadjusted odds
ratios estimated only the effect of nitrate on the dependent

variables without controlling (adjusting) for the effect of

117

other independent variables. Adjusted odds ratios estimated
the effect of nitrate while controlling (adjusting) for the
effect of other independent (predictor) variables on sow
health and productivity. The association between nitrate and
decreased sow'productivity was assessed as the odds ratios for
1) below-median farm average litter size, 2) above-median farm
percentage stillborn, and 3) above-median farm percentage born
mummified (Table 4.1a). Similarly, the association between
nitrate and sow health was measured by the odd ratios for
above-median (>0) percent illness and mortality for each of
the categories (4.1a) . Because only 25 farms had nitrate
levels 2100 ppm, the human limit of 45 ppm was used during
stratified analysis when control variables were included.

Multiple linear and logistic regression were utilized to
examine the effect of nitrate on sow productivity and health.
This allowed.the use of nitrate as a«continuous variable while
controlling for the effect.of other predictor variables on the
dependent variables. While it is difficult to control for
more than a few variables with stratified analysis (due to
vacant cells), multivariate regression procedures can
simultaneously control many independent variables. In doing
so, joint confounding can be controlled. With multiple
logistic regression, odds ratios were calculated for the
association of nitrate with sow productivity and health by
exponentiating the maximum likelihood-derived coefficients

(Hosmer and Lemeshow, 1989).

118

RESULTS

Results of water analysis revealed that the mean
concentration of nitrate in well-water during the NSS was 17.9
ppm with a median of 2.1 ppm (Table 4.2). No nitrate was
detected in 46.8% (267/571) of the well-water samples. Tables
4.3a-4.3b describe the size of swine farms, years of swine
experience, and frequency of selected management practices on
farms which participated in the NSS. The general state of
farrowing swine productivity and health of the study farms is
presented in Table 4.3c.

Spearman correlations among the independent and dependent
variables are presented in Tables 4.4a-4.4f. There are
statistically significant correlations (P50.05) between
nitrate and several other independent variables (ammonia,
nitrite, the total number of swine on the farm and vaccination
against atrophic rhinitis and pseudorabies) . However, the
largest correlation between these independent variables is
-.251, providing little evidence that multicollinearity among
the independent variables would be a problem. Numerous
independent variables were correlated (P5.20) with the
dependent variables. Nitrate and independent variables
correlated with the dependent variables were included in
stratified analysis and multiple logistic regression.

Univariate logistic regression did not disclose any additional

119
independent variables to include in stratified analysis and
multiple logistic regression.

A comparison of swine farms stratified by drinking-water
nitrate levels (45 ppm) revealed no significant.differences in
the distribution of other independent variables except for
ammonia and vaccination of the sow against parvovirus or
leptospirosis (Tables 4.5a-4.5b). There are no significant
differences in sow health or productivity between farms with
levels of nitrate 245 ppm and those <45 ppm (Table 4.5c).

With stratified analysis, the unadjusted odds ratios for
the various measures of sow health and productivity on farms
exposed.to nitrate 245 ppm, and nitrate 2100 ppm.are displayed
in Table 4.6. Neither concentration of nitrate demonstrates
any significant association with sow health or productivity.

The results of stratified analyses for the associations
between nitrate 245 ppm.and the various measures of sow'health
and productivity while controlling (adjusting) for other
factors are contained in Tables 4.7a-4.7k. Mantel-Haenszel
estimators provided adjusted summary odds ratio estimators
which did not significantly differ from the unadjusted odds
ratios, thereby providing no evidence of confounding. The
Breslow-Day statistic tests the null hypothesis that there is
no difference in odds ratios between any of the strata. Lack
of significance of the Breslow-Day tests implies uniformity of
the stratum-specific odds ratios providing evidence that

effect modification is not occurring (Breslow and Day, 1980).

120

Multiple linear regression models did not reveal any
association between nitrate contained in drinking water and
sow productivity as assessed by the farm average litter size
(Table 4.8a), or percentage of the litter stillborn (Table
4.8b) . This study could have detected if nitrate in the
drinking water explained 3% of the variation in either farm
average litter size or percent stillborn, with a power of .90
at. a = .05. Multiple logistic regression revealed. no
association between nitrate and the risk of a farm
experiencing an above median percentage of the litter born
mummified (Table 4.8c). Similarly, no effect was seen on the
health of sows as measured by the risk of a farm experiencing
above median farrowing swine morbidity and mortality due to
farrowing problems (Tables 4.8d-4.8e), reproductive problems
other than farrowing problems (Tables 4.8f-4.8g), other known
health problems (Tables 4.8h-4.8i) or unknown health.problems
(Tables 4.8j-4.8k).

The statistical power of this study was determined by
utilizing the tables published by Cohen (1988) for linear
regression and those by Hsieh (1989) for logistic regression.
In regards to logistic regression, with the sample size of
this study (571), the smallest detectable difference (odds
ratio) for several levels of power can be found in Table 4.9.
The level of statistical power varies with the probability of
the event occurring; With a sample size of 571, an odds ratio

as low as 1.3 for illness due to farrowing problems could have

121
been detected (if one existed) at a=0.05 with a statistical
power of 80% (Table 4.9). Similarly, statistical power was
sufficient to have detected an odds ratio (at a=0.05, B=.20)
as low as 1.4 for illness due to reproductive, known, or
unknown problems; 1.5 for death due to farrowing or known
problems; 1.8 due to unknown problems; and 2.5 for death due

to reproductive problems.

DISCUSSION

The NSS was a large swine study designed to reflect the
swine population.and swine farms of the U.S. IHowever, not all
swine farms initially selected for the study, chose to
participate. A comparison of respondents versus
nonrespondents (based on data previously reported to NASS
regarding’ herd. size, litter' size, and litter' mortality)
revealed no significant differences in regards to litter size
or mortality. However, both total herd and farrowing herd
sizes were larger with NSS participants compared to
nonparticipants. Therefore, the herd sizes reported with the
NSS are larger than those which would have been reported, had
all selected farms chosen to participate.

Investigators have reported dystocia affecting 2.9% (103
farrowings on 5 farms) (Randall, 1972a), 0.25% (772
farrowings) (Jones, 1966), and 1.54%-2.50% (70 farms) (Lingaas

and Ronnigen, 1991) of farrowing sows. The overall farm

122

average percentage of sows ill due to farrowing problems in
this investigation is 2.1%. Because this category of illness
includes dystocia plus other farrowing-related problems (i.e.
prolapsed uterus), this figure is not immediately comparable
with the previously mentioned investigations. However, it is
thought that dystocia is by far the largest portion of
farrowing related problems.

Previous studies have reported 1.8% (24/125) (Randall,
1972b) and 1.6% (228/14,390) (Billie et al., 1974) of all pigs
born were mummified; 5% (67/125) (Randall, 1972b) and 4.3%
(622/14,390) (Billie et al., 1974) stillborn. Another study
reported that a total of 7.6% (4,366/57,195) of the pigs born
were stillborn or mummified (Partlow et al., 1993). These
findings are similar to the average farm rates of 1.4%
mummified and 6.9% stillborn' observed in this study.
Comparing my study with other investigations is somewhat
tenuous in that there are several differences between the
studies. The previous studies were conducted in England,
Canada, Norway, and Denmark.while this investigation was done
in the U.S. This study is much larger than the other studies
as it involves 27,207 farrowings on 571 swine farms. Also,
unlike the previously mentioned studies which were performed
on an individual animal basis, this study utilized the farmlas
the unit of analysis.

The presence of water samples with no nitrate detected is

an impediment to statistical analysis. The occurrence of

123

samples with values below detection limits is common in
environmental water sampling and can be handled in a variety
of ways (Newman et al., 1989; Hurd, 1993). In this study,
values below the detection level were recorded as 0. This
method will, to some extent, bias the estimate of the mean
downward while increasing the estimate of the standard
deviation.

Nitrate levels ‘measured. in ‘this study exceeded. the
E.P.A.’s maximum concentration of 45 ppm for human drinking
water in 12.1% (69/571) of the wells sampled. This figure is
higher than estimates of 6.4% by the U.S. Geological Survey or
2.4% by the E.P.A. (U.S.D.A., 1991). However, the level of
12.1% found in this study compares favorably with the 10%
reported by the Monsanto Agricultural Products Company (USDA,
1991). The differences in estimates of nitrate contamination
of wells is probably related to the different criteria for
well selection. The U.S. Geological Survey and the E.P.A.
sampled household wells, whereas the NSS and the Monsanto
study sampled wells on farms.

In order to assume that no difference exists when there
is insufficient evidence to reject the null hypothesis, the
statistical power (power = 1-B where B is the type II error)
of a study must be sufficiently large. Statistical power is
the probability of detecting a difference if, in fact, one
exists. Without sufficient power a study may not detect any

effect from an exposure, although in reality the factor does

124
affect the outcome of interest. Adjustment for covariates in
logistic regression either has no effect or results in a loss
of precision (Robinson and Jewell, 1991). To maintain the
same level of precision, the sample size may need to be
increased. The amount of increase in sample size is directly
related to the amount of correlation between the covariate of
interest (nitrate) and the remaining covariates. To calculate
the increase in sample size needed, the following formula may

be used (Hsieh, 1989):

n, = no / (1"P2)

n0 = number of samples needed when only the covariate of
interest is included in the model,

n, = number of samples needed after adjustment for other
covariates and

p = multiple correlation coefficient relating the specific
covariate of interest to the remaining covariates.

From the formula, it can be seen that the greater the
correlations among covariates, the larger the sample size must
be to achieve the same level of power to detect an identical
amount of difference (difference in this study refers to the
odds ratio). Because the amount of correlation between,
covariates in this study is small, p2 is also small.
Consequently, no modification of these tables was deemed

necessary to adjust for covariates.

125

Had.any of the.covariates been.effect modifiers, the odds
ratios would.not have remained constant across the strata" No
evidence of confounding was seen as the odds ratios did not
appreciably change with adjustment for other covariates.

When included in multivariate analysis, nitrate was not
associated with farrowing sow health or productivity.
Controlling for potentially confounding and/or effect
modifying factors did not change the results. ‘The power level
achieved. with this study' was sufficiently' high to 'have
detected even a moderate level of effect from nitrate, if such
an effect existed.

The lack of any detectable effect from nitrate in.a study
this powerful, indicates that the concentration of nitrate
seen in well-water during the NSS does not affect the health
or productivity of farrowing swine as measured by the criteria
utilized in this investigation. These findings agree with
studies that found no effect from nitrate on sow productivity
(Seerley et al., 1965; Tollett et al., 1960). A previous
study linking nitrate to decreased productivity (Garner et
al., 1958) was conducted with a small number of animals, so
those results may be unreliable.

Because of the paucity of information on the effects of
low levels of nitrate on swine health and productivity,
collaborative studies would serve to solidify these
conclusions. In addition, this study only examined the

effects of nitrate contained in drinking water on farrowing

126
sow health. Further research is needed to determine the

effects of nitrate on swine during other stages of production.

127

 

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128

 

 

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129

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130

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131

Table 4.3b: Prevalence of selected practices on
swine farms: National Swine Survey, 1989 - 1991.

 

 

Variable %
Vaccinate for:
Actinobacillus 9.5
Atrophic Rhinitis 45.2
Parvovirus 69.5
Leptospirosis 77.8
Pseudorabies 22.2
E. coli 52.9
Rota virus 18.9
Clostridium 26.3
Erysipelas 68.7
Transmissible
gastroenteritis 29.9
Other vaccines 18.7
Type Farrowing
(all-in all-out) 55.5
(continuous) 44.5
Use of Antibiotics
in the Feed 41.2

Use of Anthelmintics

86.2

 

132

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Asoo.v

coon.v

AmHo.C

 

Ammo.c Anoo.v Asnm.c ooHuHees:
Hoo.u emo. moo. ooo.u moo.n oeo. A o>¢
xooo.c Ammo.v .oom.v .emn.. Home.c AmHo.c ouonHHHum
soo.u ooo.- seo.u Heo.u Hno. oHo.u A o>¢
Ammm.c Amms.v .moo.. AHHo.v .uoo.. .ooo.. eNHn
smo. mHo. mso.u Hmo.u mHH. moo. nouuHH o><
Homo.c xeoo.v Ammo.c .nuo.. Ammo.c noon.c
noo.u oHo. oHo. Hoo.u sHo. eno.n ououqu
u oHnHuoc AHonuoe > neouo sesame ounce condo

 

.HeoHteooH .he>usm enHtm HeeOHuez «hquHuosooum see no
seasoned one eeHceHue> useoseoevaH neesuec nQOHueHeuuoo senueeom «oe.v eHcea

136

.~.owm um uCMOHMHcon euo oecHoc .esHo> m u memecuceuom cH muecesc e .ovm u z A

 

 

Amos.c x~o~.c .ono.. .oHo.. .ooo.. .oHo.. Ammo.c Asso.c omsoo
«Ho.u moo. Hoo.u ooH. oHH. ooH. omo. ooo.u .sxcouunom A
.voH.. Amoe.v Anom.e .Hoo.. .uoH.. xemA.C Asoo.v .ooo.. omsoo noose
ooo.n omo.u oeo.u oeH. moo. mmo.u omo.u ~so.u noououuuoz A
.oHH.. .soH.. “moo.c coon.c Asso.c .vnH.. xeo~.c meo.c .nonm .ouoom
moo. ooo.u ooo.u ono. sHo.u moo. oeo.u «No.1 nocuouunos A
xoo~.c .ooo.. .AoH.. .Hoo.. .HoH.. .nHo.. «oso.v Ao-.v ocHsonuom
soo.u moo. moo. eoH. moo. ooH. emo.u ooo.u tuna: A
Ao-.c .oHo.. .voo.. .Hoo.. Ions.v .Hse.c stm.V «noo.v noosHHH
Hoo.u HHH. mso.n omH. eHo. ono. ~oo.n moo. ceases: A
xo~5.c coco.c xoeo.v .voo.. .ooo.. Amss.c .onH.c .«no.. mmooHHH 830:2
oHo.u omo. oHo.n omH. oHH. «Ho. moo.u ooo.u nosuo A
.ooH.. Homo.c xHos.C xoo~.C .ouH.. ons.c xHoo.c Ammo.e none .ouoom
ooo.u Hoo. nHo.u eeo. eoo. oHo. mmo.: mmo.n nonuouHHH A
.Hso.. .uoo.. moao.c .uoo.. .oHH.. coho.c xoo.Hc Anoo.c ooHsouuoo
oso.u moo. ooo.u omH. moo. poo. oo.ou Hoo.u nHHH A
Homenha +uemo>¢ sienna athua euHuqu eHcoa34 asHuem eueuqu

 

.HeeHIeoaH .heeusm ecHsm HecoHuez
son no meanneel one neHceHue> uaeonemeonH seesuec enouueHeuuoo seaweemm

«flquOfl
«vvoe CHA‘B

137

.~.owm um ucooHuHcmHm euo oHoc .esHo> m n memecuceuoc cH oneness e

 

 

.Hno.. .AAH.. Ammo.c .uoH.. mes.c .ooH.. Amoo.c omsoo
ooo. moo. omo. mmo. HHo.n omo. ooo.: csocxoouunoz A
Amms.v .eoH.. .«oo.. .oem.c Amse.v Asmo.v Ammn.v omsoo ozone
mHo. mmo. nso.u ooo.- ooo.: ooo.: meo.n nocuonunoz A
.AAH.. xos~.V loom.c Amee.v xeno.c Amme.v Amos.c .nonm .ouoem
moo. ooo. ooo. moo.n ooo.: moo.u oHo.u nocuouuuoz A
on~.C .Hno.. Ao-.C noes.v coho.v xoom.c Asmm.v osHsounom
mmo. ooo. Hmo. «Ho. soo.u ono. moo. uuuom A
Amsm.. .ooo.. .Aoo.. xemm.v xeos.c meA.C x~o~.v mmooHHH
moo. mHH. Hso. ooo.- «Ho.u Amo. ooo.: escorts A
.ooH.. .omH.. Ammo.c .meo.v “ooo.c sto.c AnHm.V noosHHH noose
mmo. ooo. mno.n moo. o~o.u soo. Aeo.n noouo A
comm.c Amoe.v ano.v meo.c xeo~.c cosm.v Anom.c .nouo .ouoom
omo.u moo. ooo.: Hmo. ooo.: ooo.- «no.1 nocuouHHH A
xom~.C .Amo.. .Amo.. Amo~.v Hemo.v .ooo.. Amoo.c osHsouuom
seo. Hoo. moo. poo. ooo.: sso. moo. uHHH A
oHanHnm mHamum osHuos uoHo > anon HHoo.m mos

 

.HooHuoooH .»e>uso eano HanoHuez
zoo no neuuueel one eeHceHHe> udecdeneocH deesuen unoHueHeuuoo deaueenm

«fluddcfl
«cv.¢ OHAUB

138

.N.owm um ucnoHuHcmHm euo cecHoc .esHo> m u memecuceuem CH oneness « .mmo u z A

 

 

.oom.v Ammo.v .mHo.v .Hoo.. .Hmo.. .Hmo.. moose
mmo. moo. omo. HoH. ooo. moo. csosxconuno: A
comm.c AomA.C sto.C xoom.v Homo.. moms.v omsoo ososm
omo.: omo.: omo.: moo. omo. mHo. mocuouuuoz A
.nmm.c comm.v xsem.c Anem.v Ammo.v Ammo.c .nomm .ouoom
omo. soo.a mmo.: ooo.: omo. ooo.: mosuouuuoz A
.oHH.. Amos.c xeeo.c .omo.. .mHo.. AHHm.c ooHsonmom
soo. «Ho. mHo. Hoo. moH. mmo. tumor A
.omm.v .ooH.. Ammo.v .omo.. .mHH.c comm.v omeoHHH
Heo.n Amo.a omo.: moo. ooo. omo. ssocxoo A
onm.c Aoom.v mem.C Aooo.c .moo.. AHmo.c mmosHHH ozosx
mmo.- soo.u mHo.u mmo. oso. mmo. monoo A
homo.c Aesm.v Amnm.c Amom.c coho.v Ammo.c .nouo .ouoom
moo. omo. ooo.: ooo.: ooo. moo. mosuoaHHH A
Aooo.c Homo.c Amoo.c Home.c .moH.. .an.. osHsoumom
Hoo. oHo.n AHo. HHo. ooo. Hoo. uHHH A
m oHnHAsm AHonAam > nonoo oosonm cocoa o>uam

 

.Hoouuooou .mopuso eanm HesoHuez
no nonuneea use neHneHHe> unocceme0dH deetuec ncOHueHeHuoe ceaueeem

«cuHeec ton
«uv.v OHACB

139

“nanny no u 2 H “manna one u z A “umeu eon xceu

.GOHHHHS nee euume u see

coxooHHz ecu you esHo> m u coxooHHz

 

 

 

 

Hoo. o.Huo H.o m.o o.Huo o H.o caddy sanom
Hoo. o.Hno o H.o o.mHuo o m.o Anode oHcoes<
mom. ouo o o o.mHuo o H.o Anode euHmqu
n A.ooouo.me m.os e.om m.eouo o m.o Anode euouqu
ooo. o.suH s.m Am.m HHuH o.m Am.m suHuom 30m o>¢
ems. onHuH om mm omHuo om mm msHeuom muse»
Hmm. omm.euHm moo ems mms.esHum m.moo omm.H esHsm Houoe
QONOOHfiB annex deuce: nee: oucem neHoex coo: eHneHue>
naueu so euouqu Sen va onus“ «on eueuqu See mvv

 

.HooHuoooH .meeusm eanm HesOHuez
He>eH ecu ou msHouoooe veHuHueuuo manna eann no neHunHHeueeueco oeuoeHem «em.v eHaea

«House udHccHHo ecu sH eueuqu no

.mfiuou me n 2 H “manna mom n z u Humeu aan xcou soxooHHz ecu How esHo> m n coxouHHs

 

140

 

new. m.mm m.om OwucHEHecucd HO emD
one. o.me o.He deem ecu cH
moHuoHcHucc no em:
eon. m.m¢ w.m¢ AnsoscHucoov
eon. >.om m.om AnsonHHm :HuHHoc
msHsouuom eeee
bmH. o.MH m.mH mecH00e> Hecuo
mum. m.om H.m~ mHuHueuceouunmm
eHchmHEmcoua
on. m.oo m.om moHeeHmaum
mam. m.m~ h.mm EsHoHuumoHO
bmH. m.om H.wH msuH> ouom
omm. m.om ¢.~m HHoo .m
mHm. >.Hm n.~m meHcouocsemm
vuo. ¢.wm m.oh eHnouHemoueeH
Hmo. h.mb H.me usuH>o>uem
mom. o.mo o.mv mHuHchm oHceouuc
an. m.¢ N.0H msHHHochCHuOC
"you eumcHuoo>
couooHHB A A eHceHue>

 

 

Heueuqu See va +eueuqu See mev

 

.HmnH I eoeH shebuum ecHtm HecOHuez «Mount uchcHuo ecu cH eueHuHc Ho He>eH ecu ou
ucHoHoooe oeHuHueuun naueu ecth co ueOHuosHm oeueeHee no eeceHebeum Acm.v eHcea

141

ueeu Bum xcou coxooHHs

ecu you esHm> m u coxouHHz

 

emseo c3occcat

 

moo. muo o H.o o.ouo o H.o suHHmuuoz A
OmHHflU §OCM

mos. oHuo o m.o m.ono o m.o necuousuHHouuoz A
.oumem uecuo:

omo. o.muo o H.o A.Ano o H.o suHHounoz A
mcHzouunmt

omo. omuo o o.o H.Ano o m.o suHHouuoz A
mmecHHH

ooo. mmuo o H.H m.oono o o.o ceases: A
mmecHHH

emm. oHno o o.o mmuo o m.o cease necuo A
.oumem uecuo:

mom. s.>no o o.o o.nono o m.H mmocHHH A
mcHsouunml

oom. o.omuo o o.H ooHuo o m.m noesHHH A
ceHuHeesz

ooo. m.oono o.o m.m m.oHuo o.o m.H ooouceonom
:nonHHHum

emm. m.mmuo o.o s.o omuo o.o o.o eomuceomom
emHm

omm. mHue.m o.oH o.oH H.oHum m.oH m.oH neuuHH eonuo><

cOMOOHHB emcem ceHoea ceHcec nee: eHceHue>

 

 

 

.HooHuoooH .mopusu esHso HesoHuez
ucHouoooe oeHuHueuun eaueu ecHse co cuHeec one huueHuosuoue son uo een—Anus: ”on; eHcea

«House ochcHuo ecu cH eueHuHc no HebeH ecu ou

142

.HHeO ouem ou esc eueHsoHoO ou eHcmcs A .Ho>ueucH eosecwucoo Amm u .H.O

 

 

no.mthv. no.~ mm.mlom. eo.H huHHeuuoz emseo c3ochD AOA

mm.oumm. om. Hm.muem. mm. muHHounoz ozone nocuo AoA
, AAAHoumo:

n A oe.oumm. Ae.H o>Huosoouoom nonuo AoA

I A m>.HIhm. mo. wuHHouuoz mcHsouuom AOA

H¢.ntm¢. mN.H mw.HIhe. mm. mmecHHH :3OCKCD AOA

no.NI~H. me. o¢.HImN. mm. mmecHHH c3o:& uecuo AOA
mmecHHH

NN.NImH. we. me.mlwm. m~.H e>Huoocoumem uecuo ADA

HH.HueH. on. om.HuHe. HA. mmosHHH osHsouuoo AoA
AcoHoea e>ocev

o>.HImm. mo. mo.Htmm. mm. ceHuHeas: A
AceHcea e>ocov

o~.Htem. mm. mm.Htw¢. ow. :uocHHHum A

AanceE soHecV
we.mtmo. mm.H me.HI~m. om. emHm ueuuHH emoue><
.H.o OHuem mono .H.O OHuem mono eHceHHe>
Sam ooHA eueuqu emu nvA eueuqu

 

.HooHuoomH .mepuso cause
Hecouuez «cuHeec one huH>Huoscouo son ocHsouueu so See ooHM eueuuHc ace See m.m
eueuuHc .eueuuHc no nHe>eH eHceuoeueo no ethHeae ueHuHueuun oeunsnoecb no; eHcea

143

.AoeuecHooe> .oeuecHooe> uocv

me ecoHuecHooe> HHe “AesoscHucoo .usouHHe sHtHHev mcHsouueu no eezu “Amm .mv ou mm .mv
0» mm .mvc AuHmoo moouo>o axoooHA .ooo.Hw ou oomA .oomw ou ooA.mowc ocHsm Houou onA.oc
.oHueH moco ensue e .oHueu mono hueeesanfiu

eHcoSEe me oemHuomeueo eue meHceHue> ece

 

 

mod. os.H moo. Am. mo. u n on. oo.H > umcuo
Hon. Hm. AHo. om. mo. u u mo.H om. oHommm
mms. mH. Amm. oo. mo. u .. me. mo. .38
mom. mo. son. mo. no. u I mm. mm. ouseq
Rem. oH.H Hmo. oH. oo. n u oo.H mm. o>uom
mom. on. mom. mo. oo. 1 n He. mm. someone
ooo. mH. mom. mm. on. Am. 2. mm. on. Home:
moo. om. moo. mo. 3. mm. on mo. 2.. 8.639
omm. oH.H ooo. oH. Hm. a u mo.m mo. oHcossm

mmm. mm. «om. a u t a ocoz
m x m Ax do :8 .mo Nso .mo nochHue>
gamer.” HennseectHeusec Houucoo

 

eueuqu eeetuec sOHueHooeee ecu Hon eenMHese eeHuHueuue eeuesnee no hue—ease

.HoomuoooH .me>usm ooHso HeooHuez

AenHm ueuuHc eoeuebc use aan no»
men . v edcea

144

.AoeuecHooe> .oeuecHooe> uocv me mCOHuecHOOe>
HHe 2mm .mv ou AM .AV ou m.m .mvv auHuee emoue>e ZmX .m.w ou oA .3 .3353 “SA
.3 .EsHuec we cemHuooeueo eue meHceHue> ecu. .oHueu ecco ensue A. .OHueu moco whefiasmndo

 

 

0mm. mm.H mom. 2.. ow. I I an mm. mOHO
mum. moH NHe. he. Hm. I I no.4” m.m. HHOO .m
hmH. mmJo mom. 2.. om. mH. mm.H H5. H0. Hemm><
emu. No. men. Hm. on. I mm. mm. mm. gwuem
mHm. No. wmm. mm. on. I I mm. mm. .Eswuem
man. no. «cm. I I I I ecoz

m x m Ax .mo Jo 0so «no .mo neHceHug
a HeunceemIHeuce: Houusoo

 

.HeAHIooeH .he>usm esHbm HesoHuez AncuuHcHHHum no emeuseouem ecu use aan mom
eueuqu seetuec coHueHooeee ecu How eeehHese eeHuHueuue eeunsnee no hues-5m «cs; eHcee

145

.AceuecHooe>

.ceuecHooe> uosv me coHuecHooe> «Am.0Hw .m.OHAV me mHm uHH o>e «AnsoscHucou .usOIHHe sH
IHHev msHsouueu mo eewu “AmN .mv ou mm .mv ou NM .mvv huHuee emeue>e «AOOOHA .ooo.Hw ou
oo? .83 cu ooA .oowc ooHsm H33 2m.m .m.v on on .8 .5380. ZoA .8 .8933 23.3
.OHueu moco ecsuo A .oHueu mono huegsmudo

eHcane me cemHuomeuee eue meHceHue> eca

 

 

new. no. mom. Ho. mm. I I em. no.H mfluHCHcm
«mm. on. 5mm. No. cm. I I OH.H mm. mam u..H_H 9,4
35. OH. omo. Ho. mm. I I Hm. moH Mohegan.
mom. mn.~ Nmm. Ho. Ho.H oo.m mw. om. mm. Hemmerfl
5mm. mm. mom. Ho. no...“ wH.H mm. mm. oo.N Esq—Hum.
ow». mm. mom. No. no. I nH .H Hm. No2" seafluem
Sb. no. mNm. Ho. mm. I I em. No.H gwhcm
Rm. on. new. no. em. I I no. Ho.H euncog

mmo. Ho. «mm. I I I I ecoz
m x m Ax .mo .mo 9.8 Nso :8 «39.3..»
a HemnceecIHeucec Houucoo

 

.HomHImooH .mo>uso eano HeooHuez
eueuqu ceetuec cOHueHoonee ecu no... eeehHece eeHuHueHue eeunsfiee no hue-ism

«eeHaasc no eueuceouem ecu use Ian me»

«Oh . v CHARM.

146

.AceuecHooe> .ceuecHooe> uocv we ecowuosHOOe>

HHe “AmsoscHucoe .usOIHHe cHIHHeV oCH3onnen no emhu “Amm .mv ou mm .mvv auHuem eoeue>e
NAOQOHA .ooo.Hw ou oomA .oomwv ecHsn Heuou “AOA .ov euHuuchme cemHnomeueo.ene meHceHne>

 

 

ecB .HHeo onem ou esc ceueHsoHeo uoc w .oHuen mono eosno A .oHueu mono mueeesmuJfio
8H. oo.m soH. Ho.H so. I mm. oo.m oudeq
moo. on. H3. :4 mo. I Ho. ooH 25nd
AR. mo. mom. moé mo. I ms. om. dnmsnm
o8. no. mom. ooH on. I Ho. ms. noose:
SA. 3. on. mm.H os. I oo. oo. HHoo .m
mm». AH. osm. om.H mm. I mo. om. someone
Bo. oo.H mom. oH.H ms. oo. oo. om.H Homo:
mum. mH.H Hon. oo. 2.. ms. mo. om.H 5639
o mum. AH.H me. I o A». ouHuqu

mom. mm.H AHA. I I I esoz

m x m «on do do Nso .mo noHceH ue>
Nan—IUHMOHM HOHHHOO

 

 

.HeaHIeomH .hebusm ecHtm HecoHuez
eueuqu seesuec eonueHoonee ecu non nenhHece eeHnHueuue eeuesnee no fine—ease

AnaeHcoum mcHeouHeh ou eso onecHHH use See mom

«Us. I v Canada.

147

enuan muee» «Amm
Heuou

one

«non

.mv ou mm
.ov ouuuuuc Aon
.HHee ouem ou esn neueHseHee uoc @

.mvv >uHuem eeoue>e NAOOOHA
.ov Esnuec “AOA.ov MHGOSEM we nemHuooeuee eue neHceHue>
. oHueu nnno >ueeesmudo

.ooo.Hw ou oomA

.oHueu nnno ensue A

.Aeea .ocv we neen
cH neHuoHcHuce “Aneuesueee> .neuecHeee> uocv we ecoHuecHeee> HHe “AOHN .OHVV osueuen

.oonwv enusm

 

 

one. mH. mmo. oo. oo. I oo. om. .m oHnHuo<
moo. oo.m omo. mo. no. I om. HH.o oudoc
mso. Hm. omo. no. mo. I on. so. cosmos
moo. S. mom. 2.. oo. I Ho. mm. oumuum
sou. oH.m ooo. om. oo. I mm.H mm. canons
Boo. om. mmo. oo. HA. om. mo. oH.H unoo><
Rom. oo.m Hom. mm. A». Am. Hm.H mo.m csmHue
o omo. om. HA. I A He. ouuuuuz

mHH. oo.m Hoo. mm. 2.. I om. om.H asuuom
ems. mo. mmo. mm. on. I oo. ms. succeee
ooo. om. Aoo. I I I ocoz

u «x nmo 8no .mo uoHceHue>

NUDIBOH MGM” HOHHQOO

 

 

.HaeHImeoH .hebusm ecHem HecoHuez
eueuqu neesuec coHueHeoene ecu uon eeehHese neunHueuue neuesnne no hues—use

AnaeHcoum ucnsouueu ou eso auHHeuuoc use see mom

«eh.v CHAIR

148

.Amsossuucoe
.usoIHHe sHIHHeV ocuaouuen no egu “3A .3 euHuuHc we nemHuomeuee eue meHceHue>
ecu. .HHee ouem ou esn neueHseHee uo: @ .oHueu mnno ensue A .oHueu mnno xueeesmuio

 

 

Hum. mo. moo. oo. HA.H Ho.H sm.H unmodse
o mom. ms. mm.H o mm.H euuuuuz

omo. Ho. Aom.H I I ocoz

m Ax "no .mo ueHceHue>
flGl’OH MOHQ HONHQOU

 

 

.HomHIoomH .uopuso
ecnsm Hedonuez .mcHsouueu secs uecuo eleHcoum ebnuesnoumem ou esn neecHHH nee lee no».

eueuqu ceeauec couueHeoeee ecu uon eeehHece neHnHueuue neuesnne no hues—esn «no; 2239

149

.Aneuecueee> .neuecHeee> uocv we coHuecHeee> «AusoscHucoe .usOIHHe sHIHHev
oeusouuen no eexu :mN .mvv huHuee eoeue>e zodov once—Sue we nemuuomeuee eue meHceHue>
ecu. .HHee ouem ou esn neueHseHee uoc e .oHueu nnno ensue A .oHueu mnno .muefiasmudo

 

 

mum. ms. moo. mm. om.H oA.H oo. mHuHsHsm
moo. mo. ooo. AH. oo.H mo. os.m someone
pom. mm.H mso. AH. om.H oo.o oo. unoo><
mmm. mo.H Hoo. om. HA.H oo.m oo. mucossm
mmo. om. Aso.H I I osoz

o x o Ax .ao Nso :8 333.23
eOIboHneum HennceecIHeucez Houucoe

 

.HooHIoooH .ue>uso
ecnrm HecOHuez «ocutouueu cecn. uecuo nSeHcoum e>Huesnoumem ou esn huHHeuuoc one fine no»

eueuunz ceetuec coHueHeoeee ecu uon nenhHece neHnHueuue neuesnne no hue—ease «on; eHcen.

150

ecB .HHee ouem ou esn neueHseHee uoc @

..AneuecHeee> .neuecueee> uocv me ecoHuecHeee> HHe “AOOOHA .ooo.Hw ou
oomA .oomwv ecusm Heuou NAOA .ov euHuuHc NAOA .ov esHuec me nemHuooeuee eue meHceHue>

.oHueu mnno ensue A

. oHueu mnno huessssmuio

 

 

 

 

mom. 2.. 23. 2.4 mm. I mm. oo.H ouooq
mmm. ooH oom. om.H no. I mm. so. mHuHoHcm
mos. mH. omm. mo.H Ho. I mo. oo. duosum
Hom. Sim oom. mo. oo. mm. mm. mo.H 5.639
o mom. oo.H mo. I o mo. euHuqu

ooo. oH. omm. AHA oo. I oo. ms. ssuuom
omm. on.H Amo. I I I osoz

m x u «x .mo Jo Nso .co noHceHue>

IAIIBIBHmoBHI. Houunoo
.HooHIoooH

.hebusm ecntm Heconuez

uneasee crock uecuo ou esn neecHHH use See mom eueuunz ceeeuec

cOHueHeonee ecu uon .neuesnnec eenhHece neHnHueuue nounsnne no hueflasm Ach.v eHcea

151

.Aneuecueee> .neuecHeee> uocv no mcouuecneee> HHe “Amsoscuucoe .usOIHHe

oHIHHS oousouumn no e83 23A .oHvC oouauou mouse muse» 1887 .8on co oo.? .8me
esH3m Heuou “AOA .ov euHuuHc “AOA.ov eHcere we nemHuomeuee eue neHceHue> ecB

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152

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153

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154

Table 4.8a: Results of a multiple linear regression model for
the effect of Nitrate on the farm average litter size:
National Swine Survey, 1989-1991. -

 

 

Std. T for H0

variable Coef. Error Parameter=0 P
Intercept 10.337 .272 38.018 <.001
Nitrate 0.086 .051 1.698 .090
Ammonia -0.056 .056 -0.985 .325
TtlSwn 0.000001 .000008 0.125 .901
Angar 0.086 .051 1.698 .964
TypeFar -O.226 .126 -1.789 .955
Vaccination:

Parvo 0.042 .192 0.221 .826

Lepto 0.060 .227 0.264 .792

Clos 0.224 .151 1.487 .138

Erysip 0.054 .170 0.315 .753

Other V -0.477 .165 -2.893 .004

 

Model F-value = 1.758,

P-value

= .066, adjusted R?

155

Table 4.8b: Results of a multiple linear regression model for
the effect of Nitrate on the farm average percentage
stillborn: National Swine Survey, 1989-1991.

 

 

Std. T for Ho

Variable Coeff. Error Parameter=o P
Intercept 6.132 .530 11.564 <.0001
Nitrate -0.003 .005 -0.747 .456
Barium 1.896 .938 2.022 .044
Angar 0.304 .146 2.080 .038
Vaccination:

E. coli -0.698 .418 -1.672 .095

Clos 0.182 .471 0.387 .699

 

Model F-value = 2.269, P-value = .046, adjusted R?== .012.

156

Table 4.8c: Results of a multiple logistic regression model
for the effect of Nitrate on the farm average percentage born
mummified: National Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.00
Ammonia .96 .90, 1.13
Barium 1.44 .59, 3.56
TtlSwn 1.16 1.02, 1.31
Angar 1.00 1.00, 1.00
Avg. Litter Size 1.19 1.03, 1.38
TypeFar 1.22 .86, 1.74
Vaccination for:

Rhinitis 1.29 .91, 1.82

 

CI = Confidence Interval

157

Table 4.8d: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sows ill
due to farrowing problems: National Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.00
Nitrite 1.23 .86, 1.74
TtlSwn 1.00 1.00, 1.00
Angar 1.09 .94, 1.27
TypeFar 1.19 .81, 1.74
Vaccination for:
E. coli 1.27 .84, 1.93
Hemoph 1.40 .77, 2.57
Erysip 1.30 .77, 2.19
Parvo .88 .50, 1.56
Lepto 1.11 .56, 2.23

 

CI = Confidence Interval

158

Table 4.8e: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sow
mortality due to farrowing problems: National Swine Survey,
1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate .99 .98, 1.01
Ammonia 1.09 .89, 1.32
Barium .21 .03, 1.80
Nitrite 1.17 .92, 1.49
TtlSwn 1.00 1.00, 1.00
Angar 1.18 .95, 1.47
YrsSwn 1.01 1.00, 1.02
Vaccination for:

Erysip 1.34 .58, 3.02

Lepto 1.40 .74, 2.65

Pseudo 1.85 .62, 5.52
Antibio F 1.40 .78, 2.53

 

CI = Confidence Interval

159

Table 4.8f: Results of a multiple logistic regression model
for the effect of Nitrate on the farm.percentage of sows with
reproductive illness other than farrowing problems: National
Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI

Nitrate 1.00 1.00, 1.01
Nitrite 1.31 .95, 1.80
TypeFar 1.37 .87, 2.16

 

CI = Confidence Interval

160

Table 4.8g: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sow
mortality due to reproductive illness other than farrowing
problems: National Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .98, 1.01
Ammonia 1.08 .76, 1.52
Angar .66 .36, 1.21
TypeFar .37 .10, 1.30
Vaccination for:

Rhinitis 2.36 .67, 8.31

 

CI = Confidence Interval

161

Table 4.8h: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sows ill
‘with other known problems: National Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.00
Barium .18 .03, 1.15
Nitrite 1.58 1.06, 2.35
TtlSWn 1.00 1.00, 1.00
Vaccination for:

Rhinitis 1.33 .78, 2.28

Erysip 1.62 .75, 3.49

Lepto 1.10 .56, 2.18

 

CI = Confidence Interval

162

Table 4.8i: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sow
mortality due to other known problems: National Swine Survey,
1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.01
Nitrite 1.33 .89, 2.00
TtlSwn 1.00 1.00, 1.00
YrsSwn .99 .98, 1.01
TypeFar 1.48 .77, 2.85
Vaccination for:

Hemoph .15 .02, 1.28

Erysip 2.10 .33, 6.29

Lepto 1.27 .55, 2.96

 

CI = Confidence Interval

163

Table 4.8j: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sows ill
with unknown problems: National Swine Survey, 1989-1991.

 

 

Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.01
TtlSwn 1.00 1.00, 1.00
Angar 1.10 .92, 1.32
YrsSwn .99 .98, 1.00
Vaccination for:
Hemop 1.52 .76, 3.04
Erysip .85 .40, 1.79
Lepto 1.74 .88, 3.44
Pseudo 1.67 .99, 2.81
Anthel .63 .33, 1.17

 

CI = Confidence Interval

164

Table 4.8k: Results of a multiple logistic regression model
for the effect of Nitrate on the farm percentage of sows
mortality due to unknown problems: National Swine Survey,

 

 

1989-1991.
Variable Odds Ratio 95% CI
Nitrate 1.00 .99, 1.01
Ammonia .80 .38, 1.73
Nitrite 1.75 .94, 3.28
TtlSwn 1.00 1.00, 1.00
YrsSwn .97 .95, 1.00
Vaccination for:
E. coli .94 .31, 2.87
Clos .99 .34, 2.90
Rhinitis 2.37 .82, 6.89
Parvo .92 .21, 4.01
Erysip .83 .23, 2.96
Lepto 3.36 .30, 37.83
Pseudo 3.98 1.56, 10.13

 

CI = Confidence Interval

95% 95%*

165
90%

for an overall event proportion (Prop)
Power (1-3)

with a sample size of 571.

The approximate minimum detectable odds ratio when
80%

70%

 

 

c=0.05 (one-tailed),

Table 4.9:

0555099887766655555

3222211111111111111

05099877766555544444

32211111111111111111

55987766655554444433

22111111111111111111

059876655544444333333

321111111111111111111

508765554444433333333

221111111111111111111

.01
.02
.03
.04
.05
.06
.07
.08
.09
.10
.12
.14
.16
.18
.20
.25
.30
.35
.40
.45
.50

 

* 1-B=95%, a=0.01 (one-tailed).

166
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CHAPTER 5

DISCUSSION

171

172

Chapter 3 reviews the known effects of nitrate on animal
health and productivity. Because nitrate is frequently found
in groundwater, it is important to determine what effects the
consumption of nitrate-contaminated drinking water may have.

The objectives of this study were to investigate the
effects of nitrate contained in drinking water on farrowing
swine health and productivity. Data used in this study were
taken from a much larger study entitled the "National Swine
Survey" (NSS). The NSS is a large epidemiological study,
designed to permit inferences to be made about the swine
population of the U.S. The portion of data from the NSS used
in my analyses was collected in a cross-sectional manner.

Sow health was assessed by determining the rates of
illness or death attributed to; farrowing problems,
reproductive problems other than farrowing, "other known"
problems, and unknown problems. Sow productivity was measured
by calculating the percentage of pigs born alive, born
mummified, and stillborn.

These measures of swine health and productivity, the
nitrate concentration of drinking water, and potentially
confounding and/or effect modifying variables were examined
using descriptive statistics, correlation analysis, univariate
logistic regression, stratified analysis and multiple logistic
regression.

This study has many strengths. The study population was

selected by the NASS, an agency whose mission is to select and

173

sample agricultural populations in order to make inferences
about the U.S. It is large (571 farms sampled), powerful
study (there is a high probability of detecting an association
between nitrate and sow productivity or health if one really
did exist). With the sample size of this study, there is an
80% probability of detecting an odds ratio as low as 1.3, and
a 95% probability of detecting an odds ratio of 1.5.

All questionnaires were administered by specifically
trained personnel. These interviewers had no preconceived
ideas to be proven or dispelled by this study, thereby
minimizing interviewer bias. All water samples were collected
and analyzed at one laboratory using established protocols
diminishing measurement error.

With a cross-sectional study, exposure and disease status
are evaluated at the same time. It is assumed that the
exposure to nitrate remained at a constant level and occurred
throughout the sow’s life. The levels of nitrate measured in
the drinking water are unlikely to vary much (due to the
length of time required for surface deposits of nitrate to
enter groundwater). However, nitrate is also found in many
foodstuffs, suggesting the possibility that the swine were
exposed to higher levels of nitrate than those indicated by
the water analysis. In this case, misclassification of
exposure status would have occurred. It is doubtful that the
level of nitrate exposure has been underestimated to any

substantial degree because the foodstuffs most likely to

174
contain high levels of nitrate (green leafy vegetable
material) are not generally fed to swine in the U.S.

The concentration of the drinking water provided to the
sows was measured in the farrowing unit. Misclassification of
exposure status could also have occurred if the sows were
exposed to a different level of nitrate in drinking water when
they were not in the farrowing unit or if the level of nitrate
changed during the monitoring period. To minimize this
problem, those farms which changed the water source during the
study were excluded. It is unlikely that many sows were
exposed to more than one concentration of nitrate on the farm
because most farms had only one water source. Regarding the
length of time the sows were exposed to nitrate in the
drinking water, information concerning the origin of the sows
was not available and it is possibly that at least some of the
27,207 sows may have been.purchased and brought onto the study
farm. In this case, their exposure period would be much less
than that indicated by their parity. There is no evidence to
suggest that if misclassification of exposure status occurred,
that it differed between the exposed and nonexposed sample.
Nondifferential misclassification would tend to bias the
results towards the null. However, it seem doubtful that the
results of this study were significantly affected by
misclassification.

The nitrate contained in well-water did not adversely

effect the health and productivity of farrowing swine as

175
assessed by my criteria. While the levels of nitrate seen in
well-water during the NSS did not effect the health and
productivity of farrowing swine, this does not mean that
nitrate does not have any affect on these parameters. Rather,
it may indicate that nitrate levels were not yet high enough
to cause problems. It is widely believed that nitrate levels
are rising in the U.S. In this circumstance, repeating this
study at a future time could result in different conclusions.
Also, this study examined only the effects of nitrate on
farrowing swine health. Further research is needed to
determine the effects of nitrate on swine during other stages
of production. In addition, groundwater is the primary source
of drinking water for approximately 85% of the rural
population and 50% of the entire U.S. population. With so
many humans and animals dependent on groundwater, it is
imperative to determine the factors that contribute to nitrate
contamination in order to prevent further degradation of this

resource .

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