1 TECHNICAL REPORT DATA
1 (Please read Instructions on the reverse before completing)
p.,Sort no. 2.
1 FPA_Rnn/i_Ri_n?fi
3-R6C,p(Hjf |
v TITLE AND SUBTITLE
Sewage Sludge - Viral and Pathogenic Agents in
Soil-PIant-Animal Systems
5.	report DATE
Marrh 1QR1
6.	PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
6. T. Edds and J. M. Davidson
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADORESS
Institute of Food and Agricultural Sciences
University of Florida
Gainesville, Florida 32610
10. PROGRAM ELEMENT NO.
CAYB1B
11. CONTRACT/GRANT NO.
R804570
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final - 9/76 - 12/80
14. SPONSORING AGENCY CODE
• EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A multidisciplinary study was carried out to determine the ultimate fate of various
toxic elements or pathogens associated with Florida and Chicago municipal sludges when
applied to soil-piant-water systems and to determine physiologic, pathologic, growth,
and reproductive responses of cattle, swine, and foultry fed sludges, grains, or forages
from soils pretreated with urban liquid digested sludges as well as health effects in
mice receiving liver or kidney tissues from steers and swine exposed to such feeds or
contaminants. Minimal differences occurred in growth performance or egg production in
cattle, swine, or poultry fed forage or grain from soils pretreated with a variety of
urban sewage sludges. Cattle and swine tissues, when fed to mice, resulted in
alterations of the normal mineral balance as well as reproductive performance. Tissues
from animals intended for human consumption exposed to sarcocyst contaminated sewage
sludges may serve as health hazards for animals and humans. Application of urban sewage
sludges at 19.8 t/hectare produced equivalent plant growth stimulation for corn, barley,
wheat, and sorghum as commercial fertilizers. Certain bacteria, commonly associated
with sludges, disappear in a few days after soil or plant application; however certain
viruses and parasites were shown to persist.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFI ERS/OPEN ENDED TERMS
c. COSATI Field/Group


57U
68G
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page!
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) previous edition is obsolete

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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies of
the U.S. Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
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FOREWORD
The U.S. Environmental Protection Agency was created because of increasing
public and governmental concern about the dangers of pollution to the health and
welfare to the American people. Noxious air, foul water, and spoiled land are
tragic testimony to the deterioration of our national environment. The
complexity of that environment and the interplay between its components require
a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching for
solutions. The primary mission of the Health Effects Research Laboratory in
Cincinnati (HERL) is to provide a sound health effects data base in support of
the regulatory activities of the EPA. To this end, HERL conducts a research
program to identify, characterize, and quantitate harmful effects of pollutants
that may result from exposure to chemical, physical, or biological agents found
in the environment. In addition to the valuable health information generated by
these activities, new research techniques and methods are being developed that
contribute to a better understanding of human biochemical and physiological
functions, and how these functions are altered by low-level insults.
Recycling digested municipal sludges in agricultural systems is an
attractive alternative method for their utilization if "safe" management
techniques can be devised that do not adversely affect plant productivity or
animal and human health. This study of the ultimate fate of various toxic
elements and pathogens in sludges applied to soil-plant-animal systems will aid
decision makers in selecting such techniques.
James B. Lucas
Acting Director
Health Effects Research Laboratory
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ABSTRACT
This research was initiated to determine if digested sewage sludges could
be applied to a soil-plant-animal system to improve soil fertility, increase
forage and grain production, provide animal feeds necessary for optimal animal
growth or performance without posing a hazard to plant and animal production or
human health. The studies also included the persistence and movement of
pathogens, drugs, or chemicals in soils, plant products, or animal tissues.
Beef steers were fed digested municipal sludges incorporated into feedlot
diets and feeds (corn grain, forage, sorghum silages, and bahiagrass pastures)
produced on land treated with sludge. These studies were conducted to determine
the effects of these feeding programs on animal performance, carcass quality,
and concentrations of selected toxic elements in liver, muscle, and kidney
tissues. The performance and carcass data of treated steers in all of the
studies were generally not different from the control steers.
Feeding sewage sludge on reproductive performance in female swine during
successive gestation-lactation periods was evaluated. These studies indicate
that breeding, farrowing, and rebreeding weights were reduced. Lactation and
gestation weight changes were lower and fewer pigs were farrowed in sow groups
fed 10 and 20% sewage sludge in their diets.
Duplicate experiments of 21 days duration were conducted with day-old
broiler-type chicks and laying hens to study the influence of replacing one-half
or all of the normal dietary corn complement with corn grown on soil fertilized
with municipal sludge. Corn from the sludge-amended soil did not adversely
affect final body weights or daily feed intake. Substitution of a sludge with
high metal concentrations or equivalent levels of certain hazardous metals
altered growth and laying performance.
Toxicity from feeding dried sewage sludge included in a normal swine
starter ration, may occur from a deficiency of available protein or other
essential nutrients, or from the accumulation of hazardous chemical residues.
Cadmium exposure induced microcytic and hypochromic anemia. Cadmium also
induced differences in the activity of liver serum enzymes in pigs exposed to
aflatoxin or warfarin. This is the first demonstration of the cadmium
blocking effect on the microsomal enzyme system in pigs. Of 7 pigs fed 10%
Gainesville sludge, 4 had Sarcosporidia in the myocardium, and the hearts of 2
of the 4 pigs fed 20% contained the parasite. Among cattle fed Pensacola sludge,
19 of 32 contained Sarcosporidia in the cardiac muscle, while the cardiac muscle
of 6 of 17 controls was parasitized. The presence of Sarcosporidia in hearts of
swine and cattle fed sludge may be of public health significance.
iv

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Land spreading of sewage sludge is probably the most practical means of
disposal for municipalities and cities. Uptake of certain metals by forage and
grain crops from land treated with sludge may create health risks. Pre-1978
sludge from Chicago contained large quantities of copper, zinc, lead, and
cadmium. The Pensacola sludge was high in zinc. Metal uptake of the corn plant
was directly associated with soil pH. The higher the soil pH the smaller the
quantity of metal uptake.
Sludge application at the 24 ton/ha rate compared favorably with mineral
fertilizer as a source of plant nutrients for bermudagrass.
Samples of sludge, feed, feces, and animal tissues (kidney, liver, spleen,
and blood were analyzed for pathogenic bacteria. Pathogenic bacteria were not
found to be a significant hazard.
Viruses were not detected in topsoils 8 months after spreading Pensacola
sludge. Enteroviruses represent a minimal hazard, either through translocation
through grain or forage or with regard to groundwater contamination.
Samples of sludge, soil/sludge mixture, feed, and animal tissues (kidney,
liver, fat, muscle) were analyzed for chlorinated hydrocarbon pesticide
residues and also polychlorinated biphenyls. Little, if any, pesticide
residues were present in sludges used in this research project.
This report was submitted in fulfillment of Grant No. R804570 by the
University of Florida under the sponsorship of the U.S. Environmental Pro-
tection Agency. This report covers the period September 1, 1976 through
December 31, 1980 and work was completed December 31, 1980.
v

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CONTENTS
Foreword .
Abstract .
Conclusions
Recommendations
Acknowledgement
EFFECTS OF LIQUID DIGESTED SLUDGE ON SOILS AND PLANTS .
The Nutrient Status of Six Field Crops Grown on Soils
Treated with Liquid Digested Sludge ....
The Utilization of Liquid Digested Sludge on Agricultural
Land ..*•••*•..«
The Uptake of Certain Metals by Corn Grown on Soil Treated
with Chicago or Pensacola Sewage Sludge ....
Sludge Effects on Yield and Cadmium Uptake of Coastcross 1
Bermudagrass .........
Transport of Sewage Sludge Constituents in a Sandy Soil
and Uptake of Metals by Bermudagrass ....
CATTLE FEEDING TRIALS WITH SLUDGES OR FEED GROWN ON SLUDGE
TREATED LAND 	
Dried Pensacola Liquid Digested Sludge in the Diet of
Steers
Winter Annual Pastures Fertilized with Pensacola Liquid
Digested Sludge and Grazed by Growing Beef Steers .
Dried Pensacola Liquid Digested Sludge (DPS) in the Diets
of Feedlot Steers ........
Dried Chicago Sludge and Corn from Soil Fertilized with
Liquid Pensacola Sludge in Diets of Beef Steers
Forage Sorghum Silages Grown on Soil Treated with Liquid
Digested Sludge and Fed to Beef Steers ....
Preceding page blank
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X
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1
2
5
7
8
15
20
21
27
30
38
44

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CONTENTS (Continued)
Pensacola Bahiagrass Pastures Fertilized with Pensacola
Liquid Sludge and Grazed by Beef Steers ....
CATTLE - OTHER HEALTH EFFECTS 	
The Effects of Recycled Cattle Manure on the Health and
Performance of Steers .......
SWINE FEEDING TRIALS WITH SLUDGES 	
Performance and Tissue Minerals of Swine Fed Sewage Sludge
Di ets . . . . . . . . . .
Effect of Feeding Sewage Sludge Diets on Reproduction,
Growth, and Tissue Mineral Accumulation ....
Effect of Feeding Digested Sewage Sludge on Long-term Sow
Reproduction Performance . . . . . .
SWINE - OTHER HEALTH EFFECTS 	
Feeding Trials of Dried Urban Sludge and the Equivalent
Cadmium Level in Swine .......
Toxicology of Aflatoxin B]_, Warfarin, and Cadmium in
Young Pigs .........
High Performance Liquid Chromatographic Determination of
Sulfonamides of Ionic Suppression .....
Comparison of the Pharmacokinetics of Sulfisoxazole in
Humans and Two Monogastric Species .....
Comparison of the Potential Toxicity of Bilirubin in
Humans and Two Monogastric Species after a Single
Administration of Sulfisoxazole .....
POULTRY 	
Health Effects of Sewage Sludge and Grain from Sludge
Treated Soils Versus Equivalent Metal Levels in Poultry
HEALTH EFFECTS IN MICE FED DIETS THAT CONTAINED TISSUES FROM
CATTLE OR SWINE FED SEWAGE SLUDGE RATIONS ....
MICROBIOLOGY 	
General Proceedings ........
Bacteriological Analysis of Feed, Grass, and Sludge
vii i

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CONTENTS (Continued)
FATE OF VIRUSES FOLLOWING APPLICATION OF MUNICIPAL SLUDGE
TO LAND 	
PESTICIDE RESIDUES IN FEED, SLUDGE, SOIL/SLUDGE, AND
ANIMAL TISSUES 	
PUBLICATIONS 	
ix

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CONCLUSIONS
Land spreading of urban sewage sludge is probably the most practical
means of disposal. Sewage sludge was shown equivalent or superior to
commercial fertilizers for production of certain crops under Florida con-
ditions. However, uptake of metals by forage and grain crops may create
certain risks. Metal uptake by the corn plant was directly related to the soil
pH; higher pH levels reduced their uptake. Levels present in grain were less
than in the forage. Cadmium levels in forage from soils pretreated with
certain sludges resulted in high levels in liver and kidney tissues of cattle
consuming such forage. However, performance and carcass data of treated
steers in these studies were not different from the data obtained with the
control steers. Clinical chemistry tests and pathologic lesions suggested
cumulative toxic effects including liver damage.
The 1979 steer trial, where animals grazed on forage from soils
pretreated with Pensacola sludge and spraying of the sludge on the growing
plants, resulted in presence of Sarcosporidia sp. in the cardiac and skeletal
muscles. This may be of public health significance.
Incorporation of dried sewage sludge at ten to 20 percent of swine
rations produced depressed weight gains and the 21 day weaning weights were
lower in pigs from sows consuming the sludge-containing diets. The kidney
cadmium levels of sows receiving the ten and 20 percent sludge levels were
increased significantly, i.e., four ppm for controls and 17 and 24 ppm for the
sludge rations; both lead and cadmium were increased in the liver and kidneys
of weanling pigs. Reproductive performance was more suppresssed in the second
generation sows than in the first.
Growth trials with Cobb broiler chicks compared the effects of poultry
rations with 0, three, and six percent dried Chicago sludge. Increased levels
of cadmium in the liver and kidneys occurred in those chicks receiving the
increased levels of the sludge. However, none of the production criteria,
i.e., production, daily feed intake, feed efficiency, egg weights, nor body
weights, were adversely affected in Leghorn hens receiving such modified
diets.
Having demonstrated that increased cadmium levels occurred in tissues
from cattle and swine consuming feeds from sludge amended soils, these liver
and kidney tissues were dried, ground, and incorporated into mouse diets. The
finished diets contained a 15 percent level of protein and five percent levels
of kidney and liver tissue. Metals were translocated through the cattle and
swine tissues with increased levels of cadmium, nickel, chromium, and lead in
liver and kidney tissues of mice. These increases in mice were associated
x

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with decreases in number of mice weaned in the treated versus the control
groups.
Analysis for pesticide residues in the various sludges indicated that
little, if any, chlorinated residues were present. It was concluded that
these sludges presented no hazard from the aspect of pesticide residues.
Samples of sludge, feed, feces, and animal tissues (kidney, liver,
spleen, and blood, were analyzed for pathogenic bacteria. Contamination was
a major problem, both when collecting specimens during the trials as well as
at slaughter. No enteric pathogens or Mycobacteria were isolated from these
samples. There was one isolation of Staphylococcus aureus, and two isolations
of Streptococcus pyogenes during the cattle and swine trials. Two group B
Salmonella enteritides isolates were obtained from the feces of animals on a
sludge amended diet plus three isolates at a later date from the same group.
The very few positive isolates suggested that these three types of digested
sewage sludges posed no significant health hazards from bacteria.
Finally, when digested sludge was added to a lagoon at Jay, Florida,
enteroviruses were readily detected in grab samples from the lagoon. The
level of sludge-associated viruses dropped to low or undetectable levels
following disposal of sludge on land and during periods when addition of
digested sludge to the lagoon was suspended. Enteroviruses were not detected
in wells located on the sludge disposal site or near the lagoon.
Overall average quality of sludges used in the project follows:
TABLE 1. CONCENTRATIONS OF METALS IN SEWAGE SLUDGE
PPM-MEANS-DRY MATTER BASIS
1976-1979

Dried Pensacola
Sludge (DPS)
Dried Florida (UF)a
Sludge (DFS)
Dried Chicago'3
Sludge (DCS)
Cadmium (Cd)
12
13
163
Cobalt (Co)
0.6
7.9
22
Chromium (Cr)
220
218
2,888
Copper (Cu)
548
517
1,365
Iron (Fe)
4,619
9,367
37,267
Lead (Pb)
485
465
774
Mercury (Hg)
7.9
82
5
Nickel (Ni)
35
32
376
Zinc (Zn)
2,440
1,217
2,501
a University of
13 Donated by H.
Florida
J. Baker and Bros.,
Inc., Temple Terrace,
xi
FL

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RECOMMENDATIONS
Present EPA guidelines on allowable levels of certain contaminants,
including metals, would assure availability of homogeneous urban sewage
sludges which could be utilized in soil enrichment programs for crop or
forestlands. Further research is necessary to assure safe rates and
frequency of application of sewage sludges, along with other essential
elements, to enhance crop production. If urban sewage sludges for pro-
duction of certain crops are shown to be contraindicated, this information
should be made available. Since certain metals, including cadmium, lead,
nickel, and chromium, have been shown to be accumulative in animals con-
suming forage or grain from sludge-amended soils and therefore have
potential hazard to animal health and mankind, it is proposed that further
research be done to establish safe guideline levels in feeds intended for
meat producing animals.
The presence of Saroooystis sp. in muscle from cattle and swine con-
suming sludge or forage and grain fertilized with sewage sludge incor-
porated into their diets suggest that this potential animal and human
health hazard may be associated with consumption of urban sludges. Methods
to eliminate this hazard or prevent its infectivity must be established
prior to utilization of sludges for crop or animal production. Other
parasites, including infective stages of ascarids, may persist in sludges.
Destruction of parasites or preventive programs to eliminate them from
sludge must also be developed. Therefore, it is recommended that research
to establish the incidence, diagnosis and factors predisposing to sarco-
cystis infection in cattle and swine associated with sludge utilization
be initiated.
Finally, since viruses hazardous to animal production and human health
have been shown to be present in certain urban sewage sludges, further
research to characterize these viruses and assure their reduction to non-
hazardous levels should be continued and completed expeditiously to allow
land application of sewage sludges as plant nutrients.
xi i

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ACKNOWLEDGMENT
The following individuals served as project leaders for the many aspects
of this research and may be contacted on specific questions:
Davidson, J. A., Soils Department, Coordinator, Assistant Dean, Institute
of Food and Agricultural Sciences, University of Florida, Gainesville,
FL 32610, 904-392-1786
Edds, G. T., Toxicology, Preventive Medicine, College of Veterinary Medi-
cine, University of Florida, Gainesville, FL 32610, 904-392-1841
Bertrand, J. E., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, Jay, FL, 904-994-5215
Bitton, G., Environmental Engineering, University of Florida, Gainesville,
FL 32610, 904-392-0838
Breland, H. L., Analytical Research, Soils Department, University of
Florida, Gainesville, FL (Retired)
Devore, J. M., Analytical Research, University of Florida, Gainesville, FL
(replacing H. L. Breland), 904-392-1950
Cornell, J. A., Statistics, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1946
Damron, B. L., Poultry Science, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1931
Gifford, G. E., Medical Microbiology, College of Medicine, University of
Florida, Gainesville, FL 32610, 904-392-3319
Hammell, D. L., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, (Resigned)
Hammond, L. C., Soils Department, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL 32610, 904-392-1951
Hoffmann, E. M., Microbiology, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1906
Hortenstine, C. C., Soils Department, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL 32610, 904-392-1804
Kelley, K., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1924
xi i i

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Lutrick, M. C., Soils Department, Institute of Food and Agriculutral
Sciences, University of Florida (Jay, FL), 904-994-5215
Nguyen, H. T., Pathology, College of Veterinary Medicine, University of
Florida, Gainesville, FL 32610, 904-392-1221
Osuna, 0., Preventive Medicine, College of Veterinary Medicine, University
of Florida, Gainesville, FL (Resigned)
Overman, A. R., Agricultural Engineering, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL 32610, 904-392-1864
Richter, M. F., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, Ona, FL, 813-735-3121
Shirley, R. L., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1924
Simpson, C. F., Preventive Medicine, College of Veterinary Medicine,
University of Florida, Gainesville, FL 32610, 904-392-1841
Thompson, N. P., Food Science, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32610, 904-392-1786
White, C. E., Animal Science, Institute of Food and Agricultural Sciences,
University of Florida, LiveOak, FL, 904-362-1725
xiv

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EFFECTS OF LIQUID DIGESTED SLUDGE ON SOILS AND PLANTS
SECTION A. The Nutrient Status of Six Field Crops Grown on Soils Treated
with Liquid Digested Sludge. M.C. Lutrick
SECTION B. The Utilization of Liquid Digested Sludge on Agricultural Land.
M.C. Lutrick
SECTION C. The Uptake of Certain Metals by Corn Grown on Soil Treated
with Chicago or Pensacola Sewage Sludge. M.C. Lutrick
SECTION D. Sludge Effects on Yield and Cadmium Uptake of Coastcross 1
Bermudagrass. C.C. Hortenstine
SECTION E. Transport of Sewage Sludge Constituents in a Sandy Soil and
Uptake of Metals by Bermudagrass. L.C. Hammond
1

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A. THE NUTRIENT STATUS OF SIX FIELD CROPS GROWN
ON SOILS TREATED WITH LIQUID DIGESTED SLUDGE
INTRODUCTION
The disposal of liquid digested sludge (LDS) has become a major problem
for many cities. The application of LDS on agricultural land is probably
the most practical means of disposal for cities and municipalities. This
method eliminates the high cost of alternate means (drying processes) of
disposal and an increase in air pollution.
The most extensive studies of LDS used on cropland were reported by
Hinesly et at. (1971). They concluded that N was the main constituent to
consider when using LDS in large quantities. LDS was shown to be an effec-
tive source of N, P, and micronutrients. Heavy metals are a ubiquitous
constituent of LDS and after application to the soil remain in the plow
layer, according to the above authors. Earlier research in Florida by
Hortenstine and Rothwell (1973) showed that all of the plant nutrients
measured except Mn were increased by compost applications on sorghum.
This study was initiated to measure the effects of liquid Pensacola
sludge (LPS), a source of LDS, on the nutrient content of field crops and
to establish limits for annual application rates of LDS to crops and soils
of west Florida. The results formed a basis for later trials under the
EPA project.
METHODS AND MATERIALS
Experiment 1 was a greenhouse study on three Paleudult soils (Red Bay,
Dothan, and Troup). Grain sorghum (Sorghum bicolor (L.) Moench) was used as
the indicator crop. Experiment 2 was also a greenhouse study on a previously
limed and well fertilized Red Bay top soil. Corn (Zea mays L.), grain sor-
ghum, wheat [Tr-itiaum aestivum L.), bahiagrass (Paspalum notation Fluegge),
peanuts (Avaohis hypogaea L.), and soybeans (Glycine max L.) were grown to
determine the nutrient uptake from the LPS.
A field experiment was established on Troup fine sandy loam with 0 and
31.7 metric tons per hectare (t/ha) of LPS applied and incorporated into
the soil, on which corn was planted March 23, 1973, and harvested
August 30, 1973. The earleaf from the corn of the field experiment was taken
when the grain was in the hard dough stage of maturity. Grain samples were
taken at harvest. Soil samples were taken at the same time as leaf samples
and extracted with double acid so that the micronutrients could be deter-
mined as well as P, K, and Mg.
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Fresh LPS samples were taken from each delivery and sent to the Analy-
tical Research Laboratory, Soil Science Department, University of Florida,
Gainesville, Florida, for analyses.
The analyses of LPS (Table 1) indicate that Zn would be the element
most likely to reach levels toxic to plants if large amounts of LPS were
added to the soil. The pH range of the LPS was from 6.4 to 7.0. Thus, the
LPS would not be expected to have much influence on soil pH except at
high rates of application on very acid soils. The importance of LPS as
an N source is indicated by the fact that 342 kg/ha of N would be supplied
by an application of 6.6 t/ha of LPS.
TABLE 1. ELEMENTAL COMPOSITION OF LIQUID DIGESTED SLUDGE AND AMOUNTS OF
ELEMENTS IN A 6.6 T/HA APPLICATION
CONTENT*
ELEMENTS	mg/1	kg/ha
N
1366
342.0
Ca
539
135.0
P
491
123.0
A1
446
112.0
Zn
104
26.0
Fe
77
19.0
Na
67
17.0
Mg
56
14.0
K
50
13.0
Si
46
12.0
Cu
19
4.8
Pb
15
3.8
Cr
8
2.0
Ni
3
0.8
Mn
2
0.5
Ti
2
0.5
Cd
0.55
0.13
Hg
0.55
0.04
* Each value is the average of 5 samples taken at different times.
RESULTS AND DISCUSSION
In Experiment 1, the application of up to 80 t/ha of LPS increased the
dry matter production of grain sorghum. The most significant effect on
forage composition was the reduction of the K concentration in the plants
grown on soils to which LPS was applied. This indicates that K probably
should be added to the soil to supplement the K added from the application
of LPS.
3

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In Experiment 2, corn, grain sorghum, wheat, bahiagrass, peanuts,
and soybeans were grown with and without LPS. Dry matter production and
uptake of N increased for all crops grown with LPS. The large growth
response of soybeans and peanuts suggests the possibility that the LPS must
add some element other than N that was deficient in the soil. The elements
Na, Fe, A1, and Pb, that could have become toxic when LPS was added to the
soil, were generally reduced in the herbage of these crops.
In a field test with corn, the treatments of 0 and 31.7 t/ha of LPS
produced 6020 and 7460 kg/ha of grain, respectively. There was very little
difference in the protein content of the leaves of the corn grown on the
LPS-treated soil compared with the corn grown where no LPS was added. The
same was true of the grain.
4

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B. THE UTILIZATION OF LIQUID DIGESTED SLUDGE ON AGRICULTURAL LAND
INTRODUCTION
The application of LPS prior to planting increased the yield of soy-
beans when compared with commercial fertilizer treatments.
This study was initiated to determine the crop responses, nutrient
uptake, and heavy metal accumulation on land treated with LPS.
METHODS AND MATERIALS
Experiments were begun in 1974 using corn (Zea mays L.), grain sorghum
(Sorghum biaolor (L.) Moench), and soybeans (Glycine max L.) to determine
the effects of liquid Pensacola sludge (LPS) on plant growth and yield.
The accumulation of nutrients, certain microorganisms, and heavy metals in
plants and soils was determined. The treatments were 0, 20, and 40 t/ha
of LPS applied on an annual basis. The 0 LPS treatment received 500 kg/ha
of 0-10-20 commercial fertilizer at planting for corn and grain sorghum
and 250 kg/ha of ammonium nitrate as a sidedress application. The 0 LPS
treatment for soybeans received 500 kg/ha of 0-9-17 commercial fertilizer
at planting time. Other LPS treatments received no commerical fertilizer.
The experimental design was a randomized complete block with four replica-
tions for each crop. Pioneer hybrid '3369A1 corn, Funks hybrid 1G-5221
grain sorghum, and 'Ransom' soybean were planted on three different Pale-
udult soils (Troup, Lucy, and Orangeburg, respectively). The soils were
sampled to a depth of 150 cm at 30 cm increments. Whole plant samples
were taken when the corn, sorghum, and soybeans were at anthesis. Grain
samples were taken from all plots at maturity.
Fresh LPS samples were taken periodically, dried, and sent to the
Analytical Research Laboratory, Soil Science Department, Gainesville, Florida,
for analyses. The composition of the LPS is shown in Table 1. All plant
samples were weighed and ashed, and the ash dissolved in 0.1 N HC1. Soil
samples were extracted with double acid (0.05 N HC1 + 0.02 N H2S0J. The P
was determined colorimetrically using ammonium molybdate and ascorbic acid
as indicated by Watanabe and 01 sen (1965). All other determinations were
made with either the emission or absorption flame spectrophotometer.
RESULTS AND DISCUSSION
The response of corn and grain sorghum to the amount and method of
LPS application was reduced by lack of moisture throughout the growing
season. Rainfall was less than normal from March through July, 1974.
5

-------
The amount of LPS affected the corn and soybean yields but had no significant
effect on the yield of grain sorghum.
TABLE 2. THE PRODUCTION OF CORN, GRAIN SORGHUM, AND
SOYBEAN ON LPS TREATED SOIL IN 1974
Amount of
LPS	Corn forage	Sorghum Soybean
applied	Green wt	Dry wt	grain	seed
t/ha

- 		kg/ha


0
29,790
9,350
4020
2750
20
29,340
10,100
4090
3370
40
24,190
8,600
3590
2830

sig quadratic
NS
sig quadratic
Rainfall was adequate for the production of soybeans. Soybeans grown
with a commercial fertilizer treatment averaged 15 cm taller than soybeans
grown on 20 t/ha LPS treatment but produced less seed (2750 kg/ha, compared
with 3370 kg/ha, as shown in Table 2. Half of the 40 t/ha treatment was
applied to the plots while the crop was growing. This foliar application
coated the small plants and the lack of rainfall allowed the coating to
remain on the plants thus reducing the yield of corn, grain sorghum, and
soybeans compared with the 20 t/ha treatment, which was all applied prior to
planting. The LPS applied at 20 t/ha caused all three crops to mature
approximately 1 week earlier than the commercial fertilizer treatment. Con-
centrations of N, P, and Zn are high in the LPS.
In general, micronutrient and heavy metal composition was higher in
plant tissue from LPS-treated plots on all three crops tested. Zinc seemed
to be the element most likely to be taken up in the plant and seed in excess
since it was found to be very high in the LPS. The analyses of soil treated
with LPS indicated that LPS reduced the soil pH and increased P and Zn
content.
Analyses of water from Pond Creek and a local spring located near the
test site indicated that no detectable amount of nutrients occurred in the
runoff waters from the LPS treated land. Water from all wells was low in
nutrients, particularly nitrate nitrogen (N03-N). The N03-N level remained
consistently at 0.6 ppm. Water samples taken from the well at the lagoon
site (a means of on-farm storage of LPS) indicated that there was some
contamination from the LPS in the lagoon.
These preliminary data indicate that LPS can be used on agricultural
lands, up to a point, without detrimental effects on crops or cattle con-
suming forages grown on LPS-treated land. Additional data are needed to
determine the amount of LPS required to utilize LPS as a fertilizer.
6

-------
C. THE UPTAKE OF CERTAIN METALS BY CORN GROWN ON SOIL TREATED
WITH CHICAGO OR PENSACOLA SEWAGE SLUDGE
SUMMARY
Land spreading of sewage sludge is probably the most practical means
of disposal for municipalities and cities. However, uptake of certain
metals by forage and grain crops from land treated with sludge creates
health risks. A 3-year study was conducted to determine the uptake of
copper (Cu), zinc (Zn), manganese (Mn), lead (Pb), and cadmium (Cd) by corn
leaves and grain from soil treated with Chicago and Pensacola sewage sludge.
The dried Chicago sludge (DCS) contained large quantities of Cu, Zn, Pb,
and Cd. The Pensacola sludge was high only in Zn.
The quantities of metals extracted from sludge-treated soil were pro-
portional to the quantities added from the sludges. The metal uptake by
the corn plant was directly associated with soil pH. The higher the
soil pH the smaller the quantity of metal uptake. The quantity of metals
extracted from the siudge-treated soil after 1 year was somewhat less than
from the soil where sludge had been recently applied. The quantity taken
up by the corn leaves was much less from the residual sludge treatment than
from the same treatment where sludge had been recently applied.
The concentration of metals in the grain was always much less than
the concentration found in the corn leaves from the same sludge treatment.
Corn plants from the DCS treatment probably would have contained too much
Cd to be utilized for forage. The Pb and Cd concentration in the grain was
below detectable limits from all treatments.
7

-------
D. SLUDGE EFFECTS ON YIELD AND CADMIUM UPTAKE
OF COASTCROSS 1 BERMUDAGRASS
There is widespread interest in the United States in the utilization
of municipal sewage sludges as a soil amendment or source of plant nutrients.
However, potential hazards exist that must be resolved before sludges can
be used to any great extent on agricultural soils. This is particularly true
of metals that may enter the human food chain with more or less disastrous
effects. Cadmium is especially important in this respect as it can
accumulate within various body organs in amounts that can produce diseases
or fatalities (Shroeder, 1965; Carroll, 1966; Axelsson and Piscator, 1966).
A case in point was the incidence of the "itai itai" disease several years
ago in Japan (Tsuchiya, 1969). The occurrence of this disease in a large
population of the Jintsu Valley resulted from the daily intake of rice
grown on soil which was contaminated by Cd release in effluent from
surrounding industries.
The objectives of this study were to:
1.	measure Cd movement in and adsorption by a soil treated
with sewage sludge of known Cd content,
2.	measure uptake of Cd by Coastcross 1 bermudagrass (Cynodon
daotyton L. Pers.), and
3.	compare the sludge to a mineral fertilizer as a source of
plant nutrients.
EXPERIMENTAL PROCEDURES
Field plots (5 x 7 m) were established in a 4 x 4 Latin square on
Lake sand (Typic Quartzipsamments, hyperthermic, coated) which was sampled
prior to sludge or fertilizer applications at 30-cm increments to 360-cm
depth. Treatments were:
1.	10-4.4-8.3 (N-P-K) fertilizer applied at 6 t/ha,
2.	sewage sludge applied at 12 t/ha,
3.	sewage sludge applied at 24 t/ha, and
4.	sewage sludge applied at 48 t/ha.
The fertilizer and sludge were disked into the soil and the bermuda-
grass was sprigged in June, 1977. The fertilizer and sludge were to be
reapplied each year after 1977 for a total of 5 years. As sludge is
8

-------
notoriously low in K, additional K was applied to the siudge-treated
plots at the rate of 560 kg/ha. The bermudagrass was harvested at about
6-week intervals for yield and chemical analyses. Soil was sampled
annually at the 0 to 15 and 15 to 30 cm depths for chemical analyses.
Plant tissue samples were dried at 70C, ground in a stainless steel
Wiley mill, and ashed for 6 hours at 450C. The ash was dissolved in 1 N
HN03, transferred to volumetric flasks, and made to volume with distilled
deionized water. The solution was analyzed for K, Cu, Mn, Zn, Cd, Ni, and
Pb by atomic absorption. Total N was determined by the semimicro-Kjeldahl
method (Bremner, 1965) and N03-N was determined in a water extract with a
specific ion electrode. Phosphorus was determined by the phosphomolybdate
stannous chloride method (Jackson, 1958).
RESULTS AND DISCUSSION
The soil used in this study is a deep sand which is generally infertile
in the virgin state. As shown in Table 3, the soil was quite low in avail-
able plant nutrients with an acid pH to the 360 cm depth. The absence of
detectable Cd and extremely low Zn in the extractant used by the University
of Florida Soil Science Laboratory indicated that Lake sand is an ideal
soil on which to conduct this study as any buildup of these two elements
over a period of several years would have to result from fertilizer or
sludge applications. Although not shown in Table 3, Pb and Ni were also
not detectable in the original soil samples.
The soil in each plot was sampled just prior to the annual application
of sludge and fertilizer. Data from DA extracted soil (Table 4) indicate
that there was an appreciable increase in most of the elements considered
at the end of the second year of the study. Of particular interest was
the considerable increase in soil Ca in the 0 to 15 cm depth from sludge
applications as compared to the apparent decrease from mineral fertilizer.
Sludge applications also increased soil Zn, Cd, Cu, Pb, and Ni with some
evidence of movement of these elements below 15 cm depth.
There is presently some controversy in the United States as to the
most appropriate extractant for heavy metals in soil. As the available
phase of a metal with respect to plant uptake is the determining factor
in crop response, we compared the DA method with the method described by
Lindsay (1972). Significant (c.001) increases in heavy metal contents
of the 0 to 15 cm depth were recorded with both extractants (Tables 4 and
5) and both extractants indicated that some movement of these metals into
the 15 to 30 cm depth occurred. Correlations will be calculated between
plant uptake and soil content at the end of this study to determine which
extractant is better for this type of soil. Heavy metal extraction with
4 M HN03 was conducted to give an idea of total metal content (Table 5).
The same type of differences was revealed as with the other extractants.
The first full year of growth by the bermudagrass is reported in
Table 6. The Cd con ents of the bermudagrass in Table 6 are of particular
interest. The 30 May harvest was removed before the annual sludge applica-
tion and Cd contents increased greatly in the second harvest (17 July)
9

-------
after the one-half sludge application in May as compared to the first
harvest. The second sludge applications were on 1 August and the third
harvest on 21 August reflected a still larger increase in Cd uptake.
The yields of each harvest in 1979 and total yields indicate that
sludge applied at the 24 ton rate compared favorably with mineral fertilizer
as a source of plant nutrients. The highest rate (48 t/ha) of sludge was
superior to mineral fertilizer, whereas the 12 ton rate was apparently too
low to sustain Coastcross 1 bermudagrass. In fact, the bermudagrass in the
12 ton rate plots died out while bahiagrass became established again.
10

-------
TABLE 3. INITIAL CHEMICAL CHARACTERISTICS OF LAKE SAND TO A DEPTH OF
360 CM BEFORE FERTILIZER AND SEWAGE SLUDGE APPLICATIONS
Depth
cm
pH*
TSS*
Ca
Mg
P
K
Na
Fe
yg/g - -
Zn
Cu
Mn
Mo
Cd
0-15
5.1+
30
134
21
56
18
40
29
0.95
0.40
5.4
27
0
15-30
4.8
19
78
5
52
8
39
31
0.80
0.40
6.2
17
0
30-60
4.9
20
101
10
34
9
36
32
0.51
0.20
6.4
16
0
60-90
4.8
19
83
7
26
7
36
30
0.63
0.20
6.6
12
0
90-120
4.9
17
104
9
28
10
36
31
0.35
0.20
7.1
18
0
120-150
5.0
17
136
10
33
12
37
31
0.43
0.27
5.5
19
0
150-180
5.0
19
137
14
41
11
34
34
0.40
0.27
4.2
8
0
180-210
5.3
19
168
29
39
15
36
30
0.35
0.27
2.1
8
0
210-240
5.3
25
146
25
33
13
36
30
0.51
0.20
2.5
18
0
240-270
5.2
24
180
76
37
16
39
29
0.60
0.27
1.8
15
0
270-300
4.9
22
214
145
45
18
36
26
0.66
0.20
1.8
7
0
300-330
4.7
25
226
174
45
18
36
28
0.40
0.33
1.9
14
0
330-360
4.5
31
208
158
49
15
35
32
0.51
0.40
1.9
20
0
* pH and total soluble salts were in water (soil + water = 1:2). DA (0.05 N HC1 in 0.025 N H2S04)
extractant used for all other determinations.
+ Each entry is an average of three samplings.

-------
TABLE 4. CHEMICAL CHARACTERISTICS OF LAKE SAND (DA EXTRACTED) AT THE
END OF 2 YEARS (SAMPLED BEFORE THE THIRD APPLICATION OF
FERTILIZER AND SLUDGE).
Sludge
applied
tons/ha
PH
P
K
Ca
Mg
Fe
0-15
ug/g
Zn
cm
Cd
Cu
Pb
Ni
0*
5.35+
137
41
69
15
17
3
0
0.2
0
0
12
5.95
81
57
198
41
30
15
0.9
4.0
0.8
0.8
24
5.95
95
66
217
51
44
29
2.0
7.7
1.1
1.6
48
5.83
136
87
402
92
86
84
6.3
18.3
1.5
4.3
s
0.27
27
21
128
30
28
33
2.6
7.1
0.6
1.8






15-30
cm




0*
5.30
132
42
37
7
15
1
0
0.2
0
0
12
5.90
68
59
63
15
16
3
0.1
1.0
0
0
24
5.73
75
46
78
20
18
5
0.3
1.5
0.2
0.4
48
5.55
93
34
114
28
29
13
0.8
3.7
0.6
0.9
s
0.24
21
14
39
9
7
6
0.4
1.6
0.3
0.5
* Received 6 tons/ha of 10-4.4-8.3 (N-P-K).
+ Each entry is an average of four samples.

-------
TABLE 5. HEAVY METAL CONTENT OF LAKE SAND (HN03 AND DTPA EXTRACTED)
AT END OF 2 YEARS (SAMPLED BEFORE THE THIRD APPLICATION OF
FERTILIZER AND SLUDGE)
SIudge
applled
tons/ha
4 M HNO;
DTPA

Fe
Mn
Zn
Cu
Cd
Pb
Ni
ug/g
0-15
Fe
cm
Mn
Zn
Cu
Cd
Pb
Ni
0*
660+
32
5
1
0.03
1.8
0.5
49
3.1
1.2
0.3
0
0.1
0
12
1040
39
28
13
1.78
9.9
3.3
46
2.1
8.0
3.1
0.63
0.9
0.3
24
1315
39
42
20
2.89
14.8
5.6
61
3.0
15.9
6.0
1.25
1.6
1.6
48
2865
44
118
58
8.13
36.9
18.9
112
6.6
41.0
15.4
3.25
3.6
5.0
s
930
5
46
23
3.31
15.3
7.9
28
1.8
15.8
6.0
1.28
1.3
2







15-30
cm






0*
660
32
3
1
0.02
1.7
0.5
28
2.0
0.6
0.3
0
0

12
750
33
9
3
0.31
2.0
0.7
28
1.4
1.9
0.9
0.13
0.2

24
760
33
11
5
0.51
3.0
1.3
27
1.5
3.0
1.4
0.22
0.2

48
960
36
24
11
1.45
6.8
2.9
55
2.8
8.9
3.5
0.64
0.5
0
s	150 4 9 4 0.62 2.5 1.1 16 0.7 4.0 1.4 0.30 0.2
* Received 6 tons/ha of 10-4.4-8.3 (N-P-K).
+ Each entry is an average of four samples.

-------
TABLE 6. DRY MATTER (70C) YIELD AND SELECTED MINERAL CONTENT OF COASTCROSS 1
BERMUDAGRASS GROWN ON LAKE SAND IN 1978
SIudge
applied
Yi el d
P
K
Ca
Mg
Cu
Mn
Zn
Cd
tons/ha
kg/ha

0
/
'/
0



uq/g






30 May



0*
12
24
48
Yield
date
lost
0.11+
0.11
0.13
0.14
1.27
1.29
1.53
1.79
0.26
0.28
0.27
0.29
0.11
0.13
0.15
0.16
3.8
4.4
5.0
5.9
65
60
70
120
20.3
27.2
28.1
33.3
0.35
1.15
1.42
1.81
s

0.02
0.24
0.02
0.02
0.9
8
7.6
0.61





21 August



0*
12
24
48
No yield
data due
to army-
worms
0.34
0.29
0.30
0.34
2.75
3.95
3.43
3.35
0.53
0.51
0.41
0.48
0.09
0.16
0.16
0.15
5.5
8.8
9.0
9.5
23
70
70
87
113
178
115
151
0.63
3.63
6.05
6.47
s

0.03
0.39
0.15
0.03
1.8
26
67
2.72





12 December



0*
12
24
48
2685
1750
2950
4040
0.27
0.22
0.27
0.30
2.39
1.65
2.44
2.87
0.31
0.31
0.33
0.34
0.14
0.14
0.20
0.26
5.0
6.8
8.4
9.5
279
121
193
215
51
65
86
103
0.14
0.96
2.33
4.30
s
1010
0.04
0.47
0.02
0.05
1.8
63
25
1.68
* Received 6 tons/ha of 10-4.4-8.3 (N-P-K).
Each entry is an average of four samples.

-------
E. TRANSPORT OF SEWAGE SLUDGE CONSTITUENTS IN A
SANDY SOIL AND UPTAKE OF METALS BY BERMUDAGRASS
Commercial Chicago sewage sludge (dried) was applied to bermudagrass
growing on a well-drained sandy soil (coated, hyperthermic Typic Quartzip-
samment) at Gainesville, Florida. Annual rates for 3 years on 4 respective
treatments were 0, 12, 24, and 48 tons/ha. The no-sludge treatment received
6 tons/ha annually of a chemical fertilizer (10-4.4-8.3, N-P-K). Forage was
harvested for yield and chemical analysis at about 6-week intervals during
the growing season. Frost-killed sod remained undisturbed during the winter.
Rainfall, irrigation, and estimated evapotranspiration data were used in a
computer model to estimate the leaching potential for the soil-piant-climate
system over the 3-year period.
Forage yields increased each year and in 1979 they were 20, 14, 20, and
26 thousand kg/ha for the 0, 12, 24, and 48 tons/ha sludge rates. For
these respective treatments in 1979, uptake of Cd was 3, 22, 46, and 101
mg/ha and uptake of Zn was 413, 501, 788, and 1530 mg/ha.
Metals have accumulated in the 0 to 15 cm soil profile; Cd = 6.3 yg/g
and Zn = 83 yg/g at the highest sludge rate. Transport into the 15 to 30 cm
zone has occurred; Cd = 0.8 yg/g and Zn = 12 yg/g. These results are in
line with the low leaching potential calculated from the rainfall amounts
and distribution over the 3 year period. The following tables (7-9)
illustrate these results.
15

-------
TABLE 7. OVEN-DRY YIELDS OF COASTCROSS 1 BERMUDAGRASS
(Cynodon daotylon L. Pers.) ON LAKE SAND (TYPIC
QUARTZIPSAMMENTS, HYPERTHERMIC, COATED)
Material applied
Year 10-10-10 (6)* Sludge (12)* Sludge (24)* Sludge (48)*
	 kg/ha 	 ¦
1977




1st cut
2451
1017
649
0
2nd cut
3800
4046
3760
2769
3rd cut
1260
1240
1334
2671
TOTALS
7511
6303
5743
5440
1978




1st cut
6260
5343
5963
5797
2nd cut
6049
4715
6760
7049
3rd cut
2683
1746
2949
4037
TOTALS
15092
11804
15672
16883
* Metric tons/ha.

-------
TABLE 8. MINERAL CONSTITUENTS IN COASTCROSS 1 BERMUDAGRASS ON LAKE SAND
Year
K
Ca
Mg
Cu
Mn
Zn
Cd

- - %
- - - -
- - -
- -
- -
ppm - ¦
- - -
1977







1st cut:







10-10-10
6.33
0.46
0.29
9
231
80
0.35
SIudge-12
6.57
0.53
0.38
18
275
116
1.67
SIudge-24
5.99
0.51
0.39
31
313
114
2.45
SIudge-48
--
--
—
—
—
—
—
2nd cut:







10-10-10
4.66
0.56
0.30
8
202
16
0.33
SIudge-12
5.35
0.54
0.34
9
221
23
0.35
SIudge-24
5.41
0.54
0.32
10
247
26
0.69
SIudge-48
6.00
0.51
0.34
11
465
32
1.58
3rd cut:







10-10-10
5.70
0.85
0.45
15
277
29
0.35
SIudge-12
5.34
0.80
0.43
17
251
46
0.35
SIudge-24
5.70
0.78
0.43
18
288
49
0.44
SIudge-48
6.10
0.62
0.41
19
395
65
2.20
1978







1st cut (5/30):







10-10-10
1.27
0.26
0.11
3.8
65
20
0.35
SIudge-12
1.29
0.28
0.03
4.4
60
27
1.15
SIudge-24
1.53
0.27
0.15
5.0
70
28
1.42
SIudge-48
1.79
0.29
0.16
5.9
120
33
1.81
2nd cut (7/17):







10-10-10
2.27
0.15
0.11
5.4
103
104
0
Sludge-12
2.07
0.16
0.12
6.2
74
225
2.60
SIudge-24
2.46
0.16
0.13
7.5
89
174
3.30
SIudge-48
2.85
0.18
0.16
9.5
133
149
3.50
3rd cut (8/21):







10-10-10
2.75
0.53
0.09
5.5
123
162
0.62
SIudge-12
2.95
0.51
0.16
8.8
70
178
3.62
SIudge-24
3.43
0.41
0.16
9.0
70
115
6.05
SIudge-48
3.35
0.48
0.15
9.5
87
151
6.47
4th cut (10/25):







10-10-10
1.76
0.33
0.14
6.7
218
99
--
SIudge-12
1.39
0.23
0.13
7.4
85
73
--
SIudge-24
1.97
0.28
0.16
8.5
268
90
	
SIudge-48
2.16
0.29
0.18
8.5
172
87
--
5th cut (12/12):







10-10-10
2.39
0.31
0.14
5.0
279
52
--
Sludge-12
1.65
0.31
0.14
6.8
121
65
	
SIudge-24
2.44
0.33
0.20
8.4
193
100
--
SIudge-48
2.87
0.34
0.26
9.5
215
103
	

-------
TABLE 9.
SELECTED SOIL CONSTITUENTS IN LAKE SAND AT END OF FIRST YEAR (1977)
Treatment
PH
Ca
Mg
K
Na
NO3-N
Cu
Zn
Cd
0-15 cm




ppm




10-10-10
5.6
103
22
41
10
5.4
0.7
2.8
0.1
SIudge-12
5.8
160
40
43
13
6.0
4.1
10.5
0.9
SIudge-24
6.0
201
49
44
14
5.4
6.3
16.3
1.0
SIudge-48
6.0
243
77
55
18
6.6
11.2
29.5
2.0
15-30 cm









10-10-10
5.3
25
6
26
9
4.4
0.5
1.8
0.1
SIudge-12
5.4
25
6
34
13
4.2
0.6
1.9
0.1
SIudge-24
5.4
26
8
28
11
4.1
0.6
1.7
0.1
SIudge-48
5.2
27
11
21
9
4.8
1.2
2.2
0.1

-------
REFERENCES
1.	Hinesly, T. D., 0. C. Braids, and J. E. Molina. Agricultural Benefits
and Environmental Changes Resulting from the Use of Digested Sewage
Sludge on Field Crops. U.S. Environmental Protection Agency (SW-30d),
1971.
2.	Hortenstine, C. C., and D. F. Rothwell. Pelletized Municipal Refuse
Compost as a Soil Amendment and Nutrient Source for Sorghum. Environ.
Qua!. 2(3):343-345, 1973.
3.	Watanabe, F. S., and S. R. Olsen. Test of an Ascorbic Acid Method for
Determining Phosphorus in Water and NaHC03 Extracts from Soil. Soil
Sci. Soc. Am. Proc., 29:677-678, 1965.
19

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CATTLE FEEDING TRIALS WITH SLUDGES OR FEED GROWN ON SLUDGE TREATED LAND
SECTION A. Dried Pensacola Liquid Digested Sludge in the Diet of Steers.
J. Bertrand, M. C. Lutrick, and G. T. Edds, 1974
SECTION B. Winter Annual Pastures Fertilized with Pensacola Liquid Digested
Sludge and Grazed by Growing Beef Steers. J. Bertrand,
M. C. Lutrick, and H. T. Nguyen, 1976
SECTION C. Dried Pensacola Liquid Digested Sludge (DPS) in the Diets of
Feedlot Steers. J. Bertrand, H. T. Nguyen, and H. Breland
SECTION D. Dried Chicago Sludge and Corn from Soil Fertilized with Liquid
Pensacola Sludge in Diets of Beef Steers. J. Bertrand,
M. C. Lutrick, H. T. Nguyen, and H. Breland, 1977
SECTION E. Forage Sorghum Silages Grown on Soil Treated with Liquid
Digested Sludge and Fed to Beef Steers. J. Bertrand,
M. C. Lutrick, 0. Osuna, G. T. Edds, S. West, and J. Devore,
1978
SECTION F. Pensacola Bahiagrass Pastures Fertilized with Pensacola Liquid
Digested Sludge and Grazed by Beef Steers. J. Bertrand,
M. C. Lutrick, 0. Osuna, and J. Devore, 1979
The initial research at Jay, Florida on the comparative effectiveness
of Pensacola liquid digested sludge (LPS) and commerical fertilizers for
soil improvement indicated equal effectiveness. However, it was not known
whether certain agents or elements in the sludge would be translocated into
the plant forage or grain with potential hazard(s) to animals consuming
this feed. Therefore, a series of trials were run by Bertrand, et al.,
in beef steers under feedlot or grazing operations to determine whether
growth performance or carcass quality would be adversely affected.
20

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A. DRIED PENSACOLA LIQUID DIGESTED SLUDGE IN THE DIET OF STEERS
ABSTRACT
Six steers were randomly allotted and fed one of two diets for 219 days
to determine the effects of dried Pensacola liquid digested sludge (DPS) on
animal performance and concentrations of selected elements in liver, muscle,
and kidney tissues. The experimental diets consisted of the control diet
and the treated diet (100 grams per head daily of DPS incorporated into the
control diet prior to feeding). Steers fed the control diet gained slightly
faster and were slightly more efficient in converting feed to gain than
those fed the treated diet. Feed consumption was higher for steers on the
control diet. No significant differences in performance between the diets
were detected.
The concentrations of a number of the elements, aluminum (Al), copper
(Cu), iron (Fe), magnesium (Mg), manganese (Mn), nickel (Ni), and zinc (In),
were lower in the livers of steers receiving DPS in their diet. The Al,
cadmium (Cd), and lead (Pb) concentrations were higher in the kidneys of
steers fed DPS.
EXPERIMENTAL PROCEDURES
Six steers, average 192 kilograms (kg), of Angus and Hoi stein breeding
were weighed and allotted at random to two experimental groups of 3 steers
each. The control group received the control diet listed in Table 1; while
the treated group received the control diet plus 100 grams (g) per head
daily of DPS incorporated into the diet prior to feeding. The DPS was sun-
dried to approximately 6% moisture in shallow vats located in greenhouses.
After an overnight shrink, individual animal weights were obtained at
the beginning and end of the experiment. Group weights were obtained every
28 days during the period.
The steers were fed ad libitum once daily for a total of 219 days
(April 19 to November 24, 1974).
At the end of the experimental period, the steers were slaughtered at
the University of Florida Meats Laboratory, Gainesville. Liver, muscle,
and kidney tissues were collected at slaughter.
The DPS and tissue samples (liver, muscle, and kidney) were sent to the
Analytical Research Laboratory, Gainesville, for elemental analyses. Con-
centrations of selected elements in the DPS are listed in Table 2.
21

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Analyses of variance for animal performance data and concentrations
of selected elements in tissues were conducted according to the method of
Snedecor (1946).
RESULTS AND DISCUSSION
Performance data with steers fed the two diets for a 219-day feeding
period are shown in Table 3. Steers fed the control diet gained slightly
faster than steers fed the treated diet (1.03 and 0.93 kg/head daily,
respectively). The differences were not significant. Steers fed the control
diet were also slightly more efficient in converting feed to gain than those
fed the treated diet (8.5 versus 8.8 units of feed per unit, respectively).
Feed consumption was higher for the steers on the control diet.
The concentrations of a number of the elements A1, Cu, Fe, Mg, Mn,
Ni, and Zn were lower in the livers of steers receiving DPS in their diet
(Table 4). These reduced elemental concentrations in livers of steers
receiving DPS in their diet were all statistically significant except for
Fe and Ni. There were no differences in the concentrations of the selected
elements in muscle tissues between the two diets. The A1, Cd, Pb concentra-
tions were higher (P < 0.01) in the kidney tissues of steers fed DPS and
were a matter of concern.
22

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TABLE 1. CONTROL DIET CONSUMPTION3

Ingredients
Inter-
national
Ref. No.
Percent
Sorghum, Milo (Sorghum vulgare)
grain, pound
4-04-444
68.03
Urea - 45% N

0.45
Soybeans (Glycine max)
seeds, solvent-extracted, ground, 44%
5-05-604
4.50
Trace mineral salt*3

0.45
Phosphate - defluorinated, ground
6-01-780
1.34
Oats (Avena sativa) - hay, s-c
1-03-280
25.23
Antibiotic supplement0

+
Vitamin A palmitate^
7-05-143
+
a As-fed basis.


b Contained not less than 0.350% Zn, 0.340%
and 0.005% Co.
Fe, 0.200% Mn, 0.033% Cu,
0.007% I,
0 Zinc bacitracin added at the level of 19.E
) mg/kg of diet.

^ Supplied 3308 IU/kg of diet.

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TABLE 2. CONCENTRATIONS OF SELECTED ELEMENTS IN DRIED
PENSACOLA LIQUID DIGESTED SLUDGE3
Elements	ug/g*3
A1
13,500
Ca
16,300
Cd
20
Co
10
Cr
240
Cu
570
Fe
2,330
Hg
5
K
1,510
Mg
1,690
Mn
50
N
41,400
Na
2,030
Ni
80
P
14,900
Pb
460
Si
1,400
Ti
50
Zn
3,150
a Solids averaged 3.3%.
k Dry matter basis.
24

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TABLE 3. PERFORMANCE DATA WITH STEERS RECEIVING THE FEEDLOT DIETS
Item
Control diet
Treated diet9
Number of animals
3
3
Length of trial, days
219
219
Average initial weight, kg
190
194
Average final weight, kg
416
398
Average gain/animal, kg
226
204
Average daily gain, kg
1.03
0.93
Feed/gain ratio
8.5
8.8
Feed/animal/day, kg
8.8
8.3
a DPS was added at the level of 100 g/animal/day to the control diet.
25

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TABLE 4. CONCENTRATIONS OF SELECTED ELEMENTS IN LIVER,
MUSCLE, AND KIDNEY TISSUES9
	Li ver	 	Muscle	 	KT dney	
Elements Control Treated Control Treated Control Treated
yg/g (dry matter basis)
A1
36.06C
16.74b
57.42
70.25
38.37d
57.27e
As
0
0
0
0
0
0
Ba
0
0
0
0
0
0
Ca
72.94
80.68
63.17
65.21
219.71
242.55
Cd
0.27
0.28
0.03
0.04
1.39d
1.93e
Co
0
0
0
0
0
0
Cr
0.06
0.06
0.54
0.67
0.43
0.55
Cs
7.39
7.37
8.16
8.46
8.60
9.44
Cu
91.98e
15.74d
4.08
3.57
19.41
19.27
Fe
43.03
27.31
85.52
83.19
243.54
261.99
Mg
455.81e
192.24d
773.81
799.18
721.69
782.61
Mn
6.96C
4.66b
0.85
1.05
5.53
5.82
Mo
2.81
2.29
0
0
1.60
1.79
Ni
0.46
0.15
0.09
0.33
0.31
0.10
Pb
1.79
1.83
1.08
1.27
2.59d
6.90e
Se
0
0
0
0
0
0
Si
1.49
1.14
0.55
0.62
0.95
1.10
Sr
0
0
0
0
0
0
Ti
0
0
0
0
0
0
Zn
111.98e
54.07d
203.71
179.11
84.11
86.88
a Data presented as means of three observations.
K C
Means in a row for a particular tissue with different superscripts
differ significantly (P < 0.05).
d £
' Means in a row for a particular tissue with different superscripts
differ significantly (P < 0.01).
26

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B. WINTER ANNUAL PASTURES FERTILIZED WITH PENSACOLA LIQUID
DIGESTED SLUDGE AND GRAZED BY GROWING BEEF STEERS
Winter annual pastures (a mixture of wheat and crimson clover) rota-
tional ly grazed by growing beef steers were fertilized with normal amounts
of fertilizer (control) or Pensacola liquid digested sludge (LPS) applied
by two methods: 1. LPS #1 - 19.8 t/ha of LPS incorporated into the soil
prior to the planting, and 2. LPS #2 - 6.6 t/ha of LPS incorporated into
the soil prior to planting and 13.2 t/ha of LPS topdressed on the soil and
forage during the grazing season. The steers grazing the pastures receiving
the LPS #1, Control, and LPS #2 treatments had average daily gains of 0.97,
0.88, and 0.84 kg per head, respectively. The gains per hectare were 487,
417, and 359 kg for the steers. Incorporation of LPS into the soil prior
to planting was beneficial to forage growth while topdressing with LPS
on the soil and forage was detrimental due to trampling by the tractor and
sludge spreader, decreased growth by LPS-coated leaves, and the initial
killing of much of the young crimson clover.
The wheat was planted with a grain drill at the rate of 140 kg/ha. The
crimson clover was seeded over the wheat with a cultipacker-seeder at the
rate of 9 kg/ka. The 1.0 ha plots were planted on October 23 and 24, 1975;
while the 0.5 ha plots were planted on November 5, 1975.
The trial was initiated on December 19, 1975, and terminated when the
forage was essentially grazed out. Due to a long and consistent cold period
and slow forage growth, each group of steers had to be removed from the
experimental plots for a short period during the early part of 1976 to allow
the forage to recover.
Individual animal weights were taken at the beginning and end of the
trial period after an overnight shrink. Group weights were obtained every
28 days at approximately the same time of day. Additional grazer animals
were added and removed as needed to keep the forage uniformly grazed. Each
experimental group of steers was rotated between the two pasture plots
assigned to it as required for best utilization of good quality forage.
A mineral mixture (consisting of 2 parts defluorinated rock phosphate
and 1 part trace-mineralized salt), plain salt, and clean drinking water
were available to the animals at all times.
One group of 4 steers from each treatment was slaughtered on April 21,
1976; while 1 group of 4 steers from each treatment was slaughtered at
the University of Florida Meats Laboratory, Gainesville, on May 15, 1976.
27

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An analysis of variance on the average daily gain data was conducted
according to the method of Snedecor (1946).
RESULTS AND DISCUSSION
The performance data of growing beef steers grazing winter annual
pastures fertilized with LPS are presented in Table 5. Steers grazing the
pastures receiving the LPS #1 treatment had an average daily gain of 0.97 kg
per head for a period of 128 days, followed by an average daily gain of
0.88 kg per head for steers grazing the control pastures for a period of
121 days, and an average daily gain of 0.84 kg per head for steers grazing
the pastures receiving the LPS #2 treatment for a period of 121 days. There
were no significant differences in the average daily gain between treatments.
The larger gain per hectare for steers grazing the pastures receiving
the LPS #1 treatment was due to a longer grazing period. Steers grazing the
pastures receiving the LPS #2 treatment had a low gain per hectare, mainly
because of a low daily gain and a low stocking rate per hectare. The top-
dressing with 13.2 t/ha of LPS during the grazing period appeared to be
detrimental to the forage on these pastures from the standpoint of trampling
by the tractor and sludge spreader and poor forage growth. Coating the
surfaces of the leaves with LPS appeared to decrease forage growth; this
was apparently due to decreased photosynthesis. Thus, the use of LPS as a
fertilizer on agricultural land intended for use as winter annual pastures
for grazing by growing beef steers was beneficial if the LPS was incorporated
into the soil prior to planting.
28

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TABLE 5. PERFORMANCE DATA OF GROWING BEEF STEERS GRAZING WINTER
ANNUAL PASTURES FERTILIZED WITH PENSACOLA LIQUID DIGESTED
SLUDGE
Item

Treatments

Control
LPS #lb
LPS #2C
Initial number of animals
8d
8
8
Length of grazing, days
12 le
128f
12 le
Average initial weight, kg
202
190
198
Average final weight, kg
309
314
300
Average gain/animal, kg
107
124
102
Average daily gain, kg
0.88
0.97
0.84
Animal days/ha9
474
502
427
Stocking rate/ha9
3.9
3.9
3.5
Gain/ha, kg*1
417
487
359
Gain/ha/day, kg*1
3.43
3.78
2.94
Rotational grazing of a wheat and crimson clover mixture - 560 kg/ha
of 14-12-12 fertilizer at planting time and 3 applications of 112 kg/ha
each of ammonium nitrate during the grazing season.
k Same pasture mixture as above - fertilized with 19.8 t/ha of LPS prior
to planting.
c Same pasture mixture as above - fertilized with 6.6 t/ha of LPS prior
to planting and topdressed with 13.2 t/ha of LPS during the grazing
season.
Initially, 2 groups of 4 steer calves each.
e One group of calves grazed for 109 days; while the other group grazed
for 133 days.
f
One group of calves grazed for 116 days; while the other group grazed
for 140 days.
9 Additional grazer animals of the same type and size were added and
removed as needed to keep the forage uniformly grazed.
^ Grazer steer gain was considered at the same rate as that of the
experimental animal gain.
29

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C. DRIED PENSACOLA LIQUID DIGESTED SLUDGE (DPS) IN
THE DIETS OF FEEDLOT STEERS
ABSTRACT
Twenty-four beef steers of British breeding were randomly allotted and
fed 1 of 3 diets for 119 days to determine the effects of dried Pensacola
liquid digested sludge (DPS) incorporated into the diets at feeding time
on animal performance, carcass quality, and concentrations of selected,
potentially toxic metals in liver, muscle, and kidney tissues. The 3 levels
of dried DPS were 0, 250, and 500 grams (g) per head daily. The performance
and carcass data of steers fed the 250 and 500 g per head daily were not
significantly different from those of the control steers. The cobalt (Co),
copper (Cu), nickel (Ni), and zinc (Zn) concentrations in liver tissues
were significantly lower, while the lead (Pb) concentrations were signi-
ficantly higher, for steers receiving the high level of DPS. There were
some accumulations of Pb in kidney tissues of DPS-treated animals.
EXPERIMENTAL PROCEDURES
Twenty-four beef steers, average 290 kilograms (kg), of British
breeding (Angus and Hereford) were allotted at random from breed groups to
6 experimental groups of 4 steers each. The 5 experimental groups, 2
groups (replicates) per treatment, were assigned to 3 DPS diet levels of
0, 250, and 500 g per head daily.
The control diet consisted of 73.5% corn [Zea mays) - aerial part,
ensiled, mature, well-eared mx 50% mn 30% dry matter, International
reference no. 3-08-153 (41.5% dry matter), 23.9% ground corn, dent yellow
(zea mays indentata) - grain, gr 2 US, International reference no. 4-02-931
(88.4% dry matter), and 3.1% concentrate supplement mixture (87.7% dry
matter) on an as-fed basis. The diet was 56% corn silage, 39% ground
corn, and 5% concentrate supplement mixture on a dry matter basis. The
ingredients of the concentrate supplement mixture are listed in Table 6.
Individual animal weights were obtained at the beginning and end of the
trial after an overnight shrink. Group weights were obtained every 28
days during the period.	These weights were obtained in order to
periodically check on the performance of the steers. Blood, fecal, feed
ingredient, and DPS samples were collected each time for analysis.
The trial was initiated on August 19 and terminated on December 15,
1976 (119 days). The steers were fed ad libitum twice daily. The DPS was
30

-------
incorporated into the diets at each feeding. The amounts of feed for
each group of steers were recorded at each feeding.
The DPS was sun-dried to approximately 6% moisture in shallow vats
located in greenhouses.
The steers were slaughtered at the University of Florida Meats Labora-
tory, Gainesville. Liver, muscle, and kidney tissues were collected at
slaughter for metal analysis. Carcass measurements were secured.
The DPS, feed ingredients, blood, fecal, and tissue (liver, muscle,
and kidney) samples were sent to the Analytical Research Laboratory, Soil
Science Department, University of Florida, Gainesville, for metal analyses
and the blood was also evaluated as to hematology and clinical chemistry.
Concentrations of selected metals in DPS and feed ingredients in the diets
are listed in Table 7.
Analyses of variance for animal performance and carcass quality data
were conducted according to the method of Snedecor (1946). The computer
package (Statistical Analysis System, North Carolina State University)
outlined by Barr et at. (1976) was utilized for the statistical analyses
of the metal data in blood, feces, and tissues.
RESULTS AND DISCUSSION
Performance and carcass data with beef steers fed the 3 DPS levels
(0, 250, and 500 g/head/day) in their feedlot diets for a 119-day period
are listed in Table 8. Steers fed the control diet had an average daily
gain of 0.80 kg/head, followed by 0.77 kg/head for steers receiving
250 g/head daily, and 0.72 kg/head for steers receiving the 500 g/head
daily. There were no differences among treatments in average daily gain.
Steers receiving the 250 and 500 g/head daily in their diets were
2.4 and 6.7% less efficient in converting feed to gain than the control
steers (Table 8). The high level of DPS slightly depressed feed intake.
On a dry matter basis, the diets containing 250 and 500 g/head daily
contained 3.3 and 6.8% of DPS. Studies at Maryland indicated that as
much as 5 to 6% of the diet, on a dry matter basis, of animals grazing
liquid sludge-treated pastures could consist of sludge adhering to edible
plant surfaces (A. M. Decker, personal communication). This amount would
depend upon the prevalence of rain following each application.
There were no differences among treatments as far as any of the
carcass parameters were concerned.
Concentrations of selected metals in feces are shown in Table 9.
There were no differences among treatments. However, significant differ-
ences existed for metals in fecal material. Metal concentrations increased
as the level of DPS incorporated into the diets increased. Excretion in
the feces played an important part in the elimination of excessive amounts
of metals in the diets.
31

-------
Concentrations of selected metals in liver and kidney tissues are
shown in Table 10. The cobalt (Co), copper (Cu), nickel (Ni), and zinc
(Zn) concentrations in liver tissues were significantly lower for steers
receiving the high level of DPS. The high level of DPS may have had a
detrimental effect on the liver storage mechanism for these metals.
However, the lead (Pb) concentrations were significantly higher in the
livers of steers receiving the high level of DPS. Cobalt was the only
metal significantly higher in muscles of steers receiving DPS than in
control animals. Lead levels increased in kidney tissues of DPS-treated
animals.
32

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TABLE 6. CONCENTRATE
SUPPLEMENT MIXTURE3

Ingredients
International
Reference
Number
Percent
Soybean (Glycine max) - seeds,
solvent extracted, ground, 44%
5-04-604
60.5
Urea - 45% N

12.6
Phosphate - defluorinated, ground
6-01-780
17.9
Trace mineral salt'5

9.0
Vitamin A palmitatec
7-05-143
+
a As-fed basis.
k Contained not less than 0.350% Zn, 0.340% Fe, 0.200% Mn, 0.033% Cu,
0.007% I, and 0.005% Co.
Supplied 64,500 IU/kg of concentrate supplement mixture.
33

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TABLE 7. CONCENTRATIONS OF SELECTED METALS IN DPS AND
THE FEED INGREDIENTS IN THE FEEDLOT DIETS
Pensacola Ground Corn Concentrate
Metals	sludge	corn	silage	supplement mixture
	 Ug/gb		 	
Cd
7
<.025
<.025
2.88
Co
10
o
rH
V
o
t-H
V
12.00
Cr
192
< .10
o
r-H
V
15.90
Cu
309
1.25
3.00
25.52
Hg
5
__c
—
—
Ni
20
< .10
< .10
10.50
Pb
193
< .20
A
ro
o
4.00
Zn
2570
21.5
18.4
283.8
a Solids averaged 3.3%.
i-
Dry matter basis.
c Not analyzed.

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TABLE 8. PERFORMANCE AND CARCASS DATA WITH BEEF STEERS RECEIVING
VARIOUS LEVELS OF DPS IN FEEDLOT DIETSa
Treatments (g/head/day)b
Item
0
250
500
Number of animals
8C
8
8
Length of trial, days
119
119
119
Average initial weight, kg
288
292
290
Average final weight, kg
383
384
376
Average daily gain, kg
0.80 + 0.16
0.77 + 0.07
0.72 + 0.21
Feed/gain ratio
16.5
16.9
17.6
Feed/animal/day, kg
13.2
13.2
12.7
Average carcass quality grade^
15.5 + 3.3
14.5 + 2.3
15.4 + 2.7
Average carcass yield grade
2.8 + 0.4
2.7 + 0.5
2.7 + 0.4
Data presented as means + SD where appropriate for 8 observations.
k Grams per head daily of dried Pensacola liquid digested sludge incorporated
into the diet.
c
Two groups of 4 steers each.
14 = average good, 15 = high good, 16 = low choice.

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TABLE 9. METALS IN FECES9
	Treatments (q/head/day)
250
0
500
Number of observations	24	19	23
Metals in feces	_______ yg/g (dry matter basis) - - - ¦
Cd
0.72f
+
0.28
1.939 + 0.77
2.43h + 0.66
Co
2.53c
+
0.65
3.71d + 1.18
3.75d + 1.18
Cr
5.54f
+
2.05
30.899 + 8.83
43.73h + 13.44
Cu
12.97f
+
3.02
58.919 + 15.24
90.00h + 20.19
Hg


.01f
0.689 ± 0.48
1.48h + 0.63
Ni
3.77
+
1.65
7.95 + 4.51
9.11 + 3.93
Pb
2.75°
+
3.36
45.57d + 19.23
63.27e + 43.63
Zn
96.8f
+
29.9
250.79 + 54.1
345.5h + 87.2






Data presented as means + SD where appropriate.
Grams per head daily of dried Pensacola liquid digested sludge incorporated
into the diet.
n A a
' ' Means in a row with different superscripts differ significantly (P < 0.05).
¦f rt h
'9' Means in a row with different superscripts differ significantly (P < 0.01).

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TABLE 10. CONCENTRATIONS OF SELECTED METALS IN LIVER AND KIDNEY TISSUES'
Treatments (g/head/day)*3



0

250
500
Number of observations

8

8
8
Metals
in liver tissues



- - yg/gc


Cd
0.14
± 0.04

0.20 + 0.06
0.16 + 0.04

Co
0.359
+ 0.01

0.36g ± 0.03
0.14f + 0.02

Cr

< .10

< .10
< .10

Cu
12.67d,e
± 9.06

17.07e + 5.88
8.34d + 4.24

Hg

< .01

< .01
< .01

Ni
0.32 9
+ 0.09

0.299 + 0.11
0.14f ± 0.02

Pb
0.27f
± 0.19

0.26f + 0.16
0.589 ± 0.07

Zn
44.019 + 11.10

44.919 + 7.74
18.00f + 11.90
Metals
in kidney tissues






Cd
0.27
+ 0.10

0.38 + 0.08
0.40 + 0.09

Co
0.24
± 0.13

0.13 + 0.09
0.20 ± 0.10

Cr

< .10

< .10
< .10

Cu
3.83
+ 0.60

4.03 + 0.26
3.79 + 0.48

Hg

< .01

< .01
< .01

Ni

< .10

< .13 + 0.08
< .16 + 0.11

Pb
1.40d
± 0.14

1.95e + 0.49
1.66d'e + 0.32

In
18.79
+ 2.22

19.66 + 0.97
19.09 + 2.16
Data presented as means + SD where appropriate,
porated into the diet. c Fresh tissue basis. ®
superscripts differ significantly (P < 0.05). f
superscripts differ significantly (P < 0.01).
b
,e
>g
Grams per head daily of DPS incor-
Means in a row with different
Means in a row with different

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D. DRIED CHICAGO SLUDGE AND CORN FROM SOIL FERTILIZED WITH
LIQUID PENSACOLA SLUDGE IN DIETS OF BEEF STEERS
ABSTRACT
Twenty-four steers were randomly allotted and fed 1 of 3 diets for 141
days to determine the effects of digested municipal sludges on animal per-
formance, carcass quality, and concentrations of selected metals in liver,
muscle, and kidney tissues. The experimental diets consisted of (1) con-
trol corn diet, (2) "spiked" corn diet - control com diet plus 500 grams
(g) per head daily of dried Chicago digested sludge (DCS) high in cadmium
(Cd), and (3) LPS corn diet - corn produced from soil fertilized with sur-
face applications totaling 19.8 metric tons/hectare (t/ha) of Pensacola
liquid digested sludge (LPS) prior to planting.
Inclusion of DCS in the diet or corn grown from soil fertilized with
LPS had no effect on growth performance nor carcass quality measurements
with beef steers. No significant differences in concentrations of selected
metals were detected in livers and kidneys of steers fed the control corn
diet and the LPS corn diet. The Cd, copper (Cu), iron (Fe), and lead (Pb)
concentrations in livers were higher (P < 0.01 for Cd, Cu, and Fe;
P < 0.05 for Pb) with steers fed the "spiked" corn diet. Accumulations
(P < 0.01) of Cd, Fe, mercury (Hg), and Pb occurred in kidneys of steers
fed the "spiked" corn diet compared with the other 2 experimental diets.
Higher (P < 0.01) Cd concentrations in livers and kidneys of steers
were observed from feeding the diet containing DCS. Since Cd exposure can
cause kidney damage, the Cd content of a sewage sludge could determine the
amount that may be safely applied to agricultural land.
INTRODUCTION
The purpose of this study was to determine the effects of dried
Chicago digested sludge (DCS), high in Cd, and corn produced on soil
fertilized with Pensacola liquid digested sludge (LPS) in feedlot diets of
beef steers on animal performance, carcass quality, and concentrations of
selected metals in liver, muscle, and kidney tissues.
EXPERIMENTAL PROCEDURES
Twenty-four beef steers, average 299 kilograms (kg), of British
breeding (Angus and Angus X Hereford crosses) were allotted at random
from breed groups to 6 experimental groups of 4 steers each. The 6
experimental groups, utilizing 2 groups (replicates) per treatment, were
assigned to 3 corn diets: (1) control corn, dent yellow (Zea mays indentata)
38

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- grain, gr 2 US, International Reference No. 4-02-931; (2) control corn
plus 500 g per head daily of DCS incorporated into the diet prior to feeding;
and (3) corn produced from soil fertilized with surface applications of LPS
totaling the equivalent of 19.8 t/ha of dry material prior to planting. The
sun-dried sludge was plowed into the soil prior to planting.
The 3 corn diets each contained 35% sorghum (Sorghum vulgare) - aerial
part with head, ensiled, mature, International Reference No. 3-04-322
(36.7% dry matter), 61% ground corn (88.3% dry matter), and 4% concentrate
supplement mixture (90.9% dry matter) on an as-fed basis. These diets were
18% sorghum silage, 77% ground corn, and 5% concentrate supplement mixture
on a dry matter basis. The ingredients of the concentrate supplement mix-
ture are listed in Table 11.
Individual animal weights were obtained at the beginning and end of
the experiment after an overnight shrink. Group weights were obtained
every 28 days during the period. These weights were obtained in order to
periodically check on the performance of the steers on each of the 3 diets.
Blood, fecal, feed ingredient, and sludge samples were collected each time
the steers were weighed. One steer had to be removed from one of the
control groups during the course of the experiment due to sickness; the
data for that steer were not used in analyses of the results.
The steers were fed ad libitum twice daily for a total of 141 days
(March 23 to August 11, 1977). The amounts of feed for each diet were
recorded at each feeding. The DCS, obtained from a commercial source,
was incorporated into the appropriate diet at feeding time. The DCS
contained 6.4% moisture.
At the end of the experimental period, the steers were slaughtered at
the University of Florida Meats Laboratory, Gainesville. Liver, muscle,
and kidney tissues were collected at slaughter. Carcass measurements were
obtained.
The DCS, LPS, feed ingredients, blood, fecal, and tissue samples were
sent to the Analytical Research Laboratory, Soil Science Department, Uni-
versity of Florida, Gainesville, for metal analyses. Concentrations of
selected metals in the sludges and feed ingredients are listed in Table 12.
Analyses of variance for animal performance and carcass quality data
were conducted according to the method of Snedecor (1946). The computer
package (Statistical Analysis System, North Carolina State University) out-
lined by Barr et at. (1976) was utilized for the statistical analyses of
the metal data in blood, feces, and tissues.
RESULTS AND DISCUSSION
Performance and carcass data with beef steers fed 3 experimental diets
for a 141 day feeding period are shown in Table 13. Steers fed the LPS
corn diet had an average daily gain of 1.04 kg. This compared to an average
39

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daily gain of 0.98 and 0.91 kg with steers fed the control corn diet and
the diet containing DCS. There were no significant differences among
treatments in average daily gain.
Steers fed the LPS corn diet were 8.8 and 15.7% more efficient in
converting feed to gain than those fed the control corn and "spiked" corn
diets (Table 13). The "spiked" corn diet did not appear unpalatable as
evidenced by similar feed intakes.
There were no differences among treatments for any of the carcass
quality measurements (Table 13).
The DCS was considerably higher in a number of the metals, Cd, chromium
(Cr), copper (Cu), iron (Fe), nickel (Ni), and lead (Pb), than dried LPS
(Table 12). The Cd concentration in the concentrate supplement mixture,
probably supplied by commercial calcium phosphate, was also higher than
expected. Thus, the "spiked" corn diet contained a substantial amount of
Cd.
Table 14 shows the concentrations of selected metals in liver and kidney
tissues. The Cd, Cu, Fe, and Pb concentrations in livers were higher
(P < 0.01 for Cd, Cu, and Fe; P < 0.05 for Pb) from steers fed the "spiked"
corn diet than respective metal concentrations in livers of steers fed the
control corn diet or the LPS corn diet. This was contrary to results
reported by Bertrand et at. (1978), which showed that a high level of DPS
in the diet reduced the liver storage of cobalt (Co), Cu, Ni, and zinc (Zn).
However, the 2 sludges differed considerably in concentrations and ratios
of most of the metals. This may account for the differences in results
obtained for the 2 experiments.
The higher (P < 0.01) Cd content in livers and kidneys of steers fed
the "spiked" corn diet when compared with the other 2 diets was a matter
of concern. Ryan (1978) reported that Cd tended to accumulate in visceral
organs (liver, kidney, and pancreas). Renal tubular damage was the most
important chronic effect of Cd exposure. A high concentration of Cd in
the soil can cause an increased concentration of Cd in crops (Chaney and
Hornick, 1978). Also, livestock grazing pasture could ingest a sludge
adhering to edible plant surfaces. Therefore, the Cd concentration of
a sewage sludge could be one of the limiting factors in determining the
amount of the material that can be safely applied to agricultural land
for the production of food and feed crops.
40

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TABLE 11. CONCENTRATE SUPPLEMENT MIXTURE

Inter-
national
Ref. No.
Percent
Soybeans (Glycine max) - seeds, solvent-
extracted, ground, 44%
5-04-604
61.2
Urea - 45% N

11.2
Calcium phosphate -
dibasic, commerical
6-01-080
7.5
Limestone - ground, mn 33% calcium
6-02-632
11.2
Trace mineral salt9

8.9
Vitamin A palmitate'3
7-05-143
+
a Contained not less than 0.350% Zn, 0.340% Fe, 0.200% Mn, 0.033% Cu, 0.007% I,
and 0.005% Co.
k Supplied 66,120 IU/kg of concentrate supplement mixture.

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TABLE 12. CONCENTRATIONS OF SELECTED METALS IN DRIED CHICAGO DIGESTED SLUDGE, PENSACOLA LIQUID
DIGESTED SLUDGE, AND FEED INGREDIENTS IN THE EXPERIMENTAL DIETS
Pensacola
Chicago liquid Control LPS, Sorghum Concentrate
Metals	siudge	siudge	corn	corn	silage	supplement mixture
		 yg/gC 		
Cd
163
7
<.025
<.025
<.025
1.63
Co
22
10
< .10
< .10
< .10
7.10
Cr
2,888
192
A
h—4
O
A
I—1
O
< .10
9.63
Cu
1,365
309
1.25
2.50
3.75
19.89
Fe
37,267
3,000
236
214
622
482
Hg
2
5
__d
—
—
--
Ni
376
20
< .10
< .10
o
V
7.13
Pb
774
193
A
ro
o
A
ro
o
A
ro
o
2.20
Zn
2,501
2,570
21.5
29.5
23.8
198.5
a Solids averaged 3.3%.
k Corn produced on soil fertilized with surface applications totaling 19.8 t/ha of Pensacola
liquid digested sludge prior to planting.
c Dry matter basis.
^ Not analyzed.

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TABLE 13. PERFORMANCE AND CARCASS DATA WITH BEEF STEERS RECEIVING DRIED
CHICAGO DIGESTED SLUDGE AND CORN PRODUCED FROM SOIL FERTILIZED
WITH PENSACOLA LIQUID DIGESTED SLUDGE IN FEEDLOT DIETS9
"Spiked"	LPS
Item	Control	corn diet" corn diet
J
Number of animals
7
8
8
Length of trial, days
141
141
141
Average initial weight,
kg
292
301
303
Average final weight,
kg
430
429
449
Average daily gain, kg
0.98 + 0.18
0.91 + 0.07
1.04 + 0.20
Feed/gain ratio
11.1
11.8
10.2
Average carcass quality
gradee
17.0 + 1.3
15.9 + 1.7
16.1 ± 1.5
Average carcass yield
grade
3.4 + 0.4
2.8 + 0.5
3.2 ± 0.6
a Data presented as means + SD for 7 or 8 observations where appro-
priate.
k Control corn plus 500 g/head/day of DCS incorporated into the diet.
c Diet containing corn produced from soil fertilized with surface
applications totaling 19.8 t/ha prior to planting.
d Two groups of 4 steers each.
e 15 = high good, 16 = low choice, 17 = average choice, 18 = high choice.
43

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E. FORAGE SORGHUM SILAGES GROWN ON SOIL TREATED WITH LIQUID
DIGESTED SLUDGE AND FED TO BEEF STEERS
ABSTRACT
Processed sewage sludges have potential as fertilizers on agricultural
land. Forage sorghum (Sorghum bisolor (L.) Moench) silages, grown on soil
treated with Pensacola liquid digested sludge (LPS) turned under prior to
planting, were fed as the main ingredient in the diet of beef steers to
determine the effects on animal performance, carcass quality, and concen-
trations of selected potentially toxic metals in liver, muscle, and kidney
tissues. The experimental diets consisted of: (1) control (normal ferti-
lization), (2) LPS #1, i.e., 40.4 metric tons/ha of LPS, and (3) LPS #2,
i.e., 110.6 metric tons/ha of LPS. Inclusion of silages grown on soil
treated with LPS in the diets had no effect on animal performance and car-
cass quality measurements of beef steers. The cadmium (Cd) concentrations
were lower (P < 0.05) in the livers of steers fed the LPS #1 diet. The
reason for this was not apparent. However, the concentrations of copper
(Cu) and iron (Fe) in livers of steers fed the LPS diets were lower (P <
0.01) than those of steers fed the control diet. This indicated that the
metals in LPS, even when the material was used as a fertilizer for growing
silage, were accumulated to some extent in the sorghum plants and had a
detrimental effect at the absorption site and/or at the liver storage
site for Cu and Fe. There were no differences among treatments in the
concentrations of selected metals in muscle and kidney tissues.
INTRODUCTION
Forage sorghum (Sorghum hioolor (L.) Moench) silage, properly supple-
mented, is a feed with good potential for growing calves or finishing
steers in northwest Florida (Bertrand et al., 1974; Bertrand et al., 1975).
Sorghum, especially the forage varieties from which two harvests (a first
crop and a ratoon crop) per growing season can normally be obtained, can
produce substantially more forage per unit of land than corn (zea mays L.)
(Dunavin, 1973).
Sludges have potential as a fertilizer on cropland (Hammond, 1974;
Lutrick and Bertrand, 1974). Performance was not affected when crops grown
on land receiving applications of digested sewage sludge were fed to live-
stock (Hartman, 1975; USEPA, 1972). However, metal contaminants contained
in sludges can accumulate in plant tissues (Chaney et al., 1978). This
could be of concern from a human health standpoint when human and industrial
wastes are applied to land intended for the production of forage for con-
sumption by food animals (CAST, 1976).
44

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The purpose of this study was to evaluate the effects of forage sorghum
silages, grown on soil treated with various levels of Pensacola liquid
digested sludge (LPS) prior to planting, in diets of beef steers on per-
formance, carcass quality, and concentrations of selected potentially toxic
metals in liver, muscle, and kidney tissues.
MATERIALS AND METHODS
Twenty-four steers (average 178 kg) were weighed and allotted at
random from breed groups to 6 experimental groups of 4 steers each. The 6
experimental groups, utilizing 2 groups (replicates) per treatment, were
assigned to the 3 following treatments:
1.	Control - diet containing	forage sorghum silage grown on soil
fertilized with 448 kg/ha	of 8-24-24 fertilizer at planting
time and sidedressed with	224 kg/ha of ammonium nitrate at the
first cultivation.
2.	LPS #1 - diet containing forage sorghum silage grown on soil
treated with surface applications totaling 15 cm/ha (39 metric
tons/ha of dry material) of LPS turned under prior to planting.
3.	LPS #2 - diet containing forage sorghum silage grown on soil
treated with surface applications totaling 22.5 cm/ha (59 metric
tons/ha of dry material) of LPS turned under prior to planting.
The feedlot diets consisted of 95% forage sorghum silage (31% dry
matter) and 5% concentrate supplement mixture {89% dry matter) on an as-fed
basis. These diets were 87% forage sorghum silage and 13% concentrate
supplement mixture on a dry matter basis. The ingredients of the concen-
trate supplement mixture are listed in Table 15.
After an overnight shrink, individual animal weights were obtained at
the beginning and end of the experiment. Group weights were obtained every
28 days during the experiment. These weights were obtained in order to
periodically check on the performance of the steers on each of the 3
diets. Blood, fecal, and feed ingredient samples were collected each time
the steers were weighed.
The trial was initiated on February 22 and terminated on August 9, 1978
(168 days). The steers were fed ad libitum once daily. The amounts of feed
for each diet were recorded at each feeding.
At the end of the feeding period, the steers were slaughtered at the
University of Florida Meats Laboratory, Gainesville. Liver, muscle, and
kidney tissues were collected at slaughter. Carcass measurements were
obtained by trained personnel at the Meats Laboratory.
The LPS, feed ingredients, blood, fecal, and tissue samples were sent
to the Analytical Research Laboratory, University of Florida, Gainesville
for metal analyses. Samples were measured by volume (blood) or weight,
45

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dried where appropriate (feed ingredients, LPS, and feces), wet-ashed,
dissolved in 0.2% nitric acid, and determinations were made with an emission
or absorption flame spectrophotometer. Concentrations of selected metals
in the sludge and feed ingredients are listed in Table 16.
Analyses of variance for animal performance and carcass quality data
were conducted according to the method of Snedecor (1946). The metal
concentration data for blood, feces, and tissues (liver, muscle, and kidney)
between treatments were tested for significance of variances by the SAS
computer method (Statistical Analysis System, North Carolina State Uni-
versity) (Barret at., 1976).
RESULTS AND DISCUSSION
Performance and carcass data of steers receiving the 3 forage sorghum
silage diets for the 168 day period are shown in Table 17. Steers fed the
control diet had an average daily gain of 0.55 kg per head, followed by the
average daily (0.54 kg/head) of steers fed the LPS #2 diet and the average
daily gain (0.45 kg/head) of steers fed the LPS #1 diet. There were
no significant differences among treatments in average daily gain. This
was probably due to the limited amount of observations and the large varia-
tion in individual gain within treatment groups. Steers receiving the
control diet were 22.4 and 2.4% more efficient in converting feed to gain
than those receiving the LPS #1 and LPS #2 diets, respectively. Feed
consumption was not affected by the LPS treatments. The average carcass
yield grade was lower (P < 0.05) for steers receiving the control diet
than that of steers receiving the LPS diets. This was probably due to
chance and did not represent true treatment differences. There were no
differences among treatments as far as any of the other carcass parameters
were concerned.
Concentrations of selected metals in blood and feces are listed in
Table 18. The copper (Cu) and iron (Fe) concentrations in blood of steers
fed the LPS diets were lower (P < 0.01) than those of steers fed the
control diet. Earlier studies with dried LPS fed in the diets of feedlot
steers also showed slightly lower concentrations of Cu in the blood of
steers receiving LPS (Bertrand et al., 1978). Underwood (1971) stated
that interactions exist between metals, such as Fe, zinc (Zn), cadmium
(Cd), and Cu, which interfere with absorption. Apparently, these metals
compete for protein-binding sites in the intestinal mucosa. This may
account for the lower concentrations of Cu and Fe in blood of steers
consuming forage sorghum silages grown on soil treated with LPS. The Cd,
Cu, and Zn concentrations in feces of steers fed the LPS diets were higher
(P < 0.05 for Cu; P < 0.01 for Cd and Zn) than those of steers fed the
control diet. These results were not in complete agreement with the con-
centrations of the metals in the feed ingredients (Table 16).
Concentrations of selected metals in liver and kidney tissues are
shown in Table 17. The Cd concentrations were lower (P <0.05) in livers
of steers receiving the LPS #1 diet. The reason for this was not apparent.
However, the concentrations of Cu and Fe in livers of steers fed the LPS
diets were lower (P < 0.01) than those of steers fed the control diet.
46

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This agreed with previous studies with steers fed diets containing LPS
(Bertrand et at.* 1978). This again indicated that the metals in LPS,
even when the material was used as a fertilizer for growing the silages,
were accumulated to some extent in the sorghum plants and had a detrimental
effect at the absorption site and/or at the liver storage site for Cu and
Fe. There were no differences among treatments in the concentrations of the
selected metals in kidney tissues (Table 19).
47

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TABLE 14. CONCENTRATIONS OF SELECTED METALS IN LIVER AND MUSCLE TISSUES3
Control	"Spiked"^	LPS
	corn diet	corn diet	corn diet
Number of observations	7	8	8
Metals in liver tissues	- — ______	— - - ug/g^ - — ________
Cd	< .079 + 0.03	3.50h + 1.10	< .05g + 0.01
Cu	16.619 + 8.39	40.72h + 20.77	18.099 + 3.62
Fe	87.639 + 19.50	262.66h + 135.61	93.819 + 18.37
Pb	< .10®	< .26f +0.24	< .10e
Metals in kidney tissues
Cd	0.279 + 0.06	13.81h + 3.29	0.339 + 0.11
Cu	4.59 + 0.86	4.98 + 0.64	4.59 + 0.46
Fe	81.039 + 15.20	131.25h + 28.33	77.28g + 6.57
Pb	< .109	0.72h + 0.22	< .139 + 0.08
a Data presented as means + SD where appropriate.
^ Control corn plus 500 g/head/day of DCS incorporated into the diet.
c Diet containing corn produced from soil fertilized with surface applications totaling
19.8 t/ha.
j
Fresh tissue basis.

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Table 14.	(Continued)
e'^ Means	in a row with different superscripts differ significantly (P < 0.05).
9''1 Means	in a row with different superscripts differ significantly (P < 0.01).
KO

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TABLE 15. COMPOSITION OF THE CONCENTRATE SUPPLEMENT MIXTURE
Ingredients
Percentage
Soybean meal (44% protein)
82.3
Urea - 45% N
6.4
Dicalci um/monocal ci um phosphate
7.5
Salt (trace-mineralized)9
3.8
Vitamin A supplement*3
+
a Contained not less than 0.350% Zn, 0.340% Fe, 0.200% Mn,
0.033% Cu, 0.007% I, and 0.005% Co.
Rovimix A-650 (vitamin A supplement containing 650,000
IU/g) added at the level of 22.05 million IU/metric ton
or 22,050 IU/kg of concentrate supplement mixture.
50

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TABLE 16. CONCENTRATIONS OF SELECTED METALS IN PENSACOLA LIQUID DIGESTED
SLUDGE AND FEED INGREDIENTS IN THE EXPERIMENTAL DIETS
Pensacola Control	LPS	LPS	Concentrate
a	bC
Metals	siudge	silage	#1 silage	#2 silage	supplement mixture
	 (pg/g dry wt) 	
Cd
12
<0.17
0.25
0.31
0.38
Co
68
< 0.99
1.56
< 0.10
2.19
Cr
282
2.19
< 2.21
< 0.68
2.81
Cu
5 87
9.94
7.88
5.82
14.25
Fe
5,416
782
1,188
1,019
641
Ni
51
2.19
< 0.39
< 0.39
3.75
Pb
526
< 0.39
< 0.10
< 0.39
< 0.39
Zn
3,393
63.13
47.81
79.06
113.13
a Solids averaged 2.6%
Forage sorghum silage grown on soil treated with surface applications totaling 15 cm/ha of
Pensacola liquid digested sludge prior to planting. Each feed ingredient mean was based on
4 observations.
c Forage sorghum silage grown on soil treated with surface applications totaling 22.5 cm/ha
of Pensacola liquid digested sludge prior to planting.

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TABLE 17. PERFORMANCE AND CARCASS DATA OF BEEF STEERS RECEIVING DIETS CONTAINING FORAGE SORGHUM
SILAGES GROWN ON SOIL TREATED WITH VARIOUS LEVELS
OF PENSACOLA LIQUID DIGESTED SLUDGE PRIOR TO PLANTING3
	Item	Control diet	 	LPS #1 dietb	LPS #2 dietc
Number of animals
8d
8
8
Length of trial, days
168
168
168
Average initial wt, kg
176
179
179
Average final wt, kg
269
254
269
Average daily gain, kg
0.55 + 0.18
0.45 + 0.08
0.54 + 0.06
Feed/gain ratio
28.6
35.0
29.3
Average carcass yield grade
1.^+ 0.2
1.9f + 0.1
1.8f + 0.2
Average dressing percent
53.2 + 1.8
53.1 + 1.6
53.9 + 4.7
a Data presented as means + SD where appropriate for 8 observations.
k Diet containing forage sorghum silage grown on soil treated with surface applications totaling
15 cm/ha of LPS prior to planting.
c Diet containing forage sorghum silage grown on soil treated with surface applications totaling
22.5 cm/ha LPS prior to planting.
j
Two groups of 4 steers each.
e'^Means in a row with different superscripts differ significantly (P < 0.05).

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TABLE 18. CONCENTRATIONS OF SELECTED METALS IN BLOOD AND FECES9
	Treatments 	
Metals	Control diet	LPS #1 diet*3	LPS #2 diet^
No. of observations	40	40	40
____________ (Blood, g/ml) - -- -- -- -- -- --
Cu	0.829 + 0.14	0.52f + 0.22	0.59f + 0.23
Fe	4829 + 65	458f'g + 76	419f + 62
Pb	<0.10	< 0.10	< 0.10
Zn	4.18 + 0.51	3.91 + 0.61	3.96 + 0.92
No. of observations	34	33	31
	 (Feces, g/g dry wt) 	
Cd	0.29f + 0.19	0.569 + 0.28	0.54g+0.19
Co	1.00 + 1.06	1.32 + 0.80	1.78 + 1.59
Cr	4.58 + 2.97	4.02 + 1.55	3.77 + 1.73
Cu	14.57d + 7.52	16.28e + 4.63	17.lle + 5.12
Fe	2,947 + 860	2,994 + 823	2,705 + 957
Pb	2.27 + 1.35	1.88 + 1.44	1.84 + 1.96
Zn	96.9f + 33.0	205.49 + 54.0	224.5g+90.5
3 Data presented as means + SD where appropriate.

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TABLE 18. Continued
k Diet containing forage sorghum silage grown on soil treated with surface applications totaling
15 cm/ha of LPS prior to planting.
c Diet containing forage sorghum silage grown on soil treated with surface applications totaling
22.5 cm/ha of LPS prior to planting.
d 0
' Means in a row with different superscripts differ significantly (P < 0.05).
*F Q
'9 Means in a row with different superscripts differ significantly (P < 0.01).

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TABLE 19.
Metals
CONCENTRATIONS OF SELECTED METALS IN LIVER AND KIDNEY TISSUES3
	Treatments	
Control diet	LPS #1 diet'3	LPS #2 diet0
No. of observations 8	8	8
-	- - - 		(Liver, yg/g wet wt)	-- — - - - -
Cd 0.12e + 0.01	0.08d + 0.01	0.11e+0.03
Cu 8.619 + 6.27	2.11f + 0.42	3.14f + 1.61
Fe 368g + 111	3509 + 120	185f + 73
-	- - — - -	(.Kidney, yg/g wet wt)	--------
Cd 0.46 + 0.18	0.30 + 0.11	0.40 + 0.24
Cu 3.23 + 0.32	2.90 + 0.35	3.20 + 0.60
Fe 113.9 + 11.0	96.9 + 31.5	89.3 + 17.7
Ni 17.9 + 1.6	18.0 + 1.8	17.6 + 1.3
a
Data presented as means + SD where appropriate for 8 observations.
^ Diet containing forage sorghum silage grown on soil treated with surface
applications totaling 15 cm/ha of LPS prior to planting.
c Diet containing forage sorghum silage grown on soil treated with surface
applications totaling 22.5 cm/ha f LPS prior to planting.
d g
' Means in a row with different superscripts differ significantly
(P < 0.05).
f Q
'9 Means in a row with different superscripts differ significantly
(P < 0.01).
55

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F. PENSACOLA BAHIAGRASS PASTURES FERTILIZED WITH PENSACOLA
LIQUID DIGESTED SLUDGE AND GRAZED BY BEEF STEERS
ABSTRACT
Forty-eight beef steers were randomly allotted and grazed 1 of 3
Pensacola bahiagrass pastures for 168 days to determine the effects of
pasture treatments with Pensacola liquid digested sludge (LPS) on animal
performance, carcass quality, and concentrations of selected potentially
toxic metals in liver, muscle, and kidney tissues. The experimental pas-
tures consisted of: (1) control, i.e., normal fertilization, (2) LPS #1,
i.e., a total of 16 metric tons/ha of LPS, and (3) LPS #2, i.e., a total of
32 metric tons/ha of LPS. There were no differences among treatments for
any of the animal performance and carcass parameters. The copper (Cu)
and iron (Fe) concentrations in blood of steers grazing the LPS pastures
were lower (P < 0.05) than those of steers grazing control pastures.
Significant differences existed among treatments with respect to most of
the metals in fecal material. Metal concentrations in fecal material
increased as the level of LPS applied to pasture was increased. The Cu
concentrations in liver tissues were lower (P < 0.01) for steers grazing
the LPS pastures. There were no differences among treatments in the con-
centrations of selected metals in muscle tissues. The estimated sludge
content on the grazed forages for the LPS #1 and LPS #2 pastures was 2.21
and 4.74%, respectively.
INTRODUCTION
Warm-season perennial grass pastures, such as Pensacola bahiagrass
(Paspalum notation, Flugge), provide a long grazing period and relatively
good gains with long yearlings in northwest Florida (Bertrand and Dunavin,
1968). These pastures appear to be an ideal year-round site for the
disposal of processed sewage sludges.
Sludges have a potential as a fertilizer on cropland (Hammond, 1974;
Lutrick and Bertrand, 1974). Cattle grazing siudge-treated pastures could
ingest metal contaminants contained in the sludges and accumulated in plant
tissues or sludge adhering to edible plant surfaces (Chaney et al., 1978;
Fitzgerald, 1977). This could be of concern from a human health standpoint
when human and industrial wastes are applied on pasture land intended for
grazing by food animals (CAST, 1976). However, Kienholz et al. (1976),
reported that aged cows grazed siudge-treated pastures for 5 years and
found no marked differences in heavy metal concentrations in their tissues
when compared to control cows.
56

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The purpose of this study was to determine the effects of Pensacola
liquid digested sludge (LPS) applied to Pensacola bahiagrass pastures
grazed with beef steers on animal performance, carcass quality, and con-
centrations of selected potentially toxic metals in liver, muscle, and
kidney tissues.
EXPERIMENTAL PROCEDURE
Forty-eight beef steers (average 289 kg) of mixed breeding were
weighed and allotted at random to 6 experimental groups of 8 steers each.
The 6 experimental groups, utilizing 2 groups (replicates) per treatment,
were assigned to 3 pasture treatments as follows: (1) control - rotational
grazing of Pensacola bahiagrass pastures treated with 280 kg/ha of 8-24-24
fertilizer prior to grazing and 5 applications of 112 kg/ha each of
ammonium nitrate during the grazing season, (2) LPS #1 - same pastures as
above treated with 7.5 cm/ha (16 metric tons/ha of dry material) of LPS
applied at the rate of 3.75 cm/ha during the winter prior to grazing and
3.75 cm/ha during the grazing season, along with 112 kg/ha of muriate of
potash prior to grazing, and (3) LPS #2 - same pastures as above treated
with 15 cm/ha (32 metric tons/ha of dry material) of LPS applied at the
rate of 7.5 cm/ha during the winter prior to grazing and 7.5 cm/ha during
the grazing season, along with 112 kg/ha of muriate of potash prior to
grazi ng.
Each pasture replicate was subdivided into 2 1.0 ha plots which were
grazed rotationally by 8 steers initially. The trial was initiated on
May 23, 1979, and terminated on November 7, 1979 (168 days).
After an overnight shrink, individual animal weights were obtained at
the beginning and end of the trial period. Group weights were obtained
every 28 days during the experiment to periodically check on performance
of the steers on each of the 3 pasture treatments. Blood, fecal, forage,
and LPS samples were collected each time the steers were weighed. The
blood and fecal samples were collected from the same 4 steers each time
from each experimental group of 8 steers. Additional grazer animals of
the same type and size were added and removed as needed to keep the forages
uniformly grazed. Each experimental group of steers was rotated between
the 2 pasture plots assigned to it as required for best utilization of
good quality forage.
The LPS was applied with a Calumet sludge spreader initially during
the winter and during the grazing season on the LPS pasture plots as soon
as possible after the steers were rotated to the other rotational plots.
Each LPS application on the soil and forage was performed at the rate of
0.83 cm/ha. Certain proximate components for each of the 3 Pensacola
bahiagrass forages are shown in Table 20.
At the end of the experimental period, the steers were slaughtered
at the University of Florida Meats Laboratory, Gainesville. Liver, muscle,
and kidney tissues from the 4 selected steers in each experimental group
were collected at slaughter. Carcass measurements were obtained by trained
personnel at the Meats Laboratory.
57

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The LPS, forage, blood, fecal, and tissue samples were sent to the
Analytical Research Laboratory, Soil Science Department, University of
Florida, Gainesville for metal analyses. Samples were measured by volume
(blood) or weight, dried where appropriate (forages and feces), wet-ashed,
dissolved in 0.2% nitric acid and determinations were made with an emission
or absorption flame spectrophotometer. Concentrations of selected metals
in LPS and forages are listed in Table 21.
Analyses of variance for animal performance and carcass quality data
were conducted according to the method of Snedecor (1946). The metal con-
centration data for blood, feces, and tissues (liver, muscle, and kidney)
between treatments were tested for significance of variance by the SAS
computer method (Statistical Analysis System, North Carolina State Univer-
sity) outlined by Barr et al. (1976).
The estimated sludge content on the grazed forages was calculated by
the method of Decker et al. (1977).
RESULTS AND DISCUSSION
Performance and carcass data of beef steers grazing the Pensacola
bahiagrass pastures for the 168 day period are shown in Table 22. Steers
grazing pastures receiving the 3 treatments had identical average daily
gains (0.30 kg). However, steers grazing the LPS pastures had more avail-
able forage and higher stocking rates than those grazing the control
pastures and thus had higher gains per hectare (235 kg for LPS #1 and
LPS #2 versus 199 kg for control). There were no differences among treat-
ments for any of the animal performance and carcass parameters.
Concentrations of selected metals in blood and feces are listed in
Table 23. The copper (Cu) and iron (Fe) concentrations in blood of steers
grazing the LPS pastures were lower (P < 0.05) than those of steers grazing
control pastures. Earlier studies with DPS fed in the diets of feedlot
steers also showed slightly lower concentrations of Cu in the blood of
steers receiving DPS (Bertrand et al.* 1978). Underwood (1971) stated
that interactions exist between metals, such as Fe, zinc (Zn), cadmium
(Cd), Cu and others, which interfere with absorption. Apparently, these
metals compete for protein-binding sites in the intestinal mucosa. This
may account for the lower concentrations of Cu and Fe in blood of steers
grazing the forages treated with LPS.
Significant differences existed among treatments with respect to most
of the metals in fecal material (Table 23). Metal concentrations in fecal
material increased as levels of LPS applied to pastures were increased.
The data indicated that excretion in the feces played an important part
in the elimination of excessive amounts of metals in the diet. This agreed
with the results obtained by Bertrand et al. (1978) where various levels
of DPS were added to feedlot diets.
Concentrations of selected metals in liver, muscle, and kidney tissues
are shown in Table 24. The Cd concentrations were higher (P < 0.05) in
livers of steers grazing the LPS pastures when compared to those obtained
58

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with steers grazing the control pastures. Bertrand et at. (1980) found
high accumulations of Cd in both liver and kidney tissues of steers fed
a sludge high in Cd. The Cu concentrations in liver tissues were lower
(P < 0.01) for steers grazing the LPS pastures. This suggested that the
other metals contained in the LPS may have a detrimental effect at the
absorption site and/or at the liver storage site for Cu. The nickel (Ni)
concentrations were higher (P < 0.05) in liver tissues of steers grazing
the LPS #1 pastures.
There were no differences among treatments in the concentrations of
selected metals in muscle tissues (Table 24). The Ni concentrations were
higher (P < 0.05J in kidney tissues of steers grazing the LPS #1 pastures.
The reason for higher concentrations of Ni in both liver and kidney tissues
of steers grazing the LPS #1 pastures was not clear.
The estimated sludge content on the grazed forages on a dry matter
basis is shown in Table 25. The estimated sludge content of 2.21% on the
grazed forages for the LPS #1 pastures agreed very closely with the
estimate of 2.18% by Decker et at. (1977); while the estimated sludge
content of 4.74% on the grazed forages for the LPS #2 pastures was some-
what higher.
59

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TABLE 20. CERTAIN PROXIMATE COMPONENTS OF
THE PENSACOLA BAHIAGRASS FORAGES9
Proximate components	Control	LPS #1^	LPS #2C
Dry matter, %
32.98
32.16
29.01
Crude protein, %
4.07
4.68
4.89
Ash, %
1.78
2.27
2.71
Crude fiber, l
9.76
9.06
7.43
a Means of 14 samples collected at intervals during the trial period.
k A total of 16 metric tons/ha of Pensacola liquid digested sludge applied
prior to and during the grazing season.
c A total of 32 metric tons/ha of Pensacola liquid digested sludge applied
prior to and during the grazing season.
60

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TABLE 21. CONCENTRATIONS OF SELECTED METALS IN PENSACOLA LIQUID
DIGESTED SLUDGE AND PENSACOLA BAHIAGRASS FORAGES
Pensacola
Liquid Control LPS #1, LPS #2
Metal s	SI udge	forage	forage	forage
	 ( g/g dry wt) 	
Cd
< 7
< .15
0.36
0.78
Co
< .10
< .38
< .38
< .66
Cr
256
2.68
6.11
9.56
Cu
473
6.30
19.00
31.74
Fe
11,838
101
466
651
Ni
93
1.95
2.24
2.70
Pb
397
< .75
11.03
20.48
Zn
2,083
27.49
79.56
145.50
a Solids averaged 2.14%.
k A total of 16 metric tons/ha of Pensacola liquid digested
sludge applied prior to and during the grazing season.
0 A total of 32 metric tons/ha of Pensacola liquid digested
sludge applied prior to and during the grazing season.
61

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TABLE 22. PERFORMANCE AND CARCASS DATA OF BEEF STEERS GRAZING PENSACOLA
BAHIAGRASS PASTURES TREATED WITH PENSACOLA LIQUID DIGESTED SLUDGE3
Initial no. of animals
Length of grazing, days
Average initial wt, kg
Average final wt, kg
Average gain/animal, kg
Average daily gain, kg
Animal days/hae
Stocking rate/hae
Gain/ha, kg
Gain/ha/day, kg
Average carcass quality grade
Average carcass yield grade
Average hot carcass wt, kg
Average dressing percentage
Treatments
Control
LPS #lb
LPS #2C
16d
16
16
168
168
168
291
286
289
341
336
340
50
50
51
0.30 + 0.12
0.30 + 0.13
0.30 + 0.09
664
783
783
4.0
4.7
4.7
199
235
235
1.18
1.40
1.40
11.18 + 1.7
11.8 + 1.1
11.9 + 1.3
2.1 + 0.5
1.9 + 0.5
1.8 + 0.5
176
176
177
51.6 + 2.6
52.4 + 2.5
52.1 + 3.4

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TABLE 22. Continued
a Data presented as means + SD where appropriate.
k A total of 16 metric tons/ha of Pensacola liquid digested sludge applied prior to and
during the grazing season.
r
A total of 32 metric tons/ha of Pensacola liquid digested sludge applied prior to and
during the grazing season.
^ Two experimental groups of 8 steers each.
e Additional grazer animals of the same type and size were added and removed as needed to
keep the forage uniformly grazed.
cr»
CO

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TABLE 23. CONCENTRATIONS OF SELECTED METALS IN BLOOD AND FECES
Treaments
Metals
Control
LPS #lb
LPS #2C
No. of observations
48
48
(Blood, yg/ml) -
48
Cd
< .01
< .01
<.01
Co
< .10
< .10
< .10
Cr
0.55 + 0.12
0.50 + 0.15
0.53 + 0.14
Cu
0.84e + 0.10
0.73d + 0.23
0.76d + 0.13
Fe
514e + 78
491d'e + 69
478d + 89
Ni
< .10
< .10
<.10
Pb
< .10
<.10
<.10
Zn
3.74 + 0.94
3.95 + 0.62
3.79 + 1.05
No. of observations
41
44
[Feces, yg/ dry wt)
44
Cd
<0.12f + 0.21
0.969 + 0.76
1.35h + 1.04
Co
< 0.53 + 0.68
<0.76 + 0.99
<0.71 + 0.91
Cr
3.79f + 3.15
9.909 + 6.90
11.44h + 10.12
Cu
13.43f + 5.18
32.149 + 19.77
38.15^ + 29.85
Fe
84ld + 551
l,129e + 667
l,142e + 843
Ni
2.15f + 2.22
4.26g + 2.28
4.399 + 2.51
Pb
3.86f + 4.61
20.929 + 21.36
23.269 + 23.03
Zn
70.97 + 33.54
234.359 + 117.80
288.53h + 177.82
64

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TABLE 23. Continued
a Data presented as means + SD where appropriate.
k A total of 16 metric tons/ha of Pensacola liquid digested sludge
applied prior to and during the grazing season.
c A total of 32 metric tons/ha of Pensacola liquid digested sludge
applied prior to and during the grazing season.
d 6
' Means in a row with different superscripts differ significantly
(P < 0.05).
Means in a row with different superscripts differ significantly
(P < 0.01).
65

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TABLE 24. CONCENTRATIONS OF SELECTED METALS IN
LIVER, MUSCLE, AND KIDNEY TISSUES3
	Treatments	
Metals	Controls	LPS #lb	LPS #2C
No. of observations 8 8 8
	 _____ (Liver, yg/g wet wt) 	 - -
Cd
0.04d
+ 0.01
0.056
+ 0.02
0.07e
+ 0.03
Co

<.10

< .10

< .10
Cr
0.73e
+ 0.02
0.72e
+ 0.01
0. 71d
+ 0.01
Cu
21.739 + 12.71
6.91f
+ 5.11
6.31f
+ 4.12
Fe
82.28 + 23.80
116.13 + 29.42
125.88 + 53.11
Ni
<0.18d
+ 0.21
<0.56e
+ 0.25

< .10'
Pb
< 0.32
+ 0.62
< 0.31
+ 0.60
< 0.49
+ 1.11
Zn
38.48
+ 5.30
38.64
+ 6.96
39.50
+ 4.52



(Muscle, yg/g wet wt)


Cd

< .01

< .01

< .01
Co

< .10

< .10

< .10
Cr
0.73
+ 0.01
0.74
+ 0.03
0.73
+ 0.02
Cu
1.03
+ 0.15
0.96
+ 0.14
0.96
+ 0.11
Fe
39.18
+ 8.33
39.45
+ 5.21
48.54
+ 6.82
Ni
1 0.37
+ 0.47
< 0.58
+ 0.36

< .10
Pb

< .10
< 0.74
+ 1.36
< 0.62
+ 1.11
Zn
45.15
+ 8.70
48.88 + 12.68
53.48
+ 7.91
66

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TABLE 24.
Continued
Treatments


Control
LPS #lb
LPS #2°
No. of observations 8
8
8


(Kidney, g/g wet wt)

Cd
0.27 + 0.10
0.28 + 0.27
0.24 + 0.08
Co
< .10
< .10
A
O
Cr
0.73 +0.02
0.73 + 0.02
0.73 + 0.01
Cu
3.91 + 0.35
3.76 + 0.36
3.71 + 0.31
Fe
87.26 + 22.06
88.90 + 18.09
84.21 + 26.86
Ni
< 0.22d + 0.23
0.64e + 0.17
< .10d
Pb
< 1.83 + 3.24
< 1.89 + 2.02
< 0.76 + 1.87
Zn
22.08 + 2.20
20.66 + 2.46
20.19 + 1.12
a Data presented as means + SD where appropriate.
k A total of 22 metric tons/ha of Pensacola liquid digested sludge applied
prior to and during the grazing season.
c A total of 44 metric tons/ha of Pensacola liquid digested sludge applied
prior to and during the grazing season.
d s
' Means in a row with different superscripts differ significantly
(P < 0.05).
¦P n
'9 Means in a row with different superscripts differ significantly
(P < 0.01).
67

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TABLE 25. ESTIMATION OF SLUDGE CONTENT ON GRAZED FORAGES3
Control	LPS #1. LPS #2,	Difference	Pensacojla	Sludge on forage
Metals forage	forage	forage	LPS#1 LPS #2	sludge	LPS #1 LPS #2
		(yg/g dry Wt)	 	{%)	
Cd <0.15	0.36	0.78	0.21 0.63	7	3.00 9.00
Co <0.38	<0.38	<0.66	-C -C	.10
Cr 2.68	6.11	9.56	3.43 6.88	256	1.34 2.69
Cu 6.30	19.00	31.74	12.70 25.44	473	2.68 5.38
Fe 101	466	651	365 550	11,838	3.08 4.65
Ni 1.95	2.24	2.70	0.29 0.75	93	0.31 0.81
Pb <0.75	11.03	20.48	10.28 19.73	397	2.59 4.97
Zn 27.49	79.56	145.50	52.07 118.01	2,083	2J50 5.67
Means	2.21 4.74
a Dry matter basis,
k Taken from Table 21.
c Not used in estimation.

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REFERENCES
1.	Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig. A User's
Guide to SAS 76. Sparks Press, Raleigh, NC, 1976.
2.	Bertrand, J. E., and L. S. Dunavin. Temporary and Permanent Summer
Pastures for Beef Heifers Fed Limited Concentrate With and Without
MGA (Melengestrol Acetate). Soil and Crop Sci. Soc. Fla. Proc.,
28:14, 1968.
3.	Bertrand, J. E., F. S. Baker, Jr., D. W. Beardsley, H. L. Chapman, Jr.,
T. J. Cunha, J. F. Hentges, Jr., and W. K. Mathis. Growing Calves in
Florida. Fla. Agr. Exp. Sta. Bull. 761, 1974.
4.	Bertrand, J. E., M. C. Lutrick, and L. S. Dunavin. Silages in Finishing
Rations for Beef Steers. Fla. Agr. Exp. Sta. Bull. 779, 1975.
5.	Bertrand, J. E., M. C. Lutrick, H. L. Breland, and R. L. West. Dried
Liquid Digested Sludge in the Ration of Feedlot Steers. Proc. First
Ann. Conf. of Applied Research and Practice on Municipal and Industrial
Waste, Madison, WI, p. 441, 1978.
6.	Bertrand, J. E., M. C. Lutrick, H. L. Breland, and R. L. West. Effects
of Dried Digested Sludge and Corn Grown on Soil Treated with Liquid
Digested Sludge on Performance, Carcass Quality, and Tissue Residues
in Beef Steers. J. Anim. Sci., 50:35, 1980.
7.	CAST. 1976. Application of Sewage Sludge to Cropland: Appraisal
of Potential Hazards of the Heavy Metals to Plants and Animals. Rep.
No. 64, Council for Agricultural Science and Technology, Ames, IA.
8.	Chaney, R. L., and S. B. Hornick. Accumulation and Effects of Cadmium
on Crops. First International Cadmium Conf. Proc., Metals Bull.,
London, p. 125, 1978.
9.	Chaney, R. L., P. T. Hundemann, W. T. Palmer, R. J. Small, M. C. White,
and A. M. Decker. Plant Accumulation of Heavy Metals and Phytotoxicity
Resulting from Utilization of Sewage Sludge and Sludge Composts on
Cropland. Proc. 1977 Nat. Conf. Composting of Municipal Residues and
Sludges, pp. 86-97.
10.	Decker, A. M., C. H. Darrah, J. R. Hall, E. Strickling, J. P. Davidson,
R. C. Hammond, S. B. Mohanty, R. L. Chaney, and J. J. Murray.
Feasibility of Using Sewage Sludge for Plant and Animal Production.
Report on Cooperative Research Project, University of Maryland and
U.S.D.A., 1977.
11.	Dunavin, L. S. Variety Trials of Summer Annual Grasses and of Forage
Sorghums for Silage. Fla. Agr. Exp. Sta. Res. Report WF73-5, 1973.
69

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12.	Fitzgerald, P. R. Recovery and Utilization of Strip-mined Land by
Application of an Aerobically Digested Sludge and Livestock Grazing.
Proc. Symposium on Municipal Waste Water and Sludge Recycling on
Forest Land and Disturbed Land. Sponsored by Pennsylvania State Uni-
versity, School of Forest Resources, Philadelphia, PA, March 21-23, 1977.
13.	Hammond, L. C. Processed Sewage Sludge as a Fertilizer in Florida.
Soil and Crop Sci. Soc. Fla. Proc., 33:135, 1974.
14.	Hartman, J. W., Jr. An Evaluation of Land Treatment of Municipal
Wastewater and Physical Siting of Facility Installations. Department
of Army Publication, 1975.
15.	Kienholz, E. W., G. M. Ward, and D. E. Johnson. Sewage Sludge Metals
in Cattle Tissues. J. Anim. Sci., 43:230 (Abstract), 1976.
16.	Lutrick, M. C., and J. E. Bertrand. Agronomic and Cattle Studies with
Municipal Liquid Digested Sludge. Institute of Environmental Sciences
Proc. 22nd Ann. Meeting, Philadelphia, PA, p. 528, 1974.
17.	Ryan, J. A. Cadmium: Utilization and Environmental Implications.
Proc. First Ann. Conf. of Applied Research and Practice on Municipal
and Industrial Waste, Madison, WI, p. 269, 1978.
18.	Snedecor, G. W. Statistical Methods (4th Ed.), Iowa State College
Press, Ames, IA, 1946.
19.	Underwood, E. J. Trace Elements in Human and Animal Nutrition.
Academic Press, Inc., New York, NY, 1971.
20.	USEPA. Irrigation of Liquid Digested Sludge - Wealth from Waste
(movie). Office of Water Programs Operations, Washington, DC, 1972.
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CATTLE - OTHER HEALTH EFFECTS
Project Participants
G.	T. Edds
C.	F. Simpson
0.	Osuna
H.	T. Nguyen
K.	E. Ferslew
R.	L. Suber
K.	L. Kelly
R.	B. Bortell
Summary of Results - 1975-1980
71

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CATTLE - OTHER HEALTH EFFECTS
G. T. Edds, C. F. Simpson, 0. Osuna, K. E. Ferslew,
R. L. Suber, and K. L. Kelly
The 1975 preliminary trial of mixing 100 g dried Pensacola sludge
(DPS) daily in the ration for 3 steers versus a regular ration for 3
control animals was utilized to determine which health parameters should
be monitored for future experiments. The cattle were bled monthly as well
as fecals samples taken to examine for level of parasite infection. The
hematocrit, red and white blood cell counts, sedimentation rates, and
parasite egg counts were determined. Differential white cell counts and
prothrombin times were done. Pasture grass samples were secured for
cyanide and bacteriological assays. There were no significant differences
in any of the parameters measured.
The following year (1976), 24 steers were secured and assigned to 3
groups, and allowed to graze on winter annual pastures. A control group
(no treatment) and 2 groups grazed on pastures pretreated with liquid
Pensacola sludge (LPS) during the trial. Blood and fecal samples were
secured from each animal at the outset and at 28 day intervals during the
trial. The following parameters were followed: hematocrits, red and
white cell counts, prothrombin times, differential counts, and parasite
egg counts. Four representative animals from each group were slaughtered
after 5 months. There were no significant differences in growth performance
nor in the measured health parameters.
In August, 1976, in a 120 day feeding trial, another group of 24
steers were allotted to 3 groups, a control group receiving a regular
feedlot ration, one group received this ration plus 250 g dried Pensacola
sludge (DPS) mixed in their feed, and the third group received a ration
containing 500 g DPS daily in their feed. Parameters monitored at the
outset and monthly included red and white cell counts, prothrombin times,
and parasite egg counts. Parasite egg counts were initially high for
trichostrongyles, neoascarids, and coccidia. The feed samples collected
were also examined for presence of mycotoxins. Samples of the DPS were
collected for pesticide assays and samples of the blood, feces, sludge,
and feed provided to bacteriology for examination. Blood samples from
each animal were examined for serum enzyme levels including alkaline
phosphatase, AP, gamma-glytamyl transpeptidase (yGT), serum glutamate
oxaloacetate transaminase (SG0T), and serum glutamate pyruvate trans-
aminase (SGPT). Finally, at slaughter, liver, kidney, and muscle tissues
were collected and examined for pathologic changes.
72

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There were no significant differences in growth performance between
the control and 250 g/head/day groups, but there was significant growth
suppression in the growth of the group receiving 500 g daily and there
were significant increases in the AP, yGT, SGOT, and SGPT levels in the
groups of steers on the 250 and 500 g/head/day after 8, 12, and 16 weeks.
In addition, there was a significant increase in the cadmium level of
kidney tissues collected at slaughter in the animals receiving 500 g DPS
daily.
An experiment, begun in March 1977, included 3 groups of steers, con-
trols received a ration containing corn from soils treated with regular
fertilizers, one group received a ration including corn from soil pre-
treated with 7.5 cm/hectare liquid Pensacola slidge (LPS), and a third
group received the same ration as the controls but also received 500 g/head/
day of dried Chicago sludge (DCS) for 5 months. Analysis of variance
showed that yGT and glutamic oxaloacetic transaminase (GOT) levels were
significantly increased for the steers receiving 500 g DCS daily. Likewise,
the steers receiving corn from soil pretreated with LPS showed increased
in GOT, glutamic pyruvic transaminase (GPT) and white blood cell counts
(Table 1).
Clinical interpretation of these values from the "treated" steers
suggest that parenchymal liver disease was present in both groups resulting
from heavy metal intoxication or parasitic invasion by flukes. Liver fluke
ova were observed in fecal samples from all 3 groups; the 7.5 cm/ha corn
group had a greater number of animals affected (5/7) than the DCS group
(2/7). The liver sections secured at slaughter of the latter group also
displayed biliary obstruction and icterus. The control corn, 7.5 cm/ha
LPS corn and sorghum silage were examined for aflatoxin Bj and found to
be negative. Animals receiving the 3 rations were negative to tetra-
cyclines and sulfonamides.
A steer feeding trial was initiated in 1978 consisting of a control
group, one group consumed forage sorghum silage pretreated with 15 cm/ha,
and a third group consumed silage from soil pretreated with 22.5 cm/ha
LPS. Blood and fecal samples were collected for metals, pesticides, and
drug residue studies; small sections of each were fixed in formalin for
histopathologic studies. Fecal examination showed the 3 groups of animals
were moderately parasitized with trichostrongyles, coccidia, and monezia.
The serum enzyme levels for AP, yGT, GOT, and GPT remained consistent
throughout the trial although there was some anemia as the trial progressed
in the group receiving forage silage from soils pretreated at the 22.5
cm/ha.
Kidney and livers were collected from these steers, frozen, and
utilized in mouse feeding trials.
The final EPA-sponsored cattle trial at Jay included 3 groups of
cattle fed on bahiagrass pastures which had received 0, 7.5 cm/ha LPS
before grazing and 7.5 cm/ha during grazing, and the third pastures had
73

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received 15 cm/ha before and 15 cm/ha during grazing. Blood was collected
at the start and monthly during the experiments to determine red and white
cell counts, packed cell volume, hemoglobin levels, serum enzyme levels
for AP, yGT, GOT, and GPT. Feces was secured to evaluate the parasite
levels. Split samples were also provided to the bacteriology and pesticide
laboratories for analysis.
Cattle in the 2 groups fed on the treated pastures developed signi-
ficant decreases in the packed cell volumes and numbers of red blood cells
present. Finding of an increased incidence of sarcocysts in the cardiac
and skeletal muscles of the cattle in the groups feeding on the LPS treated
pastures suggested that the sludge was contaminated at the time of applica-
tion. This may pose a public health hazard to humans consuming meat
from such exposed and infected animals. The developing anemia may also
have been associated with the sarcocyst infection in the steers.
74

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TABLE 1. STATISTICAL ANALYSES OF CLINICAL CHEMISTRY AND
HEMATOLOGIC PROFILES IN BEEF CATTLE (JAY) FED DCS OR CORN1 FROM DCS TREATED SOIL
Correlation t-test
Parameter	Group	to Control2	(a)
yGT
500 g/head/day
or corn
+
0.05
GOT
500 g/head/day
or corn
+
0.025
GPT
500 g/head/day
or corn

0.05
WBC
500 g/head/day
or corn
+
0.05
1 + - above the normal mean
+ - below the normal mean
2 Soil supplemented with 8 t/ha

-------
THE EFFECTS OF RECYCLED CATTLE MANURE ON THE
HEALTH AND PERFORMANCE OF STEERS
M. F. Richter
METHODS
Two 1-year feeding trials have been completed. In each trial, 20
animals were divided into 5 groups of 4 animals: group 1, initial
slaughter group; group 2, control ration; group 3, manure silage, no
withdrawal; group 4, manure silage, 10 days withdrawal; group 5, manure
silage, 20 days withdrawal. All animals were adapted to the feed and
feeding facilities before the start of the trial. At the beginning of
the trial, the initial slaughter group was sacrificed for baseline data
on the body composition of the animals. The remaining 16 animals were
placed on treatment for approximately 200 days. Manure was withdrawn
from the rations of 2 of the groups 10 and 20 days before slaughter. The
control ration contained corn grain, citrus pulp, cottonseed meal,
pelleted bagasse, malasses, and minerals. The manure ration contained
ensiled cattle manure at a level such that 20% of the dry matter of the
ration was from manure. The silage was made by mixing raw cattle manure
with pelleted bagasse at a 60:40 ratio resulting in a dry matter of 50%.
At slaughter, liver, muscle, and kidney samples were taken and analyses
were conducted for drugs, pesticides, and heavy metals.
RESULTS
Animals on the control ration consumed an average of 7.1 kg of feed
per day and gained an average of .85 kg per day; manure fed animals con-
sumed an average of 10.7 kg of feed per day	and gained .76 kg per day.
The control animals consumed 6.1 kg of dry matter; the manure fed
animals, 6.4 kg, of which 4.4 kg was concentrate feed identical to the
control ration. The feed conversion (kg feed dry matter/kg gain) was
7.2 for the control animals and 8.4 for the manure fed animals. However,
if only concentrate feed dry matter is considered, the concentrate feed
conversion for the manure fed animals is 5.8. Therefore, the manure
silage significantly contributed to the performance of animals consuming
it (P < 0.05). The manure fed steers gained 89% as much as the control
animals while consuming only 72% as much concentrate feed.
The results of the first metabolism trial show that the manure silage
significantly (P < 0.01) reduced the digestibility of all the proximate
components. Digestibility of manure and bagasse are both low so this was
expected. The results are tabulated as follows:
76

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DIGESTIBILITY AND NITROGEN BALANCE (4 ANIMALS/TREATMENT)
Control ration	Manure silage ration
Dry matter, %
82.7
+
3.2
64.7
+
0.92
Crude protein, %
70.5
+
6.6
56.0
+
2.4
Crude fiber, %
66.7
+
8.9
41.3
+
2.6
Nitrogen free extract, %
88.7
+
2.2
74.2
+
1.1
Ether extract, %
84.8
+
5.8
51.9
+
22.2
Organic matter, I
84.2
+
3.3
66.9
+
1.3
Nitrogen balance, g/day
7.2
+
11.1
2.6
+
48.7
The nitrogen balance was not different between the 2 rations and indicated
that the animals were in positive balance. This was expected since the
animals gained weight on both treatments. These data show that the manure
silage has a feeding value approximately equal to hay.
The tissue concentrations of copper, chrome, cadmium, zinc, cobalt,
and nickel was not increased due to feeding manure silage. In addition,
there was no net effect due to withdrawal of the manure silage prior to
slaughter for either 10 or 20 days. The tissue levels of selected minerals
are presented in Table 1.
Mineral balances were measured during the metabolism trial. These
indicated no significant differences existed between animals on control
and the manure silage rations. Additional carcass examinations showed
no pathologic lesions, bacterial contamination or other differences which
would indicate adverse health or meat safety effects of the manure silage
ration.
CONCLUSIONS
Based on data from the first year's study, the feeding of the manure
silage did not adversely effect the health or the food safety of the
animals; performance will be reduced slightly on this type of ration due
to its lower energy content; feeding value of the manure silage appears
to be equal to a good quality hay, and finally, feed savings are achieved
since less concentrate feed is necessary per pound of gain.
77

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TABLE 1.
TISSUE
LEVELS OF
SELECTED MINERALS,
1978



4 animals/group




tissue + SD





M = muscle

ppm,
fresh





K = kidney










L = liver

Cu

Cr
Cd

Zn
Co

Ni
Initial
M
0.78 + 0.15
0.19
+
0.04
.04
52.;
1 + 8.6
0.05 +
0.02
a
slaughter
K
4.0 + 0.3
0.17
+
0.00
0.53 + 0.31
19.8
t :-3
0.19 +
0.03
a

L
62.4 + 18.3
0.16
+
0.02
0.05 + 0.02
26.6
+ 3.3
0.27 +
0.05
a
Group "0"
M
0.79 + 0.06


0.0
0.0
35.5
+ 14.7

0.0
0.0
(no with-
K
3.5 + 0.5


0.0
0.43 + 0.07
18.0
+ 1.7

0.0
0.0
drawal )
L
37.3 + 36.3


0.0
0.12 + 0.06
30.8
+ 0.47

0.0
0.0
Group "10"
M
0.76 + 0.06


0.0
0.05 + 1.1
31.9
+ 4.9
0.1 +
0.2
0.1 + 0.2
(10 day
K
3.8 + 0.54


0.0
0.46 + 0.44
18.7
+ 3.3
0.1 +
0.2
0.1 + 0.2
withdraw)
L
29.1 + 8.3


0.0
0.09 + 0.03
32.5
+ 5.7
0.1 +
0.2
0.1 + 0.2
Group "20"
M
0.74 + 0.03


0.0
0.0
37.7
+ 9.1

0.0
0.0
(20 day
K
4.1 + 0.90


0.0
0.45 + 0.05
20.7
+ 6.7
0.29 +
0.2
0.29 + 0.2
wi thdraw)
L
32.7 + 8.6


0.0
0.08 + 0.02
34.6
+ 1.0

0.0
0.0
Control
M
0.84 + 0.06


0.0
0.0
35.3
+ 4.0

0.0
0.0
(no
K
3.5 + 0.57


0.0
0.49 + 0.16
18.0
+ 0.28

0.0
0.0
manure)
L
40.6 + 11.6


0.0
0.21 + 0.26
27.7
+ 5.6
0.2 +
0.23
0.2 + 0.23
a Not done

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SWINE FEEDING TRIALS WITH SLUDGES
SECTION A. Performance and Tissue Minerals of Swine Fed Sewage Sludge
Diets. J. Beaudouin, R. Shirley, and D. Hammell, 1976
SECTION B. Effect of Feeding Sewage Sludge Diets on Reproduction, Growth,
and Tissue Mineral Accumulation. D. Hammell and G. Edds,
1977
SECTION C. Effect of Feeding Digested Sewage Sludge on Long-Term Sow
Reproduction Performance. F. White, D. Hammell, . Shepherd,
0. Osuna, and G. Edds, 1978; 1979
79

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A. PERFORMANCE AND TISSUE MINERALS OF SWINE FED SEWAGE SLUDGE DIETS
ABSTRACT
Twelve female swine were fed in a 3 x 4 cross-over design metabolism
trial corn-soybean grower diets that contained 0, 10, or 20% sewage sludge
over 3 19-day periods. The mean values for total digestible nutrients were
79.4, 73.7, and 55.0%; those for metabolizable energy were 3.36, 2.25, and
1.15 Meal per kg diet; and those for nitrogen retained were 42.8, 44.0,
and 25.3%, respectively. Sewage sludge (0, 10, 20%) diets were fed to
31 sows approximately equally divided in the dietary groups during their
first 2 pregnancies and to their offspring from weaning to market weight.
More live pigs were farrowed and weaned per litter from sows fed 20%
sludge diets than from the control group. However, 20 day weaning weights
of pigs were lower with sows fed the sludge-containing diets. Offspring
of both first and second litters fed growing and finishing diets containing
sludge from weaning to market weight had decreased daily weight gains and
feed efficiency. There were no increases in the 9 mineral elements (Pb,
Cd, Ni, In, Cr, Cu, Mn, Fe, and A1) in sow milk or blood. Offspring of
sows fed sludge diets showed increases of several elements in selected
tissues at weaning and after consuming sludge diets to market weight.
INTRODUCTION
Kienholz et al. (1976) found aged cows that grazed pasture for 5
years that was treated with sewage sludge had no marked differences in
heavy metal concentration in their tissues. However, steers fed 15% dried
sewage sludge in feedlot diets for 94 days had decreased body weight
gains and a 10-fold increase in lead and mercury and a 2-fold increase in
cadmium and copper in their liver and kidney. Kinzell fed 0 and 30%
levels of activated sewage sludge (AcSS) in diets to rats for 3 generations
and concluded that performance and tissue heavy metal levels were such
that AcSS seemed to have potential as a feed ingredient. The purpose of
this study was to determine the effect of sewage sludge in diets fed sows
on digestibility of nutrients, reproduction, growth of offspring, and
mineral accumulation in tissues.
EXPERIMENTAL PROCEDURE
Metabolism Trial
Twelve gilts averaging 28 kg in body weight were equally divided into
3 groups of 4 and fed corn-soybean grower diets (Table 1) containing either
0, 10, or 20% sewage sludge over 3 19-day periods in a cross-over design
metabolism trial. The pigs were randomly selected from several litters
80

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of the same age and weight that had not previously been fed sewage sludge
diets. The sludge was anaerobically digested in the University of Florida
sewage plant, sun-air dried, and replaced corn grain diets on an equal air-
dry weight basis. Chromic oxide (0.1% Cr203) was added as a reference
substance and used to calculate total fecal output by the equation:
F1 mi+.nilt = Total Cr intake
p Concentration Cr/g dry matter of feces
The swine were adapted to the diets for 9 days in pens and then confined
to individual metabolism cages where feed intake was recorded and feces
and urine collected during days 12-19. Proximate analyses constituents
were determined in the sludge, diets, feces, and urine (Table 2) by AOAC
(1975) methods.
Reproduction and Weanling Pig Performance
Thirty-three York-Hampshire crossbred gilts that weighed approximately
60 kg each were allotted randomly and equally to corn-soybean gestation-
lactation diets (Table 1) that contained either 0, 10, or 20% dried Florida
sewage sludge (DFS). During approximately 12 months of feeding the diets,
2 litters were obtained from each of the sows. The sows were fed from
troughs in open pens on the ground except just prior to farrowing and
during lactation until weaning when they were kept on concrete in farrowing
pens and hand-fed. After weaning, the sows were returned to open pens
on the ground. Milk from lactating sows and tissues from randomly selected
weanling pigs, market weight offspring, and dams from the dietary groups
were analyzed for lead, cadmium, nickel, copper, chromium, zinc, manganese,
aluminum, and iron by atomic absorption spectrophotometry (Anonymous,
1973). Milk was obtained on the seventh day of the first lactation period
from 3 sows randomly selected from each dietary group. It was dried on a
steam bath, ashed at 550° overnight, ash dissolved in 0.1% HC1, and the
above elements analyzed.
Six weanling pigs (3 male, 3 female) of each dietary group of both
first and second litters were killed by jugular incision and blood obtained
in heparinized tubes. Approximately 10 minutes later, liver, kidney,
spleen, and muscle (ham) tissues were secured with a stainless steel blade
and frozen immediately at -10°C. Samples were partially oxidized with
nitric acid followed by dry ashing at 480°C. The ash was dissolved in
aqueous nitric acid and analyzed for the selected elements. Eighteen of
the brood sows (6 from each dietary group) were randomly selected and
slaughtered after weaning their second litters. The liver, kidney, spleen,
muscle, and blood were collected and handled as above prior to analyses
of the 9 elements.
Growing and Finishing Trial
Seventy-two weanling pigs of each of the first and second litters
were randomly selected and divided equally into 3 groups and fed either
0, 10, or 20% DFS-containing growing and finishing diets (Table 1) to
market weight (approximately 80 to 90 kg). In this trial, pigs were not
81

-------
changed from the dietary sewage sludge levels of their dams. Pigs of each
set of litters were allotted randomly to 18 pens according to weight, sex,
and litter number, with 2 barrows and 2 gilts in each pen (non-1ittermates).
Individual weights and pen feed consumption data were obtained every 2
weeks. At market weight, 18 animals (9 barrows, 9 gilts), 1 from each pen,
were randomly selected, slaughtered, and blood, liver, kidney, spleen, and
muscle collected and handled as above for analyses of selected elements
(Anonymous, 1973).
Statistical Analysis
In the metabolism trial, significance of variance in values due to
treatments were analyzed using the least significant difference (LSD) method
of Steel and Torrie (1960). The data in all other treatments were analyzed
for significance of variances by the SAS computer method (Barr et at., 1976).
Duplicate analyses were made for each element in the various tissues.
RESULTS AND DISCUSSION
Digestibility Trial
The digestibility of crude protein, crude fiber, ether extract, ash,
and nitrogen-free extract as well as total digestible nutrients (TDN),
metabolizable energy (ME) and nitrogen balance data are presented in Table 3.
All proximate analyses nutrient values except ether extract were decreased
(P < 0.05) in digestibility when 20% sludge was in the diet; as well as TND,
ME, and nitrogen retained. It is not apparent why more ether extractable
material was present in the feces than in the diet which ranged from 0.3
to 1.1% (Table 2). It must have been of metabolic origin.
Reproductive and Weanling Pig Performance
The reproductive performance of sows fed 10 and 20% sewage sludge
diets was not adversely affected during their first and second pregnancies
compared to the controls. There were apparent linear decreasing weights
of pigs from dams fed 10 and 20% sewage sludge diets (Table 4). Approxi-
mately 1 to 2 more live pigs were farrowed on the average and slightly
more (0.25 to 0.45) pigs were weaned per litter with diets than contained
20% sludge. However, the 21 day old weaning weights were depressed by
dietary sludge. Dams fed the 20% sludge diets had weaning weights of
offspring that averaged 0.53 and 1.45 kg less (not significant) than controls
for the first and second litters, respectively. The slightly slower gaining
pigs appeared to be normal in vigor and overall health.
Growing-Finishing Swine Performance
In Table 5 are presented performance data on first and second litter
offpsirng fed growing-finishing diets that contained 0, 10, and 20% DFS.
The swine from the second litters fed the sludge-containing diets had less
(P < 0.05) daily weight gains and a greater (P < 0.05) feed gain ratio
than the controls. The first litter swine fed diets with sludge increased
their feed intake over the controls and made similar weight gains.
82

-------
They had greater (P < 0.05) feed:gain ratio. The failure of the second
litter swine to consume more of the sludge-containing diets than the
controls as was done by the corresponding first litter group may have been
due to the stress of summer temperature as they were born in June and the
first litters were born in January. Similar weight gain depression to that
observed with the second litter groups was reported by Kienholz et al.
(1976) upon feeding 5 or 15% dried sewage sludge diets to feedlot steers.
Mineral Accumulation in Milk and Tissue
Data obtained on mineral accumulation in milk and tissues of sows
slaughtered after weaning their second litters are presented in Table 6.
The sludge diets resulted in no increase in concentration of selected
elements in milk or blood of the sows. However, sows fed the 10 to 20%
sludge-containing diets had an increase in cadmium in the kidney and
muscle (10% sludge only); nickel in the liver, kidney (20% sludge only),
spleen and muscle; copper in the liver; chromium in the liver, kidney,
spleen, and muscle; zinc in the kidney (10% sludge only); manganese in
the liver, and aluminum in the liver, kidney, spleen, and muscle. Similar
concentrations of several elements in swine liver to those found in the
present study have been reported (Liebholz et al., 1962; Hedges and
Kornegay, 1973; Gipps et al., 1974; and Skutches et al., 1974). Apparently
there is no corresponding diet versus tissue data of swine for cadmium,
nickel, chromium, and aluminum. Kienholz et al. (1976) fed diets that
contained 15% sewage sludge for 94 days to feedlot steers and found liver
and kidney had a 10-fold increase in lead and mercury, and a 2-fold increase
in cadmium and copper, but no histological or pathological changes in
tissues.
In Table 7 are presented concentrations of various elements in selected
tissues of weanling offspring from the first and second litters of sows
fed diets that contained 0, 10, and 20% DFS. The liver of those fed sludge-
containing diets had more (P < 0.05) lead, cadmium, copper, and iron in
the first litters, but not in the second ones. Aluminum was increased
in those fed the 20% sludge level in the second sludge diet but not in the
first litters. Nickel was increased with the 10% sludge diet but not at
the 20% level with the first litters. The sludge diets had no effect on
chromium, zinc, and manganese in the liver. In the kidney, the concentra-
tion of lead, cadmium, nickel, copper, chromium, zinc, aluminum, and iron
were increased in weanling pigs of the first litters of dams fed 20%
sludge diets; and the same was true of cadmium, nickel, and chromium with
the corresponding first litters. Manganese in the kidney was not affected
by the dietary sludge. In the spleen, nickel, copper, and manganese were
greater with weanling pigs of the first litters of dams fed diets that
contained 20% sewage sludge. Manganese was also greater at the 10% level
in this treatment group. Chromium was higher in the spleen of corresponding
pigs of the second litters of dams fed 20% sludge diets. Concentrations of
lead, cadmium, zinc, aluminum, and iron in the spleen was not effected by
the sludge-containing diets. In the skeletal muscle, cadmium, nickel,
chromium, zinc, and manganese were greater in the first litters of dams
fed 20% sludge diets, but all except cadmium were not effected in the
83

-------
second litters; cadmium was less. The sludge-containing diets had no
effect on lead, cadmium, copper, aluminum, and iron in muscle.
In the blood, lead and cadmium were more concentrated in offspring
of second litters of dams fed 20% sludge diets; but not in those of the
first litters. Chromium was greater in corresponding first litter pigs
of dams fed 20% sludge diets; and iron was less in the blood of the
second litter pigs. The dietary treatments had no effect on copper,
zinc, manganese, and aluminum concentration in the blood.
Concentrations of selected elements in various tissues at time of
slaughter of the growing-finishing offspring fed diets that contained 0,
10, or 20% sewage sludge are presented in Table 8. The sludge-containing
diets fed the first litter offspring had no effect on concentrations of
any of the 9 elements in the liver. However, in the second litters, cadmium
of the 20% sludge groups and zinc of both sludge groups were greater than
the controls. The kidney of the first litters was not effected by the
sludge diets. But the kidneys of the second litter swine had more cadmium
with both sludge levels. The sludge diets had no influence on mineral
deposition in the spleen of either the first or the second litters. Lead
was greater in the muscle of the second litter pigs fed the 20% sludge-
containing diet, while zinc was lower in muscle of second litter offspring
that consumed 10 ro 20% sludge diets. None of the minerals were elevated
in blood due to the dietary sludge.
REFERENCES
1.	Anon. Analytical Methods of Atomic Absorption Spectrophotometry.
Perkin-Elmer Corp., Norwalk, CT, 1973.
2.	Barr, A. H., J. H. Goodnight, J. P. Sail and J. T. Hellwig. A User's
Guide to SAS 76. Sparks Press, Raleigh, NC, 1979. 494 pp.
3.	Gipp, W. F., W. G. Pond, F. A. Kallfelz, J. B. Tasker, D. R. VanCampen,
L. Krook, and W. J. Visek. Effect of Dietary Copper, Iron, and
Ascorbic Acid Levels on Hematology, Blood, and Tissue Copper, Iron, and
Zinc Concentrations and 61tCu and 59Fe Metabolism in Young Pigs. J.
Nutrition, 104(5):532-541, 1974.
4.	Hedges, J. D. and E. T. Kornegay. Interrelationships of Dietary Copper
and Iron as Measured by Blood Parameters, Tissue Stores and Feedlot
Performance of Swine. J. Anim. Sci., 37(5):1147-1154, 1973.
5.	Kienholz, E. W., G. M. Ward, and D. E. Johnson. Sewage Sludge Metals
in Cattle Tissues. J. Anim. Sci., 43(1):230 (Abstract), 1976.
6.	Kindzell, J. H., P. R. Cheeke, and R. W. Chen. Activated Sewage
Sludge Utilization by Rats. J. Anim. Sci., 42(6):1560 (Abstract),
1976.
84

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7.	Leibholz, J. M., V. C. Spee.r, and V. W. Hays. Effect of Dietary
Manganese on Baby Pig Performance and Tissue Manganese Levels. J.
Anim. Sci., 21(4):772-776, 1962.
8.	Skutches, C. L., D. L. Herman, and F. H. Smith. Effect of Dietary
Free Gossypol on Blood Components and Tissue Iron in Swine and Rats.
J. Nutrition, 104(4) :415-422, 1974.
9.	Smith, G. S., J. M. Caddie, P. M. Walters, and H. E. Kiesling. Sewage
Solids as Feedstuffs for Ruminants. J. Anim. Sci., 43(1): 334 (Abstract),
1976.
10.	Smith, G. S. and C. R. Staples. Heavy Metals in Rats Fed Sewage
Solids. J. Anim. Sci., 43(1):233 (Abstract), 1976.
11.	Steel, R. G. D. and J. H. Torrie. Principles and Procedures of
Statistics. McGraw Hill Book Company, New York, 1960, 481 pp.
85

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TABLE 1. BASAL FIETS FED DURING METABOLISM TRIAL,
GESTATION-LACTATION, GROWING
AND FINISHING
PERIODS9

Internat'1
Ref. No.b
Gestation and
lactation
Starting
Grower and
metabolism
Finishing
Corn, grain, yellow, grond (9.0%)
4-02-931
70.45
61.45
78.25
83.25
Oats, grain, grnd (12.7%)
4-03-315
10.0
-
0
0
Sugarcane, molasses, 79.5° brix
4-04-696
-
5.0
-
-
Soybean seed, dehulled, solv extract
5-04-612
16.0
27.5
18.75
13.75
Animal fat
4-00-409
-
3.0
-
-
Phosphate, defluorinatedc
6-01-780
2.5
2.0
1.50
1.50
Salt, iodized
6-04-151
.5
.5
.50
.50
Trace mineral premix^

.05
.05
.10
.10
Vitamin premix

.5
.5
.50
.50
Limestone, grnd
6-01-069
0
-
.40
.40
? Corn was replaced on air-dry weight basis by sewage sludge in 10 and 20% sludge diets fed swine.
^ Atlas of Nutritional Data on United States and Canadian Feeds, 1971. Nat. Acad, of Sci., Wash., DC.
, Courtesy of Occidental Petroleum Co., White Springs, FL.
Provided the following elements in mg per kg diet: zinc (ZnO), 200; iron (FeSOiJ, 100; manganese
(MnSOit), 55; copper (Cu20), 11; iodine Ca(103)2, 1.5; and cobalt (CoC03), 1.0. Courtesy of Calcium
Carbonate Company, Quincy, IL.
Provided the following vitamins per kg diet: vitamin A, 11,025 IU; vitamin D, 5,513 IU; vitamin E,
22 IU; riboflavin, 11 mg; pantothenic acid, 27 mg; niacin, 55 mg; choline chloride, 1,102 mg;
vitamin B12, 49 meg. Courtesy of Charles Pfizer and Company, Terre Haute, IN.

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TABLE 2. PROXIMATE ANALYSES AND SELECTED MINERAL COMPOSITION OF SEWAGE SLUDGE AND DIETS
Air dry 	 	 Proximate analyses9	 b	
matter Cr. protein Cr. fiber Ether extract NFE	Ash
o/ 0/	o/	0/	0/	0/
h		h	lo	/o	Jo	h
SIudgec
91.9

25.9

4.3
.46
40.6

23.8

(66-98)

(22-37)
(
.7-15)
(.01-1.1)
(9-47)
(27-57
Metabolism trial:









Control diets (grower)









Period 1
85.9

17.5

6.0
1.1
69.5

5.9
Period 2
84.5

21.9

4.6
.4
64.7

8.4
Period 3
87.2

19.5

12.7
.7
55.5

11.7
Sludge diets, 10%









Period 1
84.4

19.4

6.4
.6
66.2

7.4
Period 2
89.1

18.4

6.9
.4
66.3

8.1
Period 3
85.2

19.7

3.5
.4
64.9

11.5
Sludge diets, 20%









Period 1
83.9

25.0

4.5
.6
63.2

6.8
Period 2
84.1

20.2

6.0
.3
65.1

8.5
Period 3
82.4

21.4

5.7
.6
61.9

10.5
Gestation-lactation diets









Control
84.7

20.6

5.7
.8
66.3

6.7
Sludge, 10%
85.9

20.1

5.8
.4
65.1

8.3
Sludge, 20%
84.9

20.2

7.3
.5
61.9

11.2
Minerals, mg/kg dry matter
Pb
Cd
Ni
Zn
Cu
Cr Mn
Fe
Al

SIudge
463
13
32
1190
512
213 274
10670
4436

Diets:









Control
2
.3
4
189
12
2 75
344
419

Sludge, 10%
31
1.3
5
265
42
16 90
1011
688

Sludge, 20%
66
2.2
8
381
80
32 118
1963
1045

Percentage on dry matter basis
Nitrogen-free extract
c Mean and range of 22 samples

-------
TABLE 3. DIGESTIBILITY OF DRY MATTER, ORGANIC MATTER, CRUDE PROTEIN, ETHER
EXTRACT, CRUDE FIBER, NITROGEN-FREE EXTRACT (NFE), TOTAL DIGEST-
IBLE NUTRIENTS (TDN), METABOLIZABLE ENERGY (ME), AND NITROGEN
RETAINED IN SEWAGE SLUDGE DIETS FED SWINE
Control	Sludge, 10%	Sludge, 20%
Number of swine
12
12
12
Daily intake, kg, dm
1.59
1.57
1.74
Digestibility (%)



Dry matter
76.4b
68.3b
49.8
Organic matter
79.7b
72.5b
56.5
Crude protein3
77.8b
68.5bc
58.2
a
Ether extract
-63.6b
-42.9°
-112.5
Crude fiber9
69.7
70.3
62.9
NFE
82.8b
J3
-------
TABLE 4. REPRODUCTIVE PERFORMANCE OF SOWS DURING FIRST AND SECOND LITTERS
ON DIETS CONTAINING 0, 10, and 20 PERCENT SEWAGE SLUDGE
First litter	Second litter
Item
0%
10%
20%
0%
10 %
20%
Number mated
11
11
11
9
10
10
Number farrowed
10
10
10
7
9
8
Breeding weight, kg
152.2
148.2
145.4
164.5a
167.8a
155. l'
Gestation period, days
113.4
115.2
114.4
114.6
114.6
114.5
Gestation weight gain, kg
42.9a
40.3a
30. lb
47.4a
41.8a
31.91
Total pigs farrowed per litter
9.9
9.3
11.2
9.0
9.2
10.4
Live pigs farrowed per litter
7.4
7.6
9.5
8.3
8.7
9.4
Birth weight of live pigs, kg
1.19
1.24
1.09
1.44
1.24
1.39
Weaning weight/pig (21-day), kg
5.02
4.98
4.49
6.28
5.36
4.83
Pigs weaned/litter
6.55
6.80
7.00
8.00
7.11
8.25
a h
' Means having same superscript letter do not differ significantly (P < 0.05)
-------
TABLE 5. PERFORMANCE OF FIRST AND SECOND LITTER OFFSPRING FED GROWING
AND FINISHING DIETS THAT CONTAINED 0, 10, AND 20 PERCENT
SEWAGE SLUDGE
Final weight, kg
94.50
92.90
91.40
95.41
86.91
81.63
Initial weight, kg
8.91
8.95
9.22
10.36
8.36
8.45
Weight gain, kg
85.59
83.95
82.10
85.05
78.55
73.10
Days on test
133
133
133
128
128
128
Average daily gain, kg
.64
.63
.62
. 66a
o
cr
. 56b
Average daily feed, kg
1.94a
2.10ab
2.30b
2.03
1.99
1.86
Feed:gain ration
3.06a
3.34b
3.69b
3.05a
3. 36b
3. 39b
a'k Means in same row that vary significant! have different superscript letters (P < 0.05)
-------
TABLE 6. EFFECT OF SEWAGE SLUDGE IN DIETS FED SOWS ON
CONCENTRATION OF MINERALS IN SELECTED TISSUES9'D
Diet	Pb Cd Ni Cu Cr Zn Mn A1 Fe





Milk, mg/liter



Control

1.0
.06
.1
1.0
<.01
9
.2
25
2
SIudge,
10%
1.3
.06
.1
1.1
<.01
9
.2
26
2
SIudge,
201
1.0
.05
.1
1.2
<.01
10
.1
24
1





Liver,
mg/kg dry wt



Control

<.01
2
3
39
1
231
7
259
1217
SI udge,
10%
2
5
14
64
11
243
11
763
1133
SIudge,
20%
<.01
4
23
51
12
209
9
873
1126





Kidney,
mg/kg
dry wt



Control

<.01
4
5
26
1
122
6
397
875
SIudge,
10%
<.01
17
10
54
8
123
5
1952
392
SIudge,
201
<.01
24
20
44
15
147
6
1069
451





Spleen,
mg/kg
dry wt



Control

<.01
.4
3
10
<.01
128
<.05
483
3030
SI udge,
10%
<.01
2
17
18
15
127
<.05
1123
4736
SIudge,
20%
<.01
.03
19
17
16
123
<.05
1062
5104





Muscle,
mg/kg
dry wt



Control

<.01
1
4
12
.01
113
2
485
689
SIudge,
10%
<.01
2
11
15
12
111
.6
836
304
SIudge,
20%
<.01
<.03
21
10
14
90
<.05
1006
125





Blood,
mg/liter



Control

2
1
9
13
7
33
.5
384
2051
Sludge,
10%
<.01
2
8
12
<.01
28
<.05
139
2289
SIudge,
20%
<.01
1
9
7
.1
29
<.05
374
2181
a Milk from 3 sows/treatment during seventh day of the first lactation
period.
k Tissues from 6 sows/treatment slaughtered after second pregnancy and
lactation period.
c The 
-------
TABLE 7. MINERALS	IN WEANLING	PIG TISSUES OF FIRST	AND SECOND LITTERS
SIudge
in diet 	Lead	 Cadmium Nickel	Copper	Chromium
% 	1st3	2nda		1st 2nd		1st 2nd	1st 2nd 1st 2nd
Liver, mg/kg, dry basis
0
<-01b
7.8
<.03
.9
<.01 .9
108b
11
<.01
<.01
10
<.oi:
6.2
<.03
1.1
<.01 2.9^
101b
13
<.01
.62
20
6.5 r
5.1
1.06
1.5
<.01 1.7b
85c
12
.10
.86




Ki dney,
mg/kg dry basis




0
<.01b
8.0
<.03b
3.5b
< .lb .7b
32b
20
<.01
<.01b
10
<.01b
9.9
<.03
6.2C
< .lb 1.9°
37b
19
.16
. 22b
20
llc
8.0
4.57c
6.8C
24c 2. lc
48c
21
.71
2.02c




Spleen,
mg/kg, dry basis




0
<.01
13.6
.33
1.3
.lb 2.2
18b
8.2
.5
4b
10
<.01
11.4
.65
1.3
7.8^ 4.7
15
5.4
.8
.01b
20
<.01
7.9
.03
1.9
14.5d 3.9
30c
6.7
.1
2. lc




Muscle,
mg/kg dry basis




0
<.01
6.0
.30b
.8
< .lb .9
15
4.0
<.01b
.21b
10
<.01
5.4
. 30
.6
< .lb 1.9
9
2.5
<.01b
. 62b
20
<.01
4.4
2.22°
1.1
20.0C 1.5
17
4.2
.62°
<.01c




Blood,
mg/liter




0
7.2
10.8 J
1.12
<.03b
5.41b 3.2
9.5
14.3
<.01b
1.9b
10
9.9
11.3b
1.12
<.03
4.43 2.5
9.4
11.1
<.01b
l.lb
20
7.6
5. lc
.47
.94°
<.01c 2.6
10.0
8.1
1.03
A
O
i—»
o
-------
TABLE 7.	Continued
Sludge
in diet 	Zinc		Manganese	A1 uminum	Iron
%	1st 2nd 1st 2nd 1st 2nd	1st 2nd
Liver, mg/kg, dry basis
0
336
172
8.2
6.9 1239
441h
274b
476
10
380
160
10.2
7.5 1563
412°
327°
438
20
408
139
10.6
7.8 1336
711c
609
525




Kidney, mg/kg, dry basis


0
115b
122
2.7
6.6 1555?
561b
256l
224
10
103°
100
6.1
4.4 1691°
351£
263°
205
20
313°
108
9.2
5.8 2207
530
475
257



Spleen, mg/kg, dry
basis


0
114
132
.05°
2.0 1627
626
566
918
10
95
115
2.62
1.4 1842
458
616
1029
20
128
119
2.69°
1.8 1896
851
850
1003




Muscle, mg/kg, dry
basis


0
59b
111
<-05b
.35b 1235
368
117
52
10
66
62
• 63^
<.05u 1474
264
128
52
20
148c
94
4. 28
.30° 1529
282
345
53




Blood, mg/liter



0
25.7
21
<.05
<.05 652
440
1901
2172
10
26.8
26
<.05
<.05 584
342
1881
2186
20
29.7
20
<.05
<.05 607
344
1956
1800
a
1st and 2nd refers to first and second litters; each value is the mean of 6 pigs (3 male,
3 female) per diet; sex had no effect
'3,c'c' Means that have different superscript letters differ significantly with various
treatments (P < 0.05)
-------
SI u
in
%
0
10
20
0
10
20
0
10
20
0
10
20
0
10
TABLE 8. MINERALS IN TISSUES OF GROWING-FINISHING SWINE FROM FIRST AND SECOND
LITTERS FED DIETS CONTAINING SEWAGE SLUDGE (6 PER TREATMENT)
Lead

Cadmi um
Nickel
Copper

Chromium
1st
2nd
1st
2nd
1st
2nd
1st
2nd
1st
2nd



Liver,
mg/kg,
dry basis




7.3
1.72
1.00
.33
5.2
<.1
44
14
.63
<.01
8.6
<.01
1.19
.84
3.2
.7
31
17
.85
2.77
7.2
<.01
.56
3.18
3.4
6.6
19
17
.16
8.05



Kidney,
mg/kg dry basis




6.8
<.01
.76
1.40
4.2
<.1
19
22
<.01
<.01
11.4
<.01
2.50
8.60
7.7
.9
32
27
1.1
3.04
12.7
<.01
1.28
12.5
4.7
<.1
25
27
.22
2.18



Spleen,
mg/kg
, dry basis




12.1
1.84
1.03
.64
4.9
1.3
13
10
<.01
<.01
14.4
<.01
1.73
<.03
5.5
1.0
16
11
<.01
1.84
10.1
<.01
.67
<.03
4.6
4.7
15
6
.22
7.06



Muscle,
mg/kg
, dry basis




3.8
2.14
.79
.75
2.3
<.1
9
9
.62
<.01
4.2
<.01
.72
<.01
2.5
1.2
6
7
.43
3.05
7.0
<.01
.64
<.01
3.2
3.1
10
8
1.15
2.18



Blood
, mg/liter




5.3
11.2
.07
1.80
2.5
.7
11
9
1.01
<.01
7.1
9.7
.15
1.29
1.8
.5
11
11
.87
<.01
4.6
10.6
.16
4.47
1.2
<.1
9
13
.30
<.01
-------
TABLE 8. Continued
SIudge
in diet,	Zinc
%	1st	2nd
0	172	109^
10	143	136j
20	168	176
0	130	95
10	177	96
20	197	139
0	157	104
10	145	118
20	131	106
0	112	99j!
10	85	lb
20	137	69
0	17	24
10	17	26
20	18	24
First and second litters
K r A
' ' Values in same column
A1uminum
Manganese
Iron
1st 2nd	1st 2nd	1st 2nd

Liver, mg/kg, dry basis


6.2
9.3 441
687
519
615
5.9
6.8 472
587
614
507
5.1
8.3 401
853
558
546

Kidney, mg/kg, dry
basis


4.3
15.7 563
668
296
269
7.0
4.4 781
582
585
242
5.8
5.8 552
612
663
285

Spleen, mg/kg, dry
basis


3.3
1.6 1000
688
1081
758
3.7
.9 557
596
2507
764
3.7
.6 446
1073
1772
839

Muscle, mg/kg, dry
basis


2.4
.51 330
486
227
112
2.1
<.05 393
545
239
88
3.7
<.05 517
665
392
87

Blood, mg/liter



.6
<.05 259
479
1884
221
<.1
<.05 216
348
1676
229
<.1
<.05 259
418
1491
223
th different superscripts are different (P < 0.05)
-------
B. EFFECT OF FEEDING SEWAGE SLUDGE DIETS TO SWINE ON
REPRODUCTION, GROWTH, AND TISSUE MINERAL ACCUMULATION
ABSTRACT
A total of 39 gilts initially weighing 77 kg body weight were
allotted on the basis of weight and ancestry. Animals were allowed 1.81
kg daily of a corn-soy diet containing 0, 10, or 20% municipal sewage
sludge from the University of Florida waste treatment plant (DFS) for
2 gestation periods and were allowed ad libitum consumption of the same
diet during lactation. Gilts fed 0, 10, and 20% sludge diets weaned 7.91,
6.89, and 7.22 pigs/litter, respectively, at 21 days of age. Corresponding
number of pigs weaned/litter at 21 days of age from second litter sows
indicate sows fed 0% added sludge weaned more pigs (P 0.10) than those
fed sewage sludge diets (8.00 versus 6.27, 6.30). After weaning, gilt
offpsring were continued on the same sludge dietary treatment as their
dam. Average daily gain, average daily feed, and feed required per unit
of gain for offspring (18 per treatment, 6 replications) during the
growing-finishing period were: 0.61, 0.60, 0.56; 1.88a, 2.10'3, 2.08°;
3.06a, 3.59 , 3.70°, for those fed diets containing 0, 10, and 20% sludge
respectively. Corresponding performance values for gilt offspring (24
per treatment, 6 replications) fed sludge from Chicago which was substi-
tuted on a kg per kg basis for University of Florida sludge were: 0.65a,
0.60a, 0.49k. L84a5 L83a, 2.09b; 2.84a, 3.08a, 4.26b. Following the
termination of each growing-finishing experiment, 6 pigs per treatment
(18) were slaughtered and concentration of Hg, Cu, Fe, Zn, Cr, Ni, Cd,
Co, and Pb were determined in liver, muscle, and kidney tissue. Of
greatest concern was the accumulation of Cd in the kidney of offspring
fed diets containing dried Chicago sludge. Means with like superscripts
do not differ significantly (P < 0.05).
96
-------
TABLE 9. SLUDGE AND RATION MINERAL ANALYSIS
Grower-Finis her Diet
Mineral
ppm
Dried U of F
Sludge (DFS)
Control
10%
SIudge
20%
SIudge
Hg
81.7
0.02
3.86
8.17
Cu
555.9
12.86
41.3
81.36
Fe
4,100.0
285.5
536.25
908.75
Zn
1,216.7
204.38
216.25
346.00
Cr
217.6
2.08
11.34
25.02
Ni
24.9
1.04
2.50
5.00
Cd
9.1
.18
.938
1.39
Co
7.9
1.88
1.67
2.89
Pb
416.8
1.72
19.22
45.78
Mineral
ppm DM
Dried U of F
Sludge (DFS)
Control
10%
SIudge
20%
SIudge
Hg
81.7
-
-
-
Cu
555.9
8.65
46.43
102.10
Fe
9,366.6
269.00
3,750.00
984.00
Zn
1,216.7
123.22
568.33
303.83
Cr
217.6
1.46
18.96
37.52
Ni
24.9
1.04
3.00
7.61
Cd
9.1
.365
3.81
2.21
Co
7.9
1.67
4.17
4.17
Pb
416.8
0.83
19.58
76.25
97
-------
TABLE 10. SLUDGE AND RATION MINERAL ANALYSIS

Dried
Grower-
Finisher Diet

Mineral
ppm
Chicago
SIudge
Control
10%
SIudge
20%
Sludge
Hg
1.79
0.02
1.85
5.05
Cu
1,330
18.9
107.1
200.94
Fe
49,862
236.6
2,616
5,244.4
Zn
3,230
236.7
349.1
557.66
Cr
3,350
2.43
179.85
324.12
Ni
380
2.72
31.77
57.12
Cd
218
0.10
9.26
17.15
Co
17.91
2.0
2.74
4.31
Pb
715
1.0
38.76
86.1
Detection Limits - PPM Dry Matter
Aluminum, 0.5; Cadmium, 0.025; Cobalt, 0.10; Chromium, 0.10;
Copper, 0.05; Iron, 0.05; Lead, 0.10; Magnesium, 0.01; Manganese,
0.05; Mercury, 0.002; Nickel, 0.10; Selenium, 0.10; Zinc, 0.01
98
-------
TABLE 11. REPRODUCTIVE PERFORMANCE OF GILTS
Sewage Sludge [%)

0
10
20
Gilts mated
11
11
11
Gilts farrowed
11
9
9
Breeding wt, kg
120.6
119.4
115.4
Farrowing wt, kg
169.4
160.3
151.0
Total pigs farrowed/1 itter
10.54
9.44
9.67
Live pigs farrowed/1 itter
9.18
8.11
9.00
Birth wt/pig (live), kg
1.41
1.49
1.34
Lactation wt loss, kg
25.4
12.9
19.2
21-day wean wt/pig, kg
5.39
5.24
4.72
Pigs weaned/1itter
7.91
6.89
7.22
TABLE 12. REPRODUCTIVE PERFORMANCE OF SOWS
Sewage Sludge (%)

0
10
20
Gilts mated
11
11
11
Gilts farrowed
10
11
10
Breeding wt, kg
1.44.1
i47.4
131.7,
162.3d
Farrowing wt, kg
192.0d
190.0a
Total pigs farrowed/litter
10.6
10.0
9.1
Live pigs farrowed/litter
9.90
9.63
8.30
Birth wt/pig (live), kg
1.51
i.58
33.5a
1A7b
Lactation wt loss, kg
34.4a
16.5
21-day wean wt/pig,kg
5.11
4-86d
5.24d
Pigs weaned/litter
8.00
6.27
6.30°
L	J
' ' ' Means bearing like superscripts do not differ signifi-
cantly (P < 0.05 and P < 0.10, respectively)
99
-------
TABLE 13. GILT OFFSPRING FED CHICAGO SLUDGE



Sewaqe Sludqe (%)

0
10
20
Average daily gain,
kg
0.65a
0.60a
0.491
Average daily feed,
kg
1.84a
1.83a
2.091
Feed/Gain

2.84a
3.08a
4.261
a'b Means in same row bearing like superscripts do not
differ significantly (P < 0.05)
TABLE 14. GILT OFFSPRING FED GAINESVILLE SLUDGE



Sewage Sludge {%)

0
10
20
Average daily gain,
kg
0.61
0.60
0.56
Average daily feed,
kg
1.88a
2.10b
2.08b
Feed/Gain

3.16a
3.59b
3.70b
a'b Means in same row bearing like superscripts do not differ
significantly (P < 0.05)
100
-------
C. EFFECT OF FEEDING DIGESTED SEWAGE SLUDGE ON LONG-TERM
SOW REPRODUCTIVE PERFORMANCE
ABSTRACT
Gilts selected from 2 generations in succession were fed diets con-
taining 0, 10, or 20% dried sewage sludge (DFS) in a basal corn-soybean
mean formulation. In order to assess long-term effects, continuity in
the experiment was maintained by feeding second generation females the
same dietary regimen fed to their dams.
Data collected indicated that feeding sewage sludge to female swine
over an extended period adversely affected many criteria used in evalu-
ating reproductive performance. Breeding, farrowing, and rebreeding weights
were reduced. Lactation and gestation weight changes were lower and
fewer pigs were farrowed in sow groups receiving 10 and 20% sewage sludge
in diets. First and second generation sows fed diets containing 10 and
20% sewage sludge weaned lighter pigs at 21 days when compared with sows
fed the basal diet. In each parity, pigs farrowed by sows fed sewage
sludge in diets displayed depressed average daily gain. A comparison of
the data from both generations receiving diets containing sewage sludge
indicate that reproductive performance was more diminished in second
generation sows than in first.
INTRODUCTION
Beaudouin et at. (1980) fed diets containing 0, 10, and 20% DFS to
female swine during their first 2 pregnancies, and to their offspring
from weaning to market weight. Their data show that all proximate
analysis nutrients except ether extract were decreased in digestibility
when 20% DFS was in the diet. Total digestible nutrients, metabolizable
energy, and nitrogen retained were also decreased by the diet containing
20% DFS. Their data also indicate that breeding and gestation weights
in sows and birth and weaning weights in progeny were lower in groups
receiving 20% DFS in diets. Conversely, numbers of total and live pigs
farrowed and pigs weaned per litter were higher in sow groups fed the
diet containing 20% DFS, but means were not significant. Feeding diets
containing 10 and 20% DFS to growing-finishing pigs reduced daily gains
when compared with pigs fed the control diet.
The objectives of this experiment were:
1.	To evaluate dried University of Florida sewage sludge (DFS) as
a source of nutrients for swine.
2.	To observe the reproductive performance of female swine fed
diets containing DFS throughout 5 parities over 2 sow generations
in succession.
101
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The first objective yielded information concerning the feeding value
of DFS while the second objective revealed possible long-term effects of
sludge-containing diets on swine reproduction.
METHODS AND MATERIALS
Sixty-six gilts (33 from each of 2 successive generations) of predom-
inantly Yorkshire X Hampshire ancestry, were allotted randomly to receive
an experimental diet containing either 0, 10, or 20% DFS. Second genera-
tion gilts were selected from the second parity of first generation sows
(Figure 1). Data reporting reproductive performance of first generation
sows and postpartum performance of their progeny were collected during
3 successive farrowing. Data from second generation sows and their progeny
were collected during 2 successive farrowings. Second generation sows
received the same experimental diet fed to their dams.
Following breeding, females were moved to outside dirt lots that
were devoid of vegetation. During prebreeding, breeding, and gestation,
animals were group fed their assigned diet at the level of 1.81 kg/day.
During lactation, sows were allowed 5.4-4 kg/day consumption of the same
diet. Diets were formulated from corn-soybean meal (Table 15). Corn was
replaced by DFS on a pound to pound basis to achieve the desired treatment
ratios.
Sows were routinely dewormed with Dichlorovos 14 days postpartum and
were washed, sprayed for. external parasites, weighed, and moved into a
central farrowing barn 107 days postcoitus. Data evaluating individual
sow and progeny performance to sow dietary treatment received were collected
at parturition and weaning (21 days postpartum). Sows were rebred on
the first estrus following weaning. Differences in treatment and parity
means in both sow generations were subjected to an analysis of variance,
completely random design (Steel and Torrie, 1960) performed by a computerized
general linear model of the statistical analysis system (SAS). Differences
among treatment and parity means were calculated using the student's "t"
test. DFS was obtained from the University of Florida, Gainesville sewage
treatment plant and is the product of aerobic secondary treatment of pre-
dominantly human sewage exposed to trickling filtration, contact stabiliza-
tion, and 3-stage digestion followed by air drying over sand beds. The
proximate analysis of 22 samples of DFS from the University of Florida
sewage treatment plant were reported by Beaudouin et at. (1980) (Table 16).
RESULTS AND DISCUSSION
Breeding and Farrowing Performance
Breeding and farrowing data and gestation intervals of sows fed diets
containing 0, 10, and 20% DFS are presented in Table 17. The number of
sows bred at each parity was identical in both generations, although the
farrowing percentage of second generation sows in parities 1 and 2 was
lower than that of sows in the first generation. Sows in both generations
102
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fed diets containing DFS farrowed fewer litters than those fed the basal
diet. Following the weaning of litters, first generation sows displayed
a higher rebreeding percentage at first estrus than second generation
sows. In both first and second generations, the percentage of sows
successfully rebred was lower in groups fed diets containing DFS. Gesta-
tion length was not affected by dietary treatment and did not differ with
parity or sow generation compared.
Sow Weight Response
The effects of adding 0, 10, and 20% DFS to diets fed first and
second generation sows on weight changes measured at successive intervals
of breeding, farrowing, rebreeding, lactation, and gestation are presented
in Table 18.
Breeding weights in first generation sows were reduced by additions
of 10 and 20% DFS to diets. Breeding weights in second generation sows
were also reduced over parities 1 and 2 when DFS was added to the basal
diet. In both first and second generation sows, breeding weights increased
between each ensuing parity but were greatest between parities 1 and 2.
This observation was attributed to the compensatory weight gain that
accompanies advancing age in sows.
Farrowing weights were decreased when 10 and 20% DFS were added to
the basal diet in both sow generations but both displayed weight gains
between parities 1 and 2. This observation may also be attributed to
compensatory weight gain although sows in the first generation exhibited
a net weight reduction between parities 2 and 3.
Rebreeding weights were affected by dietary treatment received. Sows
fed diets containing 10 and 20% DFS in both generations were consistently
lighter at rebreeding between parities than those fed basal diets. As
observed with breeding and farrowing weights, means of rebreeding weight
increased with ensuing parity in both sow generations.
An inverse relationship was measured between lactation weight change
and percent DFS in diets fed first generation sows. In each of 3 successive
parities, lactation weight change was generally less in groups receiving
diets containing 10 and 20% DFS. In parity 1 of second generation sows,
lactation weight change among treatments was similar. However, in parity
2, sows receiving 10 and 20% DFS in diets lost less weight during lacta-
tion than sows receiving 0% DFS. Lactation weight change increased
between parities 1 and 2, then decreased between parities 2 and 3 in first
generation sows. Second generation sows also displayed an increase in
lactation weight change between parities 1 and 2.
The addition of 10 and 20% DFS to the sow diet in both first and
second generation sows caused a reduction in gestation weights within
each parity. Gestation weight gains were inverse to breeding, farrowing,
and rebreeding weight gains when means among successive parities were
compared. The heaviest gestation weight gains in both first and second
generation sows were measured during the first parity and were interpreted
as compensatory with age.
103
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Effects of DFS on Progeny Mortality
Table 19 presents data measuring mortality of pigs farrowed by first
and second generation sows receiving 0, 10, or 20% DFS in diets.
The total number of pigs farrowed by first generation sows fed diets
containing 0, 10, or 20% DFS was similar. However, sows fed the diet
containing 20% DFS averaged 1 pig fewer over the 3 parities. Second
generation sows fed the diet containing 10% DFS farrowed fewer pigs in
parities 1 and 2 than sows receiving the 0 or 20% diets. Litter size
remained constant between parities 1 and 2 then declined slightly between
parities 2 and 3 in first generation sows. Conversely, litter size was
increased appreciably between parities 1 and 2 in second generation sows.
Live pigs farrowed were not greatly affected by dietary treatment
fed first and second generation sows. The number of live pigs farrowed
by first generation sows increased slightly between parities 1 and 2 then
declined between parities 2 and 3. Second generation sows farrowed a
much larger number of pigs in parity 2 than in parity 1.
The number of stillborn pigs was not influenced by treatment diets
fed to first generation sows although a trend toward farrowing less
stillborn pigs was observed in the sow groups fed diets containing 10
and 20% DFS. Second generation sows fed the diet containing 20% DFS
farrowed less stillborn pigs in parity 1 but more than the other groups
in parity 2. The number of stilI born pigs farrowed decreased after
parity 1 in first generation sows but second generation sows farrowed
more stillborn pigs in parity 2 than in parity 1.
The number of pigs weaned by first and second generation sows was
not consistent with dietary treatment received. Additions of DFS to
diets fed first generation sows reduced the number of pigs weaned while
second generation sows fed 10 and 20% DFS weaned more pigs than sows fed
the basal diet. First generation sows displayed a tendency to wean fewer
pigs with each parity while second generation sows weaned more.
CONCLUSIONS
The long-term feeding of sewage sludge to female swine adversely
effects many criteria used in evaluating reproductive performance.
Breeding, farrowing, and rebreeding weights in both sow generations were
lower in groups fed 10 and 20% sludge in diets. Lactation weight change
was lower in sows fed the diets containing DFS. Gestation weight change
was lowest in sow groups fed diets containing 10 and 20% additions of DFS
First and second generation sows fed diets containing DFS farrowed
fewer pigs while the number of live, stillborn, and weaned pigs was not
influenced appreciably by treatment. The birth weights of pigs farrowed
by both generations of sows did not differ with dietary treatment but
pigs farrowed in parity 2 were heavier than those farrowed in other
parities. First and second generation sows fed diets containing 10 and
104
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20% DFS weaned lighter pigs when compared with sows fed the basal diet.
Average daily gain (ADG) was also influenced by sow dietary treatment.
In both sow generations, groups fed diets containing 10 and 20% DFS
farrowed pigs that displayed reduced ADG from birth to weaning.
A comparison of the data indicates that overall reproductive per-
formance was more diminished in second generation sows fed DFS in diets
than in first. Weight changes in first and second generation sows and
progeny weight data from each parity indicate that DFS is deficient in
content, biological value, and/or availability of an essential nutrient(s).
The mean value of crude protein in 22 samples of DFS from the Uni-
versity of Florida sewage treatment plant reported by Beaudouin et at.
(1980) shows that this nutrient partition is present in quantitatively
attractive levels. However, crude protein, calculated from nitrogen con-
tent determined by the Kjeldahl method may be based partly on nonprotein
nitrogen, nitrates, and nitrites. Swine do not utilize nonprotein nitrogen
(NPN) efficiently (Pond and Maner, 1974) and trends in reproductive
performance of sows in this study are similar to those of sows in other
studies fed regimens in which dietary protein was restricted (Pond et al.,
1968; DeGeeter et at., 1972; Hammell et at., 1976). Additions of DFS
to the basal diet appeared to dilute nutrient content and the detrimental
effects on reproductive performance in female swine may have resulted in
part from deficiencies in dietary protein.
REFERENCES
1.	Beaudouin, J., R. L. Shirley, and D. L. Hammell. Effect of Sewage
Sludge Diets Fed Swine on Nutrient Digestibility, Reproduction,
Growth, and Minerals in Tissues. J. Anim. Sci., in press, 1980.
2.	DeGeeter, M. J., V. W. Hays, D. D. Kratzer, and G. L. Cromwell. Repro-
ductive Performance of Gilts Fed Diets Low in Protein During Gestation
and Lactation. J. Anim. Sci., 35:772, 1972.
3.	Hammell, D. L., D. D. Kratzer, G. L. Cromwell, and V. W. Hays. Effect
of Protein Malnutrition of the Sow on Reproductive Performance and on
Postnatal Learning and Performance of the Offspring. J. Anim. Sci.,
43 :b89, 1976.
4.	Kienholz, E. W., G. M. Ward, and D. E. Johnson. Sewage Sludge Metals
in Cattle Tissues. J. Anim. Sci., 43(78):230 (Abstract), 1976.
5.	Pond, W. G., W. C. Wagner, J. A. Dunn, and E. F. Walker, Jr. Repro-
duction and Early Postnatal Growth of Progeny in Swine Fed a Protein-
free Diet During Gestation. J. Nutr., 94:309, 1968.
6.	Pond, W. G. and J. H. Maner. Swine Production in Temperate and
Tropical Environments. W. H. Freeman and Company, San Francisco, CA,
1974.
105
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7.	Smith, G. S., J. M. Caddie, P. M. Walters, and H. E. Kiesling.
Sewage Solids as Feedstuffs for Ruminants. J. Anim. Sci., 43:334,
1976.
8.	Smith, G. S. and C. R. Staples. Heavy Metals in Rats Fed Sewage
Solids. J. Anim. Sci., 43:233, 1976.
9.	Steel, R. G. D. and J. H. Torrie. Principles and Procedures of
Statistics. McGraw-Hill, New York, NY, 1960.
106
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FIGURE 1. DIAGRAM OF EXPERIMENTAL DESIGN USED TO EVALUATE EFFECT OF
FEEDING DIGESTED SEWAGE SLUDGE ON LONG-TERM SOW REPRODUCTIVE
PERFORMANCE
107
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TABEL 15. GESTATION-LACTATION DIETS
Ingredient (%)
Diet
1
Diet
2
Diet
3
Corn
70.45
60.45
50.45
SIudge

10.00
20.00
Ground oats
10.00
10.00
10.00
Soybean meal
16.00
16.00
16.00
Defluorinated phosphate1
2.50
2.50
2.50
Iodized salt
0.50
0.50
0.50
Trace mineral premix2
0.05
0.05
0.05
Vitamin premix3
0.50
0.50
0.50
Antibioti c
+
+
+
Provided courtesy of Occidental Petroleum Company, White Springs,
FL
2Provides the following in ppm of diet: zinc, 200; iron, 100;
manganese, 55; copper, 11; iodine, 1.5; and cobalt, 1.0. Courtesy
of Calcium Carbonate Company, Quincy, IL
3Provides per lb of complete feed: vitamin A, 5000 IU; vitamin D,
2500 IU; vitamin E, 5 IU; riboflavin, 5 mg; pantothenic acid, 12 mg;
niacin, 25 mg; choline chloride, 500 mg; and vitamin B12, 20 meg.
Courtesy of Charles Pfizer and Company, Terra Haute, IN
TABLE 15. PROXIMATE ANALYSIS9 OF DIGESTED SEWAGE SLUDGE FROM THE
UNIVERSITY OF FLORIDA, GAINESVILLE SEWAGE TREATMENT PLANT
L
	Partition	
Ni trogen
Air Dry Crude Crude Ether free
Matter Protein Fiber Extract Extract Ash
Item %	%	%	%	%	%_
DFSC 91.9	25.9	.46	.45	40.6 28.8
(66-98) (22-37) (.7-15) (.01-1.1) (9.47) (27-57)
a Data of Beaudouin etal., 1980
k Percent dry matter
r
Mean and range of 22 samples
108
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TABLE 17. REPRODUCTIVE SUCCESS OF SOWS FED DIETS CONTAINING DIGESTED SEWAGE SLUDGE
Pari ty
Percent Sewage Sludge (DFS) in Sow Diet
First Generation
0
10
20
Second Generation
0
10
20
Sows Bred
1
2
3
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
Sows farrowed
1
2
3
11
10
11
9
11
10
9
10
10
7
6
7
7
Gestation length
(days)
1
2
3
114.7
114.6
114.6
114.2
114.8
114.1
114.4
116.1
115.3
115.6
113.8
114.1
114.5
114.0
114.0
a Criteria for first and second generations do not differ (P < 0.05)
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TABLE 18. WEIGHT RESPONSE OF SOWS FED DIETS CONTAINING DIGESTED SEWAGE SLUDGE
Criterion Pari ty
First Generation
Sewage Sludge (DFS) in Sow Diet {%)
Second Generation
0
10
20
TAvgT
0
10
20
(Avg)
106.5
156.6
99.3
144.0
100.6
134.3
(102.1)P
(144.9)
131.6h
121.71
117.51

176. of]
198.3n
154.4]
167.I1
150.3]
152.6
(160.2)F
(172.7)b
187.2h
160.81
151.b1

157'4h
165. r
134.9]
143.51
128.4]
141.81
(140.2)
(150.1)
161.3J
139.2k
135.lk

"18-6h
-33.2"
"19'4hi
-23.6
-21.9.
-10.81
( 20.0)
( 22.5)
-25.9
-21.5
-16.4

67.
38.2n
56.0 J
17.31
52.5]
14.6
( 58.5)®
( 23.4)
52.7J
36.7k
u
33.6

Average body weight (kg)
Breeding
Farrowing
Re-
breeding
Lactation
Change
Gestation
Change
1
2
3
Avg
1
2
3
Avg
1
2
3
Avg
1
2
3
Avg
1
2
3
Avg
76.5
144.1
157.2
125.9
169.5
191.6
177.6
179.6
144.1
157.2
161.9
154.4
-25.4
-34.5
-15.8
-25.2
93.Oj
45.9n
20.6.
53.2J
h
h
h
77.7
156.4
127.2
161.7.
190.0r
171.5
174.4
147.3
156.4
152.1
151.9
-12.1.
-33.6
-19.5
-12.7
83.3:
38.1
14.8.
45.4
hi
hi
79.6 ( 77.8)
145.8
119.0
151.0.
162.3
hi
ghi
131.7
145.8
153.5.
143. V
-19.3
-16.5
-14.9
-16.9
71.4
28.6
14.8.
38.3*
"i
i
(153.1)"
U60.7).
(181.3)
160.6. (169.9)
158.O1
(141 • 0) P
(153.1)?
(155.8)b
(-18.9)k°
(-28.2)^
(-16.7)
( 82.6)®
( 37.5V
( 16.7)9
. .Second generation sows were offspring from the second parity of first generation sows.
£ Means within columns not followed by the same superscript are significantly different (P < 0.05)
^'•'^Means within columns not followed by the same superscript are significantly different (P < 0.01)
. . Means within rows not followed by the same superscript are significantly different (P < 0.05)
Means within rows not followed by the same superscript are significantly different (P < 0.01)
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TABLE 19. EFFECTS OF SOW DIETS CONTAINING DIGESTED SEWAGE
SLUDGE ON CORRESPONDING PROGENY MORTALITY
Sewage Sludge in Sow Diets (%)
:ion
Criterion	Parity 0 10	20 (Avg)
a
First Generation	Second Generation
Pigs farrowed/ 1 10.6 9.4 9.7 (9.9)
litter (no.) 2 10.6 10.0 9.1 (9.9)
Total	3 9.0 10.9 8.1 (9.3)
(8-7)
Live	2 9.9 9.6 8.3 (9.3)
(8.5)
Parity
0
10
20
1
10.6
9.4
9.7
2
10.6
10.0
9.1
3
9.0
10.9
8.1
Avg
10.1
10.1
9.0
1
9.1
8.1
9.0
2
9.9
9.6
8.3
3
7.6
10.1
7.8
Avg
8.9
9.3
8.4
1
1.3
1.2
.7
2
.7
.4
.8
3
1.0
.8
.3
Avg
1.0
.8
.6
1
7.9
6.9
7.2
2
8.0
6.3
6.3
3
6.5
6.5
6.6
Avg
7.5
6.5
6.7
(1-1)
Still-	2 .7 .4	.8 ( .6)
born	3 1.0 .8	.3 ( .7)
(7.3)
Pigs	2 8.0 6.3 6.3 (6.9)
weaned	3 6.5 6.5 6.6 (6.5)
0
10
20
(Avg)
8.5f
13.9T
8.3
10.89
11.3f
12.9 9
( 9.4)^
(12.5)
11.2d
9.6d
12. le

7.3
12.3
7.7
1U.3
10.9
10.3
( 8.6)=
(ii.or
^fq
1.8 9
:!*
.4f
2.6
( .7)®
( 1.6)
1.6
.6
1.5

4.3
7.4
5.9
8.7
7.6
6.9
( 5.9)
( 7.7)
5.9
7.3
7.3

? Second generation sows were offspring from the second parity of first generation sows.
^'c Means within columns not followed by the same superscript are significantly different (P < 0.01)
f'e Means within columns not followed by the same superscript are significantly different (P < 0.05)
Means within rows not followed by the same superscript are significantly different (P < 0.05)
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TABLE 20. BIRTH TO WEANING3 PERFORMANCE OF PROGENY FROM SOWS FED DIGESTED SEWAGE SLUDGE
Sewage Sludge in Sow Diet (%)	,
First Generation	Second Generation
Weaning
ADG
Cri terion	Parity 0	10	20 (Avq)	0	10	20 (Avq)
Average body wt
(kg)
Bi rth
1
1.41
1.49
1.34
(1.41)^
1.41
1.44
1.44
(1.43)
2
1.55
1.58
1.47
(1.54)5
1.51
1.62
1.66
(1.59)
3
1.39
1.39
1.37
(1.39)
-
-
-

Avg
1.45
1.49
1.39

1.46
1.53
1.55

1
5.39
5.23
4.72
(5.14)
5.06
4.95f
4.67f
(4.89)
2
5.11
4.87
5.24
(5.07)
6.22
4.98
5.15
(5.52)
3
5.54
4.94
5.18
(5.24)
-

"f
-
Avg
5.35
5.01
5.05

5,64e
4.97
4.91

1
.187
.179
.168
(.179)
.172
.166f
.154f
(.164)
2
.218
.165
.163
(.170)
.218
.165
. 163
(.182)
3
.193
.167
.178
(.181)
A


(.182)
Avg
.199
.170
.170

. 195
. 166
. 159

a 21 days post partum
k Second generation sows were the offspring of the second parity of first generation sows
c d
' Means within each column not sharing a common superscript are significantly different (P < 0.05)
e'^ Means within each row not sharing a common superscript are significantly different (P < 0.05)
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SWINE - OTHER HEALTH EFFECTS
6. T. Edds, C. F. Simpson, J. A. Popp, 0. Osuna, K. E. Ferslew,
R. L. Suber, and K. L. Kelly
Research was initiated in growing and breeding animals to determine
whether inclusion of various levels of urban sewage sludge in the rations
would adversely affect animal performance or gestation or result in hazar-
dous residues of metals or other toxicants that might affect human health.
The data reported in this chapter will describe the variety of trials that
influenced the other health effects in swine.
Several parameters were included in the early trials on possible
health hazards of including 10 and 20% dried University of Florida sewage
.sludge (DFS) in the rations of swine. It was suspected that bacteria and/or
viruses might persist in the DFS, thus, total white cell and differential
white cell counts were done on blood samples taken at the beginning and at
intervals throughout the feeding trials. Serum enzyme tests were performed
to identify infection or migratory damage from pathogens or parasites.
Performance of prothrombin times would give evidence of liver damage asso-
ciated with toxicants, especially endotoxins or aflatoxins. Blood and
tissue analyses were performed for metals or drugs suspected as hazardous
residues.
Newborn pigs were sacrificed and examined for normal blood levels of
cells and enzymes or for residue hazards transmitted from their dams
through their milk.
At the time of initiation of each trial and monthly thereafter, the
animals were bled for cellular and enzyme determinations, packed cell
volumes; blood samples were also collected for bacteriological and pesticide
evaluations. In addition, feed and fecal samples were collected for
similar studies; the latter was also checked for type and degree of para-
site infection.
Earlier trials incorporating dried University of Florida sludge (DFS)
into swine rations by Beaudouin et at., and Hammell et at., suggested that
accumulation or toxic effects developed from chronic exposure to rations
containing some elements present in the 10 or 20% dried sludge. There-
fore, a series of research trials were performed to determine which
factor(s) in sludge might be responsible for the delayed toxicity.
113
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SECTION A. Feeding Trials of Dried Urban Sludge and the Equivalent Cadmium
Level in Swine. O.Osuna, G. T. Edds, J. A. Popp, J. Monegue,
and K. E. Ferslew
SECTION B. Toxicology of Aflatoxin Bi, Warfarin, and Cadmium in Young
Pigs. 0. Osuna and 6. T. Edds
SECTION C. High Performance Liquid Chromatographic Determination of Sul-
fonamides by Ionic Suppression. R. L. Suber and G. T. Edds
Abstract
SECTION D. Comparison of the Pharmacokinetics of Sulfisoxazole in Humans
and Two Monogastric Species. R. L. Suber, C. Lee, G. Torosian,
and G. T. Edds
Abstract
SECTION E. Comparison of the Potential Toxicity of Bilirubin in Humans
and Two Monogastric Species after a Single Administration of
Sulfisoxazole. R. L. Suber, J. C. Gudat, and G. T. Edds
Abstract
114
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A. FEEDING TRIALS OF DRIED URBAN SEWAGE AND THE
EQUIVALENT CADMIUM LEVEL IN SWINE
ABSTRACT
Toxicity from feeding dried sewage sludge included in a normal swine
starter ration may occur from a deficiency of available protein or other
essential nutrients, or from the accumulation of hazardous chemical residues.
One lot of sewage sludge from Chicago was found to contain high levels
of cadmium (165 ppm). This trial compared the effects of feeding weanling
pigs a starter ration containing 50% dried, activated, Chicago sewage sludge
with a standard 18% crude protein basal diet, plus 83 ppm cadmium, for
9 weeks.
Forty-eight 4 week-old, hybrid pigs were allotted to 2 replicate
experiments of 2 treatment groups each. Body weight, feed consumption,
and blood samples were determined weekly including PCV, RBC, WBC, MCV,
Hb, and serum levels of 4 enzymes, AP, yGT, GOT, GPT. Feed and fecal
samples were collected weekly and analyzed for cadmium content. Tissue
samples were provided at the slaughter time on days 38, 42, and 56 for
metal analyses and pathologic evaluations.
Depressed growth and feed consumption were evident in pigs consuming
50% Chicago sludge and 83 ppm of cadmium.
Cadmium exposure induced microcytic and hypochromic anemia. At
necropsy, pale, white muscles and kidneys were observed.
INTRODUCTION
In swine production with modern feeding and management conditions,
cadmium toxicity is relatively rare. However, borderline toxicities are
possible where animals ingest recycled waste materials, such as urban
sewage sludge, in which cadmium may be concentrated (Neathery and Milton,
1976). Two percent sludge has been found to provide a satisfactory source
of vitamin Bi2 for the pig. Toxicity from feeding dried sewage sludge
included in a normal swine starter ration, may occur from a deficiency of
available protein or other essential nutrients, or from the accumulation
of hazardous chemical residues (Edds et al., 1978).
Cadmium interferes with the functioning of necessary elements such
as zinc in enzyme systems (Flick et al., 1979; Friberg et al., 1971).
Also, a lethal dose of cadmium may inhibit mitochondrial oxidative
115
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phosphorylation in rat liver and may be directly correlated with death
(Southard et al., 1974). General clinical symptoms of cadmium toxicity
in mammals include anemia, retarded testicular development or degeneration,
enlarged joints, scaly skin, liver and kidney damage, reduced growth and
increased mortality (Miller, 1971; Powell etal., 1964).
Studies of animal or human exposure to cadmium suggest that neither
bloodnor urine is a reliable indicator of total body burden of cadmium.
Blood is unsuitable because the sojourn of cadmium in this tissue is brief,
and its concentration in blood is extremely low (Petering et al., 1973).
Consequently, blood cadmium data have little diagnostic value. After
absorption, most cadmium is transported in plasma bound to gamma-globulin
(Shakin and Lerces, 1972). However, some may be bound with hemoglobin or
metallothionein in erythrocytes (Carlson and Friberg, 1957).
Generally, highest cadmium concentrations are in kidney, followed by
liver (Friberg et al., 1971; Miller et al., 1968; Neathery et al., 1974).
Muscle is well protected from ingested cadmium (Neathery and Miller, 1976).
The most notable finding upon examination of a reported case of
cadmium toxicity in swine was an extreme anemic condition (Alber, 1963).
It was found that oral or injected iron offered protection against cadmium
induced anemia in swine (Pond et al., 1973).
Recently, studies on the use of animal waste and sewage sludge for
animal feeds have been of interest. One lot of sewage sludge from Chicago
was found to contain high levels of cadmium (165 ppm). This trial compared
the effects of feeding weanling pigs a starter ration containing 50% dried,
activated, Chicago sewage sludge or a standard 18% crude protein basal
diet, plus 83 ppm cadmium, for 9 weeks.
MATERIALS AND METHODS
Forty-eight, 4-6 week old, hybrid pigs were allotted to 2 replicate
experiments of 2 treatments each, 12 pigs per group. The first of the 2
concurrent experiments included 12 barrows in both the control and the
83 ppm cadmium groups. The second experiment consisted of 6 males and
6 females in both the control and 50% Chicago sludge groups.
The pigs were fed a basal swine starter ration with an 18% crude pro-
tein basal diet (Table 1) as the control and as the ration to which either
the cadmium was added in the form of CdCl2 at an 83 ppm level or the
dried, activated, Chicago sewage sludge was added at 50% level.
All rations were tested weekly and found negative for aflatoxin Bi .
Feed and water were provided ad libitum for 9 weeks. Pigs were housed by
treatment in concrete floored and cement block pens with automatic
a
Mini column Technique, Southern Marketing and Nutrition Research Division,
Agricultural Research Service, U.S.D.A., P.O. Box 19687, New Orleans, LA.
116
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watering devices and standard pig self-feeders. Floors were washed daily
or when necessary to provide sanitary conditions. Feed was checked daily
and added when needed.
TABLE 1. BASAL DIET CONSUMPTION*
Ingredient	%
Yellow corn meal
71.8
Soybean meal
25.0
Dyna-fos
1.7
Limestone
0.8
Iodized salt
0.25
Trace mineral mix (ccc)
0.10
Vitamin premix (UF)
0.10
ASP-250
0.25
"k
Swine Feed Starter Ration, 18.71% protein, medicated, University of
Flori da.
Body weight (BW), blood, and fecal samples were taken daily. Blood
samples were procured by anterior vena cava puncture. Blood samples were
collected in tubes" with disodium edetate for white blood cell counts
(WBC), red blood cell counts (RBC), packed cell volumes (PCV), mean cor-
puscular volumes (MCV), and hemoglobin (Hb). Serum samples were obtained
for alkaline phosphatase (AP), gamma-glutamyl transpeptidase (yGT),
glutamate pyruvate transaminase (SGPT), & glutamate oxaloacetate transaminase
(SGOT) analyses. Enzyme analyses were performed with a centrifugal
analyzerc using standard procedures, reagents, and controls01.
Four pigs from each treatment group were slaughtered on weeks 4, 6,
and 9 (days 28, 42, and 56, respectively). Tissue samples collected
for histopathology and metal analyses included: kidney, liver, muscle,
heart, lung, spleen, stomach, small intestine, and testicle or ovary in
the second section of the trial. Liver samples were obtained from the
center hepatic lobe, left kidney, and muscle from the left ham.
L.
Baltimore Biological Laboratory, Division of B-D Laboratories, Inc.,
Baltimore, MD.
c Gemsaec Centrifugal Analyzer, Electro Nucleonics, Inc., Fairfield, NJ.
_i
Boehringer Mannheim Corp., Norcross, GA.
117
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Feed, tissue, and blood samples were slow-digested with hydrochloric
acid. Residues were then detected by atomic absorptione using standard
procedures . The various samples were tested for cadmium (Cd), chromium
(Cr), copper (Cu), iron (Fe), mercury (Hg), molybdenum (Mo), nickel (Ni),
lead (Pb), selenium (Se), and zinc (Zn).
Statistical analyses were performed by application of an analyses of
variance (ANOVA) to the data. Testing for significant differences on any
particular day between groups was done by "T" test analysis9.
RESULTS
Performance
Depressed growth and feed consumption were evident in the pigs con-
suming 83 ppm Cd (P < 0.05J in the diet. The control pigs of the first
experiment gained 0.37 pounds (lbs) more per day on the average than those
consuming Cd. Feed consumption for the same experiment averaged 0.28 lbs
per day less for the pigs receiving Cd. A decrease in feed efficiency was
also noted. The pigs receiving cadmium required 0.21 lbs of feed more
per lb of gain than those in the controls.
The pigs on the second experiment consuming 50% dried, activated,
Chicago sewage sludge (DCS) did not perform as well as the control pigs.
Weight gains of all pigs in the 50% DCS group were lower (P < 0.01) than
those in the control group as described by Edds et al., 1978.
Hematology
The RBC, MCV, Hb, and PCV of the cadmium pigs declined steadily and
significantly from days 21 through 56. An extreme microcytic, hypo-
chromic anemia was observed by day 42 of the trial (P < 0.01). There was
no significant difference in the WBC levels in this first experiment.
The RBC, MCV, PCV, and Hb of the 50% DCS group of the second experi-
ment paralleled the values of the control group throughout the experiment
with no significant differences. The WBC counts of this 50% DCS group
were significantly higher (P < 0.01) at the sixth week.
Serum Enzyme Levels
None of the 4 serum enzymes measured in the first experiment (AP,
yGT, GOT, and GPT) (Table 2) showed differences between treatments.
In the second experiment with 50% DCS, there was significant increase
in GOT (Table 3) at weeks 3, 5, and 7. There were no significant changes
with the other enzymes (AP, yGT, and GPT).
e Perkin-Elmer Atomic Absorption Unit.
f IFAS Soils Department, University of Florida, Gainesville, FL.
9 IFAS Statistics Department, University of Florida, Gainesville, FL.
118
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Metal Analyses
In the first experiment, Cd was at a higher concentration both in the
liver (P < 0.0001) and kidney (P < 0.0001) in the pigs consuming 83 ppm of
cadmium. No Cd was detected in blood or muscle of the control and Cd
treated groups. Copper was significantly higher in the kidney (P < 0.0037)
but lower in the muscle (P < 0.048), liver and blood (P < 0.034) in the
pigs consuming 83 ppm of Cd. Zinc was significantly higher in the kidney
(P < 0.0001) but lower in the liver (P < 0.0006) and muscle (P < 0.0221)
tissues of the 83 ppm Cd treated group. Iron was significantly decreased
in the liver (P < 0.0001), kidney (P < 0.0001) and blood (P < 0.0001) in
the pigs treated with 83 ppm Cd. There were no differences in the levels
of Co, Hg, Pb, Ni, or Cr in the liver, kidney, muscle, and blood between
the control and 83 ppm Cd groups. Molybdenum was significantly higher
(P < 0.017) in the kidney but not significantly different in liver, muscle,
and blood of the 83 ppm Cd treated pigs (Table 5).
In the second experiment, Cd was at a greater concentration in the
liver (P < 0.001) and kidney (P < 0.001) in the pigs consuming 50% DCS.
However, the concentrations of Cd in the liver and kidney of the 50%
DCS group were lower than the concentrations in the same organs of the
83 ppm Cd treated group. No Cd was found in either blood or muscle of
the control and 50% DCS treated groups. Copper concentration was signi-
ficantly increased in the kidney (P < 0.001) in the 50% DCS treated
group but no differences were determined between groups in the liver,
muscle, and blood. Zinc was significantly increased in the kidney
(P < 0.001) but lower in the liver (P < 0.0276) and muscle (P < 0.037)
of the 50% DCS treated group. Iron was significantly increased in the
liver (P < 0.0161) but was lower in the muscle (P < 0.049) of the 50%
DCS pigs. No iron differences were observed in the kidney or blood of
the 2 control and 50% DCS groups. There were significantly increased con-
centrations of Hg (P < 0.015), Ni (P < 0.0003), and Cr (P < 0.018) in
the kidney of the pigs treated with 50% DCS. There were no differences
in the concentrations of Fe, Co, Mo, and Pb in the kidneys of the control
and 50% DCS groups. Chromium was significantly increased in the liver
(P < 0.0274) of the 50% DCS treated group. No significant differences
between groups were observed in the liver and muscle of Cu, Co, Mo, Hg,
and Pb. No significant differences of Cr, Zn, Co, Mo, Pb, Hg, and Ni in
blood occurred between groups. More significant levels of muscle Zn
levels (P < 0.024) and blood copper levels (P < 0.0002) were observed
in the males than in the females (Table 6).
Pathology
The pathologic examination of heart, liver, intestine, kidney, and
lung tissues collected at slaughter from the 4 treatment groups revealed
only minor lesions. The tissues from the control pigs were normal except
the intestinal tracts showed increased eosinophils in the lamina propria.
The intestines from the animals receiving the Cd supplemented ration were
normal. The heart tissue of one of the pigs in the Cd supplemented group
119
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had a small zone of focal myocarditis and a lymphoid nodule protruding
into a bronchus. Two of the other pigs in this group also had focal mono-
nuclear cell infiltration into the lung tissue.
The tissues, liver, lung, and intestinal tract of the group on 50%
Chicago sludge showed acute cholangitis, hepatic cytoplasmic vacuolation,
mononuclear cell infiltration into alveolar tissues; foci of eosinophils
were localized in the intestinal lamina propria.
No lesions were observed in the kidney tissues in any of the groups.
DISCUSSION
Performance
Depressed growth and feed consumption were evident in pigs consuming
either rations containing 83 ppm of Cd or in pigs with 50% Chicago sludge.
Toxicity from feeding dried sewage sludge included in a normal swine starter
ration may occur from a deficiency of available protein or other essential
nutrients, or from the accumulation of hazardous chemical residues (Edds
et at., 1978).
Hematology
When the control values of blood between the 2 replications are com-
pared, important conclusions can be made: (1) RBC counts in both control
groups fall into the same range between 6.3 and 7.3 millions/mm3; (2) MCV
values in both control groups fall into the same range between 44 to 56
cubic microns; (3) Hb values in both control groups fall into the same
range between 11.1 to 13.2 gm%; (4) PCV values of both control groups fall
into the same range between 35 and 41. Therefore, the conditions of the
2 replications may be considered normal physiologic standards for swine
between 4 to 15 weeks of age.
The microcytic hypochromic anemias are specific for iron deficiency
or failure to utilize iron. Chronic blood loss or iron, copper, and pyri-
doxine deficiencies must be considered (Schalm, 1965). After absorption,
most Cd is transported in plasma bound to gamma-globulin (Shaik and Lerces,
1972); however, some may be bound with hemoglobin or metallothionein in
red blood cells (Carlson and Friberg, 1957).
Our results agree with Alber (1963) that cadmium toxicity in swine
induces anemia and with Pond et at. (1973) that oral iron offers protection
against cadmium induced anemia in swine. The last statement can be under-
stood when the Fe concentrations of the diets are considered. There was
a high concentration of Fe (8,846.9 ppm) in the diets of the 50% Chicago
sewage sludge treated group. The concentrations of Fe in the Cd treated
group was similar to the control group of the first experiment. There-
fore, some types of sludge could be considered as a source of iron for
young pigs to correct anemia induced by Fe deficiency.
120
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Serum Enzyme Levels
The enzyme values are in agreement with histopathologic results.
There were no indications of liver damage induced by the 83 ppm of Cd in
the diets.
The increased levels of GOT and the high WBC counts in the 50% DCS
group may have resulted from histopathologic changes, characterized by
central lobular necrosis and vacuolation as well as acute cholangitis, in
the liver of some of the pigs.
Metal Analysis
We agree with Petering et al. (1973) that blood is unsuitable for
determination of Cd concentrations and has little diagnostic value.
Neither is muscle tissue since Cd levels are not detectable at all and our
0 values are in agreement with several other reports (Neathery and Miller,
1976). Therefore, animal muscle residue levels would pose no hazard for
human health.
Higher Cd concentrations were found in the kidney, followed by the
liver as reported by others (Neathery and Miller, 1976; Friberg et al.,
1971) in both the 83 ppm Cd and the 50% DCS groups. Probably, other
elements such as Fe, Zn, and Cu present at high concentration in the 50%
DCS feed (Table 4), decrease the absorption of Cd in the gastrointestinal
tract. This may explain the lower concentrations of Cd in tissues in this
group as compared to the group receiving 83 ppm Cd. In fact, tissue Zn
levels were elevated in the 83 ppm Cd treated pigs which is in agreement
with Powell et al. (1964).
121
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TABLE 2. SERUM ENZYME LEVELS FOR CADMIUM TRIALS
AP (iu/1)	yGT	GOT	GPT
Weeks
Control
83 ppm Cd
Control
83 ppm Cd
Control
83 ppm Cd
Control
83 ppm
1
673.1
713.7
15.2
13.3
31.5
31.0
50.3
56.1
2
827.7
812.3
27.2
25.6
25.1
33.4
43.5
50.2
3
856.1
771.0
18.7
17.7
97.9
88.3
43.2
44.0
4
756.3
685.3
14.9
16.1
69.7
66.6
50.0
56.3
5
646.0
601.2
16.5
17.6
36.6
43.4
56.4
57.6
6
703.1
601.0
15.5
14.8
37.1
35.2
29.0
35.2
7
457.5
529.3
13.3
14.9
19.7
33.9
36.6
46.7
8
507.2
601.3
15.2
18.4
12.0
25.6
21.1
31.9
9
564.2
580.3
10.1
13.2
21.0
12.0
31.1
23.4
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TABLE 3. SERUM ENZYME LEVELS FOR CHICAGO SEWAGE SLUDGE TRIALS
Weeks
Control
AP (iu/1)
50% sludge
yGT
Control
50% sludge
GOT
Control
50% sludge
Control
GPT
50% sludge
1
504.8
570.8
9.0
13.3
11.4
15.3
34.7
35.1
2
604.3
561.3
13.7
16.7
24.7
27.3
32.6
20.5
3
662.8
557.2
15.7
12.8
36.1
57.3*
39.2
40.6
4
614.9
518.5
13.7
14.9
24.3
25.1
28.2
28.4
5
556.3
644.7
12.9
15.3
23.7
44.5*
29.6
25.8
6
640.9
526.6
14.9
18.2
18.4
32.0*
31.2
25.3
7
522.4
402.3
15.9
16.0
17.9
42.3*
26.0
24.8
8
571.0
423.5
13.6
10.4
16.0
40.1
22.8
13.4
9
484.3
468.8
12.2
12.6
22.7
24.0
23.3
20.6
* P < O.Ol
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TABLE 4. AVERAGE METAL VALUES IN FEED (PPM)

Control
83 ppm Cd
Control
50% Sewage Sludge
Cd
0.36
78.6s
0.12
147.32
Cu
13.9
13.4
13.9
331.6
Fe
377.7
373.5
237.2
8,846.93
Co
0.62
0.62
0.69
6.16
Zn
209.8
190.6
183.4
773.7 1
Pb
1.09
0.62
1.66
129.9
Ni
1.71
1.36
2.75
123.8"
Cr
2.65
2.27
2.76
696.4
1	P	< 0.0001
2	P	< 0.0004
3	P	< 0.0006
4	P < 0.0007
5	P	< 0.01
124
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TABLE 5. AVERAGE MEAN VALUES OF METAL CONCENTRATIONS IN TISSUES OF YOUNG PIGS TREATED WITH 83 PPM
CdCl 2 IN THE DAILY DIETS
LIVER KIDNEY MUSCLE BLOOD
Control 83 ppm Cd	Control 83 ppm Cd	Control 83 ppm Cd	Control 83 ppm Cd
Cd
0.00
12.981
0.00
61.951
0.00
0.00
0.00
0.00
Cu
7.55
6.35
8.93
15.085
0.81
0.676
1.39
1.207
Fe
118.81
23.151
31.28
16.541
7.53
7.23
478.5
273.82
Zn
53.89
41.252
20.62
34.081
14.01
10.00"
8.26
6.987
Mo
1.25
1.30
0.73
0.783
0.00
0.00
0.00
0.00
Co
0.00
0.08
0.03
0.03
0.03
0.00
0.00
0.00
Pb
0.00
0.00
0.00
0.00
0.03
0.03
0.00
0.00
Hg
0.01
0.00
0.01
0.01
0.00
0.00
0.00
0.00
Ni
0.00
0.00
0.11
0.02
0.00
0.00
0.00
0.00
Cr
0.00
0.03
0.00
0.09
0.00
0.00
0.00
0.00
1 P
< 0.0001


5 P
< 0.0037



2 P
< 0.0006


6 p
< 0.0048



3 P
< 0.0017


7 P
< 0.03



4 P
< 0.0022







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TABLE 6. AVERAGE MEAN VALUES OF METAL CONCENTRATIONS IN TISSUES OF YOUNG PIGS FED 0 AND 50%
CHICAGO SEWAGE SLUDGE (PPM)
Control 50% DCS Control 50% DCS	Control 50% DCS	Control 50% DCS
Cd

0.00
3.151
0.00
23.491

0.00
0.00
0.02
0.02
Cu

5.30
5.90
6.60
10.701

0.800
0.831
1.05
1.12
Fe

133.67
163.24^
41.03
41.03

10.40
8.30s
442
440.60
Zn

57.80
51.307
24.30
37.401

13.99
12.497
4.64
4.58
Mo

1.00
0.92
0.70
0.79

0.02
0.02
0.00
0.00
Co

0.03
0.00
0.03
0.05

0.00
0.00
0.01
0.03
Pb

0.00
0.05
0.05
0.00

0.00
0.00
0.00
0.06
Hg

0.00
0.05
0.014
0.368s

0.00
0.00
0.00
0.00
Ni

0.00
0.00
0.021
0.5652

0.00
0.00
0.04
0.04
Cr

0.056
0.165s
0.214
0.380s

0.00
0.00
0.15
0.11
1 P
<
0.0001



5 P
< 0.0180



2 P
<
0.0003



6 p
< 0.0274



3 P
<
0.0015



7 P
< 0.0376



* p
<
0.0161



8 P
< 0.049



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B. TOXICOLOGY OF AFLATOXIN Bls WARFARIN, AND CADMIUM IN YOUNG PIGS
ABSTRACT
The objectives of this experiment were to compare the toxic effects of
aflatoxin Bl9 a dihydrofuranocoumarin, and warfarin, a 3-(a-acetonyl-benzyl)
-4 hydroxycoumarin; and also to determine whether an additive effect from
either aflatoxin Bj or warfarin occurs when cadmium is present in higher
than normal levels in the diets of young pigs.
Thirty-six healthy weaned barrows, mixed breed, averaging 9 kg of body
weight, were assigned at random to 6 treatment groups, 6 pigs per group:
Group I - negative control; Group II - 0.2 mg/kg of aflatoxin Bi; Group III -
0.2 mg/kg of warfarin; Group IV - 83 yg/g of cadmium diet; Group V - 83 yg/g
of cadmium diet plus 0.2 mg/kg of aflatoxin B ; Group VI - 83 g/g of
cadmium diet plus 0.2 mg/kg of warfarin. Groups II, III, V, and VI received
5 daily doses of the chemical in gelatin capsules during the fifth week of
the experiment and the effects were followed for 10 days. Cadmium (Cd)
was provided daily through the diets (given as cadmium chloride) during the
40 days of the experiment.
The body weight loss (P < 0.0065) in the aflatoxin Bx group was asso-
ciated with decreased feed consumption. Significantly lower values in serum
total protein (P < 0.0378), alpha globulin (P < 0.0133) beta globulin
(P < 0.00119), gamma globulin (P < 0.05), and plasma fibrinogen (P < 0.0279)
were induced by aflatoxin B^
Significantly increased values of alkaline phosphatase (P < 0.016),
sorbitol dehydrogenase (P < 0.003), and aspartate aminotransferase (P <
0.05) were determined 48 hours after initiation of the dosing with afla-
toxin Bi and correlated with hepatic fatty infiltration and vacuolation
through all lobules.
Warfarin was more effective in producing earlier and higher values in
prothrombin time (P < 0.001) and activated partial thromboplastin time
(P < 0.007) than aflatoxin Bi by the second day after initiation of dosage.
Depressed growth, feed consumption, feed efficiency, white blood cell
counts (P < 0.0056), gamma globulin values (P < 0.0018), and an extreme
microcytic hypochromic anemia were evident in the pigs consuming 83 yg/g
Cd in the diet.
The Cd concentrations were highest in kidney, 42.9 yg/g, next in
liver, 7.92 yg/g, and correlated well with the loss of iron from the kidney
(P< 0.0001) and liver (P< 0.0001) in pigs treated with 83 yg/g Cd diets.
127
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Cadmium may have blocked the liver microsomal enzyme system and pre-
vented the activation of aflatoxin Bi to a toxic anticoagulant metabolite.
On the other hand, the blockade prevented the inactivation of warfarin,
enhancing its anticoagulant effects. This blockade was demonstrated
through clinical signs, hematologic, electrophoretic and clinical chemistry
changes, as well as gross and histopathologic lesions. It is concluded
that there is an inhibitory effect on the toxicities of aflatoxin Bi while
there is an enhancing synergistic effect with warfarin when Cd is present
in the diets of young pigs at 83 ug/g.
128
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C. HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATIONS
OF SULFONAMIDES BY IONIC SUPPRESSION
ABSTRACT
A high pressure liquid chromatography procedure is reported for ex-
traction and quantitation of 8 sulfonamides in stock solutions and in vitvo
plasma samples. This assay consists of a single, one-step extraction of
sulfonamides from plasma and is sensitive to 10.0 ng/ml at 254 nm without
additional concentration of the sample. Four sulfonamides (sulfamerazine,
sulfamethazine, sulfapyridine, and sulfathiozole) were separated from the
plasma matrix by either mobile phase regardless of pH. The sulfonamides
with the highest pKa, sulfanilamide (10.5) and sulfaguanidine (11.3),
were only separable from plasma in a 50% water/50% methanol mobile phase
at pH 7.45. The sulfonamide with the lowest pKa, sulfisoxazole (4.9),
and its metabolite, acetylsulfisoxazole (N4), were separated from plasma
by either mobile phase, 50/50 or 60/40 water/methanol, when acetate buffer
reduced the pH to 4.00. Standard concentration curves of peak height were
the most sensitive at 254 nm when a 60% water/40% methanol mobile phase
at pH 4.00 was used. Sulfanilamide and sulfaguanidine were the most
responsive to ultraviolet quantitation at 254 nm regardless of ionic
suppression or polarity of the mobile phase.
129
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D. COMPARISON OF THE PHARMACOKINETICS OF SULFISOXAZOLE IN
HUMANS AND TWO M0N06ASTRIC SPECIES
ABSTRACT
The pharmacokinetic profile of sulfisoxazole in dogs and swine was
found to be biexponential; humans were observed to have a single compart-
ment for this sulfonamide. The mean extrapolated unbound, unmetabolized
serum concentration of sulfisoxazole in the first compartment was 189.42
+ 38.45 yg/ml in dogs and 245.07 +44.88 yg/ml in swine, in the second
compartment, the extrapolated concentration was 2.56 +0.39 yg/ml in dogs
and 0.423 +0.088 in swine. The mean disposition rate from the first
or distribution compartment was 0.1726 +0.0604 hours"1 in dogs and faster
in swine, 0.5368 +0.0362 hours-1. The mean disposition rate from the
second or elimination compartment was much slower_in dogs and swine,
0.206 + 0.0014 hours"1, and 0.0153 + 0.0043 hours"1, respectively. Maximum
serum acetylsulfisoxazole, the N1* metabolite, were higher in humans, 30 to
35.0 yg/ml, than in swine, 12.0 to 20.0 yg/ml. However, swine were able to
acetylate sulfisoxazole at a faster rate. The acetyl N4 metabolite was
not separated from dogs1 serum.
The distribution constants of sulfisoxazole from the central to the
peripheral compartment was greatest in swine, 0.0296 hours"1, and then
dogs, 0.0140 hours"1. The distribution from the peripheral to the central
compartment was fastest in dogs, 0.0228 hours"1, and then in swine, 0.0162
hours"1. The elimination constants were greatest in swine, 0.5063 hours"1,
and then dogs, 0.1565 hours"1. The mean volume of distribution for the
central compartment was approximately the same in dogs and swine, 10.60 and
10.48 liters, respectively. The mean volume of the second compartment
was larger in swine, 19.76 liters, than in dogs, 6.55 liters.
The fraction of sulfisoxazole bound to serum proteins is capacity
limited. At concentrations greater than 200 yg/ml, the fraction bound is
reduced from 60% to 40% in vitro . This is confirmed by a reduced fraction
bound in vivo at concentrations greater than 200 yg/ml.
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E. COMPARISON OF THE POTENTIAL TOXICITY OF BILIRUBIN IN HUMANS AND TWO
MONOGASTRIC ANIMAL SPECIES AFTER A SINGLE ADMINISTRATION OF SULFISOXAZOLE
ABSTRACT
Administration of sulfonamides during periods of hepato-biliary failure
or hepatic immaturity can increase the potential toxicity of unconjugated
or indirect bilirubin. The results reported show a small but statistically
significant increase of potentially toxic, indirect, or unconjugated bili-
rubin in dogs after oral administration of sulfisoxazole (100 mg/kg). A
similar increase was not observed in swine (100 mg/kg) or humans (approxi-
mately 28 mg/kg) after oral or intravenous administration of sulfisoxazole
or in humans (approximately 28 mg/kg) after oral administration or in dogs
(100 mg/kg) after intravenous administration.
Total and conjugated bilirubin showed small but statistically signifi-
cant increases in dogs after oral and intravenous administration of sulfi-
soxazole (100 mg/kg) and in swine after oral administration of sulfisoxazole
(100 mg/kg). Total and conjugated bilirubin were significantly correlated
in dogs after oral and intravenous administration and in swine after oral
administration of sulfisoxazole. The increase in conjugated bilirubin,
along with a concomitant increase in total bilirubin, could be due to
hepatic induction of glucuronidating capacity or regurgitation of conjugated
bilirubin from the hepatocyte instead of excretion into the bile. There
was also a significant negative correlation in conjugated and indirect bili-
rubin, while total bilirubin increased, in dogs after oral and intravenous
administration of sulfisoxazole. These data illustrate a difference in
species and route of administration when attempting to assess the potential
toxicity of bilirubin.
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POULTRY
HEALTH EFFECTS OF SEWAGE SLUDGE AND GRAIN FROM SLUDGE TREATED
SOILS VERSUS EQUIVALENT METAL LEVELS IN POULTRY
B. L. Damron, 0. Osuna, R. L. Suber, and G. T. Edds
ABSTRACT
Duplicate experiments of 21 days duration were conducted with day-old
broiler-type chicks to study the influence of replacing one-half or all of
the normal dietary corn complement with corn grown on soil fertilized with
liquid Pensacola sludge (LPS).
Neither level of sludge corn had any adverse effect upon final body
weights or daily feed intake. The feed conversion values of experiment 1
were not significantly influenced by treatment; however, a statistically
significant decrease of efficiency was noted for the all-sludge corn treat-
ment of experiment 2.
In 2 studies with laying hens, the partial or total substitution
(50 or 100%) of sludge fertilized corn for that produced with commercial
fertilizer had no statistically significant effects upon any of the pro-
duction parameters measured in experiment 1. In experiment 2, the 100%
sludge corn treatment was associated with a significantly increased daily
feed intake and final body weight. Hatchability parameters and taste panel
results for eggs indicated no significant relationship to dietary treatment.
Mineral assays of blood samples and liver, kidney, and muscle tissues from
hens and broilers were not influenced by dietary treatment.
Levels of 0, 3, or 6% dried Chicago sludge (DCS) were substituted into
the diet of broiler chicks while equivalent nutrient levels were maintained.
In addition, 4 other treatments in experiment 1 and 5 in experiment 2
contained the amounts of cadmium, chromium, copper, and iron from reagent
sources equivalent to the levels of these elements coming from 6% DCS.
In experiment 1, the feeding of iron or chromium resulted in signifi-
cant body weight depressions. Only the feed intake of the birds receiving
the iron treatment was significantly below that of the control group. Both
iron levels in experiment 2 (2,993 mg/kg and 2,196 mg/kg)
depressed body weights and daily feed intake. The cadmium and iron treat-
ments of both studies resulted in elevated liver and kidney levels of these
minerals. There was also a trend of increasing cadmium residues in the
liver and kidney resulting from increasing DCS levels; however, the
utilization rate from DCS appeared to be only approximately 20%.
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Levels of 3.5 and 7% DCS sludge were fed to hens in 2 experiments.
In addition, amounts of cadmium, chromium, or copper equivalent to those
found in the 7% DCS diet were fed from reagent sources.
In the first experiment, none of the production criteria were signi-
ficantly influenced. In experiment 2, the addition of iron resulted in a
numerical depression of egg production. Daily feed intake was significantly
reduced by the iron level fed. Hatchability data was not found to be
consistently influenced by dietary treatment in either experiment.
INTRODUCTION
Activated sludge has been the subject of animal feeding trials for
well over 20 years. The material is a concentrated source of nitrogen for
the ruminant animals, and, even though the biological value of its protein
has been determined to be in the neighborhood of 50%; research with rumi-
nants has indicated that the nitrogen retention from activated sludge is
equal to that from soybean oil meal or urea (Hurwitz, 1957). Hurwitz
(1957) and Schendel and Johnson (1954) found that a level of 2% sludge
provided a satisfactory source of vitamin B12 for growth. Levels up to 3%
gave an additional response that could not be attributed to the presence
of vitamin B12 alone and was felt to indicate the action of unknown growth
factors; possibly due to the fermentation process involved. Firth and
Johnson (1955) found that dry activated sludge could be included in the
diet of the baby pig up to a level of 5% without adversely affecting growth.
In their chick trials, levels of 2 and 10% in the diet produced a growth
response in excess of 8% over the control group. Scott and Adams (1955)
found that as little as 1% sludge depressed growth when the diet contained
100 units of vitamin D, but that 4% was tolerated very well when 200 units
of vitamin D were present.
The technique of municipal sewage processing has also been applied to
the treatment of citrus waste from commerical processing plants in Florida
(McNary et at., 1953). Many commerical plants are presently using these
procedures for the handling of plant waste water. Recently, Damron et al.
(1975) have evaluated the use of activated citrus sludge in poultry feeds
and found it to be an acceptable ingredient at levels between 5 and 7.5%
of the diet. No significant differences were found in carcass weight,
percent cooking loss, shear force, or sensory evaluation of broiler meat.
The current trials were designed to measure the effects upon poultry
(chicks and hens) of feeding corn that had been fertilized with DPS
or the feeding of low levels of sludge (DCS) itself.
CHICKS RECEIVING DIETS CONTAINING CORN FERTILIZED
WITH MUNICIPAL SLUDGE
EXPERIMENTAL PROCEDURE
Duplicate experiments were conducted with day-old broiler-type chicks
(Cobb) housed in Petersime battery brooders with raised wire floors to
study the influence of replacing one-half or all of the normal dietary
133
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corn amount with corn grown on LPS treated soil. Table 1 presents the
composition of the 3 diets employed in these experiments.
The corn (Pioneer hybrid '3369A') was grown by Dr. M. C. Lutrick,
soil chemist, I.F.A.S. Agricultural Research Center, Jay, Florida on plots
from an experiment that was begun in 1974 to determine the effects of LPS
on the growth and yield. The sludge contained 2.5 to 3.5% solids and was
spread on the plots prior to planting and disked into the soil. The sludge
plots hadreceived 22.5 cm of sludge in 1976 (the year of test corn harvest)
and a total of 60 cm during the 3-year period. The control plots received
an annual application of 500 kg/ha of 0-10-20 commerical fertilizer at
planting and 250 kg/ha of ammonium nitrate as a sidedress application.
TABLE 1.
COMPOSITION
OF EXPERIMENTAL
DIETS


50%
100%
Ingredients
Control
Sludge Corn
Sludge Corn
Yellow corn
62.27
31.14
_ _
Sludge corn
__
31.14
62.27
Soybean meal (48.5%)
31.00
31.00
31.00
Alfalfa Meal (20%)
2.50
2.50
2.50
Ground limestone
1.36
1.36
1.36
Dicalcium Phosphate
1.94
1.94
1.94
Iodized salt
0.35
0.35
0.35
Micro-ingredient mix1
0.50
0.50
0.50
DL-Methionine
0.08
0.08
0.08
1 Supplied the following activities per kilogram of diet: Vitamin A,
6600 I.U.; vitamin D3, 2200 I.C.U.; menadione dimethylpyrimidinol bisul-
fite, 2.2 mg; riboflavin, 4.4 mg; pantothenic acid, 13.2 mg, niacin,
39.6 mg; choline chloride, 499.4 mg; vitamin B12, 22 meg; ethoxyquin,
0.0125%; manganese, 60 mg; iron, 50 mg; copper, 6 mg; cobalt, 0.0198 mg;
iodine, 1.1 mg; zinc, 35 mg.
Five male and 5 female chicks were assigned to each of 6 replicate
pens in each experiment. Feed and water were provided ad libitum and
lighting was continuous. At the end of the 21 day feeding period, birds
were individually weighed, total feed consumption measured for each pen, and
values for average body weight, daily feed intake, and feed per unit of body
weight calculated. Mortality was recorded on a daily basis. Performance
data were analyzed with analyses of variance and Duncan's multiple range
test procedures using the Statistical Analysis System outlined by Barr et
at. (1976). Representative samples of kidney, liver, and muscle tissue
were collected from 4 pens which received each dietary treatment of experi-
ment 2. Two male and 2 female samples were taken from each pen for mineral
analysis.
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RESULTS AND DISCUSSION
The summary of data outlined in Table 2 indicates that the use of
either level of sludge corn had no adverse effect upon final body weights
or daily feed intake.
TABLE 2. PERFORMANCE DATA FOR BROILER CHICKS FED CORN
FERTILIZED WITH MUNICIPAL SLUDGE
(Experiment 1 and 2)
Final Body Weight Daily feed, g Feed/body weight, g
Treatment	Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2X
Control
495
509
34.9
38.6
1.56
1.60a
50% Sludge Corn
495
525
35.1
39.9
1.53
1.56a
100% Sludge Corn
501
499
35.5
40.1
1.53
1.72b
1 Means without common letters are significantly different (P < 0.05)
according to Duncan's multiple range test.
Feed conversion, in terms of feed required per unit of body weight was
not affected in experiment 1, but a significant decrease in efficiency was
associated with the 100% sludge corn treatment of experiment 2. This
depression was probably due to the degeneration of corn quality which had
occurred because of prolonged storage time. Mortality was not found to be
treated related, since the 3 birds lost were from control pens.
Analyses of liver, kidney, and muscle tissues for a range of mineral
elements provided no clear-cut trends of accumulation due to sludge
fertilized corn feeding (A-PY-l; A-PY-2).
HENS RECEIVING DIETS CONTAINING CORN FERTILIZED
WITH MUNICIPAL SLUDGE
EXPERIMENTAL PROCEDURE
Two experiments were initiated with White Leghorn hens in approxi-
mately 6 months of production to study the effects of feeding various
levels of yellow corn (0, 50, and 100% of the dietary corn complement)
fertilized with LPS upon egg production and quality parameters. The
initial experiment was of 84 days duration while the second was extended
to 112 days. The composition of the 3 diets fed in which one-half or
all of the control corn was replaced with sludge fertilized corn are
summarized in Table 3. Both control and sludge treated corn samples were
produced according to procedures outlined in the previous sections con-
cerning chick feeding experiments. In each experiment, 8 replicate groups
of 5 individually caged hens were assigned to each of 3 dietary treatments.
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Records of egg production and mortality were maintained on a daily basis.
Data concerning feed efficiency and daily feed intake were summarized and
calculated, along with average egg production at the end of each 28-day
interval. A 3-day collection of eggs from each pen was used at the end of
each production period for the determination of average egg weights, Haugh
unit (interior egg quality) and specific gravity (egg shell quality) scores
TABLE 3. COMPOSITION OF DIETS CONTAINING YELLOW CORN
FERTILIZED WITH MUNICIPAL SLUDGE (EXP. 1 AND 2)
50% 100%
Ingredient	Control	Sludge Corn	Sludge Corn
Control Corn
66.97
33.48
--
Sludge Corn
--
33.48
66.97
Soybean Meal (48.5%)
20.0
20.0
20.0
Alfalfa Meal (20%)
2.5
2.5
2.5
Animal Fat
.50
.50
.50
Ground Limestone
6.88
6.88
6.88
Dicalcium Phosphate
(18.5%, 24% Ca)
2.25
2.25
2.25
Iodized Salt
.35
.35
.35
Microingredient mix1
.50
.50
.50
DL-Methionine
.05
.05
.05
1 Supplied the following activities per kilogram of diet: Vitamin A, 6600
I.U.; vitamin D3, 2200 I.C.U.; menadione dimethylpyrimidinol bisulfite,
2.2 mg; riboflavin, 4.4 mg; pantothenic acid, 13.2 mg; niacin, 39.6 mg;
choline chloride, 499.4 mg; vitamin Bi2, 22 meg; ethoxyquin, 0.0125%;
manganese, 60 mg; iron, 50 mg; copper, 6 mg; cobalt, 0.0198 mg; iodine,
1.1 mg; zinc, 35 mg
Hens were artificially inseminated with pooled semen at the end of
the second and third experimental periods in experiment 1, and third and
4th periods in experiment 2. A collection of 7 days egg production was
placed in the incubator each time for determination of fertility, fertile
hatchability, and total hatchability. All of the above data were subjected
to the analysis of variance and Duncan's multiple range test procedures
cited previously in the section concerning chick feeding trials.
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In experiment 1, 2 small triangular taste panel trials were performed
to determine if sludge fertilized corn imparted any objectionable flavor
to eggs. Panelists were asked to select the one sample that was different
from among the 3 presented. Also, blood samples were taken from experiment
1 hens at 5 intervals during the experiment in an attempt to monitor any
change in the mineral status of the hen as feeding progressed. At termina-
tion, 10 representative hens from each treatment of experiment 1, and 12
hens from each treatment of experiment 2 were sacrificed for mineral analyses
of liver, kidney, and muscle tissue.
RESULTS AND DISCUSSION
The performance data of Table 4 for the hens of experiment 1 indicate
that the partial or total substitution of LPS fertilized corn for that
produced with commercial fertilizer had no statistically significant effects
upon any of the production parameters measured.
TABLE 4. PERFORMANCE DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF CORN FERTILIZED WITH LPS (EXP. 1)
Feed/doz1 Daily feed1 Egg Specific Haugh Final
Treatment
Production
kg
g
wts
g
Gravity
Units
Body
Wt, q
Control
70.78
1.68
96.3
62.9
1.078
72.2
1644
50% Sludge
Corn
70.59
1.72
96.6
61.6
1.077
71.6
1686
100% Sludge
Corn
68.85
1.80
102.6
62.8
1.078
70.6
1773
1 Average of first 2 production periods. The third period was lost due to
a weighing error.
Increased daily feed intake and feed efficiency values noted for the
all sludge corn diet were probably the result of corn quality deterioration
during storage. It should be noted that this dietary treatment was asso-
ciated with the highest body weights of the study, indicating that some of
the extra feed went towards fat production.
Very similar trends to those outlined above are present in the results
of experiment 2 (Table 5).
Egg production, feed efficiency, egg weight, specific gravity, and
Haugh units were not significantly affected by the presence of sludge fer-
tilized corn in the diet. The increased feed intake for the 100% replace-
ment group was significantly different from the other treatment and control.
Body weight was significantly higher for the 100% treatment.
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TABLE 5. PERFORMANCE DATA OF WHITE LEGHORN HENS
FED VARIOUS LEVELS OF CORN FERTILIZED WITH MUNICIPAL SLUDGE (EXP. 2)
Treatment
% Hen-Day
egg
Production
Feed/doz
kg
Daily Feed
g
Egg
Wt
Specific
Gravi ty
Haugh
Units
Final
Body
Wt, g
Control
68.4
1.95
107.9a
67.2
1.082
74.0
1697a
50% Sludge
Corn
55.8
1.95
105.2a
66.1
1.081
71.8
1662a
100% Sludge
Corn
68.7
1.95
113.1b
66.9
1.080
72.1
1796b
1 Means without common letters are significantly different according to
Duncan's multiple range test (P < 0.05).
In both experiments, hatchability parameters were low for all groups
and not significantly affected by dietary treatment (Tables 6 and 7). Taste
panel results (Table 8) indicated that no detectable off-flavors were im-
parted to eggs by the feeding of sludge fertilized corn. Participants
were not able to successfully select the egg sample that differed from the
remaining 2 identical ones presented.
Mineral levels from blood samples taken before treatment and at 4
intervals during the first experiment were extremely variable and provided
no indication that blood mineral levels were elevated due to the inclusion
of sludge fertilized corn in the diet (A-PY-3). Results from similar
assays on liver, kidney, and muscle samples at the end of each experiment
also displayed inconsistent variation and revealed no trends indicating
a treatment effect (A-PY-4; A-PY-5).
TABLE 6. HATCHABILITY DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF CORN FERTILIZED WITH LPS (EXP. 1)
% Fertility	% Fertile Hatch % Total Hatch
Treatment
Hatch 1
Hatch 2
Hatch 1
Hatch 2
Hatch 1
Hatch 2
Control
67.4
71.3
94.1
91.5
63.3
64.7
50% Sludge Corn
67.4
77.7
86.0
88.7
58.2
68.9
100% Sludge Corn
57.5
64.4
87.0
91.4
50.1
58.8
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TABLE 7. HATCHABILITY DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF CORN FERTILIZED WITH LPS (EXP. 2)
% Fertility	% Fertile Hatch % Total Hatch
Treatment
Hatch 1
Hatch 2
Hatch 1
Hatch 2
Hatch 1
Hatch 2
Control
78.0
42.9
83.6
60.5
68.2
41.7
50% Sludge Corn
70.8
47.6
72.2
69.3
60.3
40.2
100% Sludge Corn
79.7
49.5
80.2
67.9
61.9
41.6
TABLE 8. TASTE PANEL RESULTS FOR EGGS FROM HENS FED
VARIOUS LEVELS OF CORN FERTILIZED WITH LPS (EXP. 1)
Correct responses/number of panelists
Treatment
Trial1
Trialz
Combined1
50% Sludge Corn
4/10
3/10
7/20
100% Sludge Corn
4/10
4/10
8/20
1 Significance (P < 0.05) = 7/10 or 14/20
CHICKS FED VARIOUS LEVELS OF CHICAGO SLUDGE AND MINERALS
EXPERIMENTAL PROCEDURE
In each of 2 experiments, 8 day old Cobb color-sexed broiler chicks
(4 males and 4 females) were randomly assigned to each pen of a heated
Petersime battery brooder with raised wire floor. Four replicate pens
were assigned to each of 7 dietary treatments for a 3-week feeding period.
Levels of 0, 3, or 6%, DCS were substituted into the basal diet with
attention to maintaining equivalent nutrient levels (metabolizable energy,
crude protein, calcium, phosphorus, sulfur amino acids, and lysine) across
the 3 diets (Table 9). Six percent of the material was a necessary upper
limit because this was the greatest amount that could be included in the
formulation when sludge was assumed to have no nutritional value. In
addition, 4 other treatments in experiment 1, and 5 in experiment 2 con-
tained the amounts of cadmium, chromium, copper, and iron from reagent
sources equivalent to the levels of these elements coming from 6% sludge.
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TABLE 9. COMPOSITION OF DIETS {%)
Ingredients	Control	3% Sludge	6% Sludge
Yellow Corn
48.00
48.00
48.00
Soybean Meal (48.5%)
35.30
35.30
35.30
Defluorinated phosphate
(18% P and 32% Ca)
1.91
1.91
1.91
Limestone
1.26
1.26
1.26
Iodized salt
.35
.35
.35
Microingredients1
.50
.50
.50
Animal fat
6.52
6.52
6.52
DL-Methionine
.16
.16
.16
Dried Chicago Sludge
—
3.00
6.00
Sand
6.00
3.00
—
1 Ingredients supplied per kilogram of diet: Vitamin A, 6600 IU, vitamin
D3, 2200 ICU; menadione dimethyl-pyrimidinol bisulfite, 2.2 mg; ribo-
flavin, 4.4 mg; pantothenic acid, 13.2 mg; niacin, 39.6 mg; choline
chloride, 499.4 mg; vitamin Bi2, 22 meg; ethoxyquin, 0.0125%; manganese,
60 mg; iron, 50 mg; copper, 6 mg; cobalt, 0.0198 mg; zinc, 35 mg.
Table 10 indicates the source and amount of each element added. The
additional iron level fed in experiment 2 resulted from a second analysis
of the sludge where a somewhat lower iron level was reported. Standard
parameters of body weight, feed intake, feed efficiency (g feed/g final
body weight) and mortality were evaluated along with general observation
of the condition of the birds. Samples of liver, kidney, and muscle were
taken in each experiment from 2 birds of each sex from 3 replicate pens
of each treatment.
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TABLE 10. LEVELS OF ELEMENTS ADDED TO CHICK DIETS1
Chromium from chromic sulfate
150 mg/kg
Cadmium from cadmium chloride
12 mg/kg
Copper from cupric sulfate
92 mg/kg
Iron from ferrous sulfate
2,992 mg/kg
(Exp. 2)
2,196 mg/kg
1 Levels determined from laboratory analysis of
DCS x 6%.
RESULTS AND DISCUSSION
Three-week performance data presented in Table 11 reflect a clearly
significant depression of body weight from the supplemental inclusion of
2,992 mg/kg iron in the feed. This value was also significantly lower
than any other treatment weight.
TABLE 11. PERFORMANCE OF BROILER CHICKS FED VARIOUS
LEVELS OF DCS OR MINERALS (EXP. I)1
Diets
Body Wei ght
(q)
Feed Intake
(g)
Feed Efficiency
(g intake/ g BW)
Control
560a
38.5ab
1.43b
+ 37o DCS
54 lab
38.lab
1.48b
+6% DCS
545ab
38.2ab
1.47b
+ 2,992 mg/kg Fe
450C
34.4C
1.63a
+ 12 mg/kg Cd
540ab
39.2a
1.52b
+ 92 mg/kg Cu
552b
38.8a
1.47b
+ 150 mg/kg Cr
525
36.3bc
1.45b
1 Means without common letters are significantly different according to
Duncan's multiple range test (P < 0.05).
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The dietary addition of chromium was associated with a significantly
depressed body weight. However, the level employed (150 mg/kg) was below
that found toxic (300 mg/kg) by Kunishisa et al. (1966).
Feed intake values of experiment 1 were reduced statistically only for
the group receiving iron. It is very difficult to determine if this is
the cause of effect of the depressive results seen in body weights. Feed
efficiency is a calculation involving the other variables previously dis-
cussed; therefore, only iron significantly reduced this parameter.
Again, in the second experiment (Table 12) iron was the only element
to depress body weights.
TABLE 12. PERFORMANCE OF BROILER CHICKS FED VARIOUS LEVELS
LEVELS OF DCS OR MINERALS (EXP. 2)1
Body Weight Feed Intake Feed Efficiency
Diets	(_£)	(£)	(g intake/q BW)
Control
576a
41.4a
1. 50d
+ 3% DCS
563a
40.8ab
1.53bcd
+ 6% DCS
553a
40.6ab
1.54bcd
+ 150 mg/kg Cr
559a
41.7a
1.56abcd
+ 12 mg/kg Cd
528a
41.9a
1.68a
+ 91.5 mg/kg Cu
559a
40.6ab
1.52cd
+ 2,992 mg/kg Fe
439b
35.1C
1.66ab
+ 2,196 mg/kg Fe
470b
36.9bC
1.65abc
1 Means without common letters are significantly different (P < 0.05)
according to Duncan's multiple range test.
The degree of weight reduction did appear to be correlated with the
amount of iron fed. These results are somewhat in contrast to those of
Deobald and Elvehjem (1935) where the toxic level was identified as
approximately 4,500 mg/kg. Feed intake was reduced, as in experiment 1,
by the presence of either iron level. Feed conversion values for both
iron and cadmium treatments were also significantly poorer than that of
the control group. Although cadmium did not cause significant body weight
depressions, a numerical reduction can be noted in both experiments.
It would appear that these birds were borderline to a toxic situation even
though the National Research Council (1977) indicates 20 mg/kg to be toxic.
Mortality was not a factor in either experiment.
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Tissue data of both experiments (A-PY-6 to A-PY-9) indicated no
accumulation of minerals was fostered in muscle tissue by the feeding of
DCS or supplemental elements. The cadmium and iron treatments of both
studies resulted in elevated liver and kidney levels of those minerals in
proportion to their supplementation. There was also a trend of increasing
cadmium residues in liver and kidney resulting from increasing sludge
levels; however, the utilization rate from sludge appeared to be only
approximately 20%. The liver appeared to be the greatest accumulator of
iron, with increases related to sludge addition occurring in the first
experiment but not the second. The response of kidney tissue to iron
supplementation was definite. Copper values were not altered by any of
the dietary variables and chromium in tissues was below detection limits.
HENS FED VARIOUS LEVELS OF CHICAGO SLUDGE AND MINERALS
Eight replicate pens of 5 individually caged White Leghorn hens
were assigned to each of 6 dietary treatments for an 84-day feeding period
in experiment 1, and 112 days for the second trial. Levels of 3.5 and 7%
DCS were substituted into a basal diet with attention to maintenance of
equivalent nutrient levels across the 3 diets (Table 13).
TABLE 13. COMPOSITION OF BASAL HEN DIET FOR SLUDGE ADDITIONS
(EXPERIMENTS 1 AND 2)
Ingredient	Percentage
Yellow Corn
58.29
Soybean Meal (48.6%)
21.35
Animal Fat
4.00
Limestone
6.55
Dicalcium Phosphate
1.90
DL-Methionine
.06
Iodized Salt
.35
Microingredient mix1
.50
Inert Filler
7.00
1 Ingredients supplied per kilogram of diet: Vitamin A, 6600 IU; vitamin
D3, 2200 ICU; menadione dimethyl-pyrimidinol bisulfite, 2.2 mg; riboflavin,
4.4 mg; pantothenic acid, 13.2 mg; niacin, 39.6 mg; chloride, 499.4 mg;
vitamin Bi25 22 meg; ethoxyquin, 0.0125%, manganese, 60 mg; iron, 50 mg;
copper, 6 mg; cobalt, 0.0198 mg; zinc, 35 mg.
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In addition, 3 other treatments in both studies include the provision
of amounts of cadmium, chromium, or copper from reagent sources equivalent
to those found in the 1% diet. Those reagent sources were chromic sulfate,
copper sulfate, and cadmium chloride in both experiments. Since iron had
been found to cause reduced body weights in chick experiments, it was also
included in the second study from ferrous sulfate.
Records of egg production, egg weight, daily feed intake, feed effi-
ciency, specific gravity, Haugh units, and mortality were summarized or
calculated at 28-day intervals. Also, fertility and hatchability was
determined by artificial insemination during the second and third periods
of experiment 1, and the third and fourth months of experiment 2. Birds
were individually weighed at the time of termination. Samples of eggs,
feed, kidney, muscle, liver, and feces were taken from each treatment group
for mineral analyses.
RESULTS AND DISCUSSION
In experiment 1, none of the production criteria shown in Table 14
(egg production, daily feed intake, feed efficiency, egg weights, and
final body weights) were statistically affected by the addition of up to
1% DCS to the diet or the feeding of comparable mineral levels from reagent
sources. Specific gravity (egg shell quality) was significantly improved
in eggs from hens that received 7% DCS. Although interior egg quality
(Haugh units) was significantly reduced when compared to the value asso-
ciated with 7% DCS, it was not different from the control value. This
author feels that biological variation was largely responsible for this
effect.
TABLE 14. PERFORMANCE DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF DCS OR MINERALS (EXP. 1)
% Hen-Day Feed/ Daily Egg	Final
egg doz Feed Wt Specific Haugh Body
Treatment Production (kg) (q)	(g) Gravity1 Units1 Wt (q)
Control
76.03
1.56
99.2
62.1
U
-Q
CO
CO
o
\ ]
73.4ab
1634
+ 3.5% DCS
65.81
1.56
98.5
61.2
1.0846abc
74. 3b
1634
+ 7.0% DCS
72.56
1.62
97.4
60.7
1.086la
74.6b
1654
+ 14 mg/kg Cd
73.63
1.56
95.7
61.5
1.0828C
74. lb
1527
+ 106.8 mg/kg Cu
73.85
1.60
98.5
60.3
1.0856ab
71.9a
1617
+ 175 mg/kg Cr
72.97
1.60
100.4
61.5
1.0844abc
73.6ab
1705
1 Means without common letters are significantly different (P < 0.05)
according to Duncan's multiple range test.
144
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In experiment 2 (Table 15), the addition of iron from a reagent source
produced the main significant effect. The egg production of birds receiving
iron did not differ statistically from that of controls, but was numerically
depressed, and significantly lower than all but the chromium treatment
group. A similar reaction was noted in final body weights where the iron
treatment differed from all but the control and cadmium groups.
Daily feed intake was significantly reduced for the birds whose diet
was supplemented with iron, while Haugh unit scores of eggs from all treat-
ments were numerically or statistically better than controls. Egg weight
and specific gravity data were not found to be significantly influenced
by treatment.
TABLE 15. PERFORMANCE DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF DCS AND MINERALS (EXP. 2)

% Hen-Day
Feed/
Daily
Egg


Final


egg
doz
Feed
Wts
Specific
Haugh
Body
Treatment
Production
(kg)
(g)
(g)
Gravi ty
1 Units
wt (g)1
Control

74.6ab
1.592abc
95.7a
63.01
1.077
76.25bc
1627ab
+ 3.5% DCS

75.79a
1.553°
96.02a
62.08
1.078
78.77abc
1709a
+ 7.0% DCS

77.3a
1.516C
96.2a
61.24
1.076
81.30a
168 la
+ 14 mg/kg Cd

77.27a
1.525°
96.65a
62.0
1.077
80.04ab
1576b
+ 106.8 mg/kg Cu
75.96a
1.569bc
97.17a
62.7
1.076
78.02bc
1718a
+ 175 mg/kg Cr

73.42ab
1.645ab
96.28a
63.08
1.077
79.58ab
1678a
+ 2,562 mg/kg Fe
66.83b
1.678a
89.65b
62.42
1.077
79.95ab
15 38b
1 Means without common letters are significantly different (P < 0.05)
according to Duncan's multiple range test.
Hatchability data was not found to be consistently influenced by
dietary treatment in either experiment (Tables 16 and 17). In experiment
1 (Table 16), the values approximated those seen in the industry and did
not appear to be treatment related. This is illustrated in the fertile
and total hatchability areas, where control groups were generally the
lowest of any dietary regime. The figures presented in Table 17 for the
second experiment were extremely variable and, although some depressions
are exhibited for the 7% sludge and copper treatments, they were not
consistent over the 2 hatches and were not felt to represent a true
treatment effect.
145
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TABLE 16. HATCHABILITY DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF DCS OR MINERALS (EXP. 1)
% Fertility % Fertile Hatch % Total Hatch
Treatment	Hatch 1 Hatch 2 Hatch 1 Hatch 2 Hatch 1 Hatch 2
Control
89.4
80.2
79.5
88.0
67.2
71.0
+ 3.5% DCS
89.1
76.5
80.9
86.7
72.6
71.2
+ 7.0% DCS
94.2
86.9
82.7
93.0
82.2
82.0
+ 14 mg/kg Cd
90.1
77.1
79.9
91.5
75.3
72.1
+ 106.8 mg/kg Cu
94.9
85.0
86.3
94.9
84.0
83.7
+ 175 mg/kg Cr
90.8
88.7
87.4
91.7
77.3
81.4
TABLE 17. HATCHABILITY DATA OF WHITE LEGHORN HENS FED
VARIOUS LEVELS OF DCS OR MINERALS (EXP. 2)
% Fertility % Fertile Hatch % Total Hatch
Treatment	Hatch 1 Hatch 2 Hatch 1 Hatch 2 Hatch 1 Hatch 2
Control
76.4
68.4
85.5
93.1
66.3
63.7
+ 3.5% DCS
75.4
74.1
94.0
83.6
70.5
62.1
+ 7.0% DCS
88.0
82.2
81.4
52.0
71.6
42.5
+ 14 mg/kg Cd
74.7
82.7
87.9
83.2
66.0
68.6
+ 106.8 mg/kg Cu
55.9
72.8
92.6
85.3
53.4
61.6
+ 175 mg/kg Cr
79.5
79.9
91.1
83.3
72.5
66.2
+ 2,562 mg/kg Fe
76.4
82.3
82.5
89.0
63.5
74.2
Table 18 summarizes the analytical data taken on the feeds of the
first experiment where it can definitely be seen that the addition of
sludge did contribute significant amounts of the mineral elements under
study to the diets.
Table 19 present the same type of data for fecal samples, indicating
that large amounts of these elements were being passed through unabsorbed
in proportion to the feed's content. It is felt that this is what happens
to the majority of the minerals received by the chickens through sludge
additions to the feed.
146
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TABLE 18. ANALYSES OF FEEDS CONTAINING DCS OR MINERALS


(EXP. 1)



Treatment
Cu
Fe
A1
Cd
Cr
Control
10
188
275
0
3
+ 3.5% DCS
50
725
550
2.8
100
+ 7.0% DCS
73
1525
550
1.8
163
+ 14 mg/kg Cd
10
250
325
8.1
6
+ 106.8 mg/kg Cu
93
225
175
0
1
+ 175 mg/kg Cr
10
213
250
0
125
TABLE 19. FECAL ANALYSES FROM WHITE LEGHORN HENS FED
VARIOUS LEVELS OF DCS OR MINERALS (EXP. 1)
Treatment	Cu Fe AT Cd Cr
Control
28
750
1176
.62
9
+ 3.5% DCS
183
3534
2312
.22
265
+ 7.0% DCS
414
7125
3656
.66
656
+ 14 mg/kg Cd
31.5
663
694
.58
9.5
+ 106.8 mg/kg Cu
144.5
888
944
.69
9
+ 175 mg/kg Cr
30.5
1088
1076
.62
519
Analyses of eggs from the hens of experiment 1 did not indicate any
effect from any of the dietary additions (A-PY-10). In contrast, the
tissue analyses results from experiment 1 (A-PY-11) did point out that
increased mineral stores would result from sludge and reagent mineral
feeding. In liver, both cadmium and iron were increased stepwise as
the sludge content increased. Cadmium was at its highest level when fed
from the purified source, indicating a difference in availability from
the 2 materials. Liver copper was not altered by treatment and chro ium
was within detection limits only for the supplemental chromium treat-
ment. Similar trends are present in the data concerning kidney tissue.
Muscle minerals did not seem to be influenced to any appreciable exten
by any of the dietary additions.
147
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REFERENCES
1.	Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig. A User's
Guide to SAS 76. SAS Institute Inc., P.O. Box 10066, Raleigh, NC,
1976.
2.	Damron, B. L., A. R. Eldred, S. A. Angalet, J. L. Fry, and R. H. Harms.
Evaluation of Activated Citrus Sludge as a Poultry Feed Ingredient.
Environmental Protection Agency, Corvallis, OR, 1975.
3.	Deobold, H. J., and C. A. Elvehjim. The Effect of Feeding High Amounts
of Soluble Iron and Aluminum Salts. Amer. J. Physiol., 111:118-123,
1935.
4.	Firth, J. A. and B. C. Johnson. Sewage Sludge as a Feed Ingredient
for Swine and Poultry. J. Ag. and Food Chem., 3(9):795-796, 1955.
5.	Hurwitz, E. The Use of Activated Sludge as an Adjuvant to Animal
Feeds. IN 12th Industrial Waste Conference, Purdue University, pp.
395-414, 1957.
6.	Kunishisa, Y., T. Yoname, T. Tonka, I. Fukuda, and T. Nishikava.
The Effect of Dietary Chromium on the Performance of Chicks. Japan.
Poultry Sci., 3:10-14, 1966.
7.	McNary, R. R., R. W. Wolford, and M. H. Dougherty. Methane Fermentation
of Waste Water from Citrus Processing Plants. University of Florida
Agricultural Experiment Station Annual Report, pp. 213-214, 1953.
8.	National Research Council. Nutrient Requirements for Poultry and
Toxic Levels of Inorganic Elements for Poultry. National Academy of
Sciences, Washington, DC, 1977.
9.	Schendel, H. E. and B. C. Johnson. Activated Sewage Sludge as a
Source of Vitamin Bi2 for the Pig. J. Ag. and Food Chem., 2(1):
23-24, 1954.
10. Scott, H. M. and E. G. Adams. The Effect of Feeding Graded Levels of
Activated Sludge and Vitamin D on Growth and Bone Ash of Chicks.
Poultry Sci., 34:1233, 1955.
148
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HEALTH EFFECTS IN MICE FED DIETS THAT CONTAINED TISSUES FROM
CATTLE OR SWINE FED SEWAGE SLUDGE RATIONS
K. C. Kelley, R. L. Shirley, 0. Osuna, J. Bertrand, and G. T. Edds
ABSTRACT
At the end of the swine and cattle feeding periods (Bertrand et al.,
1979; Edds et at., 1979) the animals were slaughtered and kidney and
liver samples collected and frozen. The frozen tissues were sliced and
ground in a Hobart grinder and freeze-dried. The tissue samples were
powdered by grinding and their protein content determined by Kjeldahl
technique (Official Methods of Analysis, AOAC Eleventh Edition, 1975,
William Horwitz, Editor).
Swiss mice, 45 days of age, were fed freeze-dried liver and kidney
from cattle or swine fed a control diet, sewage sludge, corn grain, or
sorghum forage from land covered with liquid sewage sludge. Mice fed
diets containing 5% cattle kidney had higher levels of Cu, Fe, Co, and
Pb in their kidney than mice fed 10% cattle liver in diets. Nickel was
higher in kidney, liver, and muscle; Fe, Co, and Pb higher in liver, Cu,
Co, and Pb higher and Zn lower in muscle; and Cr higher in kidney of
mice fed diets that contained 5% kidney from cattle. Fi females had a 6-
fold increase in liver Cd in the DCS treatment, while F2 females showed
no differences in tissue minerals due to DCS treatments. Fi females
fed liver had a decrease in number born in both sludge treatments.
F0 mice fed kidney and liver from cattle fed sorghum that received
Pensacola liquid sludge (LPS) showed no changes in minerals in kidney,
liver, and muscle due to treatment or diet. Number born per litter was
greater in F0 mice fed kidney in the LPS treatment but there was no
effect when liver was fed.
Liver from swine F0 fed 0, 10, or 20% University of Florida digested
sewage sludge (DFS) in diets of mice showed an increase in the Pb
content of mice liver and muscle at the 20% DFS level.
Mice fed liver from swine Fi_2 in diets had higher levels of Cd in
liver, kidney, and muscle tissue in the 10% DFS group. Pb in mice liver
was increased in the 10% DFS and in muscle in both the 10 and 20% DFS
groups. Mice fed kidney from swine Fi_3 had high levels of Cd in liver.
149
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INTRODUCTION
All mice utilized in the feeding trials were housed in stainless steel
cages with wire mesh bottoms.1 Water was supplied ad libitum. Diets were
fed in glass jar feeders with stainless steel tops.2 Diets were supplied
ad libitum with the feeders being changed each two or three days as needed.
Littering cages were stainless steel with solid bottoms and Sanicel3 was
used as the contact bedding.
At the termination of the feeding trial, each mouse was sacrificed by
cervical separation, and body, liver, and kidney weights determined. Liver,
kidney, and muscle samples were taken from each mouse. Within each treat-
ment, the tissue samples were pooled and selected mineral analyses of
kidney, liver, and muscle were done by atomic absorption.4
Feeding Trial Designs
FT #1 - Cattle kidney and liver tissue—Thirty-two Cox (Swiss) out-
bred mices (16 male, 16 female), 25-45 days of age, were assigned to each
treatment group. Mice of the same sex were housed two per cage. After 15
days on the diets, one male of the same treatment was introduced into each
cage of females for breeding purposes. Twenty days later, females were put
in littering cages. The number of young born and the date were recorded.
At 21 days of age, the F, offspring were counted, 16 females per treatment
were removed for continuation on the diets; the remainder of the Fj were
sacrificed along with the 32 Fn mice for tissue samples. The mice sacri-
ficed were between day 60 and aay 70 of the feeding trial period. The F^
females fed 5% kidney in their diet were continued an additional 60 days
and sacrificed. The F. females fed 10% liver in the diet had normal adult
males introduced for breeding on day 160. On day 180, females were placed
in solid bottom litter cages (two per cage) and the date of littering of
the F? generation and the number per litter recorded. The Fg generation
at 21 days of age were counted and sacrificed along with the F, parent
female. Kidney, liver, and muscle tissue samples were taken on all mice
at the end of the feeding trial for mineral assays.
FT #2 - Swine liver tissue--Forty Cox (Swiss) outbred mice5 (20
male and 20 female), 35-45 days of age, were assigned to each treatment
group. Five mice of the same sex were housed per cage. Freeze-dried
liver from swine Fg treatments were fed for a 100 day trial and
1	LC-75/SA and LC-75/SB cages. Wahmann Manufacturing Co., Timonium, MD
2	LC-207/A Mouse Feeder, Wahmann Manufacturing Co., Timonium, MD
3	Paxton Processing Co., Inc., Univarium Research Inc., White House Station,
NJ
Atomic absorption analyses performed by Soils Department, University of
Florida, Gainesville, FL
5 Laboratory Supply Co., Inc., Indianapolis, IN
150
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freeze-dried liver from swine F. ? treatments were fed for a 34 day trial.
At the end of the feeding trial period, all mice were sacrificed and
tissue samples taken. Because of limited kidney tissue availability, this
trial was performed using liver tissue only.
FT #3 - Swine kidney and liver tissue--Twe!ve female Cox (Swiss) out-
bred mice1, 45 days of age, were assigned to each kidney treatment group.
Twenty male and 20 female mice, 35-45 days of age, were assigned to each
liver treatment group. Four or five mice of the same sex were housed
together. The kidney and liver diets were fed for 15 days. On day 15,
normal males were introduced, one per cage to the females on kidney diets.
The liver diets had one male of the same treatment introduced into the
female cage. On day 35, females were placed in littering cages and the
number of young born and the date recorded. At 21 days of age, the F,
offspring were counted. The F, mice and the parent Fg mice were sacrificed
between day 60 and 70 of the feeding trial period.
FT #4 - Cattle kidney and liver tissue—Twelve female Cox (Swiss)
outbred mice1, 45 days of age, were assigned to each kidney treatment
group and housed 4 per cage. On day 15, one normal male was introduced
into each cage of females for breeding purposes. Thirty-two mice (16
male, 16 female) were assigned to each liver treatment group and housed
4 per cage. On day 15, a male of the same treatment was introduced into
each female cage. On day 35, females were placed in littering cages, the
number of young born and the date of birth recorded. At 21 days of age,
the F, offspring were counted; 16 females on each liver diet treatment
were removed for continuation on the diet. The remaining F, and the FQ
of the liver diet were sacrificed and muscle, kidney, and Itver samples
taken. All mice on the kidney treatments were sacrificed at 21 days of
age of F, and tissue samples taken. The F, females were continued on their
respective liver diets for 180 days. Normal adult males were introduced
for breeding on day 240 to 250. On day 260 to 270, females were placed
individually in solid bottom litter cages and the date of littering on
the F2 generation and the number in the litter recorded. The F~ generation
at 21 days of age, were counted and sacrificed along with the F. parent
female. Kidney, liver, and muscle tissue samples were taken from each
mouse.
FT #5 - Swine kidney and liver tissue--Twelve female Cox (Swiss)
outbred mice1, 45 days of age, were assigned to each cattle kidney dietary
group and 16 female mice were assigned to each liver treatment group.
The mice were housed 4 per cage. On day 15, one normal male was introduced
into each cage of females for breeding purposes. On day 35, females were
placed in litter cages, the number of young born and the date of birth
recorded. At 21 days of age, the F1 offspring were counted. On day 60
of the feeding trial, both the F„ and F, mice were sacrificed, and kidney,
liver, and muscle samples were taken for mineral analysis.
1 Laboratory Supply Company, Inc., Indianapolis, IN
151
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RESULTS AND DISCUSSION
Data from the feeding trials are presented in Tables 3-9. Feeding
trial 1 (Tables 3-9) data on mice fed diets containing freeze-dried kidney
and liver tissue of cattle fed dried Chicago digested sludge (DCS) or
forage from land covered with Pensacola liquid sludge (LPS) diets are
presented. Table 3 gives the final body, liver, and kidney weights as
well as liver and kidney to body weight ratios. Mice fed kidney tissue
showed changes in the FQ generation in all parameters but no clear cut
pattern emerges between sexes and treatment. There were no differences
among treatments in any of the parameters. The FQ mice fed liver tissue
showed differences among treatments and sex in all weight parameters but
no consistent pattern occurred. The F1 female mice fed liver tissue
showed no change in their liver/body weight and kidney/body weight ratios,
but there were differences among treatments and sex in the parameters of
final body weight, liver, and kidney weight.
Mice fed kidney tissue (Table 4) in the 500 g/head/day DCS group or
forage groups had no significant differences in litter size in the F~
generation due to treatment but had a decrease in the number weaned.
The F, female mice fed kidney-containing diets had no differences among
treatment groups in the number weaned. The F~ mice fed liver tissue had
no significant differences in litter size, but the number weaned was
affected by sludge treatments. The F, females fed liver had a decrease in
litter size and in the number weaned at the 500 g/head/day DCS.
Mineral analyses data on tissues of feeding trial #1 are presented
in Tables 5-9 with Tables 5 and 6 giving the data of kidney, liver, and
muscle tissue of mice fed freeze-dried kidney and liver from cattle fed
500 g/head/day DCS or forage from land receiving 7.6 cm per hectare (cm/ha)
of LPS. Mineral analyses data (Table 7) demonstrated higher levels of
Cd in kidney and liver tissue of mice that consumed kidney or liver tissue
from cattle fed 500 g/head/day DCS. No differences in Cu, Fe, Co, Zn,
and Pb were seen in mice kidney and liver tissue. In muscle tissue of
mice, there was a significant depression at Cu in both the 7.6 cm/ha LPS
and 500 g/head/day DCS treatments. When male and female mice tissue
concentrations of the Fq generation (Table 8) were averaged across feed
types and treatment groups, females' kidney tissue had a higher concentra-
tion of Cd than the males. Male liver tissue had a higher concentration
of Cu than that of females. Muscle tissue of mice showed no differences
between the sexes for any of the minerals. When mice data were pooled
across treatments and sex (Table 9), diets containing 5% cattle kidney
and 10% cattle liver were compared. Mice kidney tissue was found to
contain significantly higher concentrations of Cu, Fe, Co, and Pb when
the mice consumed 5% cattle kidney in their diet. The mice liver tissue
also was found to contain higher levels of Fe, Co, and Pb when 5% kidney
was in the diet. The muscle tissue of mice consuming 5% kidney in their
diet had significantly higher concentrations of Cu, Co, and Pb than the
mice fed 10% liver in the diet; Zn was significantly lower in muscle
tissue of mice fed 5% kidney.
152
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TABLE 1. COMPOSITION OF DIETS FED MICE
Ingredient
Percentage
gm/kilo
Protein1
15
150
Sucrose
32
320
Corn starch2
32
320
Vitamin mix3
1
10
Salt mix1*
4
40
Alphacel5
11
110
Corn oil6
5
50
1	Protein in the diet consists of 5% freeze-dried kidney
tissue or 10% freeze-dried liver tissue and Vitamin Free
Casein (ICN Pharmaceuticals, Inc., Life Sciences Group,
Cleveland, OH) to yield a total protein of 15%.
2	Corn starch plus the non-protein portion of the freeze-
dried tissue sample.
2 3 4 5 6
Ingredient source, ICN Pharmaceuticals, Inc., Life Sciences
Group, Cleveland, OH.
153
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TABLE 2. TREATMENT OF CATTLE AND SWINE CONSUMING SEWAGE SLUDGE
SLAUGHTER DATES, AND TISSUES FED MICE IN VARIOUS TRIALS
Feeding Date
Trial Tissue Source	Treatments	Slaughtered
1
Ki dney
Cattle
Control, 7.6 cm/ha LPS1 or
500 g/head/day, DCS2
08/12/77

Li ver
Cattle
Control, 7.6 cm/ha LPS or
500 g/head/day DCS

2
Ki dney
Swine
A.	F0 10% DFS3
F0 20% DFS
B.	Fi ? Control
F{_2 10% DFS
Fx_2 201 DFS
04/04/78

Liver
Swi ne
A.	Fn Control
FQ 10% DFS
Fn 10% DFS
B.	Fi p Control
Fi p 10% DFS
F1_2 20% DFS

3
Ki dney
Swine
Fi o Control
Fl'A 10% DFS
F1-3 20% DFS
08/12/78

Liver
Swine
Fi_3 Control
Fi o 10% DFS
Fx_3 20% DFS

4
Ki dney
Cattle
Control
15.2 cm/ha LP S
22.8 cm/ha LPS
08/12/78

Li ver
Cattle
Control
15.2 cm/ha LPS
22.8 cm/ha LPS

5
Ki dney
Swi ne
F1 9 , Control
FT^'i 10% DFS
F]~_2 1 Z0% DFS
04/19/79

Liver
Swine
F1 9 1 Control
F1 p'j 10% DFS
Fj"^'J 20% DFS

1	Centimeters per hectare, Pensacola liquid sludge (LPS)
2	Dried Chicago sludge (DCS)	3 Dried Florida sludge (DFS)
154
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TABLE 3. MICE FEEDING TRIAL 1. BODY AND ORGAN WEIGHTS OF MICE FED DIETS CONTAINING FREEZE-DRIED KIDNEY AND LIVER TISSUE
FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING 7.6 CM/HA1 OF LPS2 OR 500 G/HEAD/DAY DCS
Mice
para- Tissue
meter fed
Treatment
Final
body
wt, g
Li ver
wt, g
Ki dney
wt, g
Kidney
Liver
Kidney
Liver
Ki dney
Li ver
Liver/ Kidney
body wt5
I Liver
F,
F
F,
F
F,
F
F,
F
Fo
R
Test
Generation days n
60
120
60
200
60
120
60
200
60
120
60
200
60
120
60
200
16
11
16
16
16
11
16
16
16
11
16
16
16
11
16
16
Control - no sludge
7.6 cm/ha LPS
female
male
femal e
male
500 g/head/day DCS
female
32.07+0.99 11
31.86+0.91
33.91+0.89® 16
42.98±3.53
2.32+ 1.6a 11
2.32 ± ,llh
2.84±.15" 16
3.80± .18
0.53 ± .02 11
0.53 + .02
0.58 + .02* 16
0.79 ± .02
7.2	+	.3
7.4 +	.2
9.3	±	.3
8.8 ±	.4
ac
11
16
33.98+1.21 16
b 15
37.36+0.89 16
16
2.12 + .13 16
h 15
2.72 + .10° 16
16
0.75 + .04 16
15
0.76 + .03 16
16
6.2 + .5bc 16
b 15
7.2 + .2° 16
16
33.20+0.99 13 38.43+1.12
32.34+0.78	" h
36.15+0.89? 15 37.56+0.92
37.68+3.53°
2.76 + ,16b 13 2.16 ± .12a
2.39 + .09	h
3.39 i .15® 15 2.76 + .10
3.19 + .18
0.58 i .02° 13
0.49 i .02.
0.66 + .02
0.64 + .02"
be
13
8.2	+	.2
7.3	+	.2
8.3	±	.3 15
8.4	+	.4
0.78 + .04
15 0.80 + .03
5.6 + .5
7.3 ± -2b
16
3.05 + .13
16	0.54 ±- .02
5	0.54 i- .03,
16	0.56 + .02? 16
16	0.59 ± .02
16	8.0 + .2a 13
7.8 + .4
16	9.0 + .3d 16
16	7.9 + .4
ma 1 e
16 31.76+0.99 13
5 31. 3811.35
16	33.33+0.89? 16
16	38.76+3.53
16	2.51 + ,16ab 13
5	2.47 ± .16,,.
16	3.03 + . 15™ 16
ab
13
36.3611.2
34 .54+0.89L
2.10 i .12d
2.40 .101'
0./3 i .04'J
0,71 i .03°
5.7 1 .5b
6.9 + .2b
Ki dney/ Ki dney
body wt
% Liver
a,b,c
Fo
F1
Fo
H.
60
120
60
200
16
11
16
16
1.6 +	.F 11
1"6 +	-2ac
1.8 +	,ldL 16
1.8 +	.la
2.2 + .1" 16
. 15
2.0 + .1DC 16
	16
1.7 + .1" 13
1.5 ± .2,
1.7 ± .1
1.7 ± .1
ab
15
Means ± SE on the same line with different superscripts differ significantly (P < 0.05)
2.0	+ .r
2.1	+ .1L
16
5
1.7 + .1"
1.7 + .3,
13
16 1.6 + .r 16
16 1.5 + ,1D
1.9 i .1
2.0 i .1
be
1	Centimeters per hectare
2	Pensacola liquid sludge
3	Dried Chicago sludge
'*	Bertrand et al. (1978)
5 Calculated by dividing organ weight by final body weight x 100.
-------
TABLE 4. MICE FEEDING TRIAL 1. REPRODUCTIVE DATA OF MICE FED DIETS CONTAINING FREEZE-DRIED
KIDNEY AND LIVER TISSUE FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING
7.6 CM/HA1 OF LPS2 OR 500 G/HEAD/DAY DCS3'1*
Mi ce
para-
meters
Tissue
fed
Mice
generation
Test
days

Control
No sludge
7.6 cm/ha LPS
500 q/h/d DCS
n
No./litter
n
No./litter
n
No./litter
Number
Ki dney
F0
60
6
10.1 + 1.5
7
13.5 ± 1.4
8
13.7 + 1.3
born










Liver
F0
60
9
10.2 + 2.1
10
14.9 + 2.0
7
16.0 + 2.4

Li ver
Fi
200
16
8.8 + .7a
16
6.8 + .7b
11
6.1 + .7b
Number
Ki dney
F0
60
8
6.1 ± 1.4a
8
8.5 + 1.4a
8
1.6 + 1.4b
weaned










Ki dney
Fi
120
10
6.4 + 0.9
15
8.4 + 0.8
5
6.8 + 1.4

Liver
F0
60
9
5.6 + 1.5a
10
14.0 + 1.4b
7
10.9 ± 1.7ab

Li ver
Fi
200
16
8.1 + .6a
16
6.0 + .5ab
11
3.9 + .7b
a,b Means + SE on the same line with different superscripts differ significantly (P < 0.05).
1	Centimeters per hectare.
2	Pensacola liquid sludge.
3	Dried Chicago digested sludge.
**	Bertrand et al.
-------
TABLE 5. MICE FEEDING TRIAL 1. MINERAL ANALYSES DATA OF KIDNEY, LIVER, AND MUSCLE TISSUE OF MICE FED FREEZE-
DRIED KIDNEY FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING 7.6 CM/HA1 OF LPS2 OR 500 G/HEAD/DAY DCSJ-4
Treatment
Mice
Generation
Test
days
Sex
Cd
Cu
Fe
Co
Zn
Pb
Hg
Cr
Ni
Se







Mice
kidney
mg/kg) wet
basis



Control
F o
60
F
0.00
4.34
117.1
1.38
18.6
0.77
.069
0.31
2.69
. 160
(no sludge)
Fo
60
M
0.00
4.09
121.0
1.42
19.9
0.89
.178
0.53
2.67
.110


120
F
-
-
-
-
-
-
-
-
-
-

F2
21
F
0.00
2.67
58.8
2.17
18.5
16.7
.000
0.84
5.81
.170
7.6 cm/ha

60
F
0.00
4.05
105.7
1.13
16.9
0.63
.033
0.19
2. 38
.154
LPS
Fo
60
M
0.00
3.62
189.8
1.44
24.4
1.90
.065
0.28
2.84
.140

F
120
F
0.20
3.50
85.0
0.21
16.7
0.07
.070
0.07
0.71
.710


21
F
0.00
2.80
49.3
1.72
16.9
1.56
.000
0.23
2.32
.31
500 g/h/d

60
F
2.35
3.82
109.1
1.21
17.3
0.69
.032
0.37
2.92
.160
DCS
Fo
60

1.17
3.96
157.5
1.21
17.9
0.69
.038
0.31
2.71
.120

F
120
F
1.10
3.50
94.0
0.24
19.2
0.12
.120
0.10
1.20
.600


21
F
0.00
2.85
49.3
3.93
17.5
3.57
.000
2.50
7.14
.360







Mice
1iver (mg/kg) wet
basis



Control

60
F
0.00
2.55
254.0
0.54
21.1
0.35
.000
0.10
1.14
.003
(no sludge)
Fo
60

0.00
4.69
291.0
0.68
21.1
0.56
.014
0.24
1.75
.160
Fj
120
F
0.10
2.20
154.0
0.08
18.4
0.07
.080
0.10
0.76
.380

F2
21
F
0.00
2.37
126.0
0.75
18.3
0.63
.000
0.56
3.75
.19
7.6 cm/ha

60
F
0.00
3.74
379.1
0.58
41.4
0.36
.004
0.35
1.43
.065
LPS

60
M
0.00
4.86
247.5
0.55
20.7
0.28
.010
0.05
1.42
.140

120
F
0.04
2.60
172.0
0.11
19.8
0.07
.040
0.04
0.35
.360

F2
21
F
0.00
2.66
201.8
0.81
19.0
0.58
.000
0.23
2.32
.170
500 g/h/d
•* 0
60
F
0.34
2.83
350.0
0.49
20.1
0.25
.006
0.06
1.14
. 130
DCS
Fo
60

0.23
4.63
268.4
0.56
25.2
0.48
.006
0.10
1.44
. 130

r i
120
F
0.40
1.80
151.0
0.09
18.1
0.09
.090
0.09
0.89
.450

f2
21
F
0.00
2.22
87.6
1.67
20.9
1.39
.000
0.83
2.78
.010
-------
TABLE 5. Continued
Treatment
Generation
Test
days
Sex
Cd
Cu
Control
F o
60
F
0.00
1.31
(no sludge)
Fo
60
M
0.00
1.38

Fi
120
F
0.20
0.19

F2
21
f
0.00
0.96
7.6 cm/ha
Fo
60
F
0.00
1.12
LPS
Fo
60
M
0.00
1.16

F,
120
F
0.04
1.10

f2
21
F
0.00
1.03
500 g/h/d
Fo
60
F
0.00
0.92
DCS
Fo
60
M
0.00
1.12

Fi
120
F
0.90
0.71

f2
21
F
0.00
0.58
I	Centimeters per hectare
*	Pensacola liquid sludge
3 Dried Chicago digested sludge
II	Bertrand et al.> 1978
s	Weanling female mice
Fe	Co	In	Pb
Hcj		Cr	Hi	Se
Mice muscle (mg/kg) wet basis
25.2
0.53
13.0
0.37
0.003
.14
27.1
0.47
9.5
0.41
0.006
.16
31.0
0.19
17.7
0.39
.190
.20
17.9
0.96
11.0
1.20
.000
.60
18.1	0.40
28.5	0.46
24.0	0.31
18.1	1.04
21.0	0.50
26.4	0.47
28.0	0.62
7.2	0.75
10.0	0.34
11.1	0.42
16.5	0.11
11.2	1.38
12.6	0.54
10.7	0.42
20.6	0.53
52.2	0.58
0.000	.10
0.000	.11
.040	.20
.000	.02
0.003	.23
0.006	.11
0.090	.40
.000	.41
1.11	.001
1.12	.004
1.94	.960
2.49	.006
1.10	.020
1.12	,00i
0.35	.18)
2.76	.000
1.88	.00/
1.81	.00')
0.89	.450
1.74	.000
-------
TABLE 6. MICE FEEDING TRIAL 1. MINERAL ANALYSES DATA OF KIDNEY, LIVER, AND MUSCLE TISSUE
OF MICE FED FREEZE-DRIED LIVER FROM CATTLE FED A CONTROL DIET, CORN FROM LAND
RECEIVING 7.6 CM/HA1 LPS2 OR 500 G /HEAD/DAY DCS3'4
Mlce	Tes t
Treatment
Generation
days
Sex
Cd
Cu
Fe
Co
In
Pb
Hg
Se
Cr
Ni









Mice
kidney (irn/kq) wet
basis


00
0
00
Control
0
60
F
0.08
2
52
84
0
0.00
16
0
0
00
—
--
0
(no sludge)
0
60
M
0.11
3
29
102
0
0.00
19
9
0
00
—
--
0.
00
0.
00
r i
200
F
0.00
3
54
124
8
0.00
17
7
0
00
.024
.300
0
31
0.
00

F2
21
F
0.06
3
27
92
3
0.00
16
3
0
00
.008
.160
0
14
0
70
7.6 cm/ha
r o
60
F
0.25
3
28
90
0
0.00
18
4
0
00

--
0.
00
0.
00
LPS
0
60
M
0.11
3
25
116
0
0.00
22
4
0
00
—
--
0
00
0
00

r i
200
F
0.32
4
00
115
0
0.00
17
5
0
00
.010
.300
0
28
0
50

h
21
F
0.00
2
63
67
2
0.00
9
2
0
00
.008
.210
0
22
0
30
500 g/h/d
0
60
F
0.69
2
73
96
0
0.00
17
8
0
00
.60
.300
0
00
0
oo
DCS
r o
60
M
0.32
2
48
80
0
0.00
14
9
0
00
.170
.250
0
00
0
00

r i
200
F
1.61
4
00
109
0
1.04
17
6
0
00
--
--
0
00
0
00

f2
21
F
0.21
3
60
83
0
1.04
18
7
0
00
--
--
0
00
0
00









Mice
liver
(mg/kq)
wet basis





Control
' 0
60
F
0.04
2
94
214
0
0.00
ZO
F IT
BO
--
--
0
00
0
00
(no sludge)
r Q
60
M
0.04
4
55
186
0
0.00
25
5
0
00
—
--
0
00
0
00
r i
200
F
0.03
3
23
161
7
0.00
19
5
0
00
.003
.100
0
20
0
00

F2
21
F
0.00
19.7
159
8
0.00
16
3
0
00
.008
.020
0
43
0
04
7.6 cm/ha
» u
60
F
0.04
2
97
161
0
0.00
26
6
0
00
-
__
0
00
0
00
LPS
' 0
60
M
0.04
3
85
224
0
0.00
27
6
0
00
—
--
0
00
0
00

r i
200
F
0.03
3
74
204
0
0.00
21
8
0
00
.003
.120
0
14
0
00

f2
21
F
0.00
4
90
199
5
0.00
19
9
0
00
.005
.050
0
14
0
00
500 g/h/d
' 0
60
F
0.20
3
05
207
0
0.00
43
2
0
00
.040
.200
0
00
0
00
DCS
Fu
60
M
0.15
3
07
228
0
0.00
23
1
0
00
.040
.400
0
00
0
00

»" 1
200
F
0.43
3
10
230
0
0.36
17
3
0
00
--
--
0
00
0
00

f2
21
F
0.29
8
60
201
0
0.36
19
0
0
00
--
--
0
00
0
00
-------
TABLE 6. Continued

Mice
Test




Treatment
Generation
days
Sex
Cd
Cu
Fe
Control
Fo
60
F
0.00
0.79
22.0
(no sludge)
Fo
60
M
0.00
0.81
34.0

F!
200
F
0.00
1.15
36.3

F2
21
F
0.00
1.33
32.4
7.6 cm/ha
Fo
60
F
0.00
0.59
21.0
LPS
Fo
60

0.00
0.72
30.0

Fi
200
F
0.03
1.39
39.6

f2
21

0.00
1.36
30.6
500 g/h/d
Fo
60
F
0.02
0.52
24.0
DCS
Fo
60

0.02
0.68
30.0

F,
200
F
0.04
4.30
316.0

f2
21
F
0.04
1.40
83.0
> 1 Centimeters per hectare
2	Pensacola liquid sludge
3	Dried Chicago digested sludge
'' Bertrand ei al., 1978
Co	Zn	Hcj	Se	Cr	hH
Mice muscle (mg/kg) wet basis
0.00
26.6
—
—
0.00
0.00
0.00
21.4
—
--
0.00
0.00
0.00
20.5
.015
.040
0.40
0.00
0.00
19.1
.008
.030 .
0.43
0.40
0.00
19.5
__
	
0.00
0.00
0.00
19.7
--
--
0.00
0.00
0.00
22.8
.036
.030
0.36
0.70
0.00
19.7
.010
.020
0.44
0.30
0.00
26.0
.040
.200
0.00
0.00
0.00
19.4
.040
.200
0.00
0.00
0.35
25.2
--
--
0.35
0.00
0.35
22.2
--
--
0.70
0.00
-------
TABLE 7. MICE FEEDING TRIAL 1. MINERAL ANALYSES BY TREATI1ENT OF KIDNEY, LIVER, AND MUSCLE TISSUE OF MICE FED FREEZE-DRIED
KIDNEY OR LIVER TISSUE FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING 7.6 CM/HA1 LPS2 OR 500 G/HEAD/DAY
Treatment
Mice
Generation
Test
days
Cd
Cu
Fe
Co
Zn
DCS
Pb
Control, 0%
Fo
60
4
0.04+.206
3.56+.13

106.0±8.9
0.70+.03
18.60±.B7
0.415+.140
7.6 cm/ha LPS
Fo
60
4
0.09+.20
3.55+.13

125.3+8.9
0.64+.03
20.52+.87
0.632+.140
500 g/h/d DCS
Fo
60
4
1.13+.203
3.24+.13
Mice
110.6+8.9 0.60±.03
liver (ma/kg) wet basis5
16.97+.87
0. 345+. 140
Control , 0%
Fo
60
4
.020+.021
3.68+.21

236.2+19.0
0.30+.01
22.07+3.35
0.227+.034
7.6 crn/ha LPS
Fo
60
4
.020+.021
3.85+.21

252.9+19.0
0.28+.01
29.07+3.35
0.160+.034
500 g/h/d, t)CS
Fo
60
4
.230+.021b
3.39+.21
1.07+,02C
Mice
263.3+19.0 0.26+.01
muscle (mg/kg) wet basis5
27.90+3.35
0.182+.034
Control, 0%
Fo
60
4
.0001.002

27.07+.92
0.25+.84
17.62+ .84
0.195+.020
7.6 cm/ha LPS
Fo
60
4
.000+.002
0.89+.02

24.40+.92
0.21+.84
15.07+ .84
0.190+.020
500 g/h/d DCS
Fo
60
4
.010+.002
0.81+.02

25.35+.92
0.24+.84
17.17+ .84
0.240+.020
1	Centimeters per hectare
2	Pensacola liquid sludge
3	Dried Chicago digested sludge
h Bertrand et al., 1978
5	Entries are diet means averaged across sex and feed types which were 5% kidney and 10% liver
6	Means + SE
a')Cd was higher at the 500 g/head/day DCS treatment (P < 0.05)
Cu in mice muscle was higher in the controls (P < .05).
-------
TABLE 8. MICE FEEDING TRIAL 1. MINERAL ANALYSES BY TREATMENT OF KIDNEY, LIVER, AND MUSCLE TISSUE OF MICE FED
FREEZE-DRIED KIDNEY FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING 7.6 CM/HA1 LPS2 OR
500 6/HEAD/DAY DCS3 *
Treatment
Mice
Generation
Test
days
JiL
Se
Cr
Ni
Control
7.6 cm/ha
LPS
500 g/h/d
DCS
Control
7.6 cm/ha
LPS
500 g/h/d
DCS
Control
7.6 cm/ha
LPS
500 g/h/d
DCS
Fo
F.
Fo
Fo
Fo
Fo
Fo
Mice kidney (nig/kg) wet basis5
60
60
60
60
60
60
60
60
60
.123+.054
.049+.015
.035+.002
.007+.006
.007+.002
.006+.000
.004+.001
.000+.000
.004+.001
.135+.024
.147+. 006
.42+.10
.23+.04
.140+.019	.34+.02
Mice liver (mg/kg)	wet basis5
.081+.078	.17+.06
.102+.037	.20+.14
Mice muscle (mg/kg)	wet basis5
.130+.000	.08+.01
.004+.000
.014+.008
.008+.001
.15+.01
.10+.00
.17+.05
2.68+.01
2.61±.22
2.81+.10
1.44+.30
1.42+.00
1.29+.14
1.11+.00
1.11+.01
1.44+.36
'Centimeters per hectare
2Pensacola liquid sludge
3Dried Chicago digested sludge
"•Bertrand et at., 1978
5Entries are diet means averaged across sex
6Mean + SE
-------
TABLE 9. MICE FEEDING TRIAL 1. MINERAL ANALYSES BY SEX OF KIDNEY, LIVER, AND MUSCLE TISSUE OF MICE FED FREEZE-DRiED KIDNEY
OR LIVER TISSUE FROM CATTLE FED A CONTROL DIET, CORN FROM LAND RECEIVING 7.6 CM/HA1 LPS2 OR 500 G/HEAD/DAY DCS3 "
Sex
Mice
Generation
Test
days
Cd
Cu
Fe
Co
Female
Male
Female
Male
Female
o~> Male
GO
60
60
60
60
60
60
0.56+.206
0.28+.20
6	0.10+.02
6	0.07+.02
6	0.003+.002
6	0.003+.002
3.45+.12
3.44+.13
3.01+.21
4.27+.21a
0.87+.02
0.97+.02b
Mice kidney~Tmg/kg) wet basis0
100.3+8.9	0.62+.03
Zn
Pb
127.7+8.9
0.67+.03
Mice liver (nig/kg) wet basis5
260.8+19.0 0.26+.01
240.8+19.0
0.29+.01
Mice muscle (mg/kq) wet basis6
21.38+ .92 0.23+.CI
'29.33+ ,92b
0.23+.01
17.50 + .07
19.90 + .87
28.83+ 3.35
23.86+ 3.35
17.95 + .84
15.30 + .84
0.348+. 140
0.5801.140
0.160+.0.S4
0.220+.034
0.208+ .02
0.208+ .02
'Centimeters per hectare
2Pensacola liquid sludge
3Dried Chicago digested sludge
^Bertrand et al., 1978
5Entries are diets means averaged across feed types and treatment groups
6Means + SE
aCu in male mice liver was greater (P < 0.01) than in females
^Cu and Fe in male mice muscle was greater (P < 0.01) than in fernal
es
-------
MICROBIOLOGY
E. M. Hoffmann and Suzanne Hickman
FEEDING STUDIES ON ANIMALS: GENERAL PROCEDURES
Blood and feces were examined monthly from animals fed on sludge-
amended diets or on feed material which was grown on land fertilized
with sewage sludge. At the termination of the feeding experiments, the
animals were slaughtered, and kidney, liver, and spleen samples were also
examined. Only tissue samples were analyzed in experiments involving
poultry. All bacteriologic procedures were carried out in accordance
with the Manual of Clinical Microbiology, second edition, American Society
of Microbiology (1) and Baily and Scott's Diagnostic Microbiology (2).
BLOOD CULTURES
Two ml of blood were drawn from the jugular veins of cattle or
from the carotid arteries of swine into 18 ml of peptone broth using
vacutainer blood culture tubes (Becton-Dickinson, Rutherford, NJ). The
culture tubes were vented, and placed at 37°C. Observations for growth
were made daily. Gram stains were made from tubes showing turbidity,
and they were also subcultured onto blood agar plates. The plates were
incubated at 37°C, and observed for growth. Primary blood culture tubes
were kept for 2 weeks before discarding.
All bacterial colonies which appeared on blood plates were gram
stained. Gram positive cocci which grew in clumps were subjected to
additional tests (listed below) to determine if they were Staphylococcus
aureus. Likewise, gram positive cocci growing in chains (or pairs) and
gram negative rods were tested to determine if they were potentially
pathogenic Streptococci or enteric pathogens, respectively.
Method of Analysis
Gram positive cocci in clumps
1.	Coagulase tests
2.	Catalase tests
3.	Mannitol fermentation
4.	Tellurite reduction on Vogel-Johnson medium
5.	Hemolysis on 5% blood agar
164
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Gram positive cocci in chains
1.	Hemolysis on 5% blood agar (aerobic and anaerobic)
2.	Sensitivity to bacitracin (0.04 units)
3.	Sensitivity to optichin
4.	Serologic grouping: 3-hemolytic bacteria sensitive to 0.04 units
of bacitracin were grouped using specific antiserum to the group
A antigen
Gram negati ve rods
1.	Fermentation of glucose, sucrose, and lactose using triple
sugar iron agar (TSI agar)
2.	Urease production
3.	Pyruvate production from phenylalanine
4.	H2S production
5.	Bacteria which failed to ferment sucrose or lactose but which
fermented glucose, did not produce urease, and did not convert
phenylalanine to pyruvic acid were subjected to additional
testing using Roche "Enterotubes" (Enterotube II, Roche
Diagnostics, Nutley, NJ). The following tests are made with
this system:
a.	Acid and gas from dextrose
b.	Decarboxylation of lysine
c.	Decarboxylation of ornithine
d.	H2S production
e.	Production of indole from tryptophane
f.	Adonitol fermentation
g.	Lactose fermentation
h.	Arabinose fermentation
i.	Sorbitol fermentation
j. Acetoin production
k. Deamination of phenylalanine
1. Urease production
m. Citrate utilization
6.	Final identification of Salmonella and Shigella was accomplished
using specific antisera (Lee Laboratories, Grayson, GA)
FECAL SAMPLES
Fecal samples were streaked directly on XLD and SS agar plates.
Approximately 1.0 g amounts of feces were also placed into tetrathionate
broth. The media were incubated for 20-24 hours at 37 C. All of the
tetrathionate cultures were subcultured onto XLD and SS agar plates, and
the plates were incubated for another 20-24 hour period at 37°C. On XLD
medium, Salmonella bacteria usually produce red colonies with black centers,,
however, some Salmonella give red colonies without black colonies. Shigella
bacteria also give red colonies on the medium. Lactose fermenting bacteria
give red colonies on SS medium whereas non-lactose fermenting enteric bac-
teria (potentially pathogenic) have non-pigmented colonies. All suspicious
colonies were picked and subjected to preliminary screening according to
165
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the procedure described for treatment of gram negative rods isolated from
blood cultures. Bacteria which failed to ferment sucrose or lactose but
which fermented glucose, were urease negative, and did not deaminate
phenylalanine, were placed on Roche "Enterotubes" for identification.
Final identification of Salmonella or Shigella was accomplished using
specific antisera. Salmonella were placed into serologic groups, but it
was possible to identify the species of Shigella.
ORGAN SAMPLES
Organ samples were obtained from animals during slaughter. Samples
of spleen, liver, and kidney were obtained aseptically at the slaughter-
house immediately after the animals were opened. A separate sterilized
knife was used for each sample, and the organs were handled only with
sterile gloves. The samples were placed in sterile jars and transported
to the laboratory for culturing.
The tissues were minced with sterile scissors and approximately 5 g
amounts were placed in fluid thioglycholate broth (with glucose and
indicator). A small amount of minced tissue was also placed on Lowenstein-
Jensen medium. The media were incubated at 37°C. Fluid thioglycholate
cultures were observed after 24 and 48 hours. Lowenstein-Jensen cultures
were kept for 4 weeks, and examined daily for the appearance of suspicious
colonies. Gram stains were performed on bacteria appearing in the fluid
thioglycholate tubes, and additional tests were performed on selected
types of bacteria. Gram positive cocci growing in clumps, gram positive
cocci growing in chains (or pairs), and gram negative rods were subjected
to additional testing according to the protocol described for bacteria
isolated from blood cultures.
BACTERIOLOGICAL ANALYSIS OF FEED, GRASS, AND SLUDGE
The feed, dried sludge used for diet amendment, wet sludge used for
soil treatment, and grasses grown on sludge-fertilized soil were sub-
jected to analysis for pathogenic enteric bacteria. In addition, the
numbers of coliform and fecal coliform bacteria were estimated.
One gram quantities of each sample were placed in tetrathionate
broth and incubated at 37°C. The cultures were then subcultured onto
XLD and SS agar plates which were incubated at 37°C for 20-24 hours.
Suspicious colonies (see preceding section on Fecal Samples) were picked
and subjected to further analysis using the procedures that have been
listed in the section dealing with the treatment of gram negative rods
isolated from blood cultures.
Five gram quantities of the test materials were placed into 100 ml
volumes of sterile water. Grass samples were cut into fine pieces using
sterile scissors before being added to the water. The mixtures were
shaken according to the procedure described in Standard Methods for the
Examination of Water and Wastewater (3) and the numbers of coliform
bacteria per 100 ml volume were estimated using the most probable number
166
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method (MPN) of analysis (3). Replicate samples (totaling 5) of 10 ml,
1.0 ml, and 0.1 ml were taken from the 100 ml volumes of water in which
the various samples had been suspended. Fecal coliforms were also
enumerated using the MPN procedure with EC incubated at 44.5°C (3).
BACTERIOLOGICAL ANALYSIS OF GROUND WATERS,
SURFACE WATERS, AND THE SLUDGE LAGOON
Sludge from the holding pond (lagoon) at the Jay Agricultural Research
Center, water from ponds and streams draining sludge-treated fields, and
water from wells drilled in the test area were analyzed for numbers of
coliform and fecal coliform bacteria. Samples were submitted monthly.
Those samples which were turbid, or which had particulate matter were
analyzed using the MPN methods for total coliforms and fecal coliforms
(3). Clear water samples were subjected to the membrane filter technique
for estimation of total coliforms and fecal coliforms (3).
SOIL ADSORPTION STUDIES: GENERAL PROCEDURES
Limited studies were conducted to study the fate of Escherichia ooli
in the soil-water system at the Jay Agricultural Research Center. For
these studies, a double antibiotic resistant mutant of E. ooli (strepto-
mycin and ampicillin resistant) was selected from the sewage sludge used
for soil amendment on the farm at the Jay center. Five types of experi-
ments were conducted.
Extent of E. ooli adsorption to soil
Rate of E. ooli adsorption to soil
Movement of E. ooli through topsoil
Elution of E. ooli from topsoil
Survival of E. ooli in the equivalent of ground water
ANTIBIOTIC RESISTANT E. COLI MUTANT
A 1.0 g samples of Pensacola sewage sludge was cultured in tetra-
thionate broth for ca - 24 hours at 37°C. This culture was then subcultured
onto eosin methylene blue agar (EMB), and incubated again for ca - 24 hours
at 37°C. A lactose fermenting colony which demonstrated a greenish
metallic sheen was selected and cultured on an Enterotube II at 37°C for
ca - 24 hours. The isolate, identified as Escherichia ooli, was trans-
mitted to Dr. D. E. Duggan (Department of Microbiology and Cell Science,
University of Florida), who selected a mutant which was resistant to
both streptomycin (200 yg/ml), and ampicillin (20 yg/ml). All of the
experiments described in the section dealing with soil adsorption utilized
this antibiotic resistant mutant of Escherichia ooli, and it is referred
to simply as E. ooli.
167
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EXTENT OF E. COLI ADSORPTION TO SOIL
Studies were carried out to determine the numbers of E. aoli that
would adsorb to a given amount of Orangeburg topsoil (from the Jay ARC)
in a set period of time. For some experiment, the soil and bacteria
were suspended in 0.01 M CaCl 2 to approximate the ionic conditions that
would be encountered in a soil-ground water system. Other experiments
were conducted with deionized water to approximate the conditions of soil
suspended in rain water.
Soil from the top 15.0 cm was collected from a field at the Jay
Agricultural Center which had been recently cleared of trees. This area
had not been planted to crops or fertilized in the past 20 years or
longer. The soil was air dried (20-24OC) and sifted through a screen such
that the particles were equal to or less than 1.0 mm in diameter.
The earlier adsorption studies were carried out by suspending various
amounts of soil in 100 ml of a standardized suspension of E. aoli in
water or 0.01 M CaCl2- The soi1-bacteria mixtures were shaken in
Erlynmeyer flasks at about 60 passages per minute at room temperature
(22-24°C). After 60 minutes, the soil was sedimented by centrifugation
at about 125 g at room temperature. The supernatant fluids were collected,
and the number of E. aoli enumerated by plating in nutrient agar con-
taining 200 yg/ml streptomycin and 20 ug/ml ampicillin. A control con-
sisted of bacteria in water or 0.01 M CaCl2 held for 60 minutes at room
temperature without the addition of soil.
Later, static adsorption experiments were carried out using
1 part of soil to 2 parts of sterile CaCl2- The mixtures were placed in
screw-capped tubes and mixed slowly by repeated inversion for 2 hours
at room temperature on a Fisher Roto-Rack 343 shaker. The rate of mixing
was about 20 inversions per hour. As indicated above, the initial studies
of this type utilized bacteria suspended in water. Experiments were also
conducted with E. aoli suspended in 0.01 M CaCl2 and in various concentra-
tions of Pensacola sewage sludge to determine the effects of the sus-
pending medium or the extent of bacterial adsorption to soil.
We also examined the elution of trapped and adsorbed E. aoli from
Orangeburg soil under the equivalent of ground water conditions. Time
did not permit varying the physical conditions of the system to test the
effect on desorption and/or elution. Escherichia aoli were adsorbed to
the soil using 0.01 M CaCl2 as the suspending medium with 2 parts of
bacterial suspension to 1 part soil. Adsorption was for 2 hours at 22-24°C
with mixing by slow inversion (as described previously). The mixture was
rapidly filtered using a Gel man 47 mm magnetic filter holder, and a disk
of Whatman No. 1 filter paper. A control consisted of bacteria in 0.01 M
Ca Cl2 without soil. These were also filtered, and the base-line input
level was taken as the number of bacteria recoverable from this control
at time 0.
168
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The soil which was trapped by the filter paper disk was then washed
with 0.01 M CaCl . Plate counts were performed on 25 ml volumes of filtrate
using the membrane filtration technique and nutrient agar plus antibiotics
(200 g/ml streptomycin and 20 g/ml ampicillin). The control filter paper
dis was also washed in the same fashion, and plate counts were performed.
The number of E. ooli before mixing with soil were determined by
plate counting on nutrient agar plus antibiotics. The numbers of bacteria
bound (or trapped) during the adsorption steps were calculated by taking
the difference between the initial count and the count in the first fil-
trate after a correction had been made to account for bacteria non-
specifically adsorbed or trapped in the filtration system. The elution
of E. eoli from the soil was then estimated by summarizing the counts
made of the water wash fractions.
BACTERIOLOGY: CONCLUSIONS
Blood samples were obtained from cattle which were fed on a diet
which was amended with dried sewage sludge. Blood was taken monthly, and
bacteria were isolated from a high percentage of these samples even though
clinical evidence indicated that there was no disease. These bacteria
were not readily identified as pathogens, and they probably represented
contamination introduced at the time of sampling. The sampling conditions
were not conducive to obtaining uncontaminated materials. One isolation
of staphylococcus aureus was made from a single animal in the sludge trial
group (Table 1). Similar results were obtained from blood and tissue
samples from swine (data not shown).
Fecal specimens from the sludge fed cattle were tested for the
presence of enteric pathogens. A total of 5 Salmonella isolations were
made. All of these were from the sludge fed group which received 500 g
per head per day. Three of the isolates were Salmonella ent&ritidis
group A, and 2 were Salmonella enteritidis group B (Table 2). Similar
results were obtained from fecal samples obtained from swine (data not
shown).
Kidney, liver, and spleen samples were taken at the time of slaughter,
and examined for the presence of bacteria. Bacteria were isolated from
a high percentage of the samples, but very few of these were identified
as pathogens when examined by the criteria listed in the procedures section.
The microorganisms were probably contaminants introduced into the tissues
at the time of slaughter since the conditions were not ideal for bacteri-
ologic sampling.
One isolation of Staphylococcus aureus was obtained from an animal
in the control group which did not receive sludge in the diet. Three
cases were found where g-hemolytic streptococci were present. These
bacteria were not Staphylococcus pyogenes (Table 3).
169
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Bacteriologic analyses were conducted with 1 poultry trial. It
was very difficlut to obtain "clean" poultry samples, and we abandoned
attempts to isolate any bacteria other than enteric pathogens. Salmonella
entevitidis group C was recovered from 2 fecal samples. One of these
samples was from a male chicken fed on 50% sludge-grown grain, and the
other was from a female chicken fed on 100% sludge-grown grain (Table 4).
Feed, forage, and dried sludge used for supplementation of diets
were analyzed for enteric pathogens, fecal coliforms, and total coli-
forms. There were no enteric pathogens isolated using our methods.
Fecal coliform counts were negligible except in feed, and sludge amended
feed from the Live Oak ARC in 1978 (Table 5). Two samples from the Jay
ARC had substantial total coliform counts in 1976, and there were
elevated total coliform counts associated with the materials obtained
from the Live Oak ARC in 1977 (Table 5).
Fecal and non-fecal coliform analyses were carried out using water
samples and from the Jay ARC. These samples were from wells and bodies
of water in the vicinity of the sludge storage area, and the fields which
were amended with sludge. The data summarizing these analyses for 1976
and 1977 are found in Table 6. Data from 1978 are located in Table 7.
Substantial numbers of non-fecal coliforms began appearing in August
and continued through October. Fecal coliforms began appearing in some
samples in September, and in some cases they persisted into November.
Results of fecal and non-fecal coliform analysis of water samples for
1979 are found in Table 8. Both fecal and non-fecal coliforms were asso-
ciated with certain samples throughout the period of testing.
Fecal and total coliform counts were made on a variety of sludges.
The results of the tests are shown in Table 9. The material from the
sludge pond was used to amend the soil of the Jay ARC for certain trials
which were conducted as part of the project. It can be seen that the
fecal coliform numbers dropped to a very low level during the period
between May, 1978 and November, 1978. The possible significance of this
will be addressed in a paper which is being prepared in collaboration
with Dr. S. Farrah and Dr. G. Bitton. Both of these researchers are
co-investigators on this project.
SOIL STUDIES: BACTERIOLOGY
A mutant strain of Escherichia coli (resistant to streptomycin and
ampicillin) was obtained from Dr. Dennis Duggan. This organism was
maintained in streptomycin (200 iig/ml), and ampicillin (20 ug/ml) in
nutrient broth. The growth characteristics of this strain were deter-
mined at 35°C in the above medium. Most of the experiments in this
study employed mid-log phase bacteria which usually had an optical
density of 0.600-0.650 at a wave length of 550 nm.
170
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Adsorption experiments were conducted using E. eoli suspended in
water with different concentrations of soil. It was found that roughly
90% of the bacteria were removed from the fluid phase by a 10% soil sus-
pension. The mixture was shaken at 23°C for 60 minutes before analysis.
The rate of adsorption of E. eoli to soil was measured using a 10%
soil suspension at about 23°C. The suspending medium was water. Maximum
adsorption occurred at about 40 minutes under these conditions. It was
interesting to see that there was marked bacterial replication in the
control which consisted of E. eoli suspended in water. Again, there was
better than 90% maximum removal of the bacteria from the fluid phase when
compared to the control.
There was less adsorption of the mutant strain of E. eoli to soil
when the bacteria and soil were suspended in 0.01 M CaCl2 . Roughly 64%
of the input level was removed from the fluid phase by a 10% soil sus-
pension. However, more adsorption was achieved in 0.01 M CaCl2 when the
ratio of bacterial suspension to soil was changed to 2:1. Under these
conditions, about 90% removal occurred in 60 minutes at 22-24°C. In this
experiment, the bacteria in the control apparently did not replicate
during the time-course of the experiment. This was seen in each of 3
experiments. The adsorption rate experiments carried out with water
always showed an increase in bacterial number during the time-course of
the experiment. Three experiments were carried out. We have no ready
explanation for these different results.
Bacterial loading capacity of the Orangeburg type soil in water
was investigated. The objective of this experiment was to determine
the maximum number of bacteria that could be bound (per gram) to this
type soil. It was found that nearly all of the bacteria added, at any
concentration, were removed from the fluid phase from the soil, and the
expected plateau was not reached. A number of technical problems prevented
us from determining the total loading capacity of the Orangeburg soil for
E. eoli. It was interesting to note the loading capacity of the Orange-
burg soil for E. eoli. There was a linear relationship between the amount
of bacteria added to the system and the number adsorbed.
Organic matter, in the form of sewage sludge, did not seem to
influence the extent of adsorption of E. coli to Orangeburg soil. Sludge
concentrations up to 4% did not have an appreciable effect on the ability
of the soil to adsorb bacteria.
The strong adsorption of E. coli to soil was further demonstrated
by failure of the bacteria to elute from the particles when washed
with the suspending medium. Only 0.01 M CaCl2 was used in these experi-
ments. Very few bacteria could be recovered from a 2 g sample of soil
which had adsorbed about 5.2 x 106 bacteria. This suggests that the
bacteria were indeed bound to the soil and not trapped in pores and
between particles. Other experiments were carried out to determine the
conditions necessary for eluting the bacteria from the soil. No evidence
for elution could be found by increasing ionic strength or by alteration
of pH (data not shown).
171
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REFERENCES
1.	Lennette, E. H., E. H. Spaulding, and J. P. Truant. Manual of
Clinical Microbiology (2nd edition), American Society for Micro-
biology, Washington, DC, 1974.
2.	Finegold, S. M., W. 0. Martin, and E. G. Scott. Bailey and Scott's
Diagnostic Microbiology (5th edition), C. V. Mosby, St. Louis, MO,
1978.
3.	Rand, M. C., A. E. Greenberg, and M. J. Taras. Standard Methods for
the Examination of Water and Wastewater (14th edition), American
Public Health Association, Washington, DC, 1976.
172
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TABLE 1. SLUDGE FEEDING TRIALS, CATTLE, JAY ARC BLOOD CULTURES
Dates
Treatment
Total
Samples
Samples with
bact. growth
Pathogeni c
isolates*
121975
Control
28
19
0
to
Liquid digested sludge - 1
28
19
1, S. aureus
051576
Liquid digested sludge - 2
28
19
0
081976
Control
24
11
0
to
Sludge diet (250 g/h/d)
24
8
0
121676
Sludge diet (500 g/h/d)
24
10
0
032377
Control corn
56
52
0
to
Control corn + sludge
56
53
0
081177
Sludge corn
56
52
0
022278
Control
40
19
0
to
Liquid digested sludge - 1
40
22
0
080978
Liquid digested sludge - 2
40
16
0
052379
Control
56
14
0
to
Liquid digested sludge - 1
56
26
0
110779
Liquid digested sludge - 2
56
31
0
* Pathogens defined according to the criteria described in the procedures section.
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TABLE 2. SLUDGE FEEDING TRIALS, CATTLE, JAY ARC
FECAL SAMPLES
Dates	Treatment	Total Samples	Pathogens Isolated9
121975
to
051576	No samples
081976
Control
40
0
to
Sludge diet (250 g/h/d)
40
°h
121676
Sludge diet (500 g/h/d)
40
3
032377
Control corn
56
0
to
Control corn + sludge
56
0
081177
Sludge corn
56
0
022278
Control
56
0
to
Liquid digested sludge - 1
56
0
080978
Liquid digested sludge - 2
56
2C
052379
Control
56
0
to
Liquid digested sludge - 1
56
0
110779
Liquid digested sludge - 2
56
0
Enteric pathogens identified according to methods described in the
procedures section.
Salmonella entevitidis, group C. Isolated from different individuals.
Two isolates from different individuals. Salmonella entevitidis,
group A, and Salmonella entevitidis, group B.
174
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TABLE 3. SLUDGE FEEDING TRIALS, CATTLE, JAY ARC
NECROPSY CULTURES3
Dates
Treatment
Kidneys^
i • b
Li ver
Spleens'3
121975
Control
1/8
0/8
lc/8
to
Liquid digested sludge - 1
0/8
1/8
0/8
101576
Liquid digested sludge - 2
2/8
4/8
3/8
081976
Control
4/8
1/8
6/8
to
Sludge diet (250 g/h/d)
3/8
2/8
2/8
121676
Sludge diet (500 g/h/d)
2/8
1/8
0/8
032377
Control corn
4/8
6d/8
3e/8
to
Control corn + sludge
6/8
4/8
3/8
081177
Sludge corn
5/8
4/8
0/8
052379
Control
5/8
4/8
3/8
to
Liquid digested sludge - 1
4/8
3/8
5/8
110779
Liquid digested sludge - 2
5/8
5/8
4/8
Organ samples cultured in fluid thioglycholate broth, tetrathionate
broth, and Lowenstein-Jensen medium. Pathogens, when present, were
identified according to the protocols described in the procedures
section.
The denominator is the total number of samples. The numerator is
the number of samples with bacteria present.
Q
Staphylococcus aureus
A
Two isolations of gram positive, 3-hemolytic streptococci which were
non-groupable with existing antisera, and which were bacitracin-
resistant.
e One isolate same as above.
175
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TABLE 4. POULTRY FEEDING TRIAL, GAINESVILLE
Date
FECAL
Treatment
AND BLOOD SAMPLES3
Feces'3
Blood'3
102876
Control M
0/5
0/5
102876
Control F
0/5
2c/5
102876
50% M
ld/5
0/5
102876
50% F
0/5
0/5
102876
100% M
0/5
0/5
102876
100% F
ld/5
0/5
a
Samples analyzed for enteric pathogens only.
The denominator is the total number of samples, the numerator
is the number of samples with enteric bacteria present.
c
Proteus vulgavvs
d Salmonella enterit-Ldis, group C, Vi negative
176
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TABLE 5. BACTERIAL ANALYSIS - FEED, FORAGE, AND DRIED SLUDGE SUPPLEMENTS




Enteri c
Coliforms ,
Location
Code
Year
Pathogens9
Fecal
Total
Jay ARC

Cone, supplement
1976
0/2
_
< 2/2
Jay ARC

Ground corn
1976
0/2
-
2700/2
Jay ARC

Corn shucks
1976
0/2
-
1200/2
Jay ARC

Control
1976
0/1
-
< 2/1
Jay ARC

10% sludge
1976
0/1
-
< 2/1
Jay ARC

20% sludge
1976
0/1
-
< 2/1
Gainesville
Control feed
1977
0/7
3.3/7
29/7
Gainesville
50% Chicago sludge
1977
0/7
< 2/7
1.4/7
Gainesville
Dried Chicago sludge
1977
0/7
< 2/7
1.4/7
LiveOak
ARC
ASP control
1977
0/3
.7/3
6/3
LiveOak
ARC
ASP 10% sludge
1977
0/3
3/3
4.3/3
LiveOak
ARC
ASP20% sludge
1977
0/3
.7/3
8/3
LiveOak
ARC
Li neomycin control
1977
0/4
< 2/4
22/4
LiveOak
ARC
Li neomycin 10% sludge
1977
0/4
.5/4
6.8/4
LiveOak
ARC
Li neomycin 20% sludge
1977
0/4
3.5/4
.5/4
LiveOak
ARC
Dried sludge
1977
0/4
9/4
66/4
LiveOak
ARC
Control
1978
0/8
119/8
143/8
LiveOak
ARC
10% sludge
1978
0/8
74/8
644/8
LiveOak
ARC
20% siudge
1978
0/8
4/8
233/8
Jay ARC

Control silage
1978
0/5
< 2/5
< 2/5
Jay ARC

Cone, supplement
1978
0/5
118/5
83/5
Jay ARC

6 Ac. In. silage
1978
0/5
< 2/5
< 2/5
Jay ARC

9 Ac. In. silage
1978
0/5
< 2/5
< 2/5
Ona ARC

Control feed
1978
0/3
4.6/3
4.6/3
Ona ARC

Manure sludge
1978
0/3
180/3
180/3
LiveOak
ARC
F2 control
1978
0/4
732/4
2200/4
LiveOak
ARC
F2 10% siudge
1978
0/4
1224/4
2030/4
LiveOak
ARC
F2 20% sludge
1978
0/4
1263/4
1602/4
LiveOak
ARC
F3 control
1978
0/2
1/2
13/2
LiveOak
ARC
F3 10% sludge
1978
0/2
800/2
1470/2
LiveOak
ARC
F3 20% sludge
1978
0/2
466/2
>2400/2
LiveOak
ARC
Pigs control
1978
0/1
34/1
>2400/1
LiveOak
ARC
Pig sludge corn
1978
0/1
< 2/1
>2400/1
Ona ARC

Silage
1979
0/18

23/18
177
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TABLE 5. Continued
Location
Code
Year
Enteric
Pathogens3
Col i forms ,
Fecal Total
Li veOak
ARC
F2 Control
1979
0/1
130/1
>2400/1
LiveOak
ARC
F2 10% sludge
1979
0/1
>2400/1
>2400/1
Li veOak
ARC
F2 20% sludge
1979
0/1
>2400/1
>2400/1
Li veOak
ARC
F21 control
1979
0/1
2/1
2/1
LiveOak
ARC
Fgl sludge corn
1979
0/1
< 2/1
34/1
Jay ARC

Grass 54 E
1979
0/4
1/4
611/4
Jay ARC

Grass 54 W
1979
0/4
.5/4
602/4
Jay ARC

Grass 53 W
1979
0/4
.5/4
603/4
Jay ARC

Grass 53 E
1979
0/4
2.3/4
605/4
Jay ARC

Grass 59 E
1979
0/4
.5/4
612/4
Jay ARC

Grass 59 W
1979
0/4
.5/4
603/4
Jay ARC

Sludge (pond)
1979
0/4
326/4
13000/4
a Denominator is the total number of samples. The numerator is the number
containing enteric pathogens.
k Denominator is the total number of samples. The numerator is the
average coliform count per gram for the group.
178
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TABLE 6. FECAL AND NON-FECAL COLI FORM COUNTS FROM WATER SAMPLES
OBTAINED MONTHLY FROM THE JAY ARCa, 1976-1977
1976	1977

10-5b
11-2°
12-7
1-4
2-2
3-2
4-5
Northeast well
0
0/0
0/0
0/0
0/0
0/0
0/0
Middle well
0
0/0
0/0
0/0
0/0
0/0
0/0
Southwest well
0
0/0
0/0
0/0
0/0
0/0
0/0
Sludge pond well
0
0/0
0/0
0/0
0/0
0/0
0/0
Spring
3
0/0
2/3
0/0
0/0
0/1
5/4
Pond creek
0
4/0
2/150
1/0
17/3
25/15
0/ 300
Well w/o pump
2
0/0
0/0
17/2
°/°b
0/0
0/0
A-l
0
0/0
160/140
0/0
°b
0/0
0/0
A-2
0
0/0
0/0
0/0
°b
32/260
0/0
B-l
0
0/100
0/0
0/0
2b
0/0
0/0
B-2
0
0/0
0/80
0/0
ob

0/0
1977

5-3
6-7
7-6
8-3
9-7
10-5
11-2
12-7
Northeast well
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Middle well
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Southwest well
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Sludge pond well
0/0
0/0
0/0
0/0
0/0
0/0
0/0
-
Spring
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Pond creek
1/3
3/6
109/30
2/50
0/0
7/0
0/0
11/5
Well w/o pump
0/0
5/7
1/4
0/0
0/0
12/16
0/58
0/0
A-l
0/0
0/0
0/0
-
-
-
-
-
A-2
0/0
0/0
0/0
-
-
-
-
-
B-l
0/0
-
0/0
-
-
-
-
-
B-2
0/0
-
0/0
-
-
-
-
-
9	b
Membrane filtration procedure used for analysis. Non-fecal counts only. Samples were 100 ml
0 Non-fecal and fecal counts on 100 ml samples. Numerator=non-fecal counts; denominator=fecal counts.
-------
TABLE 7. FECAL AND NON-FECAL COLIFORM COUNTS FROM WATER SAMPLES
OBTAINED FROM THE JAY ARCa, 1978

1-4
2-8
4-3
5-3
6-5
7-10
8-9
9-11
10-4
11-1
12-6
Northeast well
0/0
0/0
LA°
0/0
0/0
-
TNTC/0
TNTC/13
TNTC/0
-
0/0
Middle well
0/0
TNTC
LAC
0/0
0/0
LA
TNTC/0
TNTC/0
<2b/<2
0/0
0/0
Southwest well
0/0
TNTC
LAC
-
0/0
LA
-
TNTC/1
0/0
1/0
0/0
SIudge pond wel1
0/0
-
LAC
29/1
0/0
86/2
TNTC/0
5/1
2/0
3/0
0/0
Hill top well
0/0
0/0
LAC
0/0
0/0
LA
TNTC/0
170b/<2
8/0
1/4
-
Spring
0/1
-
LA°
3/1
2/16
LA
TNTC
TNTC 220b/49
70b/<2
0/1
Pond creek
0/51
11/23
LAC
8/3
0/3
42/1
-
TNTC
50/100
79b/13
<2b/<2
Wel1 w/o pump
0/0
0/0
LAC
3/1
0/0
LA
TNTC
-
5b/<2
44b/<2
-
3-F Cb
0/0
8/0
LAC
7/19
5/4
33/13
< 2
33/33
8/5
240(1?/ 2
70b/920
6-FCb
0/0
0/0
LAC
3/0
3/2
11/7
< 2
170/130
2/<2
-
<2b/8
a
Membrane filtration procedure used for analysis. Non-fecal and fecal counts.
Numerator = non-fecal counts; denominator = fecal counts.
b MPN procedure used when samples were turbid.
LA = lab accident. Samples leaked out during transport.
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TABLE 8.
FECAL AND NON-FECAL COLIFORM
OBTAINED FROM THE JAY
COUNTS FROM WATER SAMPLES
ARC3, 1979
Sample Source
1-10
2-12
3-7
5-2
7-10
8-7
Northeast well
0
0
0
0
0
0
Middle well
0
0
0
0
0
0
Southeast well
0
0
0
1
0
2
Sludge pond well 0
0
0
0
0
0
Hill top well
0
0
0
ob/o
0
0
Spring
22
48
8
1
28
24
Pond creek'3
180/170
46/46
3/5
2
-
122
Well w/o pump'3
<2/< 2
75/4
49/<2
8/<2
337/13
>2400/8
3-FCb
-
9/<2
<2/<2
44/5
-
218/23
6-FCb
_ >
2400/23
5/<2
5/<2
535/5
>2400/23
a Membrane filtration procedure used for analysis. Fecal counts only.
b MPN procedure used when samples were turbid. Numerator = non-fecal
counts; denominator = fecal counts.
181
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Date
TABLE 9. FECAL AND TOTAL
Sludge Pond
COLI FORMS IN SLUDGE
Pensacola
SAMPLES FROM JAY ARC
Montclair
W. F1a. Util.
10/05/76
14,000b
28,000b
160,000b
-
11/02/76
400/0
154,000/11,200
78,000/26,000
20,300/12,000
12/07/76
18,000/10,000
54,000/40,000
240,000/ -
160,000/140,000
01/04/77
165,500b
3,500b
32,500b
50,000b
02/02/77
250,000/150,000
450,000/300,000
3,900,000/3,600,000
-
03/02/77
49,000/18,000
176,000/58,000
240,000/195,000
-
04/05/77
410,000/34,000
86,500/24,000
1,260,000/380,000
-
05/03/77
730,000/420,000
126,000/24,000
-
-
06/07/77
2,000/2,000
170,000/130,000
-
-
07/06/77
5,000/5,000
-
280,000/280,000
-
08/03/77
-
-
-
-
09/07/77
-
-
-
-
10/05/77
17,000/11,000
32,500/14,000
33,000/17,000
-
11/02/77
35,000/-
18,000/ -
44,000/ -
-
12/07/77
46,500/41,000
41,000/34,000
54,000/50,000
-
-------
TABLE 9. Continued
Date
SIudge Pond
Pensacola
Montclair
W. Fla. Util.
01/06/78
8,000/7,000
70,000/26,000
79,000/13,000
	
02/10/78
-
-
33,000/16,300
	
04/03/78
3,500/2,200
-
5,400/1,600
	
05/03/78
140/40
16,000/5,400
>24,000/170
	
06/05/78
130/130
110/<20
16,000/16,000
	
07/10/78
170/<20
-/<20
330/130
	
08/09/78
490/20
230/20
>24,000/>24,000
	
09/11/78
790/320
1,600/920
-
	
10/04/78
22/0
>2,400/920
> 2,400/>2,400
	
11/01/78
20/20
9,200/1,100
>24,000/>24,000
	
12/06/78
>24,000/>24,000
2,564/20
>24,000/>24,000
	
01/10/79
>24,000/540
>24,000/>24,000
>24,000/>24,000
	
02/12/79
9,200/2,400
16,000/1,100
>24,000/>24,000
	
03/07/79
24,000/790
>24,000/>24,000
>24,000/>24,000
	
04/09/79
2,200/1,100
20/20
16,000/5,400
	
-------
TABLE 9.
Date
Continued
Sludge Pond
Pensacola
Montclai r
W. Fla. Util.
05/03/79
160,000/54,000
54,000/13,000
-
	
06/05/79
-
-
-
	
07/10/79
>24,000/9,200
>24,000/>24,000
>24,000/>24,000
	
08/07/79
>24,000/9,200
16,000/16,000
>2,400/70
	
a MPN assays per 100 ml. Numerator = total coliforms; denominator = fecal coliforms.
k Total coliforms only.
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TABLE 10. PRELIMINARY FEEDING TRIALS INITIATED JANUARY, 1976
JAY AND LIVE OAK, FLORIDA
Code
Wet/Dry
Abbreviated Bacterial Analysis
MPN E. coli!gram	
2-12-76
3-2-76 4-8-76
5-13-76
Summary
Forage
55E R-l
51E R-2
52E R-l
61W R-2
52W R-l
55W R-2
Feed
1-29-76
2-20-76
1-29-76
2-16-76
2-11-76
2-20-76
2-26-76
Forage
55-W
55-E
52-W
52-E
61-W
61-E
0
0
0
1,600
0
0
0
The most common organisms encoun-
tered in feed or forage were
Enterobactev (several species),
Psuedomonas, and Citrobaeter
freundi-L
No animal pathogens identified on
1-2-76, 2-12-76, 3-2-76, 4-8-76,
or 5-13-76*
-------
TABLE 10. Continued
Code	Wet/Dry 2-12-76 3-2-76
SIudge
1-2-76
Wet
ND
2-6-76
Dry
2,400
2-6-76
Wet
11,000
4-8-76
Wet


Wet


Wet


Dry

5-13-76 Fresh/Wet
Fresh/Dry
Soil-Jay
55E R-l
61E R-2
52E R-l	2,400
61W R-2
52W R-l	1,100
55W R-2
4-8-76 5-13-76
Summary
Organisms most frequently encoun-
tered in sludge were Enterobaoter
(several species), Escherichia
24,000	aoli, and Proteus (several species)
16,000
16,000	* No animal pathogens were iden-
5,400	tified (at above periods)
24,000
16,000
600 The most common non-lactose fer-
282 menting organisms encountered in
460	- soil were Proteus and Enterobaoter.
285 E. ooli counts are total coliforms
3,500 6,000 and not fecal coliforms. They
5,400 2,300 could be from the sludge or
naturally occurring.
* No animal pathogens were identi-
fied (at above periods)
-------
FATE OF VIRUSES FOLLOWING APPLICATION OF MUNICIPAL SLUDGE TO LAND
G. Bitton, S. R. Farrah, A. R. Overman,
G. E. Gifford, 0. C. Pancorbo, and J. M. Charles
INTRODUCTION
In the U.S., the Water Pollution Control Act of 1972 (PL 92-500), as
recently amended, requires acceptable methods for the utilization and dis-
posal of sludge. It now appears that land disposal of sewage effluents
and residuals is a viable and attractive alternative (Smith and Bryan, 1975;
Sopper and Kardos, 1973; Thabaraj, 1975; Wright, 1975). Land spreading of
wastewater effluents and residuals has many advantages, including the
addition of plant nutrients, water conservation, improvement of soil
physical properties, and increased soil organic matter. However, concern
was raised over the contamination of surface and groundwater with microbial
pathogens, particularly viruses (Bitton, 1975; Bitton, 1980; Burge and March,
1978; Cliver, 1976; Foster and Engelbrecht, 1973; Gerba et al., 1975;
Sagik and Sorber, 1978).
Viruses are generally associated with wastewater solids (Cliver, 1976;
Duff, 1970; Lund, 1974; Moore et al., 1976; Ward and Ashley, 1976; Wellings
et al., 1977) and are merely transferred to sludge as a result of waste-
water treatment operations. Sludge treatment processes, such as anaerobic
digestion, do not completely remove viruses (Bertucci et al., 1977; Lund,
1974; Moore et al., 1976).
Therefore, the application of anaerobically digested sludge onto land
may lead to groundwater contamination as a result of virus transport through
the soil matrix. The movement of sludge-associated viruses is probably
limited due to the immobilization of sludge particles which have not become
associated with the sludge solids or which dissociate from the solids as
a result of changes in the physico-chemical properties within the soil
matrix. The survival and transport of these infectious particles is of
major concern in sludge application to land.
This work is divided into 4 phases. In the first phase we undertook
laboratory experiments to gain some understanding of transport of sludge-
associated viruses through Florida soils. In the second phase of the pro-
ject, we evaluated the methodology for virus detection in soils and sludges -
a new method was developed for virus detection in soils. The selected
methods were used in phases 3 and 4. In the third phase we were concerned
with the survival and transport of enteroviruses through soil cores exposed
187
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to natural conditions. The major environmental parameters (soil temperature,
rainfall, soil moisture) were monitored at the experimental site. Finally,
the fourth phase involved the monitoring of sludges, soils, and groundwater
at 2 sludge disposal sites in Florida.
MATERIALS AND METHODS
Viruses and Viral Assays
Poliovirus type 1 (Sabin) and echovirus type 1 (Farouk) were used in
this phase of the project. Stocks were prepared as previously described
(Bitton et al., 1976). The viruses were kept at -70°C until used.
The viruses were concentrated prior to use in the soil column experi-
ments by either ultracentrifugation or by the method developed by Farrah
et al. (1978) which involved blending with trichlorotrifluoroethane (Freon
113, DuPont DeNemouns Co., Wilmington, DL) followed by concentration on
Filterite filters.
Assay was by the plaque technique on AV3 (human amnion) or MA104
(simian kidney) cell monolayers grown in Eagle's minimal essential medium
(MEM) supplemented with 10% fetal calf serum (FCS), 250 units (U) of peni-
cillin per ml, and 125 yg of streptomycin per ml. Each experiment was
assayed completely on only one cell line (AV3 or MA104) using the proce-
dure previously described (Bitton et al., 1976). If necessary, samples
were diluted prior to assay in phosphate-buffered saline (PBS) containing
2% FCS, 250 U of penicillin per ml, and 125 yg of streptomycin per ml.
The numbers of viruses were expressed as plaque-forming units (PFU).
Virus Transport Through Soils: Laboratory Experiments
The procedures described by Bitton et al. (1978) have been presented
at the International Water Pollution Conference in Stockholm, Sweden.
Development of Methodology for Virus Recovery from Soils and Sludges
Virus recovery from soils—The procedures have been described in
detail by Bitton et al. (1979).
Virus recovery from sludges--A modified version of the procedure of
Hurst et al. (2978) was used for virus recovery from sludges. This method
is described elsewhere (see "Virus monitoring in sludge application sites").
Survival and Transport of Viruses Following Sludge Application to Columns
Exposed to Natural Conditions
The sludge used was a lagooned sludge sampled at the West Florida Agri-
cultural Experiment Station (Jay, Florida). This sludge was a mixture of
aerobically digested sludge (1/3) and anaerobically digested sludge (2/3)
from the Montclair and Main Street wastewater treatment plants of Pensacola,
Florida, respectively. The mixture was kept in a lagoon at the West Florida
188
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Agricultural Experiment Station before ultimately being spread on land.
It was the lagooned sludge which was sampled and used in this study.
Sludge from the Main Street, Kanapaha site, wastewater treatment
plant of Gainesville, Florida, was also used in one experiment. The sludge
used was aerobically digested for 180 days.
The sludges were collected in sterile Nalgene bottles, transported
to the University of Florida (Gainesville) laboratory and then immediately
refrigerated. At the time of use, a sludge sample was first allowed to
come to room temperature. The pH and solid content of the sludge was then
determined. The pH was measured using a Beckman Expandomatic SS-2 pH meter.
The solids content was determined by drying a measured volume of sludge
in an oven at 105°C for 24 hours and expressed as a percentage on a weight
(grams) to volume (milliliters) basis.
The soil used was a Eustis fine sand sampled at the agronomy farm,
University of Florida, Gainesville. This was classified as a Psammentic
Paleudult, sandy siliceous, hyperthermic soil (Calhoun et at., 1974).
Undisturbed cores of this soil were obtained by driving polyvinyl
chloride pipes (40 cm in length and 5 cm or 15.5 cm inside diameter) 33 cm
into the soil; thereby consisting of the Ap and A21 soil horizons. The
soil cores were placed outside the Environmental Engineering Sciences
building at the University of Florida under natural conditions. The soil
cores rested on a wooden box such that soil leachates following rainfall
could be collected (see Figure 1). The large soil cores were insulated
by surrounding them with duct insulation. Porous ceramic cups were
installed at the bottom of the large cores in order to facilitate sampling
of the soil water during periods of low rainfall. These cups were evaluated
for their retentive capacity toward viruses as described below.
The soil temperature, soil moisture, and rainfall were monitored.
The soil temperature was monitored every hour using thermocouples placed
at the soil surface and at depths of 2.5, 10, and 20 cm on one of the
large soil cores. The thermocouples were connected to an Esterline Angus
Key Programmable Data Acquisition System (model PD-2064, Esterline Angus
Instrument Corporation, Indianapolis, IN) which printed voltage (milli-
volts) at each thermocouple every hour. The voltages measured were later
converted to temperature readings with the use of a computer. The soil
moisture was determined by drying (a measured weight of wet soil from the
top inch of the soil cores) in an oven at 105°C for 24 hours and was
expressed as a percentage. The rainfall was measured next to the soil
columns with a farm rain gage (number 510, Science Associates Inc.,
Princeton, NJ) attached to the wooden box. The rainfall was measured
after each rain event in centimeters.
Poliovirus type 1 (Sabin) or echovirus type 1 (Farouk) was seeded
in liquid sludge and mixed for 10 minutes to 1 hour using a magnetic
stirrer. The sludge was assayed for virus directly (i.e., without solids
separation). The supernatant following centrifugation (12,000 rpm or
20,842 x g for 30 min at 4°C) was also assayed. This allowed
189
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the determination of the fraction of viruses associated with sludge solids
before the application of the sludge to the soil columns. Following the
adsorption period and the initial viral assays, 2.5 cm of the virus-seeded
sludge was applied to the soil columns. Sludge seeded with echovirus was
applied to 2 large soil cores (15.5 cm inside diameter). The sludge,
seeded with poliovirus, was applied similarly to a separate set of soil
cores. The seeded-sludge was allowed to soak in and dry on top of the
soil for 3 to 4 days before being mixed with the top 2.5 cm of soil. Virus
monitoring of the drying sludge solids on top of the soil was undertaken as
described below. The soil was also monitored for viruses after the sludge
was worked under 2.5 cm. The top 2.5 cm of soil was monitored using the
methods described elsewhere.
Virus Monitoring in Sludge Application Sites
Virus recovery from sludge—A modified version of the procedure of
Hurst et at. (1978) was used to recover viruses from sludge. The modified
version was described in detail by Pancorbo et al. (Canadian Journal of
Microbiology, in press).
Lagoon water--Four liters of water overlying the lagooned sludge was
adjusted to pH 2.5 by addition of 1 M, pH 2 glycine and centrifuged at
15,000 x g for 10 min. The supernatant was passed through a series of 2
0.45 ym Filterite filters in 47 mm holders. Seven ml of PBS + 10% FCS,
pH 9, was passed through the filters, neutralized and saved as the final
sample. The pellet remaining after centrifugation was mixed with an equal
volume of PBS + 10% FCS, pH 9, readjusted to pH 9 by addition of 1 M,
pH 11.5 glycine, and centrifuged at 15,000 x g for 10 min. The supernatant
was removed and neutralized by addition of 0.05 M, pH 2 glycine before
assay.
Virus concentration procedure for well water—Water from two wells
was hand pumped into 100 gallon tanks. The water was adjusted to pH
3.5 by addition of 0.2 N HC1 and adjusted to 0.0005 M aluminum chloride.
Water from the other wells was removed using deep-well pumps, added to
100 gallon tanks and adjusted as described above or was adjusted to pH
3.5 and 0.0005 M aluminum chloride by in-line injection of acid and salts
(Gerba et at., 1978). The treated water was passed through a 10 inch,
0.45 ym porosity Filterite filter (Filterite Corp., Timonium, MD). The
filters were then treated with 800 ml of 0.05 M glycine, pH 11.5. The
glycine solution was permitted to remain in contact with the filters for
one minute, removed, and neutralized by addition of 1 M, pH 2 glycine.
The neutralized samples were stored on ice for 24 to 48 hours during
transportation to the laboratory. The samples were then adjusted to pH
3.5 by addition of 1 M pH 2 glycine and centrifuged at 800 x g for 10 min.
The supernatants were passed through a series of 2 0.45 ym Filterite filters
in 47 mm holders. Next, 7 ml of 0.05 M glycine + 2% FCS was passed through
the filters, neutralized, and used as the inoculum for cell cultures.
The sediment remaining after centrifugation at pH 3.5 was mixed with 5
volumes of phosphate buffered saline (PBS) with 10% FCS at pH 9, readjusted
190
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to pH 9 by addition of 1 M, pH 11.5 glycine, if necessary. After centri-
fugation at 15,000 xg for 10 minutes, the supernatant was removed,
neutralized, and used for inoculation of cell cultures.
Virus recovery from soils--The methods of Hurst and Gerba (1979)
and of Bitton et al. (1979) were used to recover virus from soils at the
Kanapaha site and Jay site, respectively.
Weather data for the Jay site--Weather data were supplied by the
West Florida Agricultural Experiment Station at Jay. Mean monthly air
temperatures (maximum and minimum) and total monthly precipitation from
September 1977 through March 1979 are shown in Figure 2.
Cell cultures and viral assays--BGM (Baron et al., 1970) or primary
monkey kidney cells (Flow Laboratories, Inc., McLean, VA) were used for
isolation of viruses. Serial dilutions of samples were made in PBS +
2% FCS and used to inoculate cell cultures. The cells were examined for
cytopathic effects (CPE) for up to 3 weeks. Samples showing CPE were
frozen, thawed, diluted 1/100 to 1/10,000 and used to reinoculate cell
cultures. Samples that produced CPE on the second passage were frozen.
The titer of these isolates was determined using MA-104 cells with a
methyl-cellulose overlay. Neutralization with specific antisera was used
to identify isolates (Lim and Benyesh-Melnick, 1960). Tissue-culture
infective dose (TCID50) was determined according to the method of Reed
and Muench (1938).
Illtracentrifugation—Samp!es with excessive final volumes were centri-
fuged at 120,000x g for 90 minutes in a TI-60 rotor using a Beckman Model
L3-50 ultracentrifuge (Beckman Instruments, Fullerton, CA). The pellets
were suspended in FCS and assayed.
RESULTS AND DISCUSSION
Virus Transport Through Soils: Laboratory Experiments
Prior to conducting field experiments, it appeared necessary to study
the pattern of virus transport through the soil under consideration, Red
Bay sandy loam. This soil has been sampled at the Jay site. Laboratory
packed soil columns and undisturbed soil cores were employed. The results
showed that the soil under study retained more than 99% of poliovirus type
1 following sludge application. The mechanisms involved in the transport
of viruses in sludge-soil systems are discussed by Bitton et al. (1978)
and the results have been presented at the International Water Pollution
Conference in Stockholm, Sweden.
Development of Methodology for Virus Recovery from Soils and Sludges
Virus recovery from soils--Elution of poliovirus type 1 from Eustis
fine sand--The efficiency of virus elution from a sandy soil was examined
for 3% beef extract, tryptose phosphate broth, 10% FCS in PBS, a muck
solution, 0.25 M glycine in combination with 0.05 M EDTA, 0.25 M glycine,
0.2% purified casein, 0.5% isoelectric casein, and 0.5% suspension of non-
fat dry milk.
191
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Elution of poliovirus from soil ranged from 15% with the muck solution
to more than 100% with 0.5% isoelectric casein at pH 9. Glycine-EDTA, at
a pH of 11.5 (the pH dropped to 11 upon mixing with soil), displayed 61%
elution and similar efficiency was achieved with 0.25 M glycine alone (55%).
The elution efficiency was significantly higher with purified casein, iso-
electric casein, and non-fat dry milk than with glycine-EDTA or beef
extract. Further concentration of soil eluates can be achieved with 7 out
of 9 eluents examined. Four eluents (beef extract, glycine-EDTA, iso-
electric casein, and non-fat dry milk) were further examined for the
recovery efficiency of the concentration step.
The overall recovery (elution followed by a concentration step) of
poliovirus type 1 (Sabin) from soil was examined with regards to beef
extract, 0.25 M glycine + 0.05 M EDTA, 0.5% isoelectric casein, and 0.5%
non-fat dry milk. The elution was performed at pH 9 for casein, non-fat
dry milk, and beef extract, and at pH 11 for glycine-EDTA. The results are
shown in Table 1. The best eluents were 0.5% isoelectric casein and 0.5%
non-fat dry milk. However, beef extract performed best during the concen-
tration step (organic flocculation). The overall recovery of poliovirus
was 38%, 41%, 60%, and 66% with beef extract, glycine-EDTA, isoelectric
casein, and non-fat dry milk, respectively. Analysis of variance did not
show any significant difference at the 0.05 level, among the 4 methods
for overall virus recovery. Isoelectric casein was selected to further
study the recovery of poliovirus type 1 from 4 soil types as well as that
of other enteroviruses. It was found that the recovery of poliovirus
varied from 45% to 52% for the 4 soils under study (Table 2). Analysis
of variance did not show any significant difference between the 4 soil
types.
Overall recovery of poliovirus 1, coxsackievirus B3, and echovirus 4
by the casein method is displayed in Table 3. It was shown that virus
elution was generally high and ranged from 73 to more than 100%. Echo-
virus 4 was, however, not well recovered during the concentration step
and the overall recovery was only 23%. With coxsackievirus B3, the over-
all recovery was significantly higher (at the 0.05 level) than poliovirus
type 1 or echovirus 4.
Four methods have been described for virus recovery from Eustis fine
sand, which is representative of soils found in Florida. An ideal method
should include an efficient elution step followed by a concentration step
which would aid in the detection of low numbers of viruses in relatively
large amounts (100-200 g) of soil. Regarding the elution step, the purpose
was to select an eluent that would operate efficiently at pH 9 and that
would be easily amenable to further concentration. Among 9 eluents tested,
0.5% isoelectric casein and 0.5% non-fat dry milk were the most efficient
ones in desorbing viruses from soil. These eluents operate at pH 9 and
thus avoid the potential harmful effect of high pHs (pH 11-11.5) toward
enteroviruses. Exposure over 10 min to glycine buffer at pH 11.5 may
also be harmful to poliovirus type 1 (Gerba et al., 1977). Another
advantage of these eluents was allowing further concentration of viruses
by organic flocculation. This step was similar to that observed with
beef extract (Katzenelson et al., 1976) except that flocculation occurs
192
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at pH 4.5 (Jenness and Patton, 1959] instead of pH 3.5. With regard to
overall virus recovery, no significant differences were shown between
beef extract, isoelectric casein, non-fat dry milk, and glycine-EDTA
methods.
The isoelectric method was able to recover high (4.5 x 106 - 9.3 x 106)
and low (2 x 102) inputs of viruses from 100 g of soil with efficiencies of
50% and 75%, respectively. These recovery efficienciedare similar to
those reported by Gerba et at. (1977) with respect to virus recovery from
estuarine sediments. These investigators have used 0.25 M glycine -
0.05 M EDTA (pH 11) as eluting solution and reported a mean recovery of
50%. Glycine-EDTA has been shown to be effective for recovering polio-
virus 4 as well as poliovirus 1 or coxsackievirus B3. It appears (Table 3)
that echovirus 4 was efficiently eluted from soil particles (75% elution
efficiency) but it was not well recovered (30%) during the concentration
step. Research is now being undertaken to improve the concentration step.
It also appears that the recovery efficiency of the casein method is not
significantly affected by soil type and a 50% mean recovery was achieved
with 4 Florida soils.
Methodology for Virus Recovery from Sludges
Virus recovery from sludges was performed according to a modification
of the method developed by Hurst et at. (1978). The use of this method
for mixed liquor and anaerobically digested sludge resulted in recovery
efficiencies similar to those reported by Hurst et at. (1978). However,
the use of this method for aerobically digested sludge resulted in poor
recovery. Therefore, a series of experiments were undertaken to study
the effect of sludge type on virus recovery by the Hurst et at., method.
Virus association with sludge solids was studied, using mixed liquor
and wasted, aerobic and anaerobic sludges (data not shown). It was found
that the percent association of viruses with sludge solids was signifi-
cantly higher (at the 0.01 level using Duncan's test) for wasted and
aerobic sludges than for mixed liquor and anaerobic sludge. In seeded
experiments using poliovirus type 1, virus recovery was much higher for
mixed liquor and anaerobic sludge than for wasted and aerobic sludge
(Table 4). For anaerobically digested sludge, the mean percent recovery
was 60.2% whereas for aerobically digested sludge the mean recovery was
14.5%. The difference in percent recovery was significant at the 0.01
level, using Duncan's test. A relatively high recovery (72.3%) was
obtained, using mixed liquor. This probably is the type of material used
by Hurst et al. (1978) with regard to the development of their method.
These findings are significant since many types of sludge have been
used in this research project; hopefully, a high recovery was achieved
with the lagoon sludge from the Jay site. This particular sludge is
predominantly anaerobic and thus allows a relatively good virus recovery.
It is thus necessary to take in consideration the sludge type when
one contemplates the development of methods for virus recovery from
siudge.
193
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Survival and Transport of Viruses Following Sludge Application to Soil
Columns Exposed to Natural Conditions
In previous sections, virus transport through soils under controlled
laboratory conditions has been described as adequate method for virus
detection in soils and sludges. It thus appeared necessary to study virus
transport and survival under more natural conditions. Undisturbed soil
cores (33 cm long and 15.5 cm inside diameter] were used to assess virus
transport and survival under field conditions. In these experiments, envi-
ronmental parameters (temperature, soil moisture, rainfall) were monitored;
more details are given in the "Materials and Methods" section. The protocol
of sludge disposal to soil was similar to that practiced in sludge disposal
s i tes.
Virus survival and transport were monitored during the summer and fall
season, using the same columns. Survival monitoring was terminated when
viruses were not detectable in soil samples.
Summer season—This period is generally warm and wet in the Gainesville
area. Soil temperature was measured with thermocouples placed at the
surface, 2.5, 10, and 20 cm depth. Data analysis showed that there was no
significant difference between temperature readings at these depths. The
average temperature ranged from 23.5°C to 29°C during the 35-day period.
The study period was very wet with 13.6 cm of rainfall from June 2
to July 7, 1978. Poliovirus survival was monitored in 2 soil cores which
had been treated with seeded sludge (Table 5). There was some decline
in virus numbers in the sludge prior to mixing with the top 2.5 cm of soil.
Soil monitoring revealed that poliovirus could be detected up to 35 days in
both cores. It is difficult to correlate virus survival to soil moisture
since this parameter was not continuously monitored. Heavy rainfall did
not allow the soil to dry for an extended period of time and this probably
has led to prolonged virus survival.
Monitoring of soil leachates from June 5 to August 24, 1978, did not
reveal any virus, despite their concentration by membrane filtration (Table
6). Although 51 cm of rain fell during the study period, this represented
only 0.5 to 0.7 pore volume. It appears that viruses are efficiently
retained by soils under unsaturated flow conditions.
Fall season--S1udge application was undertaken again on October 11,
1979, and virus presence in the soil and leachates was monitored for 21
and 101 days, respectively.
It became apparent that with regard to transport pattern, poliovirus
type 1 (Sabin) would not be the ideal model virus since it has a high
affinity for sludge solids and is subsequently immobilized at the top
layer of soil. A virus with less affinity for sludge solids would be more
suitable for transport studies. In other studies, echovirus 1 (Farouk)
was found to be less adsorbed to soil than poliovirus type 1 (Sabin). The
association between lagooned sludge solids (sludge used at the sludge
disposal site in Jay, Florida) and poliovirus type 1 and echovirus was
194
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therefore studied (Table 7). It was found that echovirus 1 was	less ad-
sorbed (21.7%) to sludge solids than poliovirus type 1 (95.2%). Lagooned
sludge from Jay was seeded with either of these 2 enteroviruses	and then
applied to the soil cores.
During the study period the average temperature, monitored with thermo-
couples placed in soil cores, ranged from 18°C to 27 C. Only 0.13 cm of
rain fell from October 11, 1978 to November 1, 1978. This was the period
during which virus survival was monitored. Neither virus could be detected
in soil after 21 days of incubation under natural conditions. The 2 entero-
viruses were completely inactivated sometime between day 8 and 21 (Table 8).
Soil leachates were also monitored and a summary of the data is
displayed in Table 9. Neither poliovirus nor echovirus was detected in
the leachates from all the soil cores (Table 9).
It appears from these studies that, under conditions prevailing in
North Central Florida, enteroviruses are rapidly inactivated during sludge
application to soils. Their inactivation in the soil appears to be affected
more by desiccation than by soil temperature. Under ideal conditions
(warm and dry), a rapid decline of virus was observed in the sludge drying
on top of the soil. Soil leachates collected after natural rainfall were
negative for both poliovirus type 1 (Sabin) and echovirus 1 (Farouk).
Virus Monitoring at Two Sludge Disposal Sites
Two sludge disposal sites were selected for virus monitoring: Kanapaha
near Gainesville, Florida, and Jay site near Pensacola, Florida. The moni-
toring involved attempts to recover viruses from groundwater, sludge, and
soil samples from the disposal sites.
Virus monitoring at the Kanapaha sludge disposal site—During 3 con-
secutive months (December, 1977; January, 1978; and February, 1978), the
City of Gainesville sludge disposal site (10 acres) adjacent to Lake
Kanapaha, Florida, was monitored for viruses. Aerobically digested sludge
(180 days detention time) conditioned with a cationic polymer and dewatered
by centrifugation was applied to this site and immediately incorporated
into the soil (Figure 3). The groundwater, soil, and centrifuged sludge
was monitored for indigenous viruses. The centrifuged sludge applied at
the time this study was performed originated at the Main Street treatment
plant of Gainesville. Since the conclusion of our study, the sludge from
the Kanapaha treatment plant has also been disposed of at this site. The
sludge is transported to the site in tank trucks and is spread out onto
the soil. The sludge is disked into the soil as soon as it is applied
except when a cover crop is present. In the presence of a cover crop, the
sludge is applied as a top dressing on the crop. Coastal bermudagrass is
utilized during the summer months while ryegrass is in the winter months
(Gainesville-Alachua County Regional Utilities, 1976). The monitoring wells
located throughout the sludge disposal site are 60 feet in depth. The
groundwater flows in a northwesterly direction as shown in Figure 4. The
soil found at the site belongs to the Lochloosa series (Gainesville-Alachua
County Regional Utilities, 1976). This series is a member of the loamy,
195
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siliceous, hyperthermic family of Aquic Arenic Paleudults. These soils
have thick sandy A horizons; mottled light yellowish brown B1 horizons;
yellowish brown B21t horizons; and mottled gray B22tg and B3g horizons.
The typifying pedon is Lochloosa fine sand. The methodology for virus
recovery from well water, sludge, and soil is described in the "Materials
and Methods" section.
The results obtained are shown in Table 10. Enteroviruses were
recovered from waste sludge but were inactivated almost to undetectable
levels after 90 days detention in an aerobic digestor. The sludge ulti-
mately disposed of at the Kanapaha site was aerobically digested for 180
days, conditioned with a cationic polymer and dewatered by centrifugation.
Enteroviruses could not be recovered from the centrifuged sludge. Thus,
it is understandable that no viruses were detected in the soil and ground-
water at the Kanapaha site. By increasing the detention time of the sludge
at the wastewater treatment plant, the viral hazards of sludge disposal
on land were probably eliminated. Therefore, our efforts were concentrated
on virus monitoring at the Jay site.
Agricultural Research Center at Jay, Florida—Sludge originating
from Pensacola, Florida, has been applied for some years on 10 acres at
the IFAS Agricultural Research Center in Jay, Florida. The sludge con-
tained 3-4% solids and was lagooned prior to land application at a rate
of	. A virus monitoring program has been undertaken
to assess the virus load of sludge, soil, and groundwater at the Jay site.
Moreover, we investigated the survival of indigenous viruses in the sludge
lagoon.
a.	Sludge monitoring—The sludge used in Jay originated from 3
wastewater treatment plants, located in Pensacola, Florida. Two-thirds
of the sludge was anaerobically digested (Main Street and Northeast
plants) and one-third was aerobically digested (Montclair plant). Despite
the poor performance of the Hurst et dL , method with aerobic sludge,
the virus load of aerobically digested sludge was always higher than that
of anaerobically digested sludge (Table 11). These findings raise concern
over the efficiency of aerobic digestion with regard to virus destruction.
b.	Survival of enteroviruses in the sludge lagoon--The sludges were
transported by tank trucks from Pensacola to a sludge lagoon located at
the Jay site. The lagoon was approximately 60 x 100 ft, containing liquid
sludge to a depth of approximately 6 ft, and contained approximately
1 million gallons of liquid sludge. Sludge addition to and removal from
the lagoon were carried out at the opposite ends of the lagoon. At certain
stages of crop growth, land applications of sludge as well as the addition
of digested sludges to the lagoon were suspended. This practice provided
a unique opportunity to study the fate of indigenous enteroviruses in
the sludge lagoon. Enteroviruses were readily recovered from the sludge
(15 and 80 TCID 50/g of sludge) during periods when digested sludge was
added to the lagoon. The addition of fresh digested sludge to the lagoon
was suspended on April 14, 1978. For the next 6 months, viruses were
196
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found in very low numbers or were undetected in samples of lagooned sludge.
They were again detected when the addition of digested sludge to the lagoon
was resumed on November 7, 1978 (Figure 6).
In order to determine if viruses were being eluted from the lagooned
sludge solids and then transferred to the overlying water, samples of
lagooned water were obtained while sludge addition to the lagoon was sus-
pended and after it was resumed. Enteroviruses were found in the water
when sludge was being added to the lagoon and were not found when sludge
addition was suspended. The lagooned water had a large number of algae
as shown by the concentration of chlorophyll a (Table 12).
Polioviruses (type 1, 2, and 3), coxsackie B viruses (B4), and echo-
viruses (type 1, 7, and 15) were isolated from sludge and overlying water
samples taken from the lagoon (Table 13). A variation in the relative
number of isolates was observed. Polioviruses represented greater than
90% of the isolates obtained from samples collected from 2-17 to 11-6-78.
Polio-1 was the most common serotype isolated from these samples. From
12-6-78 to 1-24-79, poliovirus serotypes represented less than 50% of the
isolates obtained while greater numbers of echo and coxsackie B viruses
were recovered (Table 13).
These data (Figure 6) show that sludge lagooning may be an efficient
means of reducing viral numbers prior to land application under the warm
climate of Florida. No information could be found in the literature with
regard to virus survival in sludge lagoons.
Indigenous virus survival in lagoon sludge freshly applied to land
was monitored on October 3, 1979. Virus was detected in sludge; using 2
methods, glycine and lysine. Table 14 shows that enteroviruses could be
detected for up to 9 days in sludge prior to mixing with soil.
c. Groundwater monitoring—Monitoring wells were dug in the study
site and their position is shown in figure 7. Their depth was 70 to 80
feet. Two other wells (not shown in figure 7) were dug at 10 ft from the
sludge lagoon. All the wells were monitored at bimonthly intervals for
a period of one year. The results have been summarized in Table 15. No
virus has been detected in any of the groundwater samples.
Previous studies have dealt with virus transport through soils treated
with sewage effluents (Bitton et al., 1976; Bitton et al., 1979; Duboise
et al., 1976; Gilbert et al., 1976; Goyal and Gerba, 1979; Lance and Gerba,
1977; Lance et al., 1976; Landry et al., 1979; Schaub and Sorber, 1977;
Scheuerman et al., 1979). However, few studies have dealt with virus trans-
port in sludge-amended soils (Damgaard-Larsen et al., 1977; Moore et al.,
1978). Damgaard-Larsen et al., used lysimeters to study virus transport
in sludge-amended soils. Viruses were completely retained by the soils
under study. Moore et al. (1978) investigated virus survival in sludge-
amended soils but no groundwater monitoring was reported.
197
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d. Soil monitoring—Following storage in the lagoon, the sludge was
ultimately spread on land. The sludge was allowed to dry on top of the
soil for 2 to 14 days, and then mixed with the soil. The field used for
sludge disposal (Figure 7) was divided into 72 plots of 40 x 120 ft,
which received from 0 to 15 acre-inches of sludge per year. Plots numbered
1, 32, and 61 which received 15 acre-inches of sludge per year were moni-
tored for viruses from June, 1978 through January, 1979. In addition,
the plot numbered 42, which received no sludge, was monitored as a control.
We have shown that enteroviruses were readily recovered from sludges,
including lagooned sludge (Table 13). However, no virus could be detected
in soil samples at the Jay site. Allowing the sludge to dry on top of the
soil before being mixed with the soil results in the inactivation of all
or most of the enteroviruses present. This may be an advantage over sludge
injection into soils (Moore et al., 1978), where viruses can survive for
longer periods of time. Despite the numerous advantages of sludge injec-
tion (aesthetic acceptability, odor and runoff are minimized), surface
spreading of sludge may result in higher inactivation of viruses.
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TABLE 1. OVERALL RECOVERY OF POLIOVIRUS TYPE 1 (SABIN)
FROM 10 G OF SOIL (EUSTIS FINE SAND), USING 4 DIFFERENT METHODS
Recovery method
Total virus
input (PFU)1
Percent
elution2
Step3 Cone.
% Recovery
Overal1
recovery
percent
Mean overall
recovery
[% + SE)
3% Beef Extract
5.0 x 105
41
95
39
37.7 + 6.54
(pH = 9.0)

52
56
29



52
86
45

0.25 M Glycine
3.8 x 105
77
61
47
41.0 ± 8
+ 0.05 M EDTA

48
72
35

(pH = 11.0)





0.5% isoelectric
4.7 x 105
128
40
51
59.5 + 8
casein (pH = 9.0)

123
55
68

0.5% non-fat
6.8 x 10s
98
80
78
66.0 + 8
dry milk

92
59
54

(pH = 9.0)





1	Ten pore volumes of autoclaved secondary sewage effluent seeded with virus and passed through
10 g soil columns. Two pore volumes of non-seeded secondary sewage effluent passed through
the columns to leach out non-associated viruses. Soil leachates assayed to determine viruses
retained by 10 g of soil.
2	30 ml of eluents added to 10 g soil samples. Samples vortexed for 30 sec, shaken for 15 min
(in the presence of glycine or glycine-EDTA, the shaking time was 4.5 min), centrifuged at
4000 rpm for 4 min. Supernatants decanted and 5 ml of aliquot of supernatants brought to pH =
7.0-7.5 with 1 M glycine (pH = 1.3-1.8). Samples made isotonic and sometimes diluted in MEM
prior to assay.
-------
TABLE 1. Continued
Eluates concentrated by organic flocculation at pH 3.5 for beef extract; 4.5 for isoelectric
casein and non-fat dry milk. Glycine-EDTA eluates were flocculated at pH 3.5 with 0.06 M
A1C1 . Floes eluted with fetal calf serum in PBS at pH 9.0. Supernatants concentrated by
membrane filtration.
ANOVA. No significant difference at the 0.05 level among 4 methods.
-------
TABLE 2. RECOVERY OF POLIOVIRUS TYPE 1 (SABIN) FROM
4 SOILS (100 G) BY THE ISOELECTRIC CASEIN METHOD
Input (PFU)1
Percent2
Recovery (% ± SE)3
Eustis Fine Sand
Jay (Top Soil)
Cypress Dome Sand
3.5	x 106
1.6	x 105
49
55
37
60
39
60
46
44
48.5 + 8
52.0 + 8
7.0 x 10*
49.5 + 8
Red Bay Sandy Loan
1.5 x 105
45.0 + 8
1	Ten pore volumes secondary sewage effluent seeded with virus passed
through 100 g soil columns; 2 pore volumes sterile secondary sewage
effluent passed to ensure adsorption. Leachates assayed to determine
amount of virus retained by soil.
2	0.5% isoelectric casein (200 ml) pH 9.0 added to soil samples. Samples
were shaken 15 min, centrifuged at 10,000 rpm for 4 min. Supernatant
was decanted and centrifuged at 10,000 rpm for 4 min, brought to pH =
4.5 with 1 M glycine (pH = 2.0). Resulting floe centrifuged at
2,000 rpm for 2 min. Supernatant decanted and remaining pellet was
resuspended in 0.15 M NaaHPOi,. at pH = 9.0.
3	ANOVA. No significant difference at the 0.05 level among the 4 soils.
204
-------
TABLE 3. RECOVERY OF 3 ENTEROVIRUSES FROM SOIL (EUSTIS FINE SAND)
WITH 0.5% (W/V) ISOELECTRIC CASEIN AT pH 9.0
Virus input Overall Mean overall
PFU/10 g % Step3 Cone Recovery recovery
Virus	Soil1 Elution2 % Recovery	%	(% + SE)
Poliovirus 1
4.7
X
105
128
40
51
59.5
+
5.5b4




123
55
68



Coxsackie B3
1.6
X
105
125
74
92
88
+
5.5a




111
76
81



Echo 4
1.9
X
105
76
32
24
22.5
+
5.5c




73
28
21



1	Ten pore volumes autoclaved secondary sewage effluent seeded with virus
and passed through 10 g soil columns. Two pore volumes of non-seeded
secondary sewage effluent passed through the soil columns to leach out
non-associated viruses. Soil leachates assayed to determine viruses
retained by 10 g of soil.
2	30 ml of the eluents were added to 10 g soil samples. Samples were vor-
texed for 30 sec, shaken for 15 tnin, and centrifuged at 4,000 rpm for
4 min. Supernatants were decanted and 5 ml aliquot of the supernatants
taken and brought to pH 7.0-7.5 with 1 M glycine (pH 1.3-1.8). Samples
were made isotonic and sometimes diluted in MEM prior to assay.
3	Soil eluates concentrated by organic flocculation at pH 4.5. Following
centifugation, floes were resuspended in 0.15 M Na2HP0lf at pH 9.0.
^ Figures followed by similar letters did not differ significantly at the
0.05 level.
205
-------
TABLE 4. EFFECT OF SLUDGE TYPE ON RECOVERY OF POLIOVIRUS FROM SLUDGE


USING
MODIFICATION OF HURST ET AL.
., METHODS



Sludge parameters
Volume
K


Mean virus




of
Vi rus

Overal1
recovery



soli ds
siudge
added

virus
for each

Sludge

content
processed
to sludge
recovery
sludge type
Sludge type
used
pH
(%)

(total
PFU)
(*)
(% ± SE)
Mixed liquor
UML
6.4
1.6
100
8.4 x
10
84.3
72.3 + 12.0a

GML
6.9
0.5
1,000
1.3 x
10
60.3

Wasted
GW
6.2
2.4
1,000
7.4 x
10
8.3
—
Aerobically
UDA
4.8
1.5
500
6.5 x
10
18.9
14.5 + 2.7b
digested
UDA
4.8
1.5
500
6.5 x
10
11.9


UDA
6.1
1.3
500
2.0 x
10
8.0


UDA
6.1
1.3
500
2.0 x
10
5.9


UDA
6.5
1.3
1,000
9.6 x
10
15.5


GDA90
5.8
1.0
1,000
1.3 x
10
14.1


PDA
5.8
2.8
1,000
4.1 x
10
26.9

Anaerobically PDAN
7.2
1.4
100
6.0 x
10
59.9
60.2 + 2.1a
digested
PDAN
6.4
1.9
1,000
5.5 x
10
63.9


LAGe
7.3
2.9
1,000
1.8 x
10
56.7

a Procedure - Hurst et at. (1978) and is described in the "Materials and Methods" section.
k Virus was added to sludge while stirring with magnetic stirrer. Stirring continued for 10 min-
60 min; total sludge volume centrifuged at 1465x g for 20 min at 4°C. Sludge supernatant and
solids generated separately subjected to virus recovery methodology.
Overall virus recovery {%) values determined from viruses recovered in final concentrates (sludge
supernatant and sludge solids) were based on amount of virus (total PFU) added to sludge as 100%.
-------
TABLE 4. Continued
Mean values with different superscripts are significantly different at 0.01 level (ANOVA).
A
Lagoon sludge is mixture of aerobically digested sludge (1/3), anaerobically digested sludge
(2/3); consequently, its properties are close to those of anaerobically digested sludge.
Therefore, lagoon sludges were placed in the population of anaerobically digested sludge.
-------
TABLE 5. SURVIVAL OF POLIOVIRUS TYPE 1 (SABIN), UNDER NATURAL CONDITIONS,
FOLLOWING SUSPENSION IN LIQUID SLUDGE AND SUBSEQUENT APPLICATION
TO LARGE SOIL CORES OF A EUSTIS FINE SAND (June 2, 1978 -
July 7, 1978)



Days after
beginning
Date exper.
Cumulative Sludge
rainfall" solids
(cm) (% w/v)
Soil
moisture
{% w/v)
Number viruses0
(PFU/g dw of
sludge or soil)
Core 1 Core 2
Liquid Sludge Sample



06-02-78 0
0 2.9
_e
5.6 x 107 5.6 x 107
Drying Sludge Samples



06-03-78 1
1.80 23.1
-
2.5 x 106 4.9 x 106
4
6.03
-
1.0 x 106 5.Ox 105
Sludqe mixed with top 2.5 cm of
soil on day 4
Soil samples (top 2.5 cm)



06-06-78 4
6.03
14.7
3.2 x 10" 4.1 x 10*
06-07-78 5
7.03
0.9
8.6 x 10* 7.5 x 10*
06-09-78 7
7.28
9.9
2.5 x 103 2.4 x 103
06-23-78 21
10.45
0.2
4.6 15.1
07-07-78 35
13.63
0.2
1.3 0.9
3
2.5 cm of lagooned	sludge (sampled at West Florida Agricultural Experi-
ment Station, Jay;	sludge had pH of 6.9 and solids content of 2.9%)
seeded with total of 7.8 x 10® PFU poliovirus applied on top of 33 cm
undisturbed, large	soil cores of Eustis fine sand (15.5 cm inside dia-
meter; contains Ap	and A21 horizons). Seeded sludge was allowed to
soak in and dry on	top of soil 4 days before being mixed with top
2.5 cm of soil.
k Rainfall data was collected at the experimental site.
c Virus monitoring of drying sludge undertaken as per "Materials and
Methods" section which used 0.05 M glycine, pH 11.5 as primary eluent,
and no further concentration of eluates. Virus monitoring of soil was
undertaken by isoelectric casein method described in the "Materials
and Methods" section.
208
-------
TABLE 5. Continued
Liquid sludge was seeded with 5.6 x 10 PFU of poliovirus per gram dry
weight of sludge. A large fraction of the viruses seeded (91.5%) were
associated with sludge solids after magnetic stirring for 1 hour and
before application to the soil cores. Soil leachates from all cores
were also monitored for viruses.
e - = not done
209
-------
TABLE 6. ANALYSIS FOR PRESENCE OF POLIOVIRUS TYPE 1 (SABIN) IN SOIL
LEACHATES COLLECTED AFTER NATURAL RAINFALL FROM SOIL CORES
OF EUSTIS FINE SAND WHICH HAD BEEN TREATED WITH 2.5 CM OF
SEEDED LIQUID SLUDGE (June 2, 1978 - August 24, 1978)
Cumulative Soil Cumulative Cumulative number
rainfall core leachate of pore
Dates	(cm)	 number	volume	volumes0 eluted
06-05-78
to
08-24-78
51.05
CI
i,544
(8.2)
0.7
06-05-78
to
08-24-78
51.05
C2
1,135
(6.0)
0.5
Dates
Cumulative
virus
eluted
(total PFU)
Cumulative
percent
total vi rus
applied
Range con-
ductivity
values of
leachates
collected
(umho/cm at 25°C)
Range
pH values
1eachates
collected
06-05-78
to
08-24-78
0
0
1M - 1,200
6.3 - 7.8
06-05-78
to
08-24-78
0
0
106 - 1,360
5.7 - 7.2
2.5 cm of lagooned sludge (sampled at W. Florida Agricultural Experi-
ment Station, Jay; sludge had pH of 6.9 and solids content of 2.9%)
seeded with a total of 7.8 x 108 PFU of poliovirus applied on top of
33 cm undisturbed soil cores of Eustis fine sand (15.5 cm inside dia-
meter; contains Ap and A21 horizons). Seeded sludge was allowed to
soak in and dry on top of soil 4 days before mixing with the top
2.5 cm of soil. Soil cores were exposed to natural conditions.
k Rainfall data collected at experimental site. Rainfall (cm) values
represent total from the beginning of the experiment on 06-02-78.
One pore volume for the large soil cores = 2,178 ml.
Viruses in soil leachates were monitored by concentrating using membrane
filtration, see "Materials and Methods" section.
0
Values in parenthesis represent number of cm of cumulative leachate volume
210
-------
TABLE 7. ASSOCIATION BETWEEN 2 ENTEROVIRUSES AND LAGOONED SLUDGE3 SOLIDS
Vi rus^
used
Virus in
unfractionated
siudge
(total PFU)
Virus in
sludge d
supernatant
(total PFU)
Viable Q
unadsorbed
vi rus
(% Vuv)
Sludge
associated
vi rus
(%)
Poliovirus
type 1
(Sabin)
7.8 x 108
8.8 x 108
6.6 x 107
4.2 x 107
8.5
4.8
91.59
95.2h
Echovirus
type 1
2.9 x 106
2.3 x 106
78.3
21.7h
(Farouk)
a The sludge used was lagooned sludge from the Jay Agricultural Research
Center.
b Virus was added to sludge while magnetic stirring the suspension 1 hr.
c Sludge solids were not separated prior to assaying.
^ Sludge was clarified by centrifugation at 20,842 x gfor 30 min at 4°C;
the supernatant was subsequently assayed.
e The Vuv% values were calculated on the corresponding unfractionated
sludge assay.
f
The "sludge associated virus (%)" values were estimated on the amount of
virus recovered in the corresponding sludge supernatant.
^ The sample of sludge used had a pH of 6.9 and a solids content of 2.9%.
The virus seeded sludge was applied to soil columns.
^ The sample of sludge used had a pH of 7.0 and a solids content of 7.0%.
The virus seeded sludge was applied to soil columns.
211
-------
TABLE 8. SURVIVAL OF POLIOVIRUS TYPE 1 (SABIN) AND ECHOVIRUS TYPE 1 (FAROUK),
UNDER NATURAL CONDITIONS, FOLLOWING SUSPENSION IN LIQUID SLUDGE
AND SUBSEQUENT APPLICATION TO LARGE SOIL CORES OF A EUSTIS FINE SAND3
(October 11, 1978 - November 1, 1978)
Date
Days
after the
beginning
of
experiment
Number of viruses
Cumulative Sludge Soil (PFU/g dry weight of sludge or soil)
rainfall solids moisture Echovirus	Poliovirus
(cm)	(%, w/v) [%, w/v) Core 1	Core 2 Core 3 Core 4
Liquid sludge sample
10-11-78d 0
Dry sludge sample
10-14-78 3
Soil samples (top 2.5 cm)
10-14-78	3
10-16-78	5
10-19-78	8
11-01-78	21
7.0
38.0
8.6x10* 8.6x10* 2.6 x 107 2.6x 10'
1.6x10" 1.3x10* 1.3x 106 2.9 x 106
Sludge mixed with top 2.5 cm of soil on day 3
0
0
0
0.13
7.5	2.8x 102	1.9 x 102	4.3x 10*
3.0	6.9 x 101	1.6 x 101	3.5 x 103
1.0	5.6 x 101	6.3 x 101	3.6 x 103
1.0 0	0	0
1.9 x 10*
2.1x 102
2.1x 102
0
2.5 cm of lagooned sludge from Jay ARC (sludge had a pH of 7.0 and a solids content of 7.0%)
seeded with 8.8 x 10 PFU of poliovirus or 2.9 x 10 PFU of echovirus applied on top of 33 cm
undisturbed, large soil cores of Eustis fine sand. Seeded sludge was allowed to soak in and
dry on top of the soil for 3 days before being mixed with the top 2.5 cm of soil.
-------
TABLE 8. Continued
k Rainfall data was collected at the experimental site.
c Virus monitoring in the drying sludge was undertaken using 0.05 M glycine, pH 11.5 as primary
eluent, without further concentration of eluates. Virus monitoring of the soil was undertaken
according to the isoelectric casein method.
^ The liquid sludge was seeded with 2.6 x 10 PFU of poliovirus or 8.6 x 10 PFU of echovirus
per gram dry weight of sludge. A large fraction of the polioviruses seeded (95.2%) were asso-
ciated with the sludge solids after magnetic stirring for 1 hour and before application to
the soil cores. Echovirus, on the other hand, was found mostly in the unadsorbed state (only
21.7% of the viruses were associated with the sludge solids). In addition to the large cores, the
virus-seeded sludge was also applied (2.5 cm) to 2 undisturbed, small soil cores of Eustis fine
sand (33 cm long and 5 cm inside diameter) for the purpose of studying transport of viruses
only. One small soil core received poliovirus seeded sludge while the other received echovirus
seeded sludge. Soil leachates from all cores were monitored for viruses.
e A dash means not done or not applicable
-------
TABLE 9. ANALYSES FOR THE	PRESENCE OF POLIOVIRUS TYPE 2 (SABIN) AND
ECHOVIRUS TYPE 1	(FAROUK) IN SOIL LEACHATES COLLECTED AFTER
NATURAL RAINFALL	FROM SOIL CORES WHICH HAD BEEN TREATED WITH
2.5 CM OF SEEDED	LIQUID SLUDGE (October 11, 1978 - January 20,
1979)
Dates
Cumulative
Soil
Cumulative
Cumulative number
leachates
rainfall13
corec
leachate
of pore
col 1ected
(cm)
number
volume
volumes eluted
12-01-78
to
24.95
SCI
400 ,
1.7
01-21-79



(20.4)

12-01-78
to
24.95
SC2
385
1.6
01-21-79



(19.6)

12-01-78
to
24.95
CI
750
0.3
01-21-79



(4.0)

12-01-78
to
24.95
C2
980
0.5
01-21-79



(4.9)

12-01-78
to
24.95
C3
920
0.4
01-21-79



(4.9)

12-28-78
to
24.95
C4
410
0.2
01-21-79



(2.2)

Dates
leachates
collected
Range conductivity
Cumulative Cumulative values of	Range of pH
virus	percent leachates	values of
eluted total virus collected	leachates
(total PFU)	applied ( mho/cm at 25°C)	collected
12-01-
01-21-
12-01-
01-21-
12-01-
01-21-
12-01-
01-21-
12-01-
01-21-
12-28-
01-21-
78	to
79
78	to
79
78	to
79
78	to
79
78	to
79
78	to
79
0
0
0
0
0
140 - 625
135 - 710
375 - 800
190 - 975
280 - 1,200
560 - 875
5.8 - 7.0
6.3
6.3
6.1
5.9
6.0
7.0
6.8
6.3
6.9
6.9
214
-------
TABLE 9. Continued
a
2.5 cm of lagooned sludge (from Jay, ARC; the sludge had a pH of 7.0
and a solids content of 7.0%) seeded with a total of 8.8 x 108 (9.5
x 107 for the small core) PFU of poliovirus or 2.9 x 106 (3.1 x 105
for the small core) PFU of echovirus was applied on top of 33 cm un-
disturbed soil cores of Eustis fine sand (15.5 cm inside diameter for
CI, C2, C3, and C4; 5 cm inside diameter for SC and SC2; contains Ap
and A21 horizons). The seeded sludge was allowed to soak in and dry
on top of the soil for 3 days before being mixed with the top 2.5 cm
of soil. The soil cores were exposed to natural conditions.
k Rainfall data was collected at the experimental site. The cumulative
rainfall (cm) values represent the total rainfall from the beginning
of the experiment, October 11, 1978, to January 20, 1979.
Echovirus was seeded in the sludge applied to small core 1, core 1
and core 2, core 3, and core 4.
^ One pore volume for the large soil cores and the small soil cores was
2,178 ml and 234 ml, respectively.
e Viruses in soil leachates were monitored by concentration using
membrane filtration.
^ Values in parentheses represent the number of cm of cumulative leachate
volume.
215
-------
TABLE 10. VIRUS ASSOCIATED WITH SLUDGE DISPOSAL
AT THE KANAPAHA SITE
Sampling date
Sample
Enteroviruses
(TCID 50)
December 1977
Wasted sludge
25.0
December 1977
Digested sludge*3
1.2
December 1977,


January 1978, and
p
H
February 1978
Centrifuged sludge
ND
February 1978
Wasted sludge
11.0
February 1978
Digested sludge
0.3
December 1977
Soil ^
ND

Groundwater
ND
January 1978
Soil
ND

Groundwater
ND
February 1978
Soil
ND

Groundwater
ND
a TCID per g (dry weight) of sludge or soil or 100 gallons of groundwater.
L
From an aerobic digestor with a detention time of 90 days.
c The centrifuged sludge had undergone aerobic digestion for 180 days
before conditioning and centrifugation.
ND = not detected.
e 200 g composite soil samples.
-C
100 gallons groundwater sampled at a 60 foot deep well (well location
shown in Figure 2).
216
-------
TABLE 11. VIRUSES ASSOCIATED WITH DIGESTED SLUDGE
ADDED TO THE SLUDGE LAGOON
SIudge source
Date
Obtained
Type of Sludge
Digestion Used
Viruses
TCIDso/q
Montclair
02-17-78
Aerobic
260
Pensacola
02-17-78
Anaerobi c
7
Montclair
12-06-78
Aerobic
41
Pensacola
12-06-78
Anaerobic
2
Montclai r
02-12-79
Aerobic
14
Pensacola
02-12-79
Anaerobic
4
TABLE 12. ENTEROVIRUS
NUMBERS IN THE
OVERLYING WATER IN
THE SLUDGE LAGOON
Date
Obtained
SIudge
Addi tion
Vi ruses
TCIDso/l
Chlorophyll a
mg/m3
10-03-78
_a
< 0.5
2,370
11-06-79
-
< 0.5
1,508
12-14-78
+
9
387
01-11-79
+
55
172
a
+ indicates that sludge was being added to the lagoon at sampling times,
- indicates that sludge was not being added to the lagoon at the
sampling time.
217
-------
TABLE 13. VIRUSES RECOVERED FROM LAGOON SLUDGE AND WATER SAMPLES AT JAY, FLORIDA
Total
Sampling	isolates Polio- Polio- Polio- Echo- Echo- Echo- Cox. Non-
Sample
period

identi fied
1
2
3
1
E41
7
15
-B42
Typ
Lagooned sludge
02-17-78
to
27
63
33
0
0
0
4
0
0
0

11-06-78











Digested sludge


32
81
9
9
0
0
0
0
0
0
(Montclair and












Pensacola)












Lagooned siudge
12-06-78
to
21
24
19
0
0
0
19
5
33
0

01-24-79











Water overlaying


16
12
25
0
19
0
6
0
31
6
lagooned sludge












Digested sludge


10
20
0
30
20
0
20
0
10
0
(Montclair and












Pensacola)












Dried sludge
10-03-79
to
10
10
0
0
30
30
10
0
10
10
10-12-79











Total


116
46
17
5
7
3
8
1
12
2
1 Echo-4
2 Coxsackie
-------
TABLE 14. SURVIVAL OF VIRUSES IN SLUDGE AFTER APPLICATION
TO LAND AT JAY, FLORIDA
Date
Obtained
Days after
Spreading on land
%
Water

TCIDso/9



Glycine Method
Urea-Lysine Method
10-03-79
0
91
1.4
4.6
10-04-79
1
39
< 0.01
0.16
10-05-79
2
40
0.72
0.10
10-08-79
5
ND
< 0.01
< 0.01
10-10-79
7
15
0.05
0.06
10-12-79
9
19
< 0.01
0.02
TABLE 15. ANALYSIS OF WELL WATER FOR THE PRESENCE
OF ENTEROVIRUSES AT JAY, FLORIDA
Total Volume of
Well Water sampled9 Viruses
no.	Si te	(1 i ters)	Detected
1
Northeast quadrant
1,100
0
3
Southwest corner (in
direction of the
groundwater flow)
1,100
0
9
East-central section
1,100
0
New
1agoon
Approximately 10 ft
from sludge lagoon
2,650
0
Total

5,950
0
a Six samples of 180 to 700 liters were obtained over a one year period.
219
-------
FIGURE 1. SOIL CORES
-------

<1)
O
c
+->
fO
CL
'i—
(J
CD
s-
Q-
+->
o
		• Maximum temperature
		Mi n i mum temperatu re
--4 Precipitation
FIGURE 2. WEATHER DATA FOR THE WEST FLORIDA AGRICULTURAL EXPERIMENT STATION
JAY, FL
-------
WASTED SLUDGE
AEROBIC DIGESTION
Sludge treatment at the Main Street
Plant, Gainesville, Florida
(2 Digesters in series:
180 days detention time)
SLUDGE CONDITIONING
(Addition of Cationic Polymer)
CENTRIFUGAT10N
APPLICATION TO LAND AT THE KANAPAHA SITE
(10 acre plot)
FIGURE 3. SCHEME FOR SLUDGE DISPOSAL AT THE KANAPAHA
SITE, GAINESVILLE, FLORIDA
222
-------
E
5 ACRES
PLANTED RYEGRASS
A A
1
A ^ A
5 ACRES
UNPLANTED IN DECEMBER, 1977
AND JANUARY, 1978; PLANTED
RYEGRASS IN JANUARY, 1978
A k
k
A A
MONITORING
CENTER WELL - 60 FT
J

A Sampling points to obtain a 200 gram composite topsoil sample (40 grams
per point) in each 5 acre plot per sampling date.
The soil found at this site belongs to the Lochloosa series.
FIGURE 4. DIAGRAM OF THE KANAPAHA SLUDGE DISPOSAL SITE
-------
PENSACOLA SLUDGE
(3-4% SOLIDS)
J
SLUDGE LAGOON
(JAY, FLORIDA)
I
APPLICATION TO LAND
AT JAY, FLORIDA
1.5-37.5
HECTARE CM/YEAR
I
SLUDGE ALLOWED TO
DRY FOR 2-14 DAYS
AND THEN TURNED UNDER
FIGURE 5. SCHEME FOR SLUDGE DISPOSAL AT JAY SITE
224
-------
Addition of fresh sludge to
lagoon suspended (April 14, 1978)
Addition of fresh
sludge to lagoon
resumed (Nov. 7, 1978)
JFMAMJJAS
	1978	
0 N D
J F M
	1979—
FIGURE 6.
-------
Plot
#61
15
acre
-in
N
+
+
S
t&l
Plot
#42
A
0
acre
-in
S5
•5?'


-
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PESTICIDE RESIDUES IN FEED, SLUDGE, SOIL/SLUDGE,
AND ANIMAL TISSUES
N. P. Thompson, 0. Osuna, G. T. Edds
ABSTRACT
Samples of sludge, soil/sludge mixture, feed, and animal tissues
(kidney, liver, fat, muscle) were analyzed for chlorinated hydrocarbon
pesticide residues and also polychlorinated biphenyls. The analytical
method is typical of that used for determination of persistent pesticide
residues in environmental samples and includes Soxhlet extraction with
petroleum ether; clean-up by gel permeation chromatography, florisil
and silicic acid columns; and detection by electron capture gas chroma-
tography. The sensitivity of the method is 0.01 ppm.
Results indicate that little if any chlorinated hydrocarbon resi-
dues were present in sludge used for research in this project. The
available tables list the residues found in ppm in sludge, feed, and
various animal tissues. It can be concluded that sludge as used in
experiments associated with this project presents no hazard from the
aspect of pesticide residues.
227
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PESTICIDE ASSAYS - 1976-1980


PCB
a-BHC
Lindane
Hepta-
Chlor
Hepta-Chlor
Epoxi de
A1 dri n
Di eldri n
Endrin
D
D
E
D
D
D
D
D
T
Chi o
dane
1976













LiveOak Feed













Control

/
/
/
/
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/
/
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/
/
-
Control + 10%
DFS
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-
Control + 20%
DFS
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/
-
Jay Feed













Control

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-
Corn

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-
Silage

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-
Concentration

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Soils (9)

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~
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-
Grass (6)

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/
-
1977













Poultry Feed













50% "Corn"

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/
100% "Corn"

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/
Control

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/
LiveOak Feed













Control

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/
Control + 10%
DFS
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/
Control + 20%
DFS
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/
DFS

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/
Premi x

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/
DCS

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/
Ration + 50%

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/
Control

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/
All assays (4136) read as ppm; analyses by Varian Gas Chromatograph in Electron Capture Mode on 4% SE
30.6% OV 210. Calculations with Hewlett-Packard Integrator. Total assays performed - results negative
to minimum detectable level. A very few sludge samples contained PCB's exceeding 5 ppm FDA guideline
level.
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OTHER ASSAYS - 1977, 1978. 1979

PCB
a-BHC
Lindane
Hepta-
Chlor
Hepta-Chlor
Epoxide
A1 dri n
Die! drin
Endrin
DDE
DDD
DDT
Fat samples
/
/
/
~
/
/
/
/
/
/
/
Li veOak











Control
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/
ANS 10% DFS
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~
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/
ANS 20% DFS
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/
Jay











Control
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/
DPS:











100 g/h/d
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250 g/h/d
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500 g/h/d
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/
DCS:











500 g/h/d
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/
Jay











Fat
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Liver
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Ki dney
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/
Brain
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Muscle
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Li veOak











Sows-Pi gs:











Ki dney
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Liver
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Muscle
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Fat
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/
Total pesticide assays run (3 years) = 4136. Poultry - grain in ration - corn grown at Jay on
23 cm/hectare treated soil - 50% or 100% of corn in ration.
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PUBLICATIONS
1.	Bertrand, J. E., Lutrick, M. C., Edds, G. T., and R. L. West. Forage
Sorghum Silages Grown on Soil Treated With Liquid Digested Sludge and
Fed to Beef Steers. Soil and Crop Sci. Soc., Florida, 40:141, 1981.
2.	Bertrand, J. E., Lutrick, M. C. and H. L. Breland. Dried Liquid Digested
Sludge in the Ration of Feedlot Steers. Procs. 1st Ann. Conf., Appl.
Res., Munic. and Indust. Waste, Madison, WI, 385-386, 1978.
3.	Bertrand, J. E., Lutrick, M. D., Breland, H. L. and R. L. West. Effects
of Dried Digested Sludge and Corn Grown on Soil Treated with Liquid
Digested Sludge on Performance, Carcass Quality, and Tissue Residues
in Beef Steers. J. An. Sci., 50:35-40, 1980.
4.	Bitton, G., Pancorbo, 0. C., Overman, A. R., and G. E. Gifford. Re-
tention of Viruses During Sludge Application to Soils. Prog. Water
Tech., 10:597-606, 1978.
5.	Bitton, G., Davidson, J. M., and S. R. Farrah. On the Value of Soil
Columns for Assessing the Transport Pattern of Viruses Through Soils:
A Critical Outlook. Water, Air, and Soil Pollution, 12:449-457, 1979.
6.	Bitton, G., Charles, M. M., and S. R. Farrah. Virus Detection in
Soils: A Comparison of Four Recovery Methods. Can. J. Microbiol.,
25:874-880, 1979.
7.	Bitton, G., Farrah, S. R., Pancorbo, 0. C. and J. M. Davidson. Fate of
Viruses Following Land Disposal of Sewage Sludge. I. Survival and
Transport Patterns in Core Studies under Natural Conditions. In
International Symposium on Viruses and Wastewater Treatment, University
of Surrey, Guildford, U.K., 1979.
8.	Farrah, S. R. and G. Bitton. Fate of Viruses Following Land Applica-
tion of Sewage Sludge. II. Field Experiments (monitoring of lagooned
sludge, soil and groundwater). In International Symposium on Viruses
and Wastewater Treatment, University of Surrey, Guildford, U.K., 1980.
9.	Damron, B. L., Hall, M. F., Janky, D. M. and M. C. Lutrick. Feeding
Corn Fertilized with Municipal Sludge to Broiler Chicks and Laying Hens.
Poultry Sci., 59:1561, 1980.
10.	Johnson, W. L., and B. L. Damron. Performance of White Leghorn Hens
Fed Various Levels of Municipal Sludge or Selected Minerals. Poultry
Sci., 59:1565, 1980.
11.	Damron, B. L., Hall, M. F., Janky, D. M., Wilson, H. R., Osuna, 0.,
Suber, R. L., and M. C. Lutrick. Corn Fertilized with Municipal Sludge
in the Diet of Chicks and Hens. Submitted for publication, Poultry
Science, 1980.
230
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12.	Osuna, 0., and G. T. Edds. Toxicology of Aflatoxin B , Warfarin,
and Cadmium in Young Pigs. Ph.D. thesis, University of Florida, pp.
1-246, 1979.
13.	Osuna, 0., and G. T. Edds. (3 papers) Toxicology of Aflatoxin B ,
Warfarin, and Cadmium in Young Pigs (9802 R) I. Performance and
Hematology (9803 R). II. Clinical Chemistry and Blood Coagulation
(9804 R). III. Metal Residues and Pathology. Am. J. Vet. Res.,
1981.
14.	Osuna, 0., and G. T. Edds. Comparative Toxicity of Cadmium versus
Cadmium Chelates in Swine - 9763 R. Am. J. Vet. Res., 1981.
15.	Osuna, 0., Edds, G. T., Popp, J. A., Monague, J., and K. E. Ferslew.
Feeding Trials of Dried Urban Sludge and the Equivalent Cadmium Level
in Swine. Proc. 8th Natl. Conf. on Municipal Sludge Management,
Miami Beach, FL, pp. 201-213, 1979.
16.	Edds, G. T. and 0. Osuna. Aflatoxin B Increases Infectious Disease
Losses in Food Animals. USAHA, 80th Annual Meeting, Miami Beach, FL,
1976.
17.	HammelI, D. L., Osuna, 0., Suber, R. L., and 6. T. Edds. Health
Effects of Sewage Sludge on Long-Term Sow Reproduction Performance.
Symposium Evaluation of Health Risks Associated with Animal Feeding
and/or Land Application of Municipal Sludge, Tampa, April 29-May 1, 1980.
18.	White, C. E., Hammel1, D. L., and 0. Osuna. Effect of Feeding
Digested Sewage Sludge on Long-term Sow Reproduction Performance.
Symposium, Evaluation of Health Risks Associated with Animal Feeding
and/or Land Application of Municipal Sludge, EPA/University of
Florida, April 29-May 1, 1980.
19.	Suber, R. L. and G. T. Edds. High Performance Liquid Chromatographic
Determinations of Sulfonamides by Ionic Suppression. J. Liquid Chrom.,
3(2):257-268, 1980.
20.	Edds, G. T., Ferslew, K. E., and R. A. Bell is. Feeding of Urban
Sewage Sludge to Swine. Proc. USAHA, pp. 207-220, 1978.
21.	Edds, G. T., Osuna, 0., and C. F. Simpson. Health Effects of Sewage
Sludge for Plant Production or Direct Feeding to Cattle, Swine, Poultry,
or Animal Tissue to Mice. Proc. Municipal and Industrial Sludge
Utilization and Disposal, Alexandria, VA, pp. 180-188, May 28-30, 1980.
ZZ. Edds, G. T. Symposium , Evaluation of Health Risks Associated with
Animal Feeding and/or Land Application of Municipal Sludge. EPA/
University of Florida, April 29-May 1, 1980, Tampa, FL.
231
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23.	Hammell, D. L. and G. T. Edds. Effect of Feeding Sewage Sludge Diets
to Swine on Reproduction, Growth, and Tissue Mineral Accumulation.
Proc. Joint ADSA-ASAS, Mich. State Univ., E. Lansing, MI, 1978.
Am. Sci. Res. Report, SW-1977-2, August, 1977.
24.	Hammell, D. L., Beaudouin, J., and R. L. Shirley. Effect of Feeding
Sewage Sludge to Growing-Finishing Swine on Performance and Tissue
Mineral Accumulation. Animal Sci. Res. Report, SW-1977-2, August,
1977.
25.	Hortenstine, C. C. Growth and Cadmium Uptake by Lettuce and Radish
Fertilized with Cadmium, Zinc, and Sewage Sludge. Proc. Soil and Crop
Science Soc. of Florida, 39, October 2-4, 1979.
25. Lutrick, M. C. and J. A. Cornell. The Nutrient Status of Six Field
Crops Grown on Soils Treated with Liquid Digested Sludge. Soil
and Crop Science Soc. of Florida, 35, 183-185, 1975.
27.	Lutrick, M. C. and J. E. Bertrand. Agronomic and Cattle Studies
with Municipal Liquid Digested Sludge. Proc., Inst, of Environ.
Sci., 1976.
28.	Lutrick, M. C., Bertrand, J. E., and H. L. Breland. The Utilization
of Liquid Digested Sludge on Agricultural Land. Soil and Crop
Science Soc. of Florida, 35, 101-106, 1975.
29.	Beaudouin, J., Shirley, R. L., and D. L. Hammell. Effect of Sewage
Sludge Diets Fed Swine on Nutrient Digestibility, Reproduction,
Growth and Minerals in Tissues. J. An. Sci., 50:572-580, 1980.
30.	Beaudouin, J., Hammell, D. L., and R. L. Shirley. Sewage Sludge Diets
Fed Swine Versus Reproduction, Growth, and Tissue Minerals. Abstract
No. 199, Annual Meeting of the Am. Soc. An. Sci., July 23-27, 1977,
Madison, WI.
31.	Beaudouin, J., Shirley, R. L., and D. L. Hammell. Effect of Sewage
Sludge in Diets Fed Swine on Total Digestible Nutrients, Metabolizable
Energy, Nitrogen Balance, and Minerals in Tissues. Paper presented
at XI International Congress of Nutrition, Rio de Janeiro, Brazil,
August 27-September 1, 1978.
32.	Kelley, K. C., Shirley, R. L., Osuna, 0., Bertrand, J., and G. T. Edds.
Health Effects in Mice Consuming Organ Tissues from Sludge-Treated
Cattle and Swine. Symposium, Evaluation of Health Risks Associated
with Animal Feeding and/or Land Application of Municipal Sludge,
EPA/University of Florida sponsored, Tampa, FL, April 29-May 1, 1980.
232
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