Citation
Effects of Dietary Aluminum from Water Treatment Residuals on Phosphorus Status and Bone Density in Growing Lambs

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Title:
Effects of Dietary Aluminum from Water Treatment Residuals on Phosphorus Status and Bone Density in Growing Lambs
Creator:
VAN ALSTYNE, RACHEL
Copyright Date:
2008

Subjects

Subjects / Keywords:
Aluminum ( jstor )
Animals ( jstor )
Bone density ( jstor )
Bones ( jstor )
Lambs ( jstor )
Minerals ( jstor )
Phosphorus ( jstor )
Plasmas ( jstor )
Sand ( jstor )
Sheep ( jstor )
City of Gainesville ( local )

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University of Florida
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University of Florida
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Copyright Rachel Van Alstyne. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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7/30/2007
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74493195 ( OCLC )

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Full Text












EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS













By

RACHEL VAN ALSTYNE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Rachel Van Alstyne


































Dedicated to family and friends who stood by me through my educational journey.















ACKNOWLEDGMENTS

The author wishes to extend sincere gratitude to Dr. Lee McDowell, Dr. George

O'Connor, and Dr. Lokenga Badinga for their service on her graduate committee.

Thanks are extended to Dr. McDowell for his support, patience, and encouragement

throughout this endeavor. Thanks are extended to Dr. O'Connor for his professional

wisdom and support regarding soil science. Thanks are extended Dr. Badinga for his

valuable insight and suggestions.

The author would like to thank Dr. Paul Davis for his assistance throughout the trial

and writing processes. His hard work, loyalty, love, and support as a friend, partner, and

colleague have been paramount to the success of the author during her life while

attending the University of Florida, in the publication of this thesis, and in the life they

seek together in the future.

Much appreciation is extended to Nancy Wilkinson, Jan Kivipelto, and Dr. Maria

Silveira for aid in laboratory analyses and data interpretation. Their support, time, and

assistance have been priceless. Thanks are given to Dr. Lori Warren, Steve Vargas,

Jessica Scott, Carlos Alosilla, Kathy Arriola, Eric Fugisaki, Tom Crawford, Jose

Aparicio, and Luis Echevarria, for their hard work and support during the trial.

Additionally, the author would like to thank her best friend and roommate, Karen

Fratangelo, for her support and kind regards during stressful times. Karen and the author

have been friends since the 6th grade and have been able to stay close and lean on each

other. Karen aided in diapering sheep, ended an ear, and shared a kind heart during hard









times. Though it may seem odd, the author would like to give her kind regards not only to

the humans who aided in her success, but also to the lambs that will always have a soft

spot in her heart. Last but not least, the author would like to thank her parents, brother,

and canine companion Vixen for their love and support throughout her education and

throughout life. She would never have had the perseverance and confidence that she does

without their love, loyalty, support, encouragement and respect.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

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

ABSTRACT .............. .......................................... ix

CHAPTER

1 INTRODUCTION ............... ................. ........... ................. ... .... 1

2 REVIEW OF LITERATURE ......................................................... .............. 4

Historical Significance of Phosphorus.................... ...... .......................... 4
R e q u ire m e n ts ................................................................................................................5
Phosphorus D eficiencies.................................................. ... ....6
Phosphorus M etabolism and Transport ................................. ............. .................. 8
Aluminum and Phosphorus Interactions.................................. ....................... 10
Pollution and Phosphorus Application to Land................................................... 14
Regulations .................. ............... ...................... .............. ........... 16
W TR and Environm ental U ses ........................................................ ............. 19

3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON
PERFORMANCE AND MINERAL STATUS OF FEEDER LAMB S.....................23

Introdu action ...................................... ................................................. 2 3
M materials and M ethods ....................................................................... ..................24
Anim als, D iets, and M anagem ent ............................................ ............... 24
Sample Collection, Preparation, and Analyses........................................25
Statistical A analysis ........................................ ................... .. .. ... 26
R e su lts ...........................................................................................2 7
D isc u ssio n ............................................................................................................. 2 9
Sum m ary and C onclu sions .............................................................. .....................36
Im p location s ........................................................................... 3 7









4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT
RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF
FEEDER LAM BS ................................... .. .. ........ .. ............43

Introdu action ...................................... ................................................. 4 3
M materials and M ethods ....................................................................... ..................44
Anim als, D iets, and M anagem ent ............................................ ............... 44
Statistical A analysis ........................ ............ ................ ....... 47
R e su lts ................................................................................................... ........ . 4 7
Radiograph BM C............................................................................... 47
Bone Density via Specific Gravity .......................................... ...............47
B one M ineral A nalyses ............................................... ............................ 47
D isc u ssio n ............................................................................................................. 4 8
Sum m ary and C onclu sions .............................................................. .....................49
Im p licatio n s ................................................................5 0

5 SUMMARY AND CONCLUSIONS.......................................................................53

APPEND IX : TABLE D A TA ............................................... ...... ......................... 57

L IT E R A T U R E C IT E D ............................................................................ ....................58

B IO G R A PH IC A L SK E TCH ..................................................................... ..................63















LIST OF TABLES


Table Page

3-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments.................................... ............... 38

3-2 Effects of dietary Al concentration and source on BW of feeder lambs .................39

3-3 Effect of dietary Al concentration and source on feed intake of feeder lambs .......39

3-4 Effect of dietary Al concentration and source on plasma P of feeder lambs ..........40

3-5 Tissue mineral composition resulting from experimental diets ...........................41

4-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments ......... ..................... .... ............51

4-2 Effect of dietary Al concentration and source on bone density of feeder lambs as
determ ined by radiography ............................................ ............................. 52

4-3 Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg
for experim mental diets ...................... ................ ............................52

A-1 Effect of dietary Al concentration and source on ADG of feeder lambs ................57















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS

By

Rachel Van Alstyne

August 2005

Chair: Lee Russell McDowell
Major Department: Animal Sciences

Experiments using growing feeder lambs were conducted to gather data on 1) the

safety of a Al-water treatment residual (WTR) ingested in amounts to provide between

2,000 and 8,000 ppm Al, and 2) the bioavailability of Al in WTR when compared to a

control (910 ppm Al from sand) and a diet containing a known bioavailable form of Al

from AlC13.

The study was conducted to examine changes in performance (ADG, BW, and feed

intake), tissue mineral concentrations, plasma P concentrations, bone mineral content

(BMC), bone density, and apparent P absorption. At experimental termination, samples

of brain, liver, kidney, heart, and bone were collected and analyzed for concentrations of

Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Thirty-two wether and ten female lambs were

assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlC13),

3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand),

and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix,









and 1.29% dicalcium phosphate). Treatments 1-5 contained P at 0.25% and

concentrations of Al were 910, 2000, 4000, 8000 and 8000 ppm, respectively for the six

diets. Compared to the control, ADG, BW, and intakes were unaffected by dietary levels

of WTR (P > 0.05); however lambs fed 2,000 ppm Al from AlC13 had reduced body

weights and lower ADG (P < 0.05). The control, most often, had the highest plasma P

concentrations and the WTR treatments generally had higher P concentrations than lambs

given AlC13. During wk 6, plasma P concentrations declined for all animals but steadily

increased thereafter. Kidney P differed; control lambs had larger deposits of P than lambs

given 8,000 ppm Al from WTR (P < 0.05). Iron deposits were highest in livers from

lambs fed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Brain Al was

highest for animals receiving 2,000 ppm Al from AlC13 and lowest for lambs given 2,000

ppm Al from WTR (P < 0.05). Brain Al concentrations increased when Al from WTR

was given in amounts above 2,000 ppm. Apparent P absorption did not differ among

WTR treatments and the control (range from 11 to 32 %), but lambs fed 2,000 Al via

A1C13 had a negative (-13%) apparent absorption of P. Values of BMC and bone density

did not vary with treatments; this is likely due to the short duration of the study. This

study found no evidence of health related defects because of the administration of the

WTR. The Al as AlC13 was more bioavailable with regard to plasma P levels and

performance, than Al via WTR; animals which were given the AlC13 were negatively

affected.














CHAPTER 1
INTRODUCTION

Manure transportation for refuse is costly. This results in the primary method for

animal waste disposal being application nearby land. Repeated long-term manure land

application leads to accumulation of phosphorus (P) (Novak and Watts, 2004). In the

United States, the livestock industry produces 500 million tons of manure each year

(Lorentzen, 2004) and many coastal soils have already become saturated with P (Novak

and Watts, 2004).

The majority of the P produced as animal waste is not adequately used for plant

uptake and much of the soil used by large industrial agriculture companies has reached its

maximum capacity for P adsorption (Novak and Watts, 2004). When manure is applied

to the land and the P remains stagnant on the upper crust of the soil bed it may be washed

away with heavy rains (Haustein et al., 2000; Federal Registar, 2004).

Phosphorus lost in leaching and runoff can lead to eutrophication, causing the

overgrowth of algae, and decreasing the survival of aquatic plants and animals (Novak

and Watts, 2004). These algae lead to a reduction in the oxygen levels within the water

and result in an overgrowth of anaerobic bacteria that generate toxins such as Pfissteria

which may result in death, rashes, respiratory illness, and memory loss in people and

animals (Haustein et al., 2000). The death and decomposition of aquatic plants can lead

to depressed aquatic oxygen levels, resulting in fish mortalities.

Aluminum (Al) applied to the land has been shown to reduce the soluble P

concentrations from animal waste (O'Connor et al., 2002). Aluminum salts have been









used to help minimize the amount of P released from animal feces. The chemical

reaction which occurs between the Al from salts and the P in the manure, result in a

decrease in P loss. This method has proved to be effective but it is costly (O'Connor et

al., 2002). The reduction of available P levels from animal waste products could result in

significant decreases in leaching and runoff, lessening contamination of water supplies.

Studies conducted at the University of Florida suggest that water treatment residuals

(WTR), especially those containing Al, increase the soils capacity to retain P. Water

treatment residuals bind P, lessening its availability and decreasing water pollution

caused by runoff (Elliott et al., 2002). Water treatment residuals are derived from the

water purification processes and can vary in their mineral content and P absorption

capacities, depending on the chemical used by the water treatment facility and the age or

dryness of the WTR. Thus, WTR can be high in Al, Fe or Ca oxides and upon drying or

aging become safe for land application (O'Connor et al., 2001; Dayton et al, 2003). The

WTR used throughout this experiment (including references, unless otherwise stated) are

high in Al-oxide from a source known for its high P sobbing capacity and will be

referenced as WTR. Water treatment residuals are the solid sediments that result after

raw water is coagulated, leaving behind amorphous Al oxides (Basta et al., 2000; Dayton

and Basta, 2001). These WTR contain amorphous solids that vary in size and shape.

Most WTR look like, and have the texture of a dark soil, but have little or no nutritive

value. However, WTR usually contain 4 to 8% nonavailable Al. In general, WTR are

discarded in landfills or in waterways, but both methods of disposal are costly and may

increase the price of drinking water (Novak et al., 2004).









Application of WTR to the land may result in one solution for animal waste

discard, while eliminating the burden and expense of WTR disposal. The chemical

mixture of soil and WTR has been proven to increase the retention value of soil P by

several fold (Novak et al., 2004).

One concern posed is that WTR contains high amounts of Al leading to

contamination and ecological risk for grazing animals, wildlife, surrounding floriculture

and water systems (USEPA, 2003). High levels of Al can adversely affect P utilization

and bone deposition. Aluminum toxicity is often observed as a P deficiency resulting in

bone density impairment (Valdivia, 1977). Toxicity is primarily linked to the degree of

Al bioavailability. In WTR, the bioavailability of Al varies, but is generally low

(O'Connor et al., 2002). During grazing, ruminants naturally consume up to 10% to 15%

of their total dry matter (DM) intake as soil (Field and Purves, 1964), and soil can contain

as much as 10% Al (Valdivia, 1977). Research with livestock at the University of

Florida demonstrated that increases in dietary Al decreased voluntary feed intake, and

feed efficiency, depressed P serum concentrations, and depressed growth and gains

(Valdivia, 1977; Rosa et al. 1982). In this research Al-WTR were directly fed to sheep to

simulate grazing-like conditions and soil consumption to emulate an ingestion of soil

material in amounts of 10% of their diet, for hypothetical assessment of health related

affects if inadvertent consumption of WTR was to occur. The following experiments

compared the bioavailability of Al from WTR to an available source of Al, in AlC13. The

main focus will be on the effects of these Al sources on P status in sheep.














CHAPTER 2
REVIEW OF LITERATURE

Historical Significance of Phosphorus

Phosphorus (P) and calium (Ca) are the two primary minerals that constitute bone

matter and are actively involved in bone development. Together, Ca and P are the most

abundant minerals in an animal's body (Miller, 1983). Eighty to 85% of an animal's P is

found in the bones and teeth. Combined, Ca and P make up 70% of the minerals found in

the body (McDowell, 2003).

The essentiality of P in bone development has been known since 1769, when bone

ash was analyzed, and P was found to be a primary component of bone material

(McDowell, 2003). Much of the early research on P was instituted in areas of South

Africa where deficiencies had become a growing concern. Low P diets had been linked

to lamsiekte and botulism in much of the grazing livestock throughout the continent of

Africa. Clinical signs which appeared included: bone chewing, depressed growth,

failures in reproduction, and reduced feed intake. Chewing the bones of dead carcasses is

the most significant indicator of a low P. The P deficiency forces the animal to find any

source of P, but the ingestion of the bones can also lead to consumption of Clostridium

botulinum and death (McDowell, 2003). In areas of Piaui, Brazil, 20,000 to 30,000 cattle

die yearly because of botulism. Today, low P diets and associated diseases are

problematic in tropical regions of the world. In Latin America, 73% of all forages

evaluated in a feed table publication were P deficient (McDowell, 1997).









Requirements

Phosphorus makes up 0.12% of the earths crust by volume but is not often found in

an uncomplexed form. Phosphorus is extremely reactive and is most commonly found as

phosphate in sedimentary rock deposits (McDowell, 2003). Requirements for P vary

depending on age, sex, activity, bioavailability of P, protein and energy in the feed, stress,

interactions between feed ingredients and nutrients, digestive anatomy and reproduction

status of the animal (Miller, 1983). Ruminant animals have a lower requirement for P

and Ca than carnivores and omnivores. For strict carnivores such as felines, the

requirement for P is 0. 60% and 0.80% for Ca, non-lactating adult humans require 0.70 to

1.25% P and 0.70 to 0.90% Ca while sheep require 0.16 to 0.38% of dietary dry matter

(DM) P and from 0.20 to 0.82% of DM Ca (NRC, 1985). The NRC (1985) estimated

endogenous loses of P, using a factorial method, to range from 20 mg/kg body weight at

maintenance to 30 mg/kg body weight in growing lambs. For proper absorption, P must

be present in a bioavailable form. Ruminants, unlike mongastrics, are able to use phytin

P from plants and it is considered to be available. Only about one third of P in most

plants is available to nonruminants (McDowell, 2003). Incomplete uptake can be linked

to an unavailable chemical form of the mineral in the plant, physical barriers in the plant

wall, or antagonist elements such as oxalic acid and phytic acid which can bind P, Ca, Fe,

Mn, and Zn (McDowell, 1997; 2003).

The amount of Ca and P found in feedstuffs varies among sources. The Ca: P ratio

in legumes is between 6:1 and 10:1 and is considered to be extremely low in P. Grasses,

if mature, are often low in both Ca and P. The values depend on soil conditions and plant

species. Alkaline soils are more abundant in trace minerals than acidic and sandy soils.

Tropical soils are usually older and acidic, marked by leaching and high environmental









temperature with compromised mineral contents, both in the soil and plants (McDowell,

1997). Seeds and seed by-products are rich in P, whereas animal by-products, tankage,

and milled flours are rich in both Ca and P (NRC, 1985). Animals on pasture will

develop P deficiencies before those fed high concentrate diets, as grains are high in P

(McDowell, 2003).

Many factors can influence absorption of minerals such as age, diet, parasites,

environmental stresses, disease, and toxic constituents. Growing animals naturally have

higher requirements for Ca and P because of bone development. A high protein and

energy diet increases the need for both Ca and P, but also increases the ability of the

animal to retain these minerals. During the rainy season in areas of South Africa and

South America, incidences of P deficiencies are common because more lush forages are

being consumed. The increased intake of energy and protein rich grasses increases

mineral requirements. Without supplementation, the forages are unable to provide many

of the needed minerals in sufficient amounts (McDowell, 1997). Infections and parasites

can affect the uptake of P and Ca. Nematodes have been proven to cause

demineralization of bone tissues in sheep (Underwood and Suttle, 1999). Stress and

activity of the animal will influence mineral needs, those under more stress or those with

higher physiological need, including growth, pregnancy, and lactation have the greatest

mineral requirements (McDowell, 2003).

Phosphorus Deficiencies

The most prevalent mineral element deficiency for grazing animals worldwide is

lack of P. The requirements for P and, frequently, other minerals are often not met by

grazing ruminants, and supplementation is often required. Additionally, certain elements

found in low pH tropical soils, such as Fe and Al, can hinder P absorption in the animal.









Deficiencies in P for grazing ruminants have been reported in 46 tropical countries in

Latin America, Southeast Asia, and Africa. The soil and forages in these livestock-

grazing areas of tropical countries are low in P (McDowell, 2003).

Phosphorus deficiency is most often seen in cattle and other grazing ruminants.

Young grasses may contain 0.3% P, but mature forages may contain 0.15% P or less

(McDowell, 1997; 2003). When dietary P becomes low, an early physiological response

is a decline in inorganic plasma P. Normal plasma P levels in ruminants are between 4.5

and 6 mg/100 ml. Levels below 4.5 mg/100 ml in ruminants are considered deficient. A

normal P level in ovine whole blood is between 35 to 45 mg and in plasma is between 4

to 9 mg per 100 ml, both of which will vary with age, and sex. Anorexic conditions are

first to occur with declines in P, decreasing feed efficiency and slowing energy

metabolism in ruminants, which ultimately results in a decline in growth (McDowell,

2003). Dry matter intake was reduced 40% in lambs receiving low-P diets and DM

digestibility was less than in animal offered the high-P diets (McDowell, 2003). A

significant decline in mineral P concentrations reduces the ability of animals to properly

digest fiber, protein and carry out normal metabolic functions (Miller, 1983).

Reproductive status may be compromised, primarily in females, which is often the most

economically damaging aspect of production. Animals deficient in P have been known to

go two to three years without calving (McDowell, 1997). Ruminants deficient in P are

listless, with swollen joints, abnormal stance, lameness, and have rough, dry hair coats.

Deficiency of P or Ca is similar to that of a deficiency in vitamin D, and lack of any of

these nutrients leads to rickets. Clinical signs can include weak bones, which may

become curved, enlarged hocks and joints, dragging of hind legs, beaded ribs and









deformed thorax (McDowell, 2003). Bone density decreases and the bone matrix

becomes soft and porous. With use of a noninvasive dual photon absorptiometry

technique, Williams et al. (1990) determined that dietary levels of 0.12 to 0.13% P lead to

bone demineralization in Angus heifers. Prior to bone disruptions, an animal suffers

stunted growth, depressed appetite, and weight loss. If the skeletal system is affected, the

bones (including: ribs, vertebrae, sternum, and spongy bone material) demineralizes

quickly. The last bones to be affected are the long bones and the smaller bones of

extremities. When P is deficient, even during normal activities, the bones can bend and

fracture, (McDowell, 2003).

Phosphorus Metabolism and Transport

Calcium and P regulation occurs as a result of the hormones, 1, 25 dihydroxy

cholecalciferol, parathyroid hormone and calcitonin. Regulation of normal Ca and P

levels depend on bodily excretion, bone deposition, resorption, and intestinal absorption

(Miller, 1983).

Phosphorus and proper availability of P depends on the Ca to P ratio, of which

should be between 1:1 or 2:1 for most monogastic species. However, for ruminants,

ratios below 1:1 and over 7:1 will negatively effect growth and feed intake. If Ca and P

needs are not met, tetany will occur as the animal withdraws Ca and P from bone in order

to maintain normal blood concentrations. The status of vitamin D is important to obtain a

desirable Ca:P ratio (McDowell, 1997; 2003). Over time, if Ca and P concentrations are

low, bone becomes soft and bone density is impaired. Most of the Ca and P in bone is in

the form of calcium phosphate and hydroxyapatite. The exact make up of bone material

varies with age, sex, physical activities, and reproductive status, but consistency is seen

within species and their stages of life (McDowell, 2003).









Absorption of P in ruminants occurs throughout the intestinal tract, including the

rumen, but is optimized in the small intestine. Its uptake occurs through active and

passive diffusion and is dependent on the solubility of the membranes that it comes in

contact with. Absorption is favored when the mineral is held in solution. Factors which

effect uptake of P include: digestive system pH, age, parasites, and other mineral intakes,

particularly Ca and Al (McDowell, 1997; 2003). Large amounts of bioavailable Al form

insoluble phosphates which bind P, making it unavailable to the animal (McDowell,

2003).

Bone undergoes turnover daily and in turn affects the P plasma levels of the animal.

Osteoblasts cause new bone formation, while osteoclasts (large multinucleated cells)

reabsorb the bone tissue. Most of the nonskeletal portion of P is found in the red blood

cells, muscle tissue and nervous system. Much of this P is used to regulate oxygen and

hemoglobin in the blood. Status of P in the body can be estimated by plasma or fecal

excretion. Feces is the primary pathway for P excretion in ruminants and non-

carnivorous animals. Carnivores excrete more P in the urine over that in the feces. In

diets low in P, the body naturally conserves P, particularly in herbivores and little to no P

is excreted in the urine (McDowell, 2003).

Phosphorus is used in almost every metabolic system, including those of ruminal

microorganisms, digestion, appetite simulation, feed conversion, fatty acid transport,

metabolism of nucleoproteins, maintenance of active cells, enzymes, hormones, and for

milk, egg, and muscle synthesis (Miller, 1983; McDowell, 1997; 2003).

Evaluation of P status can be determined by the concentration in bone, since the

majority of the mineral is in bone. Heifers fed low (0.12%) P diets at had a much lower









cortical bone index, medial lateral wall thickness, breaking load, and total ash than those

receiving a 0.20% P diet (Williams et al., 1991b). Bone density of the ribs and vertebrae

was also affected by P status (Williams et al., 1990). Blood, bone, feces, rumen fluid and

saliva can all be used with various degrees of success to indicate P status of a ruminant

and reflected dietary P levels (Williams et al., 1991a,c).

Aluminum and Phosphorus Interactions

Aluminum is the third most abundant element in the earth's crust, following silicon

and oxygen, and is the most common metal found in the earth's crust (O'Connor et al.,

2002). Aluminum is highly reactive and does not normally appear in its elemental form;

instead, Al binds to other elements or compounds (McDowell, 2003). Soil Al

concentrations can range from 1 to 30% but are typically in the 0.5 to 10% range by

weight (O'Connor et al., 2002). It is not uncommon to find high amounts of Al

complexes in tropical sandy soils, binding soil P and making it unavailable for plant

uptake (McDowell, 2003).

Aluminum chloride (AlC13) was added to fields of manure covered soil and reduced

P runoff by 53% (Smith et al., 2004). Data such as this prove that Al can bind P and

increase a soils P sorption capability.

In almost all cases, Al is considered to be a toxic mineral, and is not considered to

be a required element, except possibly in female rodents (McDowell, 2003). Rosa et al.

(1982) reported that increases in dietary P in sheep increased feed intake while it was

decreased by increases in Al and Fe. Increased dietary levels of Fe and Al in sheep diets

resulted in weight losses; ADG was decreased from 156 to 97g/d in high Fe diets and

from 159 to 95g/d in high Al diets. When additional P was added to diets containing high

Fe or Al, ADG losses were minimized. The rationale for this response was that the diet









being fed was borderline to deficient in P either at 0.17% to 0.23 % (NRC, 1985).

Additionally, plasma P levels increased with Fe and decreased with Al diets (Rosa et al.,

1982). Aluminum is not added to animal diets and, in most cases, is found in feeds only

because of contamination; whereas P is often added to animal feeds and mineral

mixtures.

When Al is absorbed via lungs, skin and intestines, only small amounts are actually

retained and can be reduced further with fluorine (F) consumption. Most Al is excreted

in the feces and urine (McDowell, 2003). In a study at the University of Florida, lambs

were given 2,000 ppm of an available source of Al (AlC13) and Al tissues levels were

only mildly elevated (Valdivia et al., 1982). In a similar study, calves were given 1,200

ppm of AlC13, and performance was not influenced and changes in tissue constituents

were only mildly elevated (Valdivia et al., 1978). The kidneys, liver, skeleton and brain

are often the tissues affected by Al toxicities (McDowell, 2003).

High dietary available Al can result in unabsorbable complexes with P in the

intestinal tract. The first effect from a low dietary P level is a decline in plasma P

(Williams et al., 1991a,c) further characteristics, including bone demineralization, then

follow (McDowell, 2003). When dietary Al exceeds the maximum tolerable level

suggested by the NRC (1985) of 1,000 ppm, animals develop characteristics of Al

toxicosis. Phosphorus is the mineral primarily affected when toxic levels of Al are

administered. An insoluble complex of Al and P is formed in the digestive system of the

animal, binding P and making it unavailable, as seen in sheep fed high levels of Al, and

signs ofP deficiency resulted (Valdivia, 1977). Bone ash and bone Mg level were

reduced when 1,450 ppm Al (chloride form) was given to wether lambs (Rosa et al.,









1982). Plasma P levels in sheep given 0.15% P with no added Al were 6.9 mg/100 ml

compared to 3.6mg/100ml for those receiving 2,000 ppm Al. Lambs fed 2,000 ppm Al

also had lower gains and feed intakes. All animals apparent P absorption was negatively

impacted except those fed high P with low Al concentrations. Correspondingly, plasma

Ca levels were reduced 0.24 mg/100 ml when 2,000 ppm Al was added. Non-ruminant

species are less tolerant to Al toxicity than ruminants. If the same studies were conducted

on monogastric animals, toxicosis would develop using 2,000 ppm Al and would

ultimately lead to death. The rational is that within the rumen Al complexes with organic

anions, not affecting P radicals in the same manner as a monogastric animal (Valdivia et

al., 1982).

High Al concentrations have also been linked to the possible onset of Alzheimer's

disease. No factual evidence has been documented to prove if an Al concentration in the

brain actually does affect the disease's occurrence. Through the influence of the disease

in the medical field is elastic and is currently being methodically investigated

(McDowell, 2003).

Toxicity is primarily linked to a high Al bioavailability (McDowell, 1997; 2003;

O'Connor, 2002). When Al toxicity is observed in the ruminant, bone density is often

impaired (Valdivia, 1977). Abnormally high amounts ofbioavailable Al can also impact

the status of Fe, Zn, and Mg in sheep. Dietary amounts of AlC13 at 1,000 ppm decreased

bone and kidney concentrations of Mg, additional antagonistic affects developed for P

and Ca as well. (Rosa et al., 1982). Bone ash was reported by Valdivia et al. (1978) to

contain lower amounts of Mg for animals fed diets containing AlC13.









The binding of P to WTR brings about concerns that plants will be limited in

required minerals such as P, Ca and Mg because they will become unavailable. Crop

yields could be negatively impacted if too much P is bound to Al or if increases in heavy

metal contents are realized within the soil (Novak and Watts, 2004). If P becomes

unavailable for plant uptake, deficiencies in both plants and animals could occur. Rosa et

al. (1982) concluded that excessive bioavailable dietary Al increases P requirements.

This may be particularly true when animals are grazing on acidic tropical pastures. In

acid soils, Al and Fe become more available and both complex with P and render it

unavailable to plants.

Acid soils with a pH of 5 or less usually contain higher amounts of available Al and

Fe (USEPA, 2003). Water treatment residuals have a pH above 5, which varies

somewhat from slightly acidic to moderately basic, and alkaline sources could act as

buffers to the soil. Past research has concluded that an elevated soil pH can be

maintained with long term use of some WTR, that have a low Al solubility. It is

unknown, but has been suggested, that WTR could have an opposite effect on the living

system and could lower the pH in the digestive systems of animals which consume it

directly (O'Connor et al., 2002). It is well known that, in general, soils with an alkaline

pH have higher mineral concentrations, than acidic soils. However, Fe, Co, Cu, Mn, and

Zn are much more available in acidic compared to alkaline soils (McDowell, 1997).

Many tropical soils are acidic (< 5.0 pH) with low P concentrations in forages. Acidic

conditions often result in high concentrations of Al and Fe which bind other minerals

(McDowell, 2003). In a study used to determine the affects of sand and soil ingestion in









sheep, tropical soils from Costa Rica with a pH of 5.2, were shown to negatively affect

the animals more than soils with higher pH's (Ammerman et al., 1984).

Pollution and Phosphorus Application to Land

Both the absolute number and percentages of the U.S. population employed strictly

in farming has fallen dramatically over time. The pressure to produce enough food, with

a smaller number of farmers, has had a worldwide impact on agricultural practices,

including the expansion of agricultural into marginal lands and the over use of land in

general. The agricultural industry needs to remain steadfast in providing adequate food

supplies, but we must not compromise environmental, socio-economic, human, and

wildlife health issues. In our effort to increase food production, pollution of our water

systems has become an issue of pressing attention. In many farming practices, manure

application to the land has become environmentally problematic. The majority of P

applied to the land as manure often is converted into an insoluble form in the surface

horizon of the soil. The accumulated P is subject to erosion or runoff following heavy

rains and transported to surface water. Thus, regulations on manure application rates

have developed to avoid P pollution of surface waters. (Dayton and Basta, 2001;

O'Connor et al., 2002; Dayton et al., 2003).

Animal producers oppose new stricter regulations placed on manure use because

the cost of compliance can be high. In Okeechobee County, Florida, the state has

mandated environmental improvements for certain farms. The state shared 75% of the

cost to update dairy facilities utilizing 456 employees and 50 million dollars (Lanyon,

1994). There are management methods that can be applied to decrease P runoff, but

many are expensive when applied to large farming operations. Using an intensive

management system on an average size farm of 100 head that was feeding a high quality









pasture, reduced concentrate feeding by 16%, and resulted in a 5% lower milk yield Yet,

this system also reduced the manure application to the land, lowered feed costs, and

reduced manure handling procedures so that the farm was able to increase overall annual

profitability by $10,000, which is equivalent to $93/cow (Rotz et al., 2002). The same

method was utilized by large farms, with 800 head, and profits were increased by

$23/cow, but only with an increase in milk production (Rotz et al., 2002).

It is a necessity to protect the land from erosion and the water from P pollution

caused by manure land application practices, but it is a struggle between the better of two

interests. A study in Pennsylvania researched several species of food animals to try to

determine differences among species and P production in manure. Three soils that

contained manure from ruminants, swine, and poultry were evaluated. Differences in P

concentrations among species could not attributed to any pertinent factor and could be

assumed to be a result of initial P variations, differences in the P distribution of the soils,

or the mixing of the soils and manures. Mixing of all manure types decreased P runoff

and was deemed useful in reducing P losses during heavy rains. Mixing the soil and

manure promotes sorption of P materials and dilutes the P in the soil surface (Kleinman

et al., 2002). Ideally, soil mixing could occur on farms, but labor and machinery costs

make the process unrealistic for large scale operations. The current strategies used to

reduce P runoff and leaching are soil tillage, crop residue management, cover crops,

buffer strips, contour tillage, runoff water impoundment and terracing. These techniques

have not be proven to achieve enough success to be used solely, or cooperatively to

reduce the current environmental problem (Dayton et al., 2003).









Regulations

Scrutiny from the general public and governmental agencies has developed with the

increasing pollutants detected in water bodies throughout the United States. In 1995, a

manure spill of 144 million liters, twice the size of the Exxon Valdez oil spill, occurred in

North Carolina (USEPA, 1997). Farms in the United States are being forced to adhere to

strict laws designed to protect the general public involving issues of odor control, water

and food safety (Powers, 2003; Federal Registar, 2004). In 1969, Congress passed the

National Environmental Policy Act (NEPA). The NEPA has two major divisions; the

Council of Environmental Quality (CEQ) and the Environmental Impact Agency (EIA).

The CEQ consists of a board of three members who advise the president on

environmental issues. The EIA oversees legislation proposed for federal action on

environmental issues (Mann and Roberts, 2000). Environmental law is governed by

statutory laws and is regulated by federal, state and local administrative agencies. The

Environmental Protection Agency (EPA) is the federal agency that oversees such issues,

(Mann and Roberts, 2000), having jurisdiction with 10 regional offices nation wide

(Meyer, 2000).

According to environmental research, the sheer amount of waste generated by large

animal facilities poses risk to ground and surface water (Lorentzen, 2004). According to

the EPA, farming creates 455 million metric tons of manure each year (Lorentzen, 2004).

In 1972, Congress amended the Federal Water Pollution Control Act (FWPCA) of 1948

with the Clean Water Act (CWA) of 1972 (Powers, 2003; Lorentzen, 2004). Again in

1977, 1981, 1987, and 2002, the CWA was amended to ensure clean water for the

following: recreational use, protection of the wildlife, and to eliminate pollutants into the

ground and drinking water. Concerns that embody the agricultural industry involve









leaching and runoff of nitrogen, solids, and P into the ground water, water ways and

water beds (Lorentzen, 2004).

Violators of the CWA are subject to both civil and criminal charges. Criminal

charges only apply if the violation was intentional. If charged criminally, the fines can

range from $2,500 to 1,000,000 dollars and from one to 15 years in prison. Civil charges

pertain to all other violations. Ignorance does not preclude one from dismissal of civil or

criminal charges. Civil fines can reach a limit of $10,000 a day and an overall maximum

of $25,000 per violation (Miller, 2004).

Watersheds do not always use filtration techniques when purifying natural water

sources (Rotz et al., 2002). A prime example is the New York State watershed located in

the Catskill Mountains. This particular region of the state is primarily covered with

forests and dairy farms, and supplies 4.5 billion liters of water to people in New York

City each day (NRC, 2000). The New York watershed which provides 90% of the

drinking water to the city, is purified only chemically, and serious harm could result if

manure solids were to contaminate the water systems (Rotz et al., 2002).

As defined by the CWA, there are two sources of pollution, point and non-point

sources. Point source means there is one defined place or confined area in which the

pollution has been released. Point source regulations mandate effluent limitations, based

on technological advancements, on the amount of pollution which can be discharged

from one source into a body of water. Concentrated animal feeding operations (CAFO'

s) are often considered point source pollution candidates (Meyer, 2000), and are defined

as operating with 700 cattle or a total of 1,000 animals (Lanyon, 1994). Non-point source

pollution occurs when the source of pollution can not be traced to a single area. Non-









point source is more often the cause of agricultural pollution; in regards to land use, run

off, and leaching (Mann and Roberts, 2000). Non-point pollution may not even be

observed in the watersheds that is directly affected, but may be carried for many meters

down stream and damage areas with no direct contact with the original pollutant (Lanyon,

1994). Classically, farms have been identified as non-point sources of pollution. It has

been predicted that within the near future, with increasing regulations and, because of

public agendas and concerns, smaller farms will too, be included in point source pollution

policies (Lanyon, 1994). For any type of discharge into open water ways, permits by the

National Pollutant Discharge Elimination System (NPDES) are required each time and

stricter rules are in the near future (Meyer, 2000).

Machine and equipment regulations governing businesses involving environmental

law are determined by the notion of best available control technology (BACT). This

requires that procedures and machines in use need to meet EPA standards for pollution-

control. New businesses need to follow standards more strictly than businesses already in

existence. As technology advances, new techniques develop that make it possible to

reduce pollution. New companies are legally bound to effectively alleviating pollution

with the use of advanced technologies. Timetables for existing companies have been

applied, meaning that the replacement of old equipment is to be implemented within a

reasonable time period. The replacement equipment protocol for existing companies

should then be based upon the best practical control technology (BPCT) law by replacing,

rather than repairing, equipment, to meet the most current EPA standards (Miller, 2004).

Many of the new regulations imposed on businesses regarding environmental

safety are locally mandated by state and county polices. In Maryland, Virginia, and









Delaware, stricter polices are being implemented in regards to P application to the land.

In Maryland, all P application must abide by the Water Quality Act of 1998, which

dictate soil testing to determine if the soil is saturated with P, and if manure application

can be permitted. In Virginia, food animal practices are closely scrutinized, particularly

the poultry industry; in Delaware manure can usually be treated once every three years to

comply with soil P limitations (Penn and Sims, 2002).

Water Quality Control Boards are now being mandated to more strictly adhere to

the monitoring of N and P levels in the soils. In California, there is an overabundance in

the pollutant count in several bodies of water of both P and N. Leaching and run off from

manure enriched fertilizers is thought to be the primary cause. The reality is that this

type of fertilization is a matter of convenience, availability, and cost profitability rather

than providing the optimal nutrients for the flora or concern for the ecosystem (Farm

Press, 2004).

WTR and Environmental Uses

Currently, there is no solution for the distribution of the large quantity of manure

produced in the livestock industry. The major issue at hand is the confinement of large

operations to small areas of land. Conflicts arrive in application and concentrations of

allowable feces. Both N and P are constituents of animal waste products, and are harmful

pollutants, yet federal, state, and county standards differ in the applicable uses and

concentrations of manure for land, resulting in confusion as to how manure should be

properly applied (Lanyon, 1994).

Water treatment residuals (WTR) are by-products from water purification

procedures. They are rich in metals like Al and Fe, though the exact composition can

vary. The elemental levels of Al, Fe, and Ca vary when comparing WTR depending on









the chemical used during the water treatment process and the age (or dryness) of the

WTR. In turn, these differences will reflect different abilities to adsorb P (Dayton et al.,

2003; Ippolito et al., 2003). During the water treatment process, a chemical, called a

coagulate, is added to the water and later forms WTR. This addition of chemicals to

water will cause a reaction and form a flocculent precipitate, which coats small particles,

such as clays making them more likely to be removed by sedimentation or filtration.

Aluminum sulfate (iron sulfate, or calcium sulfate) coagulates may be added to raw

water, (the WTR of interest for all further discussion is Al based). The water is then

circulated with vigor to uniformly disperse the Al product. Aluminum reacts readily with

alkaline products within the water and produces an Al hydroxide solid, which has

entrapped impurities. The sedimentation process allows the solids to settle-out. These

solid by-products are Al oxides bound to clay size particles and are known as WTR. The

processes of coagulation and sedimentation usually precedes filtration in a water

treatment plant, and serves to reduce turbidity and increase the efficiency of bacterial

removal by filtration (Dayton and Basta, 2001; Brady and Weil, 2002; Ippolito et al.,

2003; Water Resources, 2005). The physical characteristics of these WTR are similar to

top soils (Haustein et al., 2000).

The use of WTR and metal-binding by-products could be one solution to the

accumulation of soluble P in the top layer of soil, which leads to nonpoint pollution

during heavy rains (Penn and Sims, 2002). In particular, Al containing WTR would

benefit sandy soils low in organic material. Sandy soils tend to provide little P retention

capabilities and runoff is likely (Penn and Sims, 2002). Soils that are saturated with P

may also benefit from WTR application. It has been shown that P saturated soils are









unable to hold added P and thus will result in P ground water complications (Penn and

Sims, 2002). Added Al in the form of WTR may help depress P runoff by increasing soil

P retention capabilities (O'Connor et al., 2002; Penn and Sims, 2002). Publications in

2002 indicated a reduction in P leaching with the addition of Fe and Al from biosolids,

claiming that metal oxides formed lead to increased P retention (Soon and Bates, 2002).

Research at the University of Florida concluded that increases in dietary Al levels

reduced feed intake, gains and P plasma concentrations in sheep. The Al given to these

animals was in the form of AlC13. The impact of additional Al was not positive for

animal gains as ADG was 105 and 148 g/d for those consuming a high Al or a low Al

diet, respectively. When additional dietary P was given, the ADG increased, but it was

not as high as for animals not consuming any Al (Rosa et al., 1982). These results

demonstrate the capabilities Al had to lower P status in the animal, but it is unknown

what will occur if a less bioavailable form of Al is fed. Other mineral plasma

concentrations were also impaired with increased dietary Al. Magnesium content was

depressed in the kidneys, and bone of those animals receiving the high dietary Al (Rosa

et al., 1982). Similar results using Mg have also been documented at Rutgers University

in avian species. Young chicks and mallard ducks when fed high Al diets, as AlC13, had

a high incidence of P binding, lowered P serum levels, depressed growth, lowered tibia

weights and lower bone mineralization (Capdevielle et al., 1997).

Few studies have been conducted to determine the results of P accumulation,

ground water pollution, and the quantity of Al which is capable of binding P in WTR

when consumed by ruminants. The majority of studies in regards to WTR and Al content

have been focused on the ecological risks associated with plants in acidic soils






22


(O'Connor et al., 2002). Studies involving soil P binding mechanisms have also proven

to be helpful. Phosphorus absorption capacity was increased by 20 times with the use of

WTR when compared to high Al clay (Haustein et al., 2000).

Applications involving pollution control with the use of WTR fed to sheep will be

implemented here to compare the bioavailability of Al from WTR to an available source

of Al (AlC13) and evaluate how Al affects the performance of growing sheep.














CHAPTER 3
EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON
PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS

Introduction

Ingestion of highly available dietary Al (e.g. AlC13) by livestock may result in P

deficiency. Aluminum toxicity is often observed as a P deficiency (Valdivia, 1977).

Additionally, high amounts of bioavailable Al can also impact the status of Fe, Zn, and

Mg in sheep (Rosa et al., 1982).

Under grazing conditions, ruminants typically consume 10% to 15% of their DM

intake as soil (Field and Purves 1964; Healy, 1967; 1968). In sheep dietary Al

suppressed voluntary feed intake, feed efficiency, plasma P, growth, and gains (Rosa et

al., 1982). When additional P was ingested, these negative effects were less severe but

were still evident.

Water treatment residuals (WTR) are the byproducts from a water purification

procedure, and can contain high amounts of Al, Fe or Ca; here they contain high amounts

of Al and has a high P sorption capacity. The bioavailability of Al in WTR varies, but is

generally low and thought to be harmless (O'Connor et al., 2002). Since Al is highly

reactive and has been shown to chemically bind P, the administration of WTR on manure

containing soils could be a solution for P pollution of water systems by increasing soil P

retention capabilities (Penn and Sims, 2002). Concerns occur because of possible

ingestion of the WTR by grazing animals and the reaction of Al and P in a low pH









system. No previous research has been conducted to determine the potential toxicity of

WTR when directly consumed by grazing ruminants.

The purpose of this study was to determine if feeding growing lambs a bioavailable

source of Al (AlC13) versus a less available source of Al from a WTR would affect

growth, feed intake, plasma P levels, tissue concentrations, and apparent P absorption.

Materials and Methods

Animals, Diets, and Management

Forty-two, wether (30) and female (12), five to eight-mo-old lambs, (22 Suffolk

and 14 Suffolk-crosses) were utilized in a 111-d experiment at the University of Florida

Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted

from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d

zero. Lambs were shorn on d 42 in an attempt to combat heat stress and to increase

optimum feed intake. Prior to the experiment, lambs were vaccinated with an 8-way

Clostridial given as an injection of 2-mL, four wks apart (Ultra Choice 8; Pfizer Animal

Health, Exton, PA) and were dewormed, with two 1 mL doses of Ivermectin, two wks

apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, an amprolium solution

was given as an oral drench, lambs received 1 mL daily in a six d sequence (Corid 9.6%;

Merial, Duluth, GA). On d 21 the animals were dewormed orally with 5cc of

Fenbendazole, (Panacur; Pfizer Animal Health, Exton, PA) and again drenched with 1

mL of Corid from d 21 to 26 (Corid 9.6%).

The lambs were housed (seven to each pen), in covered, earth-floored wooden pens

(24 sq. m), bedded with pine wood chips with adequate bunk space and ad libitum water

and common salt. The University of Florida Institutional Animal Care and Use

committee approved the experimental protocol (D231) used in this study.









A corn-SBM basal diet was formulated to meet NRC (1985) requirements for CP,

TDN, vitamin, and minerals for lambs of this weight and age (Table 3-1). Prior to the

experiment, during a three wk adjustment period, lambs were fed the basal diet at 1200 to

1300 g/d per animal. During the experiment, the animals were fed once daily, 1300

to1600 g-lamb-d-1.

Lambs were stratified by sex and randomly assigned to six dietary treatments; 1)

control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4)

(5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The WTR used contained 7.8% total Al on a DM basis. Ten percent of each diet was

either sand, WTR, AlC13 or a combination of the three. The diet concentrations of Al

were 910, 2000, 2000, 4000, 8000, and 8000 ppm, (DM basis) respectively, for the six

diets.

On d 91, animals were placed into individual metabolic crates (1.4m2) to determine

apparent digestibility of P. During a subsequent 21 d crate confinement, all animals were

individually fed their respective experimental diets. Fresh feed was given ad libitum each

morning. Orts were weighed back daily. Individual feed intake, ADG, and BW

differences from wk 11 to wk 14, were evaluated.

Sample Collection, Preparation, and Analyses

Blood samples (jugular venipuncture) and lamb weights were collected on d 0 and

every 14 d thereafter. Blood was collected (10mL) with a 20 x 1 vacutainer (Vacutainer;

Becton Dickinson, Franklin Lakes, NJ) needle into evacuated tubes containing sodium

heparin. Immediately after collection, blood was centrifuged at 700 x g for 30 min, and

plasma was collected and frozen at 0 oC. After a 30 min thaw period, to allow plasma to









reach ambient temperature the proteins were separated using 10% trichoroacetic acid

(Miles et al, 2001).

On d 91, wether lambs were fitted with cloth fecal collection devices for the study

of apparent digestibility of P. Feces were collected daily for 14 d and composite samples

were frozen at 0 C. Each composite sample was sub-sampled and ground in a blender

with stainless steel blades. Feces were then dried for 16 h at 1050C to determine DM.

Samples were then ashed in a muffle furnace at 6000C for 8 h, digested in HC1, filtered,

and diluted for colorimetric P determination (Harris and Popat, 1954).

On d 111, all animals were sacrificed at a USDA approved facility. The following

tissues were collected and analyzed for Al, Ca, Cu, Fe, Mg, Mn, P, and Zn contents:

blood plasma, liver, heart, kidney, and brain and Se was analyzed for the kidney. Samples

were dried, weighed, ashed, and solubilized in HNO3 acid (Miles et al., 2001). Bone was

analyzed for P, Ca, and Mg. For all samples, P was analyzed using a colorimetric

procedure (Harris and Popat, 1954). Kidney Se was determined using fluorometric

procedures (Whetter and Ullrey, 1978). Calcium, Fe, Mg, Cu, Mn, and Zn in tissues and

feed samples were analyzed by flame atomic absorption spectrophotometry (Perkin-

Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT). Aluminum concentrations were

analyzed in diets, heart, brain, liver, and kidney by atomic absorption spectrophotometer

using nitrous oxide-acetylene flame (Varian SpectrAA 220 FS; Varian Inc., Walnut

Creek, CA).

Statistical Analysis

Soft tissue, fecal, and feed intake data were analyzed for treatment effects using

PROC GLM in SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely

randomized design. PROC MIXED of SAS was used to analyze treatment effects on









BW, ADG, and plasma P as repeated measures with a variance component covariance

structure in respect to d and subplot of animal nested within treatment. Significance was

declared at P < 0.05 and tendencies were discussed when P < 0.15.

Results

Six animals died during the experiment. The cause of death was determined to be

parasite infestation of the gastrointestinal tract, and was deemed unrelated to dietary

treatment. Body weights increased for all treatments for wks 0 to 14 (Table 3-2).

Average daily gains (Table A-i) and feed intakes (Table 3-3) also increased with time (P

< 0.05). Throughout the experiment, lambs fed 2,000 ppm Al via AlC13 consistently had

numerically lower BW than all other treatments. During wk 6, lambs fed 2,000 ppm Al

via AlC13 had lower BW than control animals and, lambs fed 2,000 ppm Al, 4,000 ppm

Al or 8,000 ppm Al from WTR (P < 0.05). Lambs receiving 2,000 ppm, 4,000 ppm and

8,000 ppm Al via WTR were heavier than animals consuming 2,000 ppm Al via AlC13

during wk 11 (P < 0.05). Body weights during wk 11 differed by 11.3 kg, (P < 0.05)

between those animals consuming 2,000 ppm Al via AlC13 and those fed 8,000 ppm Al

via WTR, whereas the difference between these two groups at wk 14 was 7.4 kg (P=

0.008).

During wk 2, ADG of lambs given 8,000 ppm Al from WTR exceeded animals

given 2,000 ppm Al via AlC13 (P < 0.05). Lambs receiving 4,000 ppm Al from WTR

tended (P = 0.11) to gain more than lambs fed 2,000 ppm Al via AlC13. During wk 4

lambs receiving the control, 2,000 ppm Al via WTR and 8,000 ppm Al via WTR

treatments had higher gains than lambs in the treatment given 2,000 ppm Al via AlC13 (P

< 0.05). During wk 6, lambs consuming the control, and 4,000 ppm Al from WTR diets

gained more than lambs consuming 2,000 ppm Al from AlC13 (P < 0.05). Additionally,









during wk 6, all treatments, except 2,000 ppm and 4,000 ppm Al via WTR had gains

much lower than the control (P < 0.05).

During wk 11, animals began a 3-wk individual feeding regime to determine feed

intake. From wk 11 to wk 14, lambs fed the control, 2,000 ppm, and 4,000 ppm Al via

WTR (P < 0.05) consumed more than those fed 8,000 ppm Al via WTR.

Observations of plasma P during wk 4 (Table 3-4) showed that animals receiving

2,000 ppm Al via WTR had higher concentrations than all other treatments, except those

receiving 8,000 ppm Al via WTR plus double the minerals and vitamins (P < 0.05). The

animals receiving 8,000 ppm Al via WTR had lowest plasma P of all groups of animals

(P < 0.05). In wks 6 to 11, the lambs receiving 2,000 ppm Al via AlC13 had lower plasma

P than controls (P < 0.05). During wk 11, both the control and lambs receiving 2,000

ppm Al via WTR had higher plasma P than animals receiving 2,000 ppm Al via AlC13 (P

< 0.05). Analyses of plasma P during wk 14 showed that controls had higher P

concentrations than lambs receiving 4,000 ppm Al via WTR, 8,000 ppm Al via WTR or

8,000 ppm Al via WTR plus two times the amount of added mineral-vitamin premix, and

1.29% dicalcium phosphate (P < 0.05). Plasma evaluations of all other minerals showed,

no differences among treatments, which included the following (.g/ml): Ca 87 to 101,

Mg 17 to 21, Cu 1.3 to 1.5, Fe 0.9 to 2.0, Mn 0.05 to 0.06, and Zn 0.3 to 1.6.

Tissue mineral concentrations (Table 3-5) among treatments were deemed not to be

hazardous to animal health. With the exception of Cu, tissue mineral concentrations

remained within normal ranges (Miles et al., 2001). Liver Cu concentrations were high

for all treatments. The mineral-vitamin premix used, inadvertently contained excess Cu in

relation to sheep requirements. Levels of P showed no differences among treatments









except that animals given 4,000 ppm Al from WTR deposited more P in the kidney than

those animals receiving 8,000 ppm Al from WTR (P < 0.05). No differences (P < 0.05)

were observed in soft tissue or bone Ca concentrations. Aluminum was deposited in

lower amounts in the brain for lambs fed 2,000 ppm Al via WTR than all other treatments

except the control (P < 0.05). Kidney Al deposits were higher in lambs receiving 2,000

ppm Al via AlC13 than those receiving 8,000 ppm Al via WTR (P < 0.05), and those

receiving 8,000 ppm Al via WTR plus two times the added amount of mineral-vitamin

premix, and 1.29% dicalcium phosphate (P < 0.05). Concentrations of Mg showed no

differences in soft tissue deposition. Differences in Fe deposition were observed in liver

(P < 0.05), with lambs consuming the AlC13 treatment having lower Fe concentrations

than those receiving the two treatments of 8,000 ppm Al as WTR. Variations in heart and

kidney Mn concentrations seemed unrelated to Al source or quantity.

Apparent P absorption ranged from -12.9 to 31.8 % (Figure 3-1). The control and

all WTR treatment lambs had a greater apparent P absorption (10.9-31.89%) than the

negative absorption (-12.9%) of lambs fed 2,000 ppm Al via AlC13 (P < 0.001).

Discussion

Increases in BW, ADG and intakes were observed for all treatments and can be

likely attributed to increased appetite which occurs in growing animals. The previous

studies at the University of Florida conducted by Valdivia et al. (1978; 1982) observed an

increase in feed intake from 1.03 to 1.20 g/d, and an increase in BW gain as dietary P was

increased from 0.15 to 0.29 % in diets that contained 1,200 ppm to 2,000 ppm Al as

A1C13. Valdivia et al. (1978) and Rosa et al. (1982) concluded that the increase in P was

able to overcome the clinical signs normally observed with Al toxicosis. Diets in the

present study contained approximately 0.25% P as fed (Table 3-1), which exceeds the









requirements (0.23% dietary P) of lambs of this age and breed (NRC, 1985; McDowell,

2003). Our study showed no major losses in weight or intakes regardless of treatment,

which seems to be attributed to the proper amounts of dietary P (0.25%) supplied. This

concurs with the work of Valdivia et al. (1978) and Rosa et al. (1982).

The control lambs, which received 910 ppm Al from sand, and lambs receiving

treatments containing WTR had no declines in intake. This is likely attributed to the low

bioavailability of Al in WTR and sand (O'Connor et al., 2002; Dayton et al., 2003). A

low bioavailable Al source is much less likely to depress intake because the Al would not

readily react with the P in the gastrointestinal tract.

Aluminum from AlC13 is an available source and has been shown to depress

intakes. Declines in intakes caused by ingestions of an available Al source have been

observed in various species including: sheep (Valdivia et al., 1978; Rosa et al., 1982),

broilers and chicks (Fethiere et al., 1990), humans (Chappard et al., 2003; Rengel, 2004)

and rats (Gomez- Alonso et al., 1996). An Al toxicity results in a P deficiency

(McDowell, 2003) which can lead to serious tissue damage, lower intakes and gains.

Williams et al. (1990; 1991a,c; 1992) induced a P deficiency in heifers and observed an

11% decrease in feed intake. In the present study, there was a decrease in feed intake for

the lambs that were fed 2,000 ppm Al via AlC13. This is expected, as AlC13 is considered

to be a bioavailable source of Al (Valdivia et al., 1978; Rosa et al., 1982), and thus may

induce a P deficiency and depress feed intake.

Ingestion of Al as AlC13 by ruminants decreases bone density, plasma P levels, feed

intakes and gains (Rosa et al., 1982; Valdivia et al., 1982; Ammerman et al., 1984).

Animals receiving the AlC13 diet repeatedly had lower BW and feed intakes than animals









fed other sources of Al. Lower intakes and gains can be attributed to Al availability,

similar observations occurred when 0.75% aluminosilcate was fed to laying hens and

feed intake was significantly depressed (Fethiere et al., 1990).

One of the objectives of the present study was to compare the availability of Al in

WTR to Al in AlC13 and a control when fed to ruminants. During wk 11, body weights

ranged from 36.8 kg for lambs fed 2,000 ppm Al via AlCl3 diet to 48.1 kg for lambs fed

8,000 ppm Al via WTR. Thus, lambs receiving 8,000 ppm Al from WTR, on average,

had BW that were 11.2 kg heavier than those fed 2,000 ppm Al from AlC13 despite the

four fold difference in total Al administered. The group fed 8,000 ppm Al from WTR had

the highest amount of Al and the largest percentage of WTR (10% of the diet as fed).

Differences observed in BW, between lambs fed 8,000 ppm Al via WTR and 2,000 ppm

Al via AlCl3 validates previous studies which showed Al in Al-WTR to be high in a non-

available source of Al (O'Connor et al., 2002; Novak and Watts, 2004) and that AlC13 is

available for uptake in the small intestine (Valdiva et al., 1978; Rosa et al., 1982). It is

thought that grazing ruminants can consume up to 10-15% of their total DM intake as soil

(Healy 1967; 1968). It has also been shown that soil Al is often consumed by grazing

ruminants in amounts as high as 10% of the soil consumed. Aluminum ingested from

soil sources has not been shown to reduce performance. Ammerman et al. (1984) fed

sheep varying soils types, from Latin America, containing as much as 16,600 ppm Al.

They concluded that the soil Al sources had no significant effect on BW, gains, and

intakes of the sheep which consumed them. The soils contained various levels of Al or

Fe oxides, which is similar to the chemical form of Al from WTR. The additions of high

Fe and Al soils had no harmful effects on P utilization, feed intake, or gains.









Differences, in general, between treatments were limited throughout the trial.

Lambs receiving diets containing Al via WTR at varying levels showed no differences in

BW from the control (P < 0.05). Additions of WTR in amounts as high as 10% of the

diet, and representing 8,000 ppm Al in the diet, do not negatively impact growing lambs

in relation to BW, ADG, and feed intakes when dietary P is at least 0.25%. Thus, under

natural grazing conditions, [where 10% of the DM intake is of soil (Field and Purves,

1964; Healy 1967; 1968)], even high rates of surface applied WTR are not expected to

harm animal performance.

During wk 14, the ADG of treatments plateaued, consistent with a natural

sigmoidal growth curve. Prior to wk 14, animals were gaining at rates between 463 to

593 g per d. The rate declined during wk 14 to only 207 to 244 g per d, but the decline is

not attributed to dietary treatments. Animals appeared healthy with notable

accumulations of body fat. Lambs in both the control and AlC13 treatment continued to

gain larger amounts of weight during wk 14, because they had not reached a maintenance

weight. Lambs fed 2,000 ppm Al via AlC13 had lowered growth, intake and BW

throughout the trial and had not reached a growth plateau by wk 14. The control animals

during wk 6 experienced an illness which was attributed to parasite infestations which

suppressed ADG means thereafter. In previous studies, similar declines in ADG were

observed with AlC13 additions, and animal growth peaked at later dates than those not

receiving an Al source (Valdivia et al., 1978; Rosa et al., 1982; Fethiere et al., 1990).

Intakes, regardless of treatment, increased with time. Constituents added to the

basal diets did not cause any animals to become anorexic, a common clinical sign of Al

toxicity, or P deficiency (Williams et al., 1992; McDowell, 2003). Differences in intakes









were evaluated individually in a 3-wk period between wk 11 to 14. Prior to this date,

lambs had been group fed. Individual intake data were similar to those reported by Rosa

et al. (1982), and Valdivia et al. (1978). Lambs fed diets containing 2,000 ppm Al from

A1C13 consumed less than the control (P > 0.05), which can again be attributed to the high

bioavailability of AlC13. Intakes were the lowest for animals consuming 8,000 ppm Al

from WTR. During wk 14, these animals had the highest BW, but a decline in ADG

from wk 11 (480 g) to wk 14 (207.0 g), which was the lowest gain for that period.

Intakes for lambs receiving 8,000 ppm Al from WTR were lower than the control, 2,000

ppm and 4,000 ppm Al from WTR (P < 0.05), but higher than lambs receiving 2,000 ppm

from AlC13 or 8,000 ppm Al from WTR with additional minerals and vitamins, (P >

0.05). Prior to wk 14, lambs fed 8,000 ppm Al from WTR showed adequate performance

in relation to gains, intake and BW. Therefore, the cause of these declines seen in lambs

receiving 8,000 ppm Al via WTR are unknown and could be related to normal growing

patterns, an unknown parasite infestation, Cu toxicities, Al toxicities, or other various

environmental interactions.

During wk 4, lambs receiving 2,000 Al from WTR had the highest concentration of

plasma P and differed from the control, those receiving 2,000 ppm Al from AlC13, 8,000

ppm Al from WTR. (P < 0.05). Huff et al. (1996) administered 3.7% aluminum sulfide

to broiler chicks and observed a declines in serum P after a 3 wk period. Lambs in the

present study, had plasma P levels decline from 54.2 [g/ml to 19.6 [g/ml, between wk 4

and wk 8. Additionally, all treatments showed declines in plasma P during this period,

but the AlC13 treatment declines were most often the greatest. During wk 11 and 14,

plasma P concentrations began to increase in all treatments. One could conclude that









plasma P concentrations declined to levels which demanded the use of body stores of P

(Williams et al., 1990; McDowell, 2003). Bone mineral content was evaluated in the

long bones, with no differences among treatments and no evidence of a mineral

depression; yet research has shown that the ribs and the vertebrae are first to become

depleted in mineral concentrations (Williams et al., 1990; 1991a; McDowell, 2003).

Therefore the possibility exists that increases in plasma P levels during wk 11 to 14

occurred from bone mineral resorption. This is unlikely, but not unreasonable, because

within a long time frame of 8 wk (between wk 6 and wk 14) bone loss most likely would

have been observed in the long bone of the leg which was analyzed. Previous

experimental data have not demonstrated similar results by showing an increase in

plasma P after a decline. Therefore, observations are speculative at this time and further

research is needed to validate this theory.

Tissue mineral concentrations analyzed for this study were in the normal ranges for

lambs of this breed and age (Miles et al., 2001; McDowell 2003). Previous research

found differences in kidney, bone, liver and spleen concentrations of Al, Fe, P Mg and Zn

(Rosa et al., 1982) and Ca (Rosa et al., 1982; Zafar et al., 2004) when various amounts of

Al were fed. In the present study, Al concentrations differed in brain, heart, liver, and

kidney, Mg in bone, and Fe in the liver. Absorption of Al in mongastrics is

approximately 0.1% (Rengel, 2004) and is thought to be even lower in ruminants

(Valdivia et al., 1970; 1978). Aluminum accumulation occurs most readily in the brain.

The exact mechanism is unknown but Al can cross the blood-brain barrier (Rengel,

2004). Accumulations of Al in brain tissue were greater from lambs fed 2,000 ppm Al

from AlC13, than from lambs fed 2,000 ppm from WTR (P < 0.05). Aluminum









concentrations in brains increased when Al from WTR was fed at levels higher than

2,000 ppm, but did not differ from the control. Liver depositions of Al were highest in

lambs fed 8,000 ppm Al from WTR (P < 0.05). In the kidney, the highest concentrations

of Al were detected when lambs were fed 2,000 ppm Al from AlC13, and differed from

both treatments receiving 8,000 ppm Al from WTR and from the control and 2,000 ppm

Al via WTR (P < 0.05). Rosa et al. (1982) observed increases in Al tissue concentration

as Al consumption increased, which was not consistently observed in our study.

Additionally, soft tissues, except brain matter, that have been evaluated in past studies

have not been shown to accumulate large amounts of Al during short time periods

(Rengel, 2004), and may not prove to be useful for determination of differences of any Al

sources and levels.

Apparent P absorption from a 14 d fecal collection showed differences among all

five treatments versus the treatment containing 2,000 Al via AlC13. Studies by Valdivia

et al. (1982) observed a marked decrease in P absorption and net P retention in lambs fed

2,000 ppm Al as AlC13. Negative apparent P absorptions were observed in all groups

except those given high P with low Al. When 0.29% P was fed with no dietary Al, the

mean apparent absorption was unaffected. In our study, the control had an apparent

absorption of 22.5%, and the mean for all the WTR groups was 21.2%. This suggests

that Al in WTR did not negatively impact or reduce dietary P absorption. Valdivia et al.

(1982) found a negative apparent P absorption (-10.7%) when 0.29% dietary P and 2,000

ppm Al as AlC13 were fed to sheep. Additionally, Martin et al. (1969) conducted P

retention studies using dietary applications of a hydrated Al source and discovered that

when Al was fed to sheep, retained amounts of P decreased linearly, as Al fed increased









from 910, 2000, 4000, 8000, and 8000 ppm. Similar results were observed in our study

when 2,000 ppm Al was added via AlC13 to the basal diet which contained 0.25% P. The

apparent P absorption averaged 12.9% at wk 14 and therefore suggested a negative

impact on dietary P utilization with added Al as AlC13.

Summary and Conclusions

A 14 wk experiment was conducted using 36 lambs. Individual feeding was

recorded between wk 11 to 14. Diets, containing 0.25% P (as fed), included 1) control

(10% sand), 2) (9.7% sand and 0.3% AlC13), 3) (2.5% WTR and 7.5% sand), 4) (5%

WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, double

the added quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The Al varied from 910 to 8,000 ppm among diets. Lambs fed the control and WTR had

no decline in intake, but the AlC13 lambs repeatedly had lower BW and intakes. The

WTR contain a non-available source of Al and did not cause performance declines

Additions of this WTR respecting Al concentrations as high as 8,000 ppm, did not

negatively impact growing ruminants in relations to BW, ADG, and intakes. During wk

6, all treatments showed declines in plasma P, but the AlC13 treatment was often the

lowest, and during wk 11 plasma P began to increase.

Accumulations of Al in the brain were greatest for lambs given 2,000 ppm Al from

A1C13 and increased numerically when Al as WTR was fed at levels higher than 2,000

ppm. With the exception of the brain, soft tissues did not accumulate large amounts of Al

during this 14 wk experiment.

Apparent P absorption from a 14 d metabolic study was positive (10.9-31.8%) for

all lambs fed the control and various levels of WTR. However, lambs that received 2,000

ppm Al via AlC13 had a negative P absorption of -12.9 %. This was a lowered (P < 0.03)









P absorption compared to all other treatments. Aluminum, as AlC13, fed at 2,000 ppm

reduced dietary P retention, but varying amounts of Al as WTR had no effect on P

apparent absorption with similar absorption rates as the control. Therefore when dietary

P is supplied in amounts of 0.25% or higher, Al (as WTR) fed to lambs in amounts as

high as 8,000 ppm did not negatively impact the feed intake, gain, BW or P absorption.

Implications

Dietary administration of AlC13 has negative impacts on ADG, BW, feed intake,

apparent absorption of P, and P plasma concentrations. Lambs fed WTR had apparent P

absorption percentages that were similar to the control and were higher than the AlC13

treatment. Water treatment residuals are not harmful when consumed in amounts up to

8,000 ppm Al, when P is supplied in amounts of 0.25%, and do not negatively affect

gain, feed intake, BW, or P availability.










Table 3-1. Diet composition (as-fed) and analyses for average (n=18) concentrations
for macro- and micro-elements for treatments
Treatments


Ingredient (%,as fed)
Ground Corn
Soybean hulls
Wet molasses, unfortified
Cottonseed hulls
Corn gluten meal, 60% CP
Alfalfa meal, 17% CP
Vegetable oil (soybean)
Sandb
Water treatment residual
Aluminum chloride
Salt
Urea
Ground limestone
Ammonium chloride
Flowers of sulfur
Mineral-Vitamin-premixd
Dicalcium phosphate

Analyses (ppme)
Ca
Mg
Na
K
P
Al
Co
Cu
Fe
Mn
Zn


1
41.1
12.5
10.0
8.0
5.5
5.0
4.0
10.0


1.0
1.6
0.7
0.5
0.01
0.01



7,170
2,780
4,060
4,180
2,520
910
7
31
66
11
74


2
41.1
12.5
10.0
8.0
5.5
5.0
4.0
9.3

0.7
1.0
1.6
0.7
0.5
0.01
0.01



7,120
2,730
3,240
5,210
2,490
2,320
5
33
65
13
70


3
41.1
12.5
10.0
8.0
5.5
5.0
4.0
7.5
2.5

1.0
1.6
0.7
0.5
0.01
0.01



7,220
2,700
3,030
3,960
2,550
2,270
6
33
67
13
71


4
41.1
12.5
10.0
8.0
5.5
5.0
4.0
5.0
5.0

1.0
1.6
0.7
0.5
0.01
0.01



7,440
2,880
3,000
4,560
2,480
3,970
5
32
66
13
70


5 6
41.1 39.9
12.5 12.5
10.0 10.0
8.0 8.0
5.5 5.5
5.0 5.0
4.0 4.0

10.0 10.0

1. 1.0
1.6 1.6
0.7 0.7
0.5 0.5
0.01 0.01
0.01 0.02
1.3


7,300
2,870
3,410
4,440
2,460
7,860
5
34
66
14
67


10,000
3,020
3,110
4,380
5,020
7,790
8
42
70
19
79


aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2)
9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR +
two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3
were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and
treatments 5 and 6 were formulated to contain 8,000 ppm Al.
b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001
% Zn.
' Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn,
0.73 % S, 0.006 % Cu, and 0.002 % Zn.
dNlineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 %
Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as
oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1
IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier.
eDry matter basis.









Table 3-2. Effects of dietary Al concentration and source on BW of feeder lambsa
Treatment
1 2 3 4 5 6 SE
BW, kg
Wk
0 32.6 31.4 33.1 31.1 32.2 31.7 1.5
2 32.8cd 31.6c 34.6cd 34.4cd 37.5d 33.6cd 1.9
4 34.3cd 31.6c 37.7de 35.8cde 41.3e 35.2cd 2.3
6 38.4d 32.3c 40.7d 39.3d 41.2d 34.7cd 2.6
11 41.4cd 36.8c 46.7d 45.1d 48.1d 42.9cd 2.5
14 49.3cd 45.9c 52.8d 50.3d 53.3d 49.7cd 1.9
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"deMeans within rows lacking a common superscript differ (P < 0.05).








Table 3-3. Effect of dietary Al concentration and source on feed intake of feeder lambsa
Treatment
1 2 3 4 5 6 SE
intake, g-lamb1-ld-1
Wk
2 827 959 1170 1120 1100 1020
4 1410 876 1150 1150 1070 1120
6 954 1110 1150 1200 1210 1210
11 1610 1460 1790 1550 1910 1940
14 1940c 1870cd 1900c 1940c 1270d 1570cd 54.6
aData represent means of intake during wk 0 to 11 and least squares means during wk 14;
n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
cd Lambs were individually fed for 3 wk ending at wkl4; Means within rows lacking a
common superscript differ (P < 0.05).









Table 3-4. Effect of dietary Al concentration and source on plasma P of feeder lambsa
Treatment
1 2 3 4 5 6 SE
--g/ml,
Wk
0 48.7 44.6 51.2 40.8 45.6 43.8 3.7
2 48.3 44.7 50.5 41.5 45.7 44.1 5.3
4 54.3d 54.2d 64.4c 49.8d 39.7e 58.5cd 4.6
6 39.2c 25.2d 27.9d 28.5d 26.2d 27.0d 2.5
8 33.8c 19.6d 19.9d 26.3cd 21.9d 28.1cd 2.3
11 36.2cd 22.2e 46.3c 30.0de 29.0de 29.3de 5.0
14 38.3c 34.0cd 34.9cd 28.0de 24.5de 24.9e 3.6
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"deMeans within rows lacking a common superscript differ (P < 0.05).











Table 3-5. Tissue mineral composition resulting from experimental diets
Treatment


-Macro elements, (o%)


0.01
0.08
0.04
38.1

0.15
0.11
0.066
0.79c


0.01
0.08
0.05
39.5

0.14
0.13
0.070
0.79c


1.16 1.16
1.21cd 1.17
1.07 1.09
14.6 15.4
-Micro elements, (mg/kg)


Ca
Heart
Kidney
Liver
Bone
Mg
Heart
Kidney
Liver
Bone


0.01
0.06
0.05
39.9

0.15
0.11
0.066
0.79c

1.16
1.12c
1.07
14.2


43.1cd
6.9cd
7.2cd
15.4

8.7
38.0
4,090

152
443
212cd

1.6cd
18.3cd
12.8

1.1


0.01
0.04
0.04
39.4

0.13
0.11
0.062
0.70cd

1.03
1.15cd
1.17
14.4


52.0d
5.1 def
9.4
22.3cd

12.7
37.3
4,570

143
442
141d

1.4d
23.2cd
11.4

1.2


0.01
0.06
0.04
37.0

0.12
0.10
0.062
0.73cd

1.09
1.15 d
1.05
14.1


47.5d
7.0cd
5.4d
25.3d

10.3
31.2
3,270

162
434
262c

1.57cd
23.9cd
11.9

1.1


Heart
Kidney
Liver


71.5
83.9
48.5


63.6
110
48.4


65.7
120
44.8


69.1
110
43.5


59.8
85.1
36.8


6.65
15.0
6.91


aData represent least squares means; n = 5, 5, 7, 7, 5, and 6 for the control and treatments 1-
5, respectively
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
"defMeans within rows lacking a common superscript differ (P < 0.05).


50.4 d
7.4
7.5d
20.9cd

14.4
36.1
3,930

146
447
162cd

1.31d
23.2d
10.2

1.1


Heart
Kidney
Liver
Bone


Al
Brain
Heart
Kidney
Liver
Cu
Heart
Kidney
Liver
Fe
Heart
Kidney
Liver
Mn
Heart
Kidney
Liver
Se
Kidney


33.9
6.1 cdef
7.1cd
16.7

9.8
46.9
3,080

154
425
208cd

1.80C
16.4c
14.1

1.3


1.2 0.07


0.02
0.05
0.05
37.7

0.13
0.10
0.064
0.61d

1.01
1.14cd
1.01
15.1


48.2d
4.5ef
5.4d
18.5cd

7.4
27.9
3,900

134
434
226c

1.50cd
18.4cd
11.4


0.004
0.02
0.01
18.3

.019
0.02
0.004
0.04

0.14
0.087
0.18
0.63


4.10
0.79
1.03
2.75

2.07
9.79
713

10.9
66.4
27.8

0.13
2.4
2.0










40.0


31.8


b
22.5








12.9

-12.9


18.2


24.0 b

b
10.9



5 6


Treatments

Figure 3-1 Effect of dietary Al source on apparent P absorption. Dietary treatments were
created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3%
sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR;
6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29%
dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al,
treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al. The SE for treatments is 8.23.
aMeans lacking a common superscript differ (P < 0.05).


30.0

20.0

10.0

0.0

-10.0


-20.0














CHAPTER 4
EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS
ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS

Introduction

Animal waste, which is often applied to grazing land, contains P that can remain in

the A horizon of the soil profile and may lead to water pollution (Haustein et al., 2000).

Aluminum (Al), if applied to the land, is thought to complex with P and to reduce the

soluble P concentrations in animal waste (O'Connor et al., 2002). This type of reduction

in soluble P levels of animal waste products could result in significant decreases in

pollution by commercial livestock operations.

Previous studies suggest that the application of Al containing water treatment

residuals (WTR), a byproduct of water purification, increases the soil's capacity to bind

and retain P (Elliott et al., 2002). The chemical mixture of soil and WTR has been shown

to increase the retention value of soil P by several fold (Novak and Watts, 2004) and

could result in a possible solution to environmental concerns associated with animal

waste disposal. A major concern is that WTR, containing high amounts of Al, may

adversely affect P utilization and bone deposition in grazing livestock that inadvertently

consume WTR.

Highly available dietary Al may create unabsorbable P complexes in the intestinal

tract and, Al toxicosis is often observed as a P deficiency (Valdivia, 1977). Diets fed to

sheep containing 0.29% P and 2,000 ppm Al via AlC13 resulted in reduced bone density

(Rosa et al., 1982). Additionally, high amounts ofbioavailable Al can also negatively









impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). The bioavailability of Al

varies in different WTR, but is generally much lower than Al compounds such as AlC13

(O'Connor et al., 2002). The purpose of this study was to determine the effect of dietary

Al as WTR and AlC13 on bone mineral content (BMC) and bone density in feeder lambs.

Materials and Methods

Animals, Diets, and Management

Forty-two, (30 wether and 12 female) five to eight-mo-old lambs, (22 Suffolk and

14 Suffolk-crosses) were utilized in a 14 wk experiment at the University of Florida

Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted

from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d

zero Prior to the experiment, lambs received an 8-way Clostridial vaccination given as

injections of 2 mL four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and

were dewormed, with two 1 mL doses with Ivermectin, two wks apart (Ivomec; Merial

Ltd., Iselin, NJ). To prevent coccidiosis, amprolium (Corid 9.6%; Merial, Duluth GA)

was used as an oral drench with lambs receiving 1 mL daily in a five d sequence.

Corn-SBM basal diets were formulated to meet NRC (1985) requirements for CP,

TDN, minerals and vitamins for lambs of this weight and age (Table 4-1). Lambs were

fed the basal diet at 1200 to 1300 g-lamb--d-1 during a 3 wk adjustment period and 1300

to 1600 g-lamb-l-d- throughout the experiment. Lambs were stratified by sex and

randomly assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and

0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR

and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-

vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain

7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the









combination of the two. The diet concentration of Al were 910, 2000, 2000, 4000, 8000,

and 8000 ppm respectively for the six diets.

Lambs were housed (seven to each pen) covered, in earth-floored wooden pens (24

sq. m), which were bedded with pine wood chips. Animals were group fed in open

troughs until d 84, when animals were placed in individual raised metabolic crates. All

lambs had access to salt and adlibitum water. Dietary treatments were offered adlibitum

and DM intake was monitored daily. Diets were not reformulated during the study.

Sample Collection and Analyses

Radiographic photometry was used to estimate bone mineral content (BMC) at 28

d, 69 d and 109 d. For each lamb, the left dorsal/palmer, third metacarpal region of the

leg was radiographed with the use of a portable x-ray machine, (Easymatic Super 325;

Universal X-Ray Products, Chicago, IL). The machine was set at 97 pkv, 30 ma, and

0.067 sec. One cm below the nutrient foramen of the third metacarpal, a cross section of

the cannon bone was compared to the standard using the image analyzer and BMC was

estimated by photodensitometry. A ten-step Al wedge, taped to the cassette parallel to

the third metacarpal, was used as a standard in estimating BMC. Radiographs were taken

with a 91.5 cm distance between the x-ray machine and the cassette (Meakim et al., 1981;

Ott et al., 1987). The films were processed with an auto-radiograph processing machine,

with Kodak products, and by Kodak development procedures (Eastman Kodak Co.,

Rochester, NY).

Radiographs were evaluated with a photometer (Photvolt Corp., New York, NY);

percentage light transmissions (%T) were used to determine solid matter. The

radiographs were zeroed using the thinnest Al step that could be distinguished as a









differing shade from the next ascending step. The BMC was evaluated 2 cm descending

from the nutrient foramen. Bone diameter and medullar width were taken to the nearest

0.2 mm using a plastic transparent ruler (Meakim et al., 1981). Determination of the %T

reading was then analyzed graphically using a logarithmic calculation. The width of the

bone segment, 2 cm below the nutrient foramen, was compared to the visible segments of

the Al step wedge.

On d 111, all animals were sacrificed at a USDA inspected facility and bone from

left leg was removed for bone mineral and bone density analysis. To prepare bone for P,

Ca, and Mg analyses, bone removed the left dorsal/palmer, third metacarpal region of the

leg, was skinned, immediately wrapped in 0.9% saline-soaked cheese cloth and frozen at

0 C. After thawing, bone was cut into 2 cm sections, 2 cm below the nutrient foramen,

and marrow was carefully removed. Samples were rinsed in deionized water and blotted

dry. Specific gravity procedures (Kit ME-40290; Mettler Instruments Corp., Hightstown,

NJ) were used to determine bone density (g/cm3) as described by Williams et al. (1990,

1991c).

Bone samples were then dried at 105 oC for 16 h, extracted with an ether soxhlet

apparatus for 48 h, air dried for 10 h, and then oven dried at 1050C again for 16 h. Dry

samples were weighed, and ashed in a muffle furnace at 6000C for 8 h. All P samples

were analyzed with colorimetric procedures (Harris and Popat, 1954), while Ca and Mg

were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model

5000, Perkin-Elmer Corp., Norwalk, CT).









Statistical Analysis

Bone density and BMC data were subjected to the GLM procedure of SAS (SAS

for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized arrangement.

Significance was declared at P < 0.05, and tendencies were recognized at P < 0.15.

Results

Radiograph BMC

At d 28, lambs receiving 2,000 ppm Al from WTR tended (P = 0.07) to have

greater bone density than lambs receiving 2,000 ppm Al as AlC13 (Table 4-2). Likewise,

lambs receiving 8,000 ppm Al as WTR tended (P = 0.11) to have denser bone then those

receiving 2,000 ppm Al via AlC13. Overall, bone density was unaffected (P = 0.51) by

treatment at d 69, as only lambs receiving 4,000 ppm Al from WTR had bones which

tended to be more dense than the controls. Near the end of the study, (d 109) bone

density was again unaffected by dietary Al content. Only bones from lambs receiving

8,000 ppm Al as WTR tended to be more dense (P = 0.15) than the controls, and no other

difference or tendencies were observed.

Bone Density via Specific Gravity

Bone density as determined by specific gravity (Williams et al., 1990; 1991) was

unaffected by treatment (P = 0.43). Bone density ranged from 1.88 to 1.94 g/cm3 and

bones from lambs receiving 2,000 ppm Al as WTR tended to be more dense (P = 0.07)

than bones from lambs receiving 4,000 ppm Al from WTR.

Bone Mineral Analyses

Bone mineral percentages (Table 4-3) of P and Ca were unaffected by treatment.

Bone mineral percentages of Ca were also unaffected by treatment (P = 0.87). Bone Mg

content differed (P < 0.05) in the lambs receiving 8,000 ppm from WTR plus double the









minerals and vitamins, having lower bone deposits of Mg than those fed the control,

2,000 ppm Al via WTR, or 4,000 ppm Al via WTR (P < 0.05). Additionally, lambs

receiving 4,000 ppm Al from WTR tended to have higher bone Mg content than lambs

fed 8,000 ppm Al from WTR (P = 0.06).

Discussion

Radiographing techniques and specific gravity measurements revealed that dietary

Al content had no effect on BMC or bone density. Bone density and P deposition is

expected to decline when additional Al is ingested according to research conducted by

Valdiva et al. (1977) and Rosa et al.(1982). Diets formulated with high Al content (up to

2,000 ppm) in previous work with mallard ducks and chicks (Capdevielle et al., 1998)

and rats caused a decline (Gomez- Alonso et al., 1996; Zafar et al., 2004) in bone mineral

declines with Al dietary additions. Yet, contradictory results have been described in

much of the research conducted with ruminant species ( Valdivia et al., 1978; 1982; Rosa

et al., 1982) Valdivia (1982) concluded that ruminants are less susceptible to toxic effects

of Al than in other species. Normally, bone ash is 17.6 % P, and 37.7 % Ca (McDowell,

2003). Studies conducted by Validiva et al. (1982) showed bone mineral percentages of

P that were slightly below average, 14 to 15%, as is seen in our study. However in the

present study, differences were not observed among dietary treatments. Since differences

were not observed, there was no notable effect of dietary Al intake on bone mineral

deposition. The long bones of the appendages are often the last affected by P deficiencies

(Williams et al., 1991a,b,c; McDowell, 2003). This could be a logical justification for

the lack of treatment effect in the present study. A secondary explanation is that the

dietary levels of P (0.25%), as seen in intake studies by Rosa et al. (1982), were high

enough to compensate for the Al additions to the basal diet. Rosa et al. (1982) also









reported that bone ash Ca levels were unaffected by dietary Al and ranged from 35 to 36

%, similar to results in our study which ranged from 33.1 to 39.9 % for bone ash Ca.

Studies conducted with rodents (Cox and Dunn, 2001; Zafar et al., 2004) found that Ca

deposits in the bone declined when dietary levels of Ca were deficient and Al was fed. In

the present study, Ca levels were above the requirement and no declines in Ca bone

deposits were observed. The absence of treatment effect is attributed to proper dietary Ca

levels and Ca to P ratios.

Summary and Conclusions

A 111 d experiment was conducted to determine if the use of Al sources (AlC13 vs.

WTR) at various levels (910 to 8,000 ppm) affected BMC and bone density of growing

sheep. Forty-two, 5 to 8-mo-old lambs, (12 ewe and 30 wethers) were utilized in a

completely randomized experimental arrangement. Treatment, consisted of the following:

1) control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand),

4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), 6) (10% WTR, 0% sand,

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

Basal diets met all requirements and contained 0.25% P. The lambs weighed between 22

to 39 kg at d zero and between 45.9 to 53.3 kg on d 111. The WTR contained 7.8% Al

on a DM basis. Ten percent of each diet was either sand, WTR, AlC13 or the combination

of two. The resulting concentrations of Al were 910, 2000, 4000, and 8000 ppm,

respectively, for the six diets.

On d 28, 69, and 109, radiographs were taken. Mean bone densities from

radiographs were similar among treatments (P = 0.30). At experimental termination, d

111, animals were sacrificed. The third metacarpal was then used for specific gravity

procedures, and no differences were observed among treatments (P > 0.40). Overall,









results indicate that Al in various forms and levels fed to growing sheep provided

adequate amounts of P (0.25%) and other required dietary nutrients had no effect on bone

density over a period of 79 d or on specific gravity calculations of bone density over 111

d.

Implications

When sheep received adequate dietary concentrations of P (0.25%), Al from A1C13

or WTR had no effect on bone density or composition. In relation to bone development,

the Al-WTR that contains 7.8% Al, which was implemented, is safe for consumption by

sheep up to 10% of their total diet.










Table 4-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for
macro- and micro-elements for treatments
Treatments


Ingredient (%,as fed)
Ground Corn
Soybean hulls
Wet molasses, unfortified
Cottonseed hulls
Corn gluten meal, 60% CP
Alfalfa meal, 17% CP
Vegetable oil (soybean)
Sandb
Water treatment residual
Aluminum chloride
Salt
Urea
Ground limestone
Ammonium chloride
Flowers of sulfur
Mineral-Vitamin-premixd
Dicalcium phosphate

Analyses (ppme)
Ca
Mg
Na
K
P
Al
Co
Cu
Fe
Mn
Zn


1
41.1
12.5
10.0
8.0
5.5
5.0
4.0
10.0


1.0
1.6
0.7
0.5
0.01
0.01



7,170
2,780
4,060
4,180
2,520
910
7
31
66
11
74


2
41.1
12.5
10.0
8.0
5.5
5.0
4.0
9.3

0.7
1.0
1.6
0.7
0.5
0.01
0.01



7,120
2,730
3,240
5,210
2,490
2,320
5
33
65
13
70


3
41.1
12.5
10.0
8.0
5.5
5.0
4.0
7.5
2.5

1.0
1.6
0.7
0.5
0.01
0.01



7,220
2,700
3,030
3,960
2,550
2,270
6
33
67
13
71


4
41.1
12.5
10.0
8.0
5.5
5.0
4.0
5.0
5.0

1.0
1.6
0.7
0.5
0.01
0.01



7,440
2,880
3,000
4,560
2,480
3,9710
5
32
66
13
70


5 6
41.1 39.9
12.5 12.5
10.0 10.0
8.0 8.0
5.5 5.5
5.0 5.0
4.0 4.0

10.0 10.0

1. 1.0
1.6 1.6
0.7 0.7
0.5 0.5
0.01 0.01
0.01 0.02
1.3


7,300
2,870
3,410
4,440
2,460
7,860
5
34
66
14
67


10,000
3,020
3,110
4,380
5,020
7,790
8
42
70
19
79


aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2)
9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR +
two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3
were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and
treatments 5 and 6 were formulated to contain 8,000 ppm Al.
b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001
% Zn.
' Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn,
0.73 % S, 0.006 % Cu, and 0.002 % Zn.
dNlineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 %
Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as
oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1
IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier.
eDry matter basis.









Table 4-2. Effect of dietary Al concentration and source on bone density of feeder
lambs as determined by radiographya
Treatmentc
1 2 3 4 5 6 SE
mm,
Day
28 5.71 4.78 6.15 5.48 6.07 4.90 0.55
69 5.76 6.14 6.60 6.91 6.49 6.22 0.44
109 4.96 5.03 5.01 5.48 6.44 6.08 0.67
aData represent least squares means, and pooled SE; across treatments; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
CMeans within rows did not differ (P < 0.05).




Table 4-3. Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and
Mg for experimental diets
Treatment
1 2 3 4 5 6 SE
g/cm3C
Bone Density 1.89 1.91 1.94 1.89 1.93 1.93 0.003
mg /cm3-
P 74.5 76.1 75.2 81.1 81.1 78.3 5.9
Ca 209 209 197 211 211 191 1.3
Mg 4.13 3.72 4.08 4.24 3.78 3.89 0.07
%.d
Ash 69.9 68.8 69.1 68.9 68.9 68.9 1.56
P 14.2 14.4 14.6 15.4 14.1 15.1 0.63
Ca 39.9 39.4 38.1 39.5 37.0 37.7 18.3
Mg 0.79e 0.70ef 0.79e 0.79e 0.73ef 0.61f 0.04
aData represent least squares means and pooled SE; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AlC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
CCalculated using fresh weights
dCalculated using Ash weights
efMeans within rows lacking a common superscript differ (P < 0.05).














CHAPTER 5
SUMMARY AND CONCLUSIONS

In many developed nations, concerns about repeated application of manure to land

'has led to strict laws and regulations because of increased P levels in nearby water

bodies. Contamination with P can occur in sandy soils because of P leaching into the

ground water, and in slit or clay soils because of runoff of soluble P or erosion of P from

soil manure into local bodies of water. When P enters the water ways causes algae

growth is stimulated. When the algae die oxygen content of the water is decreased and

leads to aquatic plant and animal death.

A chemical reaction between Al and P binds soluble P making it unavailable as a

pollutant. During the water treatment process, Al is added to bind small particles, aiding

in the sedimentation processes, and resulting in the formation of water treatment residuals

(WTR). Water treatment residuals contain a nonavailable form of Al known to reduce P

runoff and leaching. Aluminum, when consumed in a bioavailable form, decreases

growth, intake, and body weight, and depresses bone deposition in several livestock

species. A major concern is that the Al in WTR, thought to be non-available, may

negatively impact an animal when ingested.

At the University of Florida, data were gathered to determine 1) if WTR are

harmful if ingested in amounts between 2,000 to 8,000 ppm Al and 2) determine the

availability of Al in WTR when compared to a control and a diet containing a

bioavailable form of Al (AlC13). A 111-d study was conducted to determine if declines in

intake, BW, ADG, bone mineral content (BMC), bone density, plasma P, tissue P, and









apparent P absorption were produced by the dietary administration of WTR to growing

lambs. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1)

control (10% sand), 2) (9.7% sand and 0.3% A1C13), 3) (2.5% WTR and 7.5% sand), 4)

(5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus

double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate).

The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was

either sand, WTR or the combination of the two. The diet concentrations of Al were 0,

2000, 4000, and 8000 ppm, respectively, for the six diets. Body weight, intake, and ADG

data were compared among three inclusion levels of Al as WTR, and one level of Al

from AlC13. Plasma samples and lamb weights were collected every 14 d. Fecal

collection (to determine apparent P absorption) occurred between d 91 and d 105, and

individual feeding occurred between d 91 and d 111. Samples of blood, brain, liver,

kidney, heart, and bone were collected upon experimental termination.

Lamb ADG, BW, and intakes were unaffected by dietary levels of WTR when

compared to the control. However, lambs fed 2,000 ppm Al from AlC13 had reduced

growth and lower ADG (P < 0.05) than other treatments.

Plasma P concentrations were unaffected by treatments at wk 0 or wk 2. The

control consistently had higher P concentrations than most other treatments, and the

WTR treatments generally had higher P concentrations than lambs given AlC13. Between

wk 6 and wk 14, plasma P concentrations began to increase after a decline at wk 6.

Currently, there is no evidence to explain this finding but this could be attributed to bone

mineral resorptions could have caused plasma P levels to increase and then stabilize.









Tissues were analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn.

Phosphorus concentrations were unchanged across treatments for all tissues and bone,

except kidney, where the control had a higher concentration of P than the lambs given

8,000 ppm Al form WTR (P < 0.05). Bone deposits of Mg were lowest for lambs fed

8,000 ppm Al from WTR with double the added mineral-vitamin premix, and 1.29%

dicalcium phosphate. All other bone mineral concentrations were unaffected by dietary

treatment. Iron concentrations were highest in the liver of lambs feed 8,000 ppm Al from

WTR and lowest in the controls (P < 0.05). Aluminum varied in most tissues, but brain

is the primary repository for Al and is the focus of much research. Concentrations of Al

in the brain were highest for animals receiving 2,000 ppm Al from AlC13 and lowest

lambs given 2,000 ppm Al from WTR (P < 0.05). Concentration, of Al increased when

Al from WTR was given in amounts above 2,000 ppm. The accumulation of Al in the

brain has not been shown to be a threat to the animal and cannot be critiqued without

further research.

On d 91, lambs were placed in metabolism crates; feed and feces were collected for

the determination of apparent P absorption. No differences in apparent P absorption were

observed among WTR treatments, however the lambs administered 2,000 Al via AlC13

had reduced apparent absorption of P. We conclude that Al in WTR does not interfere

with the apparent absorption of dietary P, but AlC13 causes apparent fecal P absorption to

decline.

There were no BMC and bone density differences in long bones collected from

lambs in any treatment. This lack of differences among treatments perhaps may be









attributed to the short duration of the study and because that bone mineral resorption had

not yet occurred in the long bones.

Based on the data collected during this trial and from previous studies, it can be

determined that 2,000 ppm Al via AlC13 results in poor animal performance and P tissue

and plasma P declines. However, intakes, BW and ADG of lambs receiving WTR in

amounts from 2,000 ppm to 8,000 ppm Al did not differ from the control. Thus, WTR

does not appear to negatively affect performance of growing sheep. The apparent P

absorption data strengthens the idea that Al in WTR is less available to the animal then

the Al in AlC13. Apparent P absorption was not altered in lambs fed WTR, but animals

fed 2,000 ppm AlC13 were negatively impacted. Additionally, plasma P and tissue

mineral levels, with the exception of brain Al, were not altered with the administration of

Al from WTR. Under these experimental conditions, dietary administration of Al from

WTR did not cause physiological tissue damages. Overall, it has been demonstrated that

Al from WTR does not negatively impact a growing lamb's health or performance and

could be administered at levels as high as 8,000 ppm Al without causing detrimental

effects. Additional research in other ruminant species should be conducted before data

can be proper applied to species other than the ovine.















APPENDIX
TABLE DATA

Table A-1. Effect of dietary Al concentration and source on ADG of feeder lambsa
Treatment

1 2 3 4 5 6 SE
ADG, g
Wk
2 125cd 9.3c 107cd 235cd 344d 134cd 109
4 116d 4.6c 227d 97.3cd 272d 116cd 68.7
6 274c 50.6df 213cd 250ce -6.4ef -32.4f 64.9
11 257de 287d 570c 593c 480cd 463ce 91.5
14 366 361 244 208 207 269 69.7
aData represent least squares means; n = 7 per treatment
bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1)
(Control) 10% sand; 2) 9.3% sand + 0.7% AIC13; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand
+ 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin
premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000
ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were
formulated to contain 8,000 ppm Al.
cdefMeans within rows lacking a common superscript differ (P < 0.05).
















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BIOGRAPHICAL SKETCH

Rachel Van Alstyne was born in Rochester, NY, on March 4, 1979, to Fred and

Andrea Van Alstyne. She was raised in the city of Rochester until the birth of her

brother, Timothy. At age seven, Rachel and her family moved to the suburbs of

Rochester, to the town ofFairport, NY.

From the age of 16, she maintained several jobs providing enough monetary

solidity to attain the education she desired after high school graduation. Rachel has

always had an incorrigible desire to be near animals. With the exception of her

employment at Brugger's Bagels as a shift manager, in all of her jobs she was able to

surround herself with animals and/or wildlife. Rachel's employment pursuits led her

from veterinary hospitals to the Seneca Park Zoo in Rochester, where she worked as an

animal care attendant better known as a "zoo keeper."

Rachel found it to be difficult during her first year as an undergraduate, since she

did not have the agricultural background many of her classmates did, but she persevered,

and maintained a near perfect GPA, proving that she was "cut out" for this lifestyle. She

desired enhancement in the field of animal care, and she began her pursuit to be a

veterinarian. During her junior year, Rachel decided that veterinary college was not on

the path for which she was best suited. She graduated from Cornell University with her

bachelor's degree in animal science in 2002. Upon graduation, she was undecided as to

which field she desired to pursue in graduate school, and began work. In 2003 Rachel

applied and was accepted to a master's program at the University of Florida with Dr. Lee






64


McDowell. After diligent days and evenings working in both the lab and barn, through

hurricanes, lost power, sick lambs, and inadequate staffing she was able to receive her

degree in 2005. While at the University of Florida she obtained a second master's degree,

concurrently, in management, from the Warrington College of Business. Unlike many

students, Rachel was unable to take even one semester to herself without enrollment in

classes. Her second degree in management from the college was both simulating and

cumbersome, but she was able to finish both degrees within a period of two years.




Full Text

PAGE 1

EFFECTS OF DIETARY ALUMINUM FR OM WATER TREATMENT RESIDUALS ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS By RACHEL VAN ALSTYNE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Rachel Van Alstyne

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Dedicated to family and friends who stood by me through my educational journey.

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ACKNOWLEDGMENTS The author wishes to extend sincere gratitude to Dr. Lee McDowell, Dr. George OConnor, and Dr. Lokenga Badinga for their service on her graduate committee. Thanks are extended to Dr. McDowell for his support, patience, and encouragement throughout this endeavor. Thanks are extended to Dr. OConnor for his professional wisdom and support regarding soil science. Thanks are extended Dr. Badinga for his valuable insight and suggestions. The author would like to thank Dr. Paul Davis for his assistance throughout the trial and writing processes. His hard work, loyalty, love, and support as a friend, partner, and colleague have been paramount to the success of the author during her life while attending the University of Florida, in the publication of this thesis, and in the life they seek together in the future. Much appreciation is extended to Nancy Wilkinson, Jan Kivipelto, and Dr. Maria Silveira for aid in laboratory analyses and data interpretation. Their support, time, and assistance have been priceless. Thanks are given to Dr. Lori Warren, Steve Vargas, Jessica Scott, Carlos Alosilla, Kathy Arriola, Eric Fugisaki, Tom Crawford, Jos Aparicio, and Luis Echevarria, for their hard work and support during the trial. Additionally, the author would like to thank her best friend and roommate, Karen Fratangelo, for her support and kind regards during stressful times. Karen and the author have been friends since the 6th grade and have been able to stay close and lean on each other. Karen aided in diapering sheep, lended an ear, and shared a kind heart during hard iv

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times. Though it may seem odd, the author would like to give her kind regards not only to the humans who aided in her success, but also to the lambs that will always have a soft spot in her heart. Last but not least, the author would like to thank her parents, brother, and canine companion Vixen for their love and support throughout her education and throughout life. She would never have had the perseverance and confidence that she does without their love, loyalty, support, encouragement and respect. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 2 REVIEW OF LITERATURE.......................................................................................4 Historical Significance of Phosphorus..........................................................................4 Requirements................................................................................................................5 Phosphorus Deficiencies...............................................................................................6 Phosphorus Metabolism and Transport........................................................................8 Aluminum and Phosphorus Interactions.....................................................................10 Pollution and Phosphorus Application to Land..........................................................14 Regulations.................................................................................................................16 WTR and Environmental Uses...................................................................................19 3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS.....................23 Introduction.................................................................................................................23 Materials and Methods...............................................................................................24 Animals, Diets, and Management.......................................................................24 Sample Collection, Preparation, and Analyses....................................................25 Statistical Analysis..............................................................................................26 Results.........................................................................................................................27 Discussion...................................................................................................................29 Summary and Conclusions.........................................................................................36 Implications................................................................................................................37 vi

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4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS......................................................................................................43 Introduction.................................................................................................................43 Materials and Methods...............................................................................................44 Animals, Diets, and Management.......................................................................44 Statistical Analysis..............................................................................................47 Results.........................................................................................................................47 Radiograph BMC.................................................................................................47 Bone Density via Specific Gravity......................................................................47 Bone Mineral Analyses.......................................................................................47 Discussion...................................................................................................................48 Summary and Conclusions.........................................................................................49 Implications................................................................................................................50 5 SUMMARY AND CONCLUSIONS.........................................................................53 APPENDIX: TABLE DATA.............................................................................................57 LITERATURE CITED......................................................................................................58 BIOGRAPHICAL SKETCH.............................................................................................63 vii

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LIST OF TABLES Table Page 3-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments...............................................................38 3-2 Effects of dietary Al concentration and source on BW of feeder lambs .................39 3-3 Effect of dietary Al concentration and source on feed intake of feeder lambs .......39 3-4 Effect of dietary Al concentration and source on plasma P of feeder lambs ..........40 3-5 Tissue mineral composition resulting from experimental diets ..............................41 4-1 Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments...............................................................51 4-2 Effect of dietary Al concentration and source on bone density of feeder lambs as determined by radiography .....................................................................................52 4-3 Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg for experimental diets ..............................................................................................52 A-1 Effect of dietary Al concentration and source on ADG of feeder lambs ................57 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON PHOSPHORUS STATUS AND BONE DENSITY IN GROWING LAMBS By Rachel Van Alstyne August 2005 Chair: Lee Russell McDowell Major Department: Animal Sciences Experiments using growing feeder lambs were conducted to gather data on 1) the safety of a Al-water treatment residual (WTR) ingested in amounts to provide between 2,000 and 8,000 ppm Al, and 2) the bioavailability of Al in WTR when compared to a control (910 ppm Al from sand) and a diet containing a known bioavailable form of Al from AlCl3. The study was conducted to examine changes in performance (ADG, BW, and feed intake), tissue mineral concentrations, plasma P concentrations, bone mineral content (BMC), bone density, and apparent P absorption. At experimental termination, samples of brain, liver, kidney, heart, and bone were collected and analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Thirty-two wether and ten female lambs were assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, ix

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and 1.29% dicalcium phosphate). Treatments 1-5 contained P at 0.25% and concentrations of Al were 910, 2000, 4000, 8000 and 8000 ppm, respectively for the six diets. Compared to the control, ADG, BW, and intakes were unaffected by dietary levels of WTR (P > 0.05); however lambs fed 2,000 ppm Al from AlCl3 had reduced body weights and lower ADG (P < 0.05). The control, most often, had the highest plasma P concentrations and the WTR treatments generally had higher P concentrations than lambs given AlCl3. During wk 6, plasma P concentrations declined for all animals but steadily increased thereafter. Kidney P differed; control lambs had larger deposits of P than lambs given 8,000 ppm Al from WTR (P < 0.05). Iron deposits were highest in livers from lambs fed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Brain Al was highest for animals receiving 2,000 ppm Al from AlCl3 and lowest for lambs given 2,000 ppm Al from WTR (P < 0.05). Brain Al concentrations increased when Al from WTR was given in amounts above 2,000 ppm. Apparent P absorption did not differ among WTR treatments and the control (range from 11 to 32 %), but lambs fed 2,000 Al via AlCl3 had a negative (-13%) apparent absorption of P. Values of BMC and bone density did not vary with treatments; this is likely due to the short duration of the study. This study found no evidence of health related defects because of the administration of the WTR. The Al as AlCl3 was more bioavailable with regard to plasma P levels and performance, than Al via WTR; animals which were given the AlCl3 were negatively affected. x

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CHAPTER 1 INTRODUCTION Manure transportation for refuse is costly. This results in the primary method for animal waste disposal being application nearby land. Repeated long-term manure land application leads to accumulation of phosphorus (P) (Novak and Watts, 2004). In the United States, the livestock industry produces 500 million tons of manure each year (Lorentzen, 2004) and many coastal soils have already become saturated with P (Novak and Watts, 2004). The majority of the P produced as animal waste is not adequately used for plant uptake and much of the soil used by large industrial agriculture companies has reached its maximum capacity for P adsorption (Novak and Watts, 2004). When manure is applied to the land and the P remains stagnant on the upper crust of the soil bed it may be washed away with heavy rains (Haustein et al., 2000; Federal Registar, 2004). Phosphorus lost in leaching and runoff can lead to eutrophication, causing the overgrowth of algae, and decreasing the survival of aquatic plants and animals (Novak and Watts, 2004). These algae lead to a reduction in the oxygen levels within the water and result in an overgrowth of anaerobic bacteria that generate toxins such as Pfissteria which may result in death, rashes, respiratory illness, and memory loss in people and animals (Haustein et al., 2000). The death and decomposition of aquatic plants can lead to depressed aquatic oxygen levels, resulting in fish mortalities. Aluminum (Al) applied to the land has been shown to reduce the soluble P concentrations from animal waste (OConnor et al., 2002). Aluminum salts have been 1

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2 used to help minimize the amount of P released from animal feces. The chemical reaction which occurs between the Al from salts and the P in the manure, result in a decrease in P loss. This method has proved to be effective but it is costly (OConnor et al., 2002). The reduction of available P levels from animal waste products could result in significant decreases in leaching and runoff, lessening contamination of water supplies. Studies conducted at the University of Florida suggest that water treatment residuals (WTR), especially those containing Al, increase the soils capacity to retain P. Water treatment residuals bind P, lessening its availability and decreasing water pollution caused by runoff (Elliott et al., 2002). Water treatment residuals are derived from the water purification processes and can vary in their mineral content and P absorption capacities, depending on the chemical used by the water treatment facility and the age or dryness of the WTR. Thus, WTR can be high in Al, Fe or Ca oxides and upon drying or aging become safe for land application (OConnor et al., 2001; Dayton et al, 2003). The WTR used throughout this experiment (including references, unless otherwise stated) are high in Al-oxide from a source known for its high P sobbing capacity and will be referenced as WTR. Water treatment residuals are the solid sediments that result after raw water is coagulated, leaving behind amorphous Al oxides (Basta et al., 2000; Dayton and Basta, 2001). These WTR contain amorphous solids that vary in size and shape. Most WTR look like, and have the texture of a dark soil, but have little or no nutritive value. However, WTR usually contain 4 to 8% nonavailable Al. In general, WTR are discarded in landfills or in waterways, but both methods of disposal are costly and may increase the price of drinking water (Novak et al., 2004).

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3 Application of WTR to the land may result in one solution for animal waste discard, while eliminating the burden and expense of WTR disposal. The chemical mixture of soil and WTR has been proven to increase the retention value of soil P by several fold (Novak et al., 2004). One concern posed is that WTR contains high amounts of Al leading to contamination and ecological risk for grazing animals, wildlife, surrounding floriculture and water systems (USEPA, 2003). High levels of Al can adversely affect P utilization and bone deposition. Aluminum toxicity is often observed as a P deficiency resulting in bone density impairment (Valdivia, 1977). Toxicity is primarily linked to the degree of Al bioavailability. In WTR, the bioavailability of Al varies, but is generally low (OConnor et al., 2002). During grazing, ruminants naturally consume up to 10% to 15% of their total dry matter (DM) intake as soil (Field and Purves, 1964), and soil can contain as much as 10% Al (Valdivia, 1977). Research with livestock at the University of Florida demonstrated that increases in dietary Al decreased voluntary feed intake, and feed efficiency, depressed P serum concentrations, and depressed growth and gains (Valdivia, 1977; Rosa et al. 1982). In this research Al-WTR were directly fed to sheep to simulate grazing-like conditions and soil consumption to emulate an ingestion of soil material in amounts of 10% of their diet, for hypothetical assessment of health related affects if inadvertent consumption of WTR was to occur. The following experiments compared the bioavailability of Al from WTR to an available source of Al, in AlCl3. The main focus will be on the effects of these Al sources on P status in sheep.

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CHAPTER 2 REVIEW OF LITERATURE Historical Significance of Phosphorus Phosphorus (P) and calium (Ca) are the two primary minerals that constitute bone matter and are actively involved in bone development. Together, Ca and P are the most abundant minerals in an animals body (Miller, 1983). Eighty to 85% of an animals P is found in the bones and teeth. Combined, Ca and P make up 70% of the minerals found in the body (McDowell, 2003). The essentiality of P in bone development has been known since 1769, when bone ash was analyzed, and P was found to be a primary component of bone material (McDowell, 2003). Much of the early research on P was instituted in areas of South Africa where deficiencies had become a growing concern. Low P diets had been linked to lamsiekte and botulism in much of the grazing livestock throughout the continent of Africa. Clinical signs which appeared included: bone chewing, depressed growth, failures in reproduction, and reduced feed intake. Chewing the bones of dead carcasses is the most significant indicator of a low P. The P deficiency forces the animal to find any source of P, but the ingestion of the bones can also lead to consumption of Clostridium botulinum and death (McDowell, 2003). In areas of Piaui, Brazil, 20,000 to 30,000 cattle die yearly because of botulism. Today, low P diets and associated diseases are problematic in tropical regions of the world. In Latin America, 73% of all forages evaluated in a feed table publication were P deficient (McDowell, 1997). 4

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5 Requirements Phosphorus makes up 0.12% of the earths crust by volume but is not often found in an uncomplexed form. Phosphorus is extremely reactive and is most commonly found as phosphate in sedimentary rock deposits (McDowell, 2003). Requirements for P vary depending on age, sex, activity, bioavailability of P, protein and energy in the feed, stress, interactions between feed ingredients and nutrients, digestive anatomy and reproduction status of the animal (Miller, 1983). Ruminant animals have a lower requirement for P and Ca than carnivores and omnivores. For strict carnivores such as felines, the requirement for P is 0. 60% and 0.80% for Ca, non-lactating adult humans require 0.70 to 1.25% P and 0.70 to 0.90% Ca while sheep require 0.16 to 0.38% of dietary dry matter (DM) P and from 0.20 to 0.82% of DM Ca (NRC, 1985). The NRC (1985) estimated endogenous loses of P, using a factorial method, to range from 20 mg/kg body weight at maintenance to 30 mg/kg body weight in growing lambs. For proper absorption, P must be present in a bioavailable form. Ruminants, unlike mongastrics, are able to use phytin P from plants and it is considered to be available. Only about one third of P in most plants is available to nonruminants (McDowell, 2003). Incomplete uptake can be linked to an unavailable chemical form of the mineral in the plant, physical barriers in the plant wall, or antagonist elements such as oxalic acid and phytic acid which can bind P, Ca, Fe, Mn, and Zn (McDowell, 1997; 2003). The amount of Ca and P found in feedstuffs varies among sources. The Ca: P ratio in legumes is between 6:1 and 10:1 and is considered to be extremely low in P. Grasses, if mature, are often low in both Ca and P. The values depend on soil conditions and plant species. Alkaline soils are more abundant in trace minerals than acidic and sandy soils. Tropical soils are usually older and acidic, marked by leaching and high environmental

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6 temperature with compromised mineral contents, both in the soil and plants (McDowell, 1997). Seeds and seed by-products are rich in P, whereas animal by-products, tankage, and milled flours are rich in both Ca and P (NRC, 1985). Animals on pasture will develop P deficiencies before those fed high concentrate diets, as grains are high in P (McDowell, 2003). Many factors can influence absorption of minerals such as age, diet, parasites, environmental stresses, disease, and toxic constituents. Growing animals naturally have higher requirements for Ca and P because of bone development. A high protein and energy diet increases the need for both Ca and P, but also increases the ability of the animal to retain these minerals. During the rainy season in areas of South Africa and South America, incidences of P deficiencies are common because more lush forages are being consumed. The increased intake of energy and protein rich grasses increases mineral requirements. Without supplementation, the forages are unable to provide many of the needed minerals in sufficient amounts (McDowell, 1997). Infections and parasites can affect the uptake of P and Ca. Nematodes have been proven to cause demineralization of bone tissues in sheep (Underwood and Suttle, 1999). Stress and activity of the animal will influence mineral needs, those under more stress or those with higher physiological need, including growth, pregnancy, and lactation have the greatest mineral requirements (McDowell, 2003). Phosphorus Deficiencies The most prevalent mineral element deficiency for grazing animals worldwide is lack of P. The requirements for P and, frequently, other minerals are often not met by grazing ruminants, and supplementation is often required. Additionally, certain elements found in low pH tropical soils, such as Fe and Al, can hinder P absorption in the animal.

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7 Deficiencies in P for grazing ruminants have been reported in 46 tropical countries in Latin America, Southeast Asia, and Africa. The soil and forages in these livestock-grazing areas of tropical countries are low in P (McDowell, 2003). Phosphorus deficiency is most often seen in cattle and other grazing ruminants. Young grasses may contain 0.3% P, but mature forages may contain 0.15% P or less (McDowell, 1997; 2003). When dietary P becomes low, an early physiological response is a decline in inorganic plasma P. Normal plasma P levels in ruminants are between 4.5 and 6 mg/100 ml. Levels below 4.5 mg/100 ml in ruminants are considered deficient. A normal P level in ovine whole blood is between 35 to 45 mg and in plasma is between 4 to 9 mg per 100 ml, both of which will vary with age, and sex. Anorexic conditions are first to occur with declines in P, decreasing feed efficiency and slowing energy metabolism in ruminants, which ultimately results in a decline in growth (McDowell, 2003). Dry matter intake was reduced 40% in lambs receiving low-P diets and DM digestibility was less than in animal offered the high-P diets (McDowell, 2003). A significant decline in mineral P concentrations reduces the ability of animals to properly digest fiber, protein and carry out normal metabolic functions (Miller, 1983). Reproductive status may be compromised, primarily in females, which is often the most economically damaging aspect of production. Animals deficient in P have been known to go two to three years without calving (McDowell, 1997). Ruminants deficient in P are listless, with swollen joints, abnormal stance, lameness, and have rough, dry hair coats. Deficiency of P or Ca is similar to that of a deficiency in vitamin D, and lack of any of these nutrients leads to rickets. Clinical signs can include weak bones, which may become curved, enlarged hocks and joints, dragging of hind legs, beaded ribs and

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8 deformed thorax (McDowell, 2003). Bone density decreases and the bone matrix becomes soft and porous. With use of a noninvasive dual photon absorptiometry technique, Williams et al. (1990) determined that dietary levels of 0.12 to 0.13% P lead to bone demineralization in Angus heifers. Prior to bone disruptions, an animal suffers stunted growth, depressed appetite, and weight loss. If the skeletal system is affected, the bones (including: ribs, vertebrae, sternum, and spongy bone material) demineralizes quickly. The last bones to be affected are the long bones and the smaller bones of extremities. When P is deficient, even during normal activities, the bones can bend and fracture, (McDowell, 2003). Phosphorus Metabolism and Transport Calcium and P regulation occurs as a result of the hormones, 1, 25 dihydroxy cholecalciferol, parathyroid hormone and calcitonin. Regulation of normal Ca and P levels depend on bodily excretion, bone deposition, resorption, and intestinal absorption (Miller, 1983). Phosphorus and proper availability of P depends on the Ca to P ratio, of which should be between 1:1 or 2:1 for most monogastic species. However, for ruminants, ratios below 1:1 and over 7:1 will negatively effect growth and feed intake. If Ca and P needs are not met, tetany will occur as the animal withdraws Ca and P from bone in order to maintain normal blood concentrations. The status of vitamin D is important to obtain a desirable Ca:P ratio (McDowell, 1997; 2003). Over time, if Ca and P concentrations are low, bone becomes soft and bone density is impaired. Most of the Ca and P in bone is in the form of calcium phosphate and hydroxyapatite. The exact make up of bone material varies with age, sex, physical activities, and reproductive status, but consistency is seen within species and their stages of life (McDowell, 2003).

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9 Absorption of P in ruminants occurs throughout the intestinal tract, including the rumen, but is optimized in the small intestine. Its uptake occurs through active and passive diffusion and is dependent on the solubility of the membranes that it comes in contact with. Absorption is favored when the mineral is held in solution. Factors which effect uptake of P include: digestive system pH, age, parasites, and other mineral intakes, particularly Ca and Al (McDowell, 1997; 2003). Large amounts of bioavailable Al form insoluble phosphates which bind P, making it unavailable to the animal (McDowell, 2003). Bone undergoes turnover daily and in turn affects the P plasma levels of the animal. Osteoblasts cause new bone formation, while osteoclasts (large multinucleated cells) reabsorb the bone tissue. Most of the nonskeletal portion of P is found in the red blood cells, muscle tissue and nervous system. Much of this P is used to regulate oxygen and hemoglobin in the blood. Status of P in the body can be estimated by plasma or fecal excretion. Feces is the primary pathway for P excretion in ruminants and non-carnivorous animals. Carnivores excrete more P in the urine over that in the feces. In diets low in P, the body naturally conserves P, particularly in herbivores and little to no P is excreted in the urine (McDowell, 2003). Phosphorus is used in almost every metabolic system, including those of ruminal microorganisms, digestion, appetite simulation, feed conversion, fatty acid transport, metabolism of nucleoproteins, maintenance of active cells, enzymes, hormones, and for milk, egg, and muscle synthesis (Miller, 1983; McDowell, 1997; 2003). Evaluation of P status can be determined by the concentration in bone, since the majority of the mineral is in bone. Heifers fed low (0.12%) P diets at had a much lower

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10 cortical bone index, medial lateral wall thickness, breaking load, and total ash than those receiving a 0.20% P diet (Williams et al., 1991b). Bone density of the ribs and vertebrae was also affected by P status (Williams et al., 1990). Blood, bone, feces, rumen fluid and saliva can all be used with various degrees of success to indicate P status of a ruminant and reflected dietary P levels (Williams et al., 1991a,c). Aluminum and Phosphorus Interactions Aluminum is the third most abundant element in the earths crust, following silicon and oxygen, and is the most common metal found in the earths crust (OConnor et al., 2002). Aluminum is highly reactive and does not normally appear in its elemental form; instead, Al binds to other elements or compounds (McDowell, 2003). Soil Al concentrations can range from 1 to 30% but are typically in the 0.5 to 10% range by weight (OConnor et al., 2002). It is not uncommon to find high amounts of Al complexes in tropical sandy soils, binding soil P and making it unavailable for plant uptake (McDowell, 2003). Aluminum chloride (AlCl3) was added to fields of manure covered soil and reduced P runoff by 53% (Smith et al., 2004). Data such as this prove that Al can bind P and increase a soils P sorption capability. In almost all cases, Al is considered to be a toxic mineral, and is not considered to be a required element, except possibly in female rodents (McDowell, 2003). Rosa et al. (1982) reported that increases in dietary P in sheep increased feed intake while it was decreased by increases in Al and Fe. Increased dietary levels of Fe and Al in sheep diets resulted in weight losses; ADG was decreased from 156 to 97g/d in high Fe diets and from 159 to 95g/d in high Al diets. When additional P was added to diets containing high Fe or Al, ADG losses were minimized. The rationale for this response was that the diet

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11 being fed was borderline to deficient in P either at 0.17% to 0.23 % (NRC, 1985). Additionally, plasma P levels increased with Fe and decreased with Al diets (Rosa et al., 1982). Aluminum is not added to animal diets and, in most cases, is found in feeds only because of contamination; whereas P is often added to animal feeds and mineral mixtures. When Al is absorbed via lungs, skin and intestines, only small amounts are actually retained and can be reduced further with fluorine (F) consumption. Most Al is excreted in the feces and urine (McDowell, 2003). In a study at the University of Florida, lambs were given 2,000 ppm of an available source of Al (AlCl3) and Al tissues levels were only mildly elevated (Valdivia et al., 1982). In a similar study, calves were given 1,200 ppm of AlCl3, and performance was not influenced and changes in tissue constituents were only mildly elevated (Valdivia et al., 1978). The kidneys, liver, skeleton and brain are often the tissues affected by Al toxicities (McDowell, 2003). High dietary available Al can result in unabsorbable complexes with P in the intestinal tract. The first effect from a low dietary P level is a decline in plasma P (Williams et al., 1991a,c) further characteristics, including bone demineralization, then follow (McDowell, 2003). When dietary Al exceeds the maximum tolerable level suggested by the NRC (1985) of 1,000 ppm, animals develop characteristics of Al toxicosis. Phosphorus is the mineral primarily affected when toxic levels of Al are administered. An insoluble complex of Al and P is formed in the digestive system of the animal, binding P and making it unavailable, as seen in sheep fed high levels of Al, and signs of P deficiency resulted (Valdivia, 1977). Bone ash and bone Mg level were reduced when 1,450 ppm Al (chloride form) was given to wether lambs (Rosa et al.,

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12 1982). Plasma P levels in sheep given 0.15% P with no added Al were 6.9 mg/100 ml compared to 3.6mg/100ml for those receiving 2,000 ppm Al. Lambs fed 2,000 ppm Al also had lower gains and feed intakes. All animals apparent P absorption was negatively impacted except those fed high P with low Al concentrations. Correspondingly, plasma Ca levels were reduced 0.24 mg/100 ml when 2,000 ppm Al was added. Non-ruminant species are less tolerant to Al toxicity than ruminants. If the same studies were conducted on monogastric animals, toxicosis would develop using 2,000 ppm Al and would ultimately lead to death. The rational is that within the rumen Al complexes with organic anions, not affecting P radicals in the same manner as a monogastric animal (Valdivia et al., 1982). High Al concentrations have also been linked to the possible onset of Alzheimers disease. No factual evidence has been documented to prove if an Al concentration in the brain actually does affect the diseases occurrence. Through the influence of the disease in the medical field is elastic and is currently being methodically investigated (McDowell, 2003). Toxicity is primarily linked to a high Al bioavailability (McDowell, 1997; 2003; OConnor, 2002). When Al toxicity is observed in the ruminant, bone density is often impaired (Valdivia, 1977). Abnormally high amounts of bioavailable Al can also impact the status of Fe, Zn, and Mg in sheep. Dietary amounts of AlCl3 at 1,000 ppm decreased bone and kidney concentrations of Mg, additional antagonistic affects developed for P and Ca as well. (Rosa et al., 1982). Bone ash was reported by Valdivia et al. (1978) to contain lower amounts of Mg for animals fed diets containing AlCl3.

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13 The binding of P to WTR brings about concerns that plants will be limited in required minerals such as P, Ca and Mg because they will become unavailable. Crop yields could be negatively impacted if too much P is bound to Al or if increases in heavy metal contents are realized within the soil (Novak and Watts, 2004). If P becomes unavailable for plant uptake, deficiencies in both plants and animals could occur. Rosa et al. (1982) concluded that excessive bioavailable dietary Al increases P requirements. This may be particularly true when animals are grazing on acidic tropical pastures. In acid soils, Al and Fe become more available and both complex with P and render it unavailable to plants. Acid soils with a pH of 5 or less usually contain higher amounts of available Al and Fe (USEPA, 2003). Water treatment residuals have a pH above 5, which varies somewhat from slightly acidic to moderately basic, and alkaline sources could act as buffers to the soil. Past research has concluded that an elevated soil pH can be maintained with long term use of some WTR, that have a low Al solubility. It is unknown, but has been suggested, that WTR could have an opposite effect on the living system and could lower the pH in the digestive systems of animals which consume it directly (OConnor et al., 2002). It is well known that, in general, soils with an alkaline pH have higher mineral concentrations, than acidic soils. However, Fe, Co, Cu, Mn, and Zn are much more available in acidic compared to alkaline soils (McDowell, 1997). Many tropical soils are acidic (< 5.0 pH) with low P concentrations in forages. Acidic conditions often result in high concentrations of Al and Fe which bind other minerals (McDowell, 2003). In a study used to determine the affects of sand and soil ingestion in

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14 sheep, tropical soils from Costa Rica with a pH of 5.2, were shown to negatively affect the animals more than soils with higher pHs (Ammerman et al., 1984). Pollution and Phosphorus Application to Land Both the absolute number and percentages of the U.S. population employed strictly in farming has fallen dramatically over time. The pressure to produce enough food, with a smaller number of farmers, has had a worldwide impact on agricultural practices, including the expansion of agricultural into marginal lands and the over use of land in general. The agricultural industry needs to remain steadfast in providing adequate food supplies, but we must not compromise environmental, socio-economic, human, and wildlife health issues. In our effort to increase food production, pollution of our water systems has become an issue of pressing attention. In many farming practices, manure application to the land has become environmentally problematic. The majority of P applied to the land as manure often is converted into an insoluble form in the surface horizon of the soil. The accumulated P is subject to erosion or runoff following heavy rains and transported to surface water. Thus, regulations on manure application rates have developed to avoid P pollution of surface waters. (Dayton and Basta, 2001; OConnor et al., 2002; Dayton et al., 2003). Animal producers oppose new stricter regulations placed on manure use because the cost of compliance can be high. In Okeechobee County, Florida, the state has mandated environmental improvements for certain farms. The state shared 75% of the cost to update dairy facilities utilizing 456 employees and 50 million dollars (Lanyon, 1994). There are management methods that can be applied to decrease P runoff, but many are expensive when applied to large farming operations. Using an intensive management system on an average size farm of 100 head that was feeding a high quality

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15 pasture, reduced concentrate feeding by 16%, and resulted in a 5% lower milk yield Yet, this system also reduced the manure application to the land, lowered feed costs, and reduced manure handling procedures so that the farm was able to increase overall annual profitability by $10,000, which is equivalent to $93/cow (Rotz et al., 2002). The same method was utilized by large farms, with 800 head, and profits were increased by $23/cow, but only with an increase in milk production (Rotz et al., 2002). It is a necessity to protect the land from erosion and the water from P pollution caused by manure land application practices, but it is a struggle between the better of two interests. A study in Pennsylvania researched several species of food animals to try to determine differences among species and P production in manure. Three soils that contained manure from ruminants, swine, and poultry were evaluated. Differences in P concentrations among species could not attributed to any pertinent factor and could be assumed to be a result of initial P variations, differences in the P distribution of the soils, or the mixing of the soils and manures. Mixing of all manure types decreased P runoff and was deemed useful in reducing P losses during heavy rains. Mixing the soil and manure promotes sorption of P materials and dilutes the P in the soil surface (Kleinman et al., 2002). Ideally, soil mixing could occur on farms, but labor and machinery costs make the process unrealistic for large scale operations. The current strategies used to reduce P runoff and leaching are soil tillage, crop residue management, cover crops, buffer strips, contour tillage, runoff water impoundment and terracing. These techniques have not be proven to achieve enough success to be used solely, or cooperatively to reduce the current environmental problem (Dayton et al., 2003).

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16 Regulations Scrutiny from the general public and governmental agencies has developed with the increasing pollutants detected in water bodies throughout the United States. In 1995, a manure spill of 144 million liters, twice the size of the Exxon Valdez oil spill, occurred in North Carolina (USEPA, 1997). Farms in the United States are being forced to adhere to strict laws designed to protect the general public involving issues of odor control, water and food safety (Powers, 2003; Federal Registar, 2004). In 1969, Congress passed the National Environmental Policy Act (NEPA). The NEPA has two major divisions; the Council of Environmental Quality (CEQ) and the Environmental Impact Agency (EIA). The CEQ consists of a board of three members who advise the president on environmental issues. The EIA oversees legislation proposed for federal action on environmental issues (Mann and Roberts, 2000). Environmental law is governed by statutory laws and is regulated by federal, state and local administrative agencies. The Environmental Protection Agency (EPA) is the federal agency that oversees such issues, (Mann and Roberts, 2000), having jurisdiction with 10 regional offices nation wide (Meyer, 2000). According to environmental research, the sheer amount of waste generated by large animal facilities poses risk to ground and surface water (Lorentzen, 2004). According to the EPA, farming creates 455 million metric tons of manure each year (Lorentzen, 2004). In 1972, Congress amended the Federal Water Pollution Control Act (FWPCA) of 1948 with the Clean Water Act (CWA) of 1972 (Powers, 2003; Lorentzen, 2004). Again in 1977, 1981, 1987, and 2002, the CWA was amended to ensure clean water for the following: recreational use, protection of the wildlife, and to eliminate pollutants into the ground and drinking water. Concerns that embody the agricultural industry involve

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17 leaching and runoff of nitrogen, solids, and P into the ground water, water ways and water beds (Lorentzen, 2004). Violators of the CWA are subject to both civil and criminal charges. Criminal charges only apply if the violation was intentional. If charged criminally, the fines can range from $2,500 to 1,000,000 dollars and from one to 15 years in prison. Civil charges pertain to all other violations. Ignorance does not preclude one from dismissal of civil or criminal charges. Civil fines can reach a limit of $10,000 a day and an overall maximum of $25,000 per violation (Miller, 2004). Watersheds do not always use filtration techniques when purifying natural water sources (Rotz et al., 2002). A prime example is the New York State watershed located in the Catskill Mountains. This particular region of the state is primarily covered with forests and dairy farms, and supplies 4.5 billion liters of water to people in New York City each day (NRC, 2000). The New York watershed which provides 90% of the drinking water to the city, is purified only chemically, and serious harm could result if manure solids were to contaminate the water systems (Rotz et al., 2002). As defined by the CWA, there are two sources of pollution, point and non-point sources. Point source means there is one defined place or confined area in which the pollution has been released. Point source regulations mandate effluent limitations, based on technological advancements, on the amount of pollution which can be discharged from one source into a body of water. Concentrated animal feeding operations (CAFO s) are often considered point source pollution candidates (Meyer, 2000), and are defined as operating with 700 cattle or a total of 1,000 animals (Lanyon, 1994). Non-point source pollution occurs when the source of pollution can not be traced to a single area. Non

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18 point source is more often the cause of agricultural pollution; in regards to land use, run off, and leaching (Mann and Roberts, 2000). Non-point pollution may not even be observed in the watersheds that is directly affected, but may be carried for many meters down stream and damage areas with no direct contact with the original pollutant (Lanyon, 1994). Classically, farms have been identified as non-point sources of pollution. It has been predicted that within the near future, with increasing regulations and, because of public agendas and concerns, smaller farms will too, be included in point source pollution policies (Lanyon, 1994). For any type of discharge into open water ways, permits by the National Pollutant Discharge Elimination System (NPDES) are required each time and stricter rules are in the near future (Meyer, 2000). Machine and equipment regulations governing businesses involving environmental law are determined by the notion of best available control technology (BACT). This requires that procedures and machines in use need to meet EPA standards for pollution-control. New businesses need to follow standards more strictly than businesses already in existence. As technology advances, new techniques develop that make it possible to reduce pollution. New companies are legally bound to effectively alleviating pollution with the use of advanced technologies. Timetables for existing companies have been applied, meaning that the replacement of old equipment is to be implemented within a reasonable time period. The replacement equipment protocol for existing companies should then be based upon the best practical control technology (BPCT) law by replacing, rather than repairing, equipment, to meet the most current EPA standards (Miller, 2004). Many of the new regulations imposed on businesses regarding environmental safety are locally mandated by state and county polices. In Maryland, Virginia, and

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19 Delaware, stricter polices are being implemented in regards to P application to the land. In Maryland, all P application must abide by the Water Quality Act of 1998, which dictate soil testing to determine if the soil is saturated with P, and if manure application can be permitted. In Virginia, food animal practices are closely scrutinized, particularly the poultry industry; in Delaware manure can usually be treated once every three years to comply with soil P limitations (Penn and Sims, 2002). Water Quality Control Boards are now being mandated to more strictly adhere to the monitoring of N and P levels in the soils. In California, there is an overabundance in the pollutant count in several bodies of water of both P and N. Leaching and run off from manure enriched fertilizers is thought to be the primary cause. The reality is that this type of fertilization is a matter of convenience, availability, and cost profitability rather than providing the optimal nutrients for the flora or concern for the ecosystem (Farm Press, 2004). WTR and Environmental Uses Currently, there is no solution for the distribution of the large quantity of manure produced in the livestock industry. The major issue at hand is the confinement of large operations to small areas of land. Conflicts arrive in application and concentrations of allowable feces. Both N and P are constituents of animal waste products, and are harmful pollutants, yet federal, state, and county standards differ in the applicable uses and concentrations of manure for land, resulting in confusion as to how manure should be properly applied (Lanyon, 1994). Water treatment residuals (WTR) are by-products from water purification procedures. They are rich in metals like Al and Fe, though the exact composition can vary. The elemental levels of Al, Fe, and Ca vary when comparing WTR depending on

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20 the chemical used during the water treatment process and the age (or dryness) of the WTR. In turn, these differences will reflect different abilities to adsorb P (Dayton et al., 2003; Ippolito et al., 2003). During the water treatment process, a chemical, called a coagulate, is added to the water and later forms WTR. This addition of chemicals to water will cause a reaction and form a flocculent precipitate, which coats small particles, such as clays making them more likely to be removed by sedimentation or filtration. Aluminum sulfate (iron sulfate, or calcium sulfate) coagulates may be added to raw water, (the WTR of interest for all further discussion is Al based). The water is then circulated with vigor to uniformly disperse the Al product. Aluminum reacts readily with alkaline products within the water and produces an Al hydroxide solid, which has entrapped impurities. The sedimentation process allows the solids to settle-out. These solid by-products are Al oxides bound to clay size particles and are known as WTR. The processes of coagulation and sedimentation usually precedes filtration in a water treatment plant, and serves to reduce turbidity and increase the efficiency of bacterial removal by filtration (Dayton and Basta, 2001; Brady and Weil, 2002; Ippolito et al., 2003; Water Resources, 2005). The physical characteristics of these WTR are similar to top soils (Haustein et al., 2000). The use of WTR and metal-binding by-products could be one solution to the accumulation of soluble P in the top layer of soil, which leads to nonpoint pollution during heavy rains (Penn and Sims, 2002). In particular, Al containing WTR would benefit sandy soils low in organic material. Sandy soils tend to provide little P retention capabilities and runoff is likely (Penn and Sims, 2002). Soils that are saturated with P may also benefit from WTR application. It has been shown that P saturated soils are

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21 unable to hold added P and thus will result in P ground water complications (Penn and Sims, 2002). Added Al in the form of WTR may help depress P runoff by increasing soil P retention capabilities (OConnor et al., 2002; Penn and Sims, 2002). Publications in 2002 indicated a reduction in P leaching with the addition of Fe and Al from biosolids, claiming that metal oxides formed lead to increased P retention (Soon and Bates, 2002). Research at the University of Florida concluded that increases in dietary Al levels reduced feed intake, gains and P plasma concentrations in sheep. The Al given to these animals was in the form of AlCl3. The impact of additional Al was not positive for animal gains as ADG was 105 and 148 g/d for those consuming a high Al or a low Al diet, respectively. When additional dietary P was given, the ADG increased, but it was not as high as for animals not consuming any Al (Rosa et al., 1982). These results demonstrate the capabilities Al had to lower P status in the animal, but it is unknown what will occur if a less bioavailable form of Al is fed. Other mineral plasma concentrations were also impaired with increased dietary Al. Magnesium content was depressed in the kidneys, and bone of those animals receiving the high dietary Al (Rosa et al., 1982). Similar results using Mg have also been documented at Rutgers University in avian species. Young chicks and mallard ducks when fed high Al diets, as AlCl3, had a high incidence of P binding, lowered P serum levels, depressed growth, lowered tibia weights and lower bone mineralization (Capdevielle et al., 1997). Few studies have been conducted to determine the results of P accumulation, ground water pollution, and the quantity of Al which is capable of binding P in WTR when consumed by ruminants. The majority of studies in regards to WTR and Al content have been focused on the ecological risks associated with plants in acidic soils

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22 (OConnor et al., 2002). Studies involving soil P binding mechanisms have also proven to be helpful. Phosphorus absorption capacity was increased by 20 times with the use of WTR when compared to high Al clay (Haustein et al., 2000). Applications involving pollution control with the use of WTR fed to sheep will be implemented here to compare the bioavailability of Al from WTR to an available source of Al (AlCl3) and evaluate how Al affects the performance of growing sheep.

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CHAPTER 3 EFFECT OF ALUMINUM-WATER TREATMENT RESIDUALS ON PERFORMANCE AND MINERAL STATUS OF FEEDER LAMBS Introduction Ingestion of highly available dietary Al (e.g. AlCl3) by livestock may result in P deficiency. Aluminum toxicity is often observed as a P deficiency (Valdivia, 1977). Additionally, high amounts of bioavailable Al can also impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). Under grazing conditions, ruminants typically consume 10% to 15% of their DM intake as soil (Field and Purves 1964; Healy, 1967; 1968). In sheep dietary Al suppressed voluntary feed intake, feed efficiency, plasma P, growth, and gains (Rosa et al., 1982). When additional P was ingested, these negative effects were less severe but were still evident. Water treatment residuals (WTR) are the byproducts from a water purification procedure, and can contain high amounts of Al, Fe or Ca; here they contain high amounts of Al and has a high P sorption capacity. The bioavailability of Al in WTR varies, but is generally low and thought to be harmless (OConnor et al., 2002). Since Al is highly reactive and has been shown to chemically bind P, the administration of WTR on manure containing soils could be a solution for P pollution of water systems by increasing soil P retention capabilities (Penn and Sims, 2002). Concerns occur because of possible ingestion of the WTR by grazing animals and the reaction of Al and P in a low pH 23

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24 system. No previous research has been conducted to determine the potential toxicity of WTR when directly consumed by grazing ruminants. The purpose of this study was to determine if feeding growing lambs a bioavailable source of Al (AlCl3) versus a less available source of Al from a WTR would affect growth, feed intake, plasma P levels, tissue concentrations, and apparent P absorption. Materials and Methods Animals, Diets, and Management Forty-two, wether (30) and female (12), five to eight-mo-old lambs, (22 Suffolk and 14 Suffolk-crosses) were utilized in a 111-d experiment at the University of Florida Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d zero. Lambs were shorn on d 42 in an attempt to combat heat stress and to increase optimum feed intake. Prior to the experiment, lambs were vaccinated with an 8-way Clostridial given as an injection of 2-mL, four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and were dewormed, with two 1 mL doses of Ivermectin, two wks apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, an amprolium solution was given as an oral drench, lambs received 1 mL daily in a six d sequence (Corid 9.6%; Merial, Duluth, GA). On d 21 the animals were dewormed orally with 5cc of Fenbendazole, (Panacur; Pfizer Animal Health, Exton, PA) and again drenched with 1 mL of Corid from d 21 to 26 (Corid 9.6%). The lambs were housed (seven to each pen), in covered, earth-floored wooden pens (24 sq. m), bedded with pine wood chips with adequate bunk space and ad libitum water and common salt. The University of Florida Institutional Animal Care and Use committee approved the experimental protocol (D231) used in this study.

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25 A corn-SBM basal diet was formulated to meet NRC (1985) requirements for CP, TDN, vitamin, and minerals for lambs of this weight and age (Table 3-1). Prior to the experiment, during a three wk adjustment period, lambs were fed the basal diet at 1200 to 1300 g/d per animal. During the experiment, the animals were fed once daily, 1300 to1600 glamb-1d-1. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR used contained 7.8% total Al on a DM basis. Ten percent of each diet was either sand, WTR, AlCl3 or a combination of the three. The diet concentrations of Al were 910, 2000, 2000, 4000, 8000, and 8000 ppm, (DM basis) respectively, for the six diets. On d 91, animals were placed into individual metabolic crates (1.4m2) to determine apparent digestibility of P. During a subsequent 21 d crate confinement, all animals were individually fed their respective experimental diets. Fresh feed was given ad libitum each morning. Orts were weighed back daily. Individual feed intake, ADG, and BW differences from wk 11 to wk 14, were evaluated. Sample Collection, Preparation, and Analyses Blood samples (jugular venipuncture) and lamb weights were collected on d 0 and every 14 d thereafter. Blood was collected (10mL) with a 20 x 1 vacutainer (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) needle into evacuated tubes containing sodium heparin. Immediately after collection, blood was centrifuged at 700 x g for 30 min, and plasma was collected and frozen at 0 C. After a 30 min thaw period, to allow plasma to

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26 reach ambient temperature the proteins were separated using 10% trichoroacetic acid (Miles et al, 2001). On d 91, wether lambs were fitted with cloth fecal collection devices for the study of apparent digestibility of P. Feces were collected daily for 14 d and composite samples were frozen at 0 C. Each composite sample was sub-sampled and ground in a blender with stainless steel blades. Feces were then dried for 16 h at 105C to determine DM. Samples were then ashed in a muffle furnace at 600C for 8 h, digested in HCl, filtered, and diluted for colorimetric P determination (Harris and Popat, 1954). On d 111, all animals were sacrificed at a USDA approved facility. The following tissues were collected and analyzed for Al, Ca, Cu, Fe, Mg, Mn, P, and Zn contents: blood plasma, liver, heart, kidney, and brain and Se was analyzed for the kidney. Samples were dried, weighed, ashed, and solubilized in HNO3 acid (Miles et al., 2001). Bone was analyzed for P, Ca, and Mg. For all samples, P was analyzed using a colorimetric procedure (Harris and Popat, 1954). Kidney Se was determined using fluorometric procedures (Whetter and Ullrey, 1978). Calcium, Fe, Mg, Cu, Mn, and Zn in tissues and feed samples were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT). Aluminum concentrations were analyzed in diets, heart, brain, liver, and kidney by atomic absorption spectrophotometer using nitrous oxide-acetylene flame (Varian SpectrAA 220 FS; Varian Inc., Walnut Creek, CA). Statistical Analysis Soft tissue, fecal, and feed intake data were analyzed for treatment effects using PROC GLM in SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized design. PROC MIXED of SAS was used to analyze treatment effects on

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27 BW, ADG, and plasma P as repeated measures with a variance component covariance structure in respect to d and subplot of animal nested within treatment. Significance was declared at P < 0.05 and tendencies were discussed when P < 0.15. Results Six animals died during the experiment. The cause of death was determined to be parasite infestation of the gastrointestinal tract, and was deemed unrelated to dietary treatment. Body weights increased for all treatments for wks 0 to 14 (Table 3-2). Average daily gains (Table A-1) and feed intakes (Table 3-3) also increased with time (P < 0.05). Throughout the experiment, lambs fed 2,000 ppm Al via AlCl3 consistently had numerically lower BW than all other treatments. During wk 6, lambs fed 2,000 ppm Al via AlCl3 had lower BW than control animals and, lambs fed 2,000 ppm Al, 4,000 ppm Al or 8,000 ppm Al from WTR (P < 0.05). Lambs receiving 2,000 ppm, 4,000 ppm and 8,000 ppm Al via WTR were heavier than animals consuming 2,000 ppm Al via AlCl3 during wk 11 (P < 0.05). Body weights during wk 11 differed by 11.3 kg, (P < 0.05) between those animals consuming 2,000 ppm Al via AlCl3 and those fed 8,000 ppm Al via WTR, whereas the difference between these two groups at wk 14 was 7.4 kg (P= 0.008). During wk 2, ADG of lambs given 8,000 ppm Al from WTR exceeded animals given 2,000 ppm Al via AlCl3 (P < 0.05). Lambs receiving 4,000 ppm Al from WTR tended (P = 0.11) to gain more than lambs fed 2,000 ppm Al via AlCl3. During wk 4 lambs receiving the control, 2,000 ppm Al via WTR and 8,000 ppm Al via WTR treatments had higher gains than lambs in the treatment given 2,000 ppm Al via AlCl3 (P < 0.05). During wk 6, lambs consuming the control, and 4,000 ppm Al from WTR diets gained more than lambs consuming 2,000 ppm Al from AlCl3 (P < 0.05). Additionally,

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28 during wk 6, all treatments, except 2,000 ppm and 4,000 ppm Al via WTR had gains much lower than the control (P < 0.05). During wk 11, animals began a 3-wk individual feeding regime to determine feed intake. From wk 11 to wk 14, lambs fed the control, 2,000 ppm, and 4,000 ppm Al via WTR (P < 0.05) consumed more than those fed 8,000 ppm Al via WTR. Observations of plasma P during wk 4 (Table 3-4) showed that animals receiving 2,000 ppm Al via WTR had higher concentrations than all other treatments, except those receiving 8,000 ppm Al via WTR plus double the minerals and vitamins (P < 0.05). The animals receiving 8,000 ppm Al via WTR had lowest plasma P of all groups of animals (P < 0.05). In wks 6 to 11, the lambs receiving 2,000 ppm Al via AlCl3 had lower plasma P than controls (P < 0.05). During wk 11, both the control and lambs receiving 2,000 ppm Al via WTR had higher plasma P than animals receiving 2,000 ppm Al via AlCl3 (P < 0.05). Analyses of plasma P during wk 14 showed that controls had higher P concentrations than lambs receiving 4,000 ppm Al via WTR, 8,000 ppm Al via WTR or 8,000 ppm Al via WTR plus two times the amount of added mineral-vitamin premix, and 1.29% dicalcium phosphate (P < 0.05). Plasma evaluations of all other minerals showed, no differences among treatments, which included the following (g/ml): Ca 87 to 101, Mg 17 to 21, Cu 1.3 to 1.5, Fe 0.9 to 2.0, Mn 0.05 to 0.06, and Zn 0.3 to 1.6. Tissue mineral concentrations (Table 3-5) among treatments were deemed not to be hazardous to animal health. With the exception of Cu, tissue mineral concentrations remained within normal ranges (Miles et al., 2001). Liver Cu concentrations were high for all treatments. The mineral-vitamin premix used, inadvertently contained excess Cu in relation to sheep requirements. Levels of P showed no differences among treatments

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29 except that animals given 4,000 ppm Al from WTR deposited more P in the kidney than those animals receiving 8,000 ppm Al from WTR (P < 0.05). No differences (P < 0.05) were observed in soft tissue or bone Ca concentrations. Aluminum was deposited in lower amounts in the brain for lambs fed 2,000 ppm Al via WTR than all other treatments except the control (P < 0.05). Kidney Al deposits were higher in lambs receiving 2,000 ppm Al via AlCl3 than those receiving 8,000 ppm Al via WTR (P < 0.05), and those receiving 8,000 ppm Al via WTR plus two times the added amount of mineral-vitamin premix, and 1.29% dicalcium phosphate (P < 0.05). Concentrations of Mg showed no differences in soft tissue deposition. Differences in Fe deposition were observed in liver (P < 0.05), with lambs consuming the AlCl3 treatment having lower Fe concentrations than those receiving the two treatments of 8,000 ppm Al as WTR. Variations in heart and kidney Mn concentrations seemed unrelated to Al source or quantity. Apparent P absorption ranged from -12.9 to 31.8 % (Figure 3-1). The control and all WTR treatment lambs had a greater apparent P absorption (10.9-31.89%) than the negative absorption (-12.9%) of lambs fed 2,000 ppm Al via AlCl3 (P < 0.001). Discussion Increases in BW, ADG and intakes were observed for all treatments and can be likely attributed to increased appetite which occurs in growing animals. The previous studies at the University of Florida conducted by Valdivia et al. (1978; 1982) observed an increase in feed intake from 1.03 to 1.20 g/d, and an increase in BW gain as dietary P was increased from 0.15 to 0.29 % in diets that contained 1,200 ppm to 2,000 ppm Al as AlCl3. Valdivia et al. (1978) and Rosa et al. (1982) concluded that the increase in P was able to overcome the clinical signs normally observed with Al toxicosis. Diets in the present study contained approximately 0.25% P as fed (Table 3-1), which exceeds the

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30 requirements (0.23% dietary P) of lambs of this age and breed (NRC, 1985; McDowell, 2003). Our study showed no major losses in weight or intakes regardless of treatment, which seems to be attributed to the proper amounts of dietary P (0.25%) supplied. This concurs with the work of Valdivia et al. (1978) and Rosa et al. (1982). The control lambs, which received 910 ppm Al from sand, and lambs receiving treatments containing WTR had no declines in intake. This is likely attributed to the low bioavailability of Al in WTR and sand (OConnor et al., 2002; Dayton et al., 2003). A low bioavailable Al source is much less likely to depress intake because the Al would not readily react with the P in the gastrointestinal tract. Aluminum from AlCl3 is an available source and has been shown to depress intakes. Declines in intakes caused by ingestions of an available Al source have been observed in various species including: sheep (Valdivia et al., 1978; Rosa et al., 1982), broilers and chicks (Fethiere et al., 1990), humans (Chappard et al., 2003; Rengel, 2004) and rats (GmezAlonso et al., 1996). An Al toxicity results in a P deficiency (McDowell, 2003) which can lead to serious tissue damage, lower intakes and gains. Williams et al. (1990; 1991a,c; 1992) induced a P deficiency in heifers and observed an 11% decrease in feed intake. In the present study, there was a decrease in feed intake for the lambs that were fed 2,000 ppm Al via AlCl3. This is expected, as AlCl3 is considered to be a bioavailable source of Al (Valdivia et al., 1978; Rosa et al., 1982), and thus may induce a P deficiency and depress feed intake. Ingestion of Al as AlCl3 by ruminants decreases bone density, plasma P levels, feed intakes and gains (Rosa et al., 1982; Valdivia et al., 1982; Ammerman et al., 1984). Animals receiving the AlCl3 diet repeatedly had lower BW and feed intakes than animals

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31 fed other sources of Al. Lower intakes and gains can be attributed to Al availability, similar observations occurred when 0.75% aluminosilcate was fed to laying hens and feed intake was significantly depressed (Fethiere et al., 1990). One of the objectives of the present study was to compare the availability of Al in WTR to Al in AlCl3 and a control when fed to ruminants. During wk 11, body weights ranged from 36.8 kg for lambs fed 2,000 ppm Al via AlCl3 diet to 48.1 kg for lambs fed 8,000 ppm Al via WTR. Thus, lambs receiving 8,000 ppm Al from WTR, on average, had BW that were 11.2 kg heavier than those fed 2,000 ppm Al from AlCl3 despite the four fold difference in total Al administered. The group fed 8,000 ppm Al from WTR had the highest amount of Al and the largest percentage of WTR (10% of the diet as fed). Differences observed in BW, between lambs fed 8,000 ppm Al via WTR and 2,000 ppm Al via AlCl3 validates previous studies which showed Al in Al-WTR to be high in a non-available source of Al (OConnor et al., 2002; Novak and Watts, 2004) and that AlCl3 is available for uptake in the small intestine (Valdiva et al., 1978; Rosa et al., 1982). It is thought that grazing ruminants can consume up to 10-15% of their total DM intake as soil (Healy 1967; 1968). It has also been shown that soil Al is often consumed by grazing ruminants in amounts as high as 10% of the soil consumed. Aluminum ingested from soil sources has not been shown to reduce performance. Ammerman et al. (1984) fed sheep varying soils types, from Latin America, containing as much as 16,600 ppm Al. They concluded that the soil Al sources had no significant effect on BW, gains, and intakes of the sheep which consumed them. The soils contained various levels of Al or Fe oxides, which is similar to the chemical form of Al from WTR. The additions of high Fe and Al soils had no harmful effects on P utilization, feed intake, or gains.

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32 Differences, in general, between treatments were limited throughout the trial. Lambs receiving diets containing Al via WTR at varying levels showed no differences in BW from the control (P < 0.05). Additions of WTR in amounts as high as 10% of the diet, and representing 8,000 ppm Al in the diet, do not negatively impact growing lambs in relation to BW, ADG, and feed intakes when dietary P is at least 0.25%. Thus, under natural grazing conditions, [where 10% of the DM intake is of soil (Field and Purves, 1964; Healy 1967; 1968)], even high rates of surface applied WTR are not expected to harm animal performance. During wk 14, the ADG of treatments plateaued, consistent with a natural sigmoidal growth curve. Prior to wk 14, animals were gaining at rates between 463 to 593 g per d. The rate declined during wk 14 to only 207 to 244 g per d, but the decline is not attributed to dietary treatments. Animals appeared healthy with notable accumulations of body fat. Lambs in both the control and AlCl3 treatment continued to gain larger amounts of weight during wk 14, because they had not reached a maintenance weight. Lambs fed 2,000 ppm Al via AlCl3 had lowered growth, intake and BW throughout the trial and had not reached a growth plateau by wk 14. The control animals during wk 6 experienced an illness which was attributed to parasite infestations which suppressed ADG means thereafter. In previous studies, similar declines in ADG were observed with AlCl3 additions, and animal growth peaked at later dates than those not receiving an Al source (Valdivia et al., 1978; Rosa et al., 1982; Fethiere et al., 1990). Intakes, regardless of treatment, increased with time. Constituents added to the basal diets did not cause any animals to become anorexic, a common clinical sign of Al toxicity, or P deficiency (Williams et al., 1992; McDowell, 2003). Differences in intakes

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33 were evaluated individually in a 3-wk period between wk 11 to 14. Prior to this date, lambs had been group fed. Individual intake data were similar to those reported by Rosa et al. (1982), and Valdivia et al. (1978). Lambs fed diets containing 2,000 ppm Al from AlCl3 consumed less than the control (P > 0.05), which can again be attributed to the high bioavailability of AlCl3. Intakes were the lowest for animals consuming 8,000 ppm Al from WTR. During wk 14, these animals had the highest BW, but a decline in ADG from wk 11 (480 g) to wk 14 (207.0 g), which was the lowest gain for that period. Intakes for lambs receiving 8,000 ppm Al from WTR were lower than the control, 2,000 ppm and 4,000 ppm Al from WTR (P < 0.05), but higher than lambs receiving 2,000 ppm from AlCl3 or 8,000 ppm Al from WTR with additional minerals and vitamins, (P > 0.05). Prior to wk 14, lambs fed 8,000 ppm Al from WTR showed adequate performance in relation to gains, intake and BW. Therefore, the cause of these declines seen in lambs receiving 8,000 ppm Al via WTR are unknown and could be related to normal growing patterns, an unknown parasite infestation, Cu toxicities, Al toxicities, or other various environmental interactions. During wk 4, lambs receiving 2,000 Al from WTR had the highest concentration of plasma P and differed from the control, those receiving 2,000 ppm Al from AlCl3, 8,000 ppm Al from WTR. (P < 0.05). Huff et al. (1996) administered 3.7% aluminum sulfide to broiler chicks and observed a declines in serum P after a 3 wk period. Lambs in the present study, had plasma P levels decline from 54.2 g/ml to 19.6 g/ml, between wk 4 and wk 8. Additionally, all treatments showed declines in plasma P during this period, but the AlCl3 treatment declines were most often the greatest. During wk 11 and 14, plasma P concentrations began to increase in all treatments. One could conclude that

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34 plasma P concentrations declined to levels which demanded the use of body stores of P (Williams et al., 1990; McDowell, 2003). Bone mineral content was evaluated in the long bones, with no differences among treatments and no evidence of a mineral depression; yet research has shown that the ribs and the vertebrae are first to become depleted in mineral concentrations (Williams et al., 1990; 1991a; McDowell, 2003). Therefore the possibility exists that increases in plasma P levels during wk 11 to 14 occurred from bone mineral resorption. This is unlikely, but not unreasonable, because within a long time frame of 8 wk (between wk 6 and wk 14) bone loss most likely would have been observed in the long bone of the leg which was analyzed. Previous experimental data have not demonstrated similar results by showing an increase in plasma P after a decline. Therefore, observations are speculative at this time and further research is needed to validate this theory. Tissue mineral concentrations analyzed for this study were in the normal ranges for lambs of this breed and age (Miles et al., 2001; McDowell 2003). Previous research found differences in kidney, bone, liver and spleen concentrations of Al, Fe, P Mg and Zn (Rosa et al., 1982) and Ca (Rosa et al., 1982; Zafar et al., 2004) when various amounts of Al were fed. In the present study, Al concentrations differed in brain, heart, liver, and kidney, Mg in bone, and Fe in the liver. Absorption of Al in mongastrics is approximately 0.1% (Rengel, 2004) and is thought to be even lower in ruminants (Valdivia et al., 1970; 1978). Aluminum accumulation occurs most readily in the brain. The exact mechanism is unknown but Al can cross the blood-brain barrier (Rengel, 2004). Accumulations of Al in brain tissue were greater from lambs fed 2,000 ppm Al from AlCl3, than from lambs fed 2,000 ppm from WTR (P < 0.05). Aluminum

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35 concentrations in brains increased when Al from WTR was fed at levels higher than 2,000 ppm, but did not differ from the control. Liver depositions of Al were highest in lambs fed 8,000 ppm Al from WTR (P < 0.05). In the kidney, the highest concentrations of Al were detected when lambs were fed 2,000 ppm Al from AlCl3, and differed from both treatments receiving 8,000 ppm Al from WTR and from the control and 2,000 ppm Al via WTR (P < 0.05). Rosa et al. (1982) observed increases in Al tissue concentration as Al consumption increased, which was not consistently observed in our study. Additionally, soft tissues, except brain matter, that have been evaluated in past studies have not been shown to accumulate large amounts of Al during short time periods (Rengel, 2004), and may not prove to be useful for determination of differences of any Al sources and levels. Apparent P absorption from a 14 d fecal collection showed differences among all five treatments versus the treatment containing 2,000 Al via AlCl3. Studies by Valdivia et al. (1982) observed a marked decrease in P absorption and net P retention in lambs fed 2,000 ppm Al as AlCl3. Negative apparent P absorptions were observed in all groups except those given high P with low Al. When 0.29% P was fed with no dietary Al, the mean apparent absorption was unaffected. In our study, the control had an apparent absorption of 22.5%, and the mean for all the WTR groups was 21.2%. This suggests that Al in WTR did not negatively impact or reduce dietary P absorption. Valdivia et al. (1982) found a negative apparent P absorption (-10.7%) when 0.29% dietary P and 2,000 ppm Al as AlCl3 were fed to sheep. Additionally, Martin et al. (1969) conducted P retention studies using dietary applications of a hydrated Al source and discovered that when Al was fed to sheep, retained amounts of P decreased linearly, as Al fed increased

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36 from 910, 2000, 4000, 8000, and 8000 ppm. Similar results were observed in our study when 2,000 ppm Al was added via AlCl3 to the basal diet which contained 0.25% P. The apparent P absorption averaged 12.9% at wk 14 and therefore suggested a negative impact on dietary P utilization with added Al as AlCl3. Summary and Conclusions A 14 wk experiment was conducted using 36 lambs. Individual feeding was recorded between wk 11 to 14. Diets, containing 0.25% P (as fed), included 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, double the added quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The Al varied from 910 to 8,000 ppm among diets. Lambs fed the control and WTR had no decline in intake, but the AlCl3 lambs repeatedly had lower BW and intakes. The WTR contain a non-available source of Al and did not cause performance declines Additions of this WTR respecting Al concentrations as high as 8,000 ppm, did not negatively impact growing ruminants in relations to BW, ADG, and intakes. During wk 6, all treatments showed declines in plasma P, but the AlCl3 treatment was often the lowest, and during wk 11 plasma P began to increase. Accumulations of Al in the brain were greatest for lambs given 2,000 ppm Al from AlCl3 and increased numerically when Al as WTR was fed at levels higher than 2,000 ppm. With the exception of the brain, soft tissues did not accumulate large amounts of Al during this 14 wk experiment. Apparent P absorption from a 14 d metabolic study was positive (10.9-31.8%) for all lambs fed the control and various levels of WTR. However, lambs that received 2,000 ppm Al via AlCl3 had a negative P absorption of -12.9 %. This was a lowered (P < 0.03)

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37 P absorption compared to all other treatments. Aluminum, as AlCl3, fed at 2,000 ppm reduced dietary P retention, but varying amounts of Al as WTR had no effect on P apparent absorption with similar absorption rates as the control. Therefore when dietary P is supplied in amounts of 0.25% or higher, Al (as WTR) fed to lambs in amounts as high as 8,000 ppm did not negatively impact the feed intake, gain, BW or P absorption. Implications Dietary administration of AlCl3 has negative impacts on ADG, BW, feed intake, apparent absorption of P, and P plasma concentrations. Lambs fed WTR had apparent P absorption percentages that were similar to the control and were higher than the AlCl3 treatment. Water treatment residuals are not harmful when consumed in amounts up to 8,000 ppm Al, when P is supplied in amounts of 0.25%, and do not negatively affect gain, feed intake, BW, or P availability.

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38 Table 3-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments Treatmentsa Ingredient (%,as fed) 1 2 3 4 5 6 Ground Corn 41.1 41.1 41.1 41.1 41.1 39.9 Soybean hulls 12.5 12.5 12.5 12.5 12.5 12.5 Wet molasses, unfortified 10.0 10.0 10.0 10.0 10.0 10.0 Cottonseed hulls 8.0 8.0 8.0 8.0 8.0 8.0 Corn gluten meal, 60% CP 5.5 5.5 5.5 5.5 5.5 5.5 Alfalfa meal, 17% CP 5.0 5.0 5.0 5.0 5.0 5.0 Vegetable oil (soybean) 4.0 4.0 4.0 4.0 4.0 4.0 Sandb 10.0 9.3 7.5 5.0 Water treatment residualc 2.5 5.0 10.0 10.0 Aluminum chloride 0.7 Salt 1.0 1.0 1.0 1.0 1. 1.0 Urea 1.6 1.6 1.6 1.6 1.6 1.6 Ground limestone 0.7 0.7 0.7 0.7 0.7 0.7 Ammonium chloride 0.5 0.5 0.5 0.5 0.5 0.5 Flowers of sulfur 0.01 0.01 0.01 0.01 0.01 0.01 Mineral-Vitamin-premixd 0.01 0.01 0.01 0.01 0.01 0.02 Dicalcium phosphate 1.3 Analyses (ppme) Ca 7,170 7,120 7,220 7,440 7,300 10,000 Mg 2,780 2,730 2,700 2,880 2,870 3,020 Na 4,060 3,240 3,030 3,000 3,410 3,110 K 4,180 5,210 3,960 4,560 4,440 4,380 P 2,520 2,490 2,550 2,480 2,460 5,020 Al 910 2,320 2,270 3,970 7,860 7,790 Co 7 5 6 5 5 8 Cu 31 33 33 32 34 42 Fe 66 65 67 66 66 70 Mn 11 13 13 13 14 19 Zn 74 70 71 70 67 79 aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001 % Zn. c Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn, 0.73 % S, 0.006 % Cu, and 0.002 % Zn. dMineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 % Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1 IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier. eDry matter basis.

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39 Table 3-2. Effects of dietary Al concentration and source on BW of feeder lambsa Treatmentb 1 2 3 4 5 6 SE BW, kg Wk 0 32.6 31.4 33.1 31.1 32.2 31.7 1.5 2 32.8cd 31.6c 34.6cd 34.4cd 37.5d 33.6cd 1.9 4 34.3cd 31.6c 37.7de 35.8cde 41.3e 35.2cd 2.3 6 38.4d 32.3c 40.7d 39.3d 41.2d 34.7cd 2.6 11 41.4cd 36.8c 46.7d 45.1d 48.1d 42.9cd 2.5 14 49.3cd 45.9c 52.8d 50.3d 53.3d 49.7cd 1.9 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdeMeans within rows lacking a common superscript differ (P < 0.05). Table 3-3. Effect of dietary Al concentration and source on feed intake of feeder lambsa Treatmentb 1 2 3 4 5 6 SE intake, glamb-1d-1 Wk 2 827 959 1170 1120 1100 1020 4 1410 876 1150 1150 1070 1120 6 954 1110 1150 1200 1210 1210 11 1610 1460 1790 1550 1910 1940 14 1940c 1870cd 1900c 1940c 1270d 1570cd 54.6 aData represent means of intake during wk 0 to 11 and least squares means during wk 14; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cd Lambs were individually fed for 3 wk ending at wk14; Means within rows lacking a common superscript differ (P < 0.05).

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40 Table 3-4. Effect of dietary Al concentration and source on plasma P of feeder lambsa Treatmentb 1 2 3 4 5 6 SE g/ml, Wk 0 48.7 44.6 51.2 40.8 45.6 43.8 3.7 2 48.3 44.7 50.5 41.5 45.7 44.1 5.3 4 54.3d 54.2d 64.4c 49.8d 39.7e 58.5cd 4.6 6 39.2c 25.2d 27.9d 28.5d 26.2d 27.0d 2.5 8 33.8c 19.6d 19.9d 26.3cd 21.9d 28.1cd 2.3 11 36.2cd 22.2e 46.3c 30.0de 29.0de 29.3de 5.0 14 38.3c 34.0cd 34.9cd 28.0de 24.5de 24.9e 3.6 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdeMeans within rows lacking a common superscript differ (P < 0.05).

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41 Table 3-5. Tissue mineral composition resulting from experimental dietsa Treatmentb 1 2 3 4 5 6 SE Macro elements, (%) Ca Heart 0.01 0.01 0.01 0.01 0.01 0.02 0.004 Kidney 0.06 0.04 0.08 0.08 0.06 0.05 0.02 Liver 0.05 0.04 0.04 0.05 0.04 0.05 0.01 Bone 39.9 39.4 38.1 39.5 37.0 37.7 18.3 Mg Heart 0.15 0.13 0.15 0.14 0.12 0.13 .019 Kidney 0.11 0.11 0.11 0.13 0.10 0.10 0.02 Liver 0.066 0.062 0.066 0.070 0.062 0.064 0.004 Bone 0.79c 0.70cd 0.79c 0.79c 0.73cd 0.61d 0.04 P Heart 1.16 1.03 1.16 1.16 1.09 1.01 0.14 Kidney 1.12c 1.15cd 1.21cd 1.17c 1.15d 1.14cd 0.087 Liver 1.07 1.17 1.07 1.09 1.05 1.01 0.18 Bone 14.2 14.4 14.6 15.4 14.1 15.1 0.63 Micro elements, (mg/kg) Al Brain 43.1cd 52.0d 33.9c 50.4d 47.5d 48.2d 4.10 Heart 6.9cd 5.1def 6.1cdef 7.4c 7.0cd 4.5ef 0.79 Kidney 7.2cd 9.4c 7.1cd 7.5cd 5.4d 5.4d 1.03 Liver 15.4c 22.3cd 16.7c 20.9cd 25.3d 18.5cd 2.75 Cu Heart 8.7 12.7 9.8 14.4 10.3 7.4 2.07 Kidney 38.0 37.3 46.9 36.1 31.2 27.9 9.79 Liver 4,090 4,570 3,080 3,930 3,270 3,900 713 Fe Heart 152 143 154 146 162 134 10.9 Kidney 443 442 425 447 434 434 66.4 Liver 212cd 141d 208cd 162cd 262c 226c 27.8 Mn Heart 1.6cd 1.4d 1.80c 1.31d 1.57cd 1.50cd 0.13 Kidney 18.3cd 23.2cd 16.4c 23.2d 23.9cd 18.4cd 2.4 Liver 12.8 11.4 14.1 10.2 11.9 11.4 2.0 Se Kidney 1.1 1.2 1.3 1.1 1.1 1.2 0.07 Zn Heart 71.5 63.6 65.7 69.1 59.8 61.2 6.65 Kidney 83.9 110 120 110 85.1 95.2 15.0 Liver 48.5 48.4 44.8 43.5 36.8 46.3 6.91 aData represent least squares means; n = 5, 5, 7, 7, 5, and 6 for the control and treatments 1-5, respectively bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdefMeans within rows lacking a common superscript differ (P < 0.05).

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42 22.5-12.918.231.824.010.9-20.0-10.00.010.020.030.040.0123456TreatmentsApparent P absorption b b b b b a Figure 3-1 Effect of dietary Al source on apparent P absorption Dietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. The SE for treatments is 8.23. abMeans lacking a common superscript differ (P < 0.05).

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CHAPTER 4 EFFECT OF DIETARY ALUMINUM FROM WATER TREATMENT RESIDUALS ON BONE DENSITY AND BONE MINERAL CONTENT OF FEEDER LAMBS Introduction Animal waste, which is often applied to grazing land, contains P that can remain in the A horizon of the soil profile and may lead to water pollution (Haustein et al., 2000). Aluminum (Al), if applied to the land, is thought to complex with P and to reduce the soluble P concentrations in animal waste (OConnor et al., 2002). This type of reduction in soluble P levels of animal waste products could result in significant decreases in pollution by commercial livestock operations. Previous studies suggest that the application of Al containing water treatment residuals (WTR), a byproduct of water purification, increases the soils capacity to bind and retain P (Elliott et al., 2002). The chemical mixture of soil and WTR has been shown to increase the retention value of soil P by several fold (Novak and Watts, 2004) and could result in a possible solution to environmental concerns associated with animal waste disposal. A major concern is that WTR, containing high amounts of Al, may adversely affect P utilization and bone deposition in grazing livestock that inadvertently consume WTR. Highly available dietary Al may create unabsorbable P complexes in the intestinal tract and, Al toxicosis is often observed as a P deficiency (Valdivia, 1977). Diets fed to sheep containing 0.29% P and 2,000 ppm Al via AlCl3 resulted in reduced bone density (Rosa et al., 1982). Additionally, high amounts of bioavailable Al can also negatively 43

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44 impact the status of Fe, Zn, and Mg in sheep (Rosa et al., 1982). The bioavailability of Al varies in different WTR, but is generally much lower than Al compounds such as AlCl3 (OConnor et al., 2002). The purpose of this study was to determine the effect of dietary Al as WTR and AlCl3 on bone mineral content (BMC) and bone density in feeder lambs. Materials and Methods Animals, Diets, and Management Forty-two, (30 wether and 12 female) five to eight-mo-old lambs, (22 Suffolk and 14 Suffolk-crosses) were utilized in a 14 wk experiment at the University of Florida Sheep Nutritional Unit located in Gainesville, Florida. The experiment was conducted from June 6th until September 25, 2004. The lambs weighed between 22 to 39 kg at d zero Prior to the experiment, lambs received an 8-way Clostridial vaccination given as injections of 2 mL four wks apart (Ultra Choice 8; Pfizer Animal Health, Exton, PA) and were dewormed, with two 1 mL doses with Ivermectin, two wks apart (Ivomec; Merial Ltd., Iselin, NJ). To prevent coccidiosis, amprolium (Corid 9.6%; Merial, Duluth GA) was used as an oral drench with lambs receiving 1 mL daily in a five d sequence. Corn-SBM basal diets were formulated to meet NRC (1985) requirements for CP, TDN, minerals and vitamins for lambs of this weight and age (Table 4-1). Lambs were fed the basal diet at 1200 to 1300 glamb-1d-1 during a 3 wk adjustment period and 1300 to 1600 glamb-1d-1 throughout the experiment. Lambs were stratified by sex and randomly assigned to six dietary treatments: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the

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45 combination of the two. The diet concentration of Al were 910, 2000, 2000, 4000, 8000, and 8000 ppm respectively for the six diets. Lambs were housed (seven to each pen) covered, in earth-floored wooden pens (24 sq. m), which were bedded with pine wood chips. Animals were group fed in open troughs until d 84, when animals were placed in individual raised metabolic crates. All lambs had access to salt and ad libitum water. Dietary treatments were offered ad libitum and DM intake was monitored daily. Diets were not reformulated during the study. Sample Collection and Analyses Radiographic photometry was used to estimate bone mineral content (BMC) at 28 d, 69 d and 109 d. For each lamb, the left dorsal/palmer, third metacarpal region of the leg was radiographed with the use of a portable x-ray machine, (Easymatic Super 325; Universal X-Ray Products, Chicago, IL). The machine was set at 97 pkv, 30 ma, and 0.067 sec. One cm below the nutrient foramen of the third metacarpal, a cross section of the cannon bone was compared to the standard using the image analyzer and BMC was estimated by photodensitometry. A ten-step Al wedge, taped to the cassette parallel to the third metacarpal, was used as a standard in estimating BMC. Radiographs were taken with a 91.5 cm distance between the x-ray machine and the cassette (Meakim et al., 1981; Ott et al., 1987). The films were processed with an auto-radiograph processing machine, with Kodak products, and by Kodak development procedures (Eastman Kodak Co., Rochester, NY). Radiographs were evaluated with a photometer (Photvolt Corp., New York, NY); percentage light transmissions (%T) were used to determine solid matter The radiographs were zeroed using the thinnest Al step that could be distinguished as a

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46 differing shade from the next ascending step. The BMC was evaluated 2 cm descending from the nutrient foramen. Bone diameter and medullar width were taken to the nearest 0.2 mm using a plastic transparent ruler (Meakim et al., 1981). Determination of the %T reading was then analyzed graphically using a logarithmic calculation. The width of the bone segment, 2 cm below the nutrient foramen, was compared to the visible segments of the Al step wedge. On d 111, all animals were sacrificed at a USDA inspected facility and bone from left leg was removed for bone mineral and bone density analysis. To prepare bone for P, Ca, and Mg analyses, bone removed the left dorsal/palmer, third metacarpal region of the leg, was skinned, immediately wrapped in 0.9% saline-soaked cheese cloth and frozen at 0 C. After thawing, bone was cut into 2 cm sections, 2 cm below the nutrient foramen, and marrow was carefully removed. Samples were rinsed in deionized water and blotted dry. Specific gravity procedures (Kit ME-40290; Mettler Instruments Corp., Hightstown, NJ) were used to determine bone density (g/cm) as described by Williams et al. (1990, 1991c). Bone samples were then dried at 105 C for 16 h extracted with an ether soxhlet apparatus for 48 h, air dried for 10 h, and then oven dried at 105C again for 16 h. Dry samples were weighed, and ashed in a muffle furnace at 600C for 8 h. All P samples were analyzed with colorimetric procedures (Harris and Popat, 1954), while Ca and Mg were analyzed by flame atomic absorption spectrophotometry (Perkin-Elmer Model 5000, Perkin-Elmer Corp., Norwalk, CT).

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47 Statistical Analysis Bone density and BMC data were subjected to the GLM procedure of SAS (SAS for Windows v9; SAS Inst., Inc. Cary, NC) in a completely randomized arrangement. Significance was declared at P < 0.05, and tendencies were recognized at P < 0.15. Results Radiograph BMC At d 28, lambs receiving 2,000 ppm Al from WTR tended (P = 0.07) to have greater bone density than lambs receiving 2,000 ppm Al as AlCl3 (Table 4-2). Likewise, lambs receiving 8,000 ppm Al as WTR tended (P = 0.11) to have denser bone then those receiving 2,000 ppm Al via AlCl3. Overall, bone density was unaffected (P = 0.51) by treatment at d 69, as only lambs receiving 4,000 ppm Al from WTR had bones which tended to be more dense than the controls. Near the end of the study, (d 109) bone density was again unaffected by dietary Al content. Only bones from lambs receiving 8,000 ppm Al as WTR tended to be more dense (P = 0.15) than the controls, and no other difference or tendencies were observed. Bone Density via Specific Gravity Bone density as determined by specific gravity (Williams et al., 1990; 1991) was unaffected by treatment (P = 0.43). Bone density ranged from 1.88 to 1.94 g/cm3 and bones from lambs receiving 2,000 ppm Al as WTR tended to be more dense (P = 0.07) than bones from lambs receiving 4,000 ppm Al from WTR. Bone Mineral Analyses Bone mineral percentages (Table 4-3) of P and Ca were unaffected by treatment. Bone mineral percentages of Ca were also unaffected by treatment (P = 0.87). Bone Mg content differed (P < 0.05) in the lambs receiving 8,000 ppm from WTR plus double the

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48 minerals and vitamins, having lower bone deposits of Mg than those fed the control, 2,000 ppm Al via WTR, or 4,000 ppm Al via WTR (P < 0.05). Additionally, lambs receiving 4,000 ppm Al from WTR tended to have higher bone Mg content than lambs fed 8,000 ppm Al from WTR (P = 0.06). Discussion Radiographing techniques and specific gravity measurements revealed that dietary Al content had no effect on BMC or bone density. Bone density and P deposition is expected to decline when additional Al is ingested according to research conducted by Valdiva et al. (1977) and Rosa et al.(1982). Diets formulated with high Al content (up to 2,000 ppm) in previous work with mallard ducks and chicks (Capdevielle et al., 1998) and rats caused a decline (GmezAlonso et al., 1996; Zafar et al., 2004) in bone mineral declines with Al dietary additions. Yet, contradictory results have been described in much of the research conducted with ruminant species ( Valdivia et al., 1978; 1982; Rosa et al., 1982) Valdivia (1982) concluded that ruminants are less susceptible to toxic effects of Al than in other species. Normally, bone ash is 17.6 % P, and 37.7 % Ca (McDowell, 2003). Studies conducted by Validiva et al. (1982) showed bone mineral percentages of P that were slightly below average, 14 to 15%, as is seen in our study. However in the present study, differences were not observed among dietary treatments. Since differences were not observed, there was no notable effect of dietary Al intake on bone mineral deposition. The long bones of the appendages are often the last affected by P deficiencies (Williams et al., 1991a,b,c; McDowell, 2003). This could be a logical justification for the lack of treatment effect in the present study. A secondary explanation is that the dietary levels of P (0.25%), as seen in intake studies by Rosa et al. (1982), were high enough to compensate for the Al additions to the basal diet. Rosa et al. (1982) also

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49 reported that bone ash Ca levels were unaffected by dietary Al and ranged from 35 to 36 %, similar to results in our study which ranged from 33.1 to 39.9 % for bone ash Ca. Studies conducted with rodents (Cox and Dunn, 2001; Zafar et al., 2004) found that Ca deposits in the bone declined when dietary levels of Ca were deficient and Al was fed. In the present study, Ca levels were above the requirement and no declines in Ca bone deposits were observed. The absence of treatment effect is attributed to proper dietary Ca levels and Ca to P ratios. Summary and Conclusions A 111 d experiment was conducted to determine if the use of Al sources (AlCl3 vs. WTR) at various levels (910 to 8,000 ppm) affected BMC and bone density of growing sheep. Forty-two, 5 to 8-mo-old lambs, (12 ewe and 30 wethers) were utilized in a completely randomized experimental arrangement. Treatment, consisted of the following: 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), 6) (10% WTR, 0% sand, double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). Basal diets met all requirements and contained 0.25% P. The lambs weighed between 22 to 39 kg at d zero and between 45.9 to 53.3 kg on d 111. The WTR contained 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR, AlCl3 or the combination of two. The resulting concentrations of Al were 910, 2000, 4000, and 8000 ppm, respectively, for the six diets. On d 28, 69, and 109, radiographs were taken. Mean bone densities from radiographs were similar among treatments (P = 0.30). At experimental termination, d 111, animals were sacrificed. The third metacarpal was then used for specific gravity procedures, and no differences were observed among treatments (P > 0.40). Overall,

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50 results indicate that Al in various forms and levels fed to growing sheep provided adequate amounts of P (0.25%) and other required dietary nutrients had no effect on bone density over a period of 79 d or on specific gravity calculations of bone density over 111 d. Implications When sheep received adequate dietary concentrations of P (0.25%), Al from AlCl3 or WTR had no effect on bone density or composition. In relation to bone development, the Al-WTR that contains 7.8% Al, which was implemented, is safe for consumption by sheep up to 10% of their total diet.

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51 Table 4-1. Diet composition (as-fed) and analyses for average (n=18) concentrations for macroand micro-elements for treatments Treatmentsa Ingredient (%,as fed) 1 2 3 4 5 6 Ground Corn 41.1 41.1 41.1 41.1 41.1 39.9 Soybean hulls 12.5 12.5 12.5 12.5 12.5 12.5 Wet molasses, unfortified 10.0 10.0 10.0 10.0 10.0 10.0 Cottonseed hulls 8.0 8.0 8.0 8.0 8.0 8.0 Corn gluten meal, 60% CP 5.5 5.5 5.5 5.5 5.5 5.5 Alfalfa meal, 17% CP 5.0 5.0 5.0 5.0 5.0 5.0 Vegetable oil (soybean) 4.0 4.0 4.0 4.0 4.0 4.0 Sandb 10.0 9.3 7.5 5.0 Water treatment residualc 2.5 5.0 10.0 10.0 Aluminum chloride 0.7 Salt 1.0 1.0 1.0 1.0 1. 1.0 Urea 1.6 1.6 1.6 1.6 1.6 1.6 Ground limestone 0.7 0.7 0.7 0.7 0.7 0.7 Ammonium chloride 0.5 0.5 0.5 0.5 0.5 0.5 Flowers of sulfur 0.01 0.01 0.01 0.01 0.01 0.01 Mineral-Vitamin-premixd 0.01 0.01 0.01 0.01 0.01 0.02 Dicalcium phosphate 1.3 Analyses (ppme) Ca 7,170 7,120 7,220 7,440 7,300 10,000 Mg 2,780 2,730 2,700 2,880 2,870 3,020 Na 4,060 3,240 3,030 3,000 3,410 3,110 K 4,180 5,210 3,960 4,560 4,440 4,380 P 2,520 2,490 2,550 2,480 2,460 5,020 Al 910 2,320 2,270 3,9710 7,860 7,790 Co 7 5 6 5 5 8 Cu 31 33 33 32 34 42 Fe 66 65 67 66 66 70 Mn 11 13 13 13 14 19 Zn 74 70 71 70 67 79 aDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. b Sand contained 0.2 % Fe, 0.01 % Al, 0.09% Ca, 0.03 % Mg, 0.1 % P, 0.005 % Mn, 0.004 % Cu, and 0.001 % Zn. c Water Treatment Residuals contained 0.30 % Fe, 7.8 % Al, 0.11 % Ca, 0.024 % Mg, 0.3 % P, 0.004 % Mn, 0.73 % S, 0.006 % Cu, and 0.002 % Zn. dMineral-Vitamin-premix contained 8.0% Mg (as oxide), 0.70% Fe (as sulfate), 2.40% S (as sulfate), 1.9 % Cu (as sulfate), 6.0 % Mn (as oxide), 0.47 % I (as iodate), 0.075 % Se (as sodium selenite), 4.5 % Zn (as oxide and sulfate), 133,363.4 IU/kg Vitamin A supplement, 412,272.7 IU/kg Vitamin D3 supplement, 259.1 IU/kg Vitamin E supplement, rice mill byproduct, and stabilized fat as a vitamin carrier. eDry matter basis.

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52 Table 4-2. Effect of dietary Al concentration and source on bone density of feeder lambs as determined by radiographya Treatmentb,c 1 2 3 4 5 6 SE mm, Day 28 5.71 4.78 6.15 5.48 6.07 4.90 0.55 69 5.76 6.14 6.60 6.91 6.49 6.22 0.44 109 4.96 5.03 5.01 5.48 6.44 6.08 0.67 aData represent least squares means, and pooled SE; across treatments; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cMeans within rows did not differ (P < 0.05). Table 4-3. Effect of dietary Al on dry fat-free bone mineral concentrations of Ca, P and Mg for experimental dietsa Treatmentb 1 2 3 4 5 6 SE g/cm3c Bone Density 1.89 1.91 1.94 1.89 1.93 1.93 0.003 mg /cm3c P 74.5 76.1 75.2 81.1 81.1 78.3 5.9 Ca 209 209 197 211 211 191 1.3 Mg 4.13 3.72 4.08 4.24 3.78 3.89 0.07 %d Ash 69.9 68.8 69.1 68.9 68.9 68.9 1.56 P 14.2 14.4 14.6 15.4 14.1 15.1 0.63 Ca 39.9 39.4 38.1 39.5 37.0 37.7 18.3 Mg 0.79e 0.70ef 0.79e 0.79e 0.73ef 0.61f 0.04 aData represent least squares means and pooled SE; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to contain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cCalculated using fresh weights dCalculated using Ash weights efMeans within rows lacking a common superscript differ (P < 0.05).

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CHAPTER 5 SUMMARY AND CONCLUSIONS In many developed nations, concerns about repeated application of manure to land `has led to strict laws and regulations because of increased P levels in nearby water bodies. Contamination with P can occur in sandy soils because of P leaching into the ground water, and in slit or clay soils because of runoff of soluble P or erosion of P from soil manure into local bodies of water. When P enters the water ways causes algae growth is stimulated. When the algae die oxygen content of the water is decreased and leads to aquatic plant and animal death. A chemical reaction between Al and P binds soluble P making it unavailable as a pollutant. During the water treatment process, Al is added to bind small particles, aiding in the sedimentation processes, and resulting in the formation of water treatment residuals (WTR). Water treatment residuals contain a nonavailable form of Al known to reduce P runoff and leaching. Aluminum, when consumed in a bioavailable form, decreases growth, intake, and body weight, and depresses bone deposition in several livestock species. A major concern is that the Al in WTR, thought to be non-available, may negatively impact an animal when ingested. At the University of Florida, data were gathered to determine 1) if WTR are harmful if ingested in amounts between 2,000 to 8,000 ppm Al and 2) determine the availability of Al in WTR when compared to a control and a diet containing a bioavailable form of Al (AlCl3). A 111-d study was conducted to determine if declines in intake, BW, ADG, bone mineral content (BMC), bone density, plasma P, tissue P, and 53

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54 apparent P absorption were produced by the dietary administration of WTR to growing lambs. Lambs were stratified by sex and randomly assigned to six dietary treatments; 1) control (10% sand), 2) (9.7% sand and 0.3% AlCl3), 3) (2.5% WTR and 7.5% sand), 4) (5% WTR and 5% sand), 5) (10% WTR and 0% sand), and 6) (10% WTR, 0% sand, plus double the quantities of the mineral-vitamin premix, and 1.29% dicalcium phosphate). The WTR was analyzed to contain 7.8% Al on a DM basis. Ten percent of each diet was either sand, WTR or the combination of the two. The diet concentrations of Al were 0, 2000, 4000, and 8000 ppm, respectively, for the six diets. Body weight, intake, and ADG data were compared among three inclusion levels of Al as WTR, and one level of Al from AlCl3. Plasma samples and lamb weights were collected every 14 d. Fecal collection (to determine apparent P absorption) occurred between d 91 and d 105, and individual feeding occurred between d 91 and d 111. Samples of blood, brain, liver, kidney, heart, and bone were collected upon experimental termination. Lamb ADG, BW, and intakes were unaffected by dietary levels of WTR when compared to the control. However, lambs fed 2,000 ppm Al from AlCl3 had reduced growth and lower ADG (P < 0.05) than other treatments. Plasma P concentrations were unaffected by treatments at wk 0 or wk 2. The control consistently had higher P concentrations than most other treatments, and the WTR treatments generally had higher P concentrations than lambs given AlCl3. Between wk 6 and wk 14, plasma P concentrations began to increase after a decline at wk 6. Currently, there is no evidence to explain this finding but this could be attributed to bone mineral resorptions could have caused plasma P levels to increase and then stabilize.

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55 Tissues were analyzed for concentrations of Ca, P, Mg, Cu, Fe, Mn, Se, and Zn. Phosphorus concentrations were unchanged across treatments for all tissues and bone, except kidney, where the control had a higher concentration of P than the lambs given 8,000 ppm Al form WTR (P < 0.05). Bone deposits of Mg were lowest for lambs fed 8,000 ppm Al from WTR with double the added mineral-vitamin premix, and 1.29% dicalcium phosphate. All other bone mineral concentrations were unaffected by dietary treatment. Iron concentrations were highest in the liver of lambs feed 8,000 ppm Al from WTR and lowest in the controls (P < 0.05). Aluminum varied in most tissues, but brain is the primary repository for Al and is the focus of much research. Concentrations of Al in the brain were highest for animals receiving 2,000 ppm Al from AlCl3 and lowest lambs given 2,000 ppm Al from WTR (P < 0.05). Concentration, of Al increased when Al from WTR was given in amounts above 2,000 ppm. The accumulation of Al in the brain has not been shown to be a threat to the animal and cannot be critiqued without further research. On d 91, lambs were placed in metabolism crates; feed and feces were collected for the determination of apparent P absorption. No differences in apparent P absorption were observed among WTR treatments, however the lambs administered 2,000 Al via AlCl3 had reduced apparent absorption of P. We conclude that Al in WTR does not interfere with the apparent absorption of dietary P, but AlCl3 causes apparent fecal P absorption to decline. There were no BMC and bone density differences in long bones collected from lambs in any treatment. This lack of differences among treatments perhaps may be

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56 attributed to the short duration of the study and because that bone mineral resorption had not yet occurred in the long bones. Based on the data collected during this trial and from previous studies, it can be determined that 2,000 ppm Al via AlCl3 results in poor animal performance and P tissue and plasma P declines. However, intakes, BW and ADG of lambs receiving WTR in amounts from 2,000 ppm to 8,000 ppm Al did not differ from the control. Thus, WTR does not appear to negatively affect performance of growing sheep. The apparent P absorption data strengthens the idea that Al in WTR is less available to the animal then the Al in AlCl3. Apparent P absorption was not altered in lambs fed WTR, but animals fed 2,000 ppm AlCl3 were negatively impacted. Additionally, plasma P and tissue mineral levels, with the exception of brain Al, were not altered with the administration of Al from WTR. Under these experimental conditions, dietary administration of Al from WTR did not cause physiological tissue damages. Overall, it has been demonstrated that Al from WTR does not negatively impact a growing lambs health or performance and could be administered at levels as high as 8,000 ppm Al without causing detrimental effects. Additional research in other ruminant species should be conducted before data can be proper applied to species other than the ovine.

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57 APPENDIX TABLE DATA Table A-1. Effect of dietary Al concentr ation and source on ADG of feeder lambsa Treatmentb 1 2 3 4 5 6 SE ADG, g Wk 2 125cd 9.3c 107cd 235cd 344d 134cd 109 4 116d 4.6c 227d 97.3cd 272d 116cd 68.7 6 274c 50.6df 213cd 250ce -6.4ef -32.4f 64.9 11 257de 287d 570c 593c 480cd 463ce 91.5 14 366 361 244 208 207 269 69.7 aData represent least squares means; n = 7 per treatment bDietary treatments were created by additions to a corn-SBM basal diet as follows: 1) (Control) 10% sand; 2) 9.3% sand + 0.7% AlCl3; 3) 7.5% sand, + 2.5% WTR; 4) 5% sand + 5%WTR; 5) 10% WTR; 6) 10% WTR + two times the added amount of mineral-vitamin premix + 1.29% dicalcium phosphate; Treatments 2 and 3 were formulated to contain 2,000 ppm Al, treatment 4 was formulated to c ontain 4,000 ppm Al, and treatments 5 and 6 were formulated to contain 8,000 ppm Al. cdefMeans within rows lacking a common superscript differ ( P < 0.05).

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LITERATURE CITED Ammerman, C.B., R. Valdivia, I.V. Rosa, P.R. Henry, J.P. Feaster, and W.G. Blue. 1984. Effect of sand or soil as a dietary component on phosphorus utilization by sheep. J. Anim. Sci. 58:1093-1099. Basta, N.T., R.J. Zupancic, and E.A. Dayton. 2000. Evaluating soil tests to predict bermuda grass growth in drinking water treatment residuals with phosphorus fertilizer. J. Environ. Qual. 29:2007-2012. Brady, N.C., and Weil, R.R. 2002. The Nature and Properties of Soils. Pearson Education, Inc. Capdevielle, M.C., L.E. Hart, J. Goof, and C.G. Scanes.1998. Aluminum and acid effects on calcium and phosphorus metabolism in young growing chickens (Gallus gallus domesticus) and mallard ducks (Anas platyrhynchos). Arch. Environ. Contam. Toxicol. 35:82-88. Chappard, D., P. Insalaco, and M. Audran. 2003. Aluminum osteodystrophy and celiac disease. Calcif. Tissue. Int. 10:223-26. Cox, K.A. and M.A. Dunn. 2001. Aluminum toxicity alters the regulation of calbindinD28k protein and mRNA expression in chick intestine. J. of Nutr. 131:2007-2013. Dayton, E.A., and N.T. Basta. 2001 Characterization of drinking water treatment residual for use as a soil constituent. Water Environ. Res. 73:52-57. Dayton, E.A., N.T. Basta, C.A. Jakober, and J.A. Hattey. 2003. Using treatment residuals to reduce phosphorus in agriculture runoff. Am. Water Works Assoc. J.95:151-159. Elliott, H.A., G.A. OConnor, P. Lu, and S. Brinton. 2002. Influence of water treatment residuals on P solubility and leaching. J. Environ. Qual. 31:1362-1369. Farm Press. 2004. Right phosphorus management can cut vegetable costs, runoff. PRIMEDIA Business Magazine & Media Inc. Tampa, FL Feb. 7. p4. 58

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59 Federal Register. 2004. Agency information collection activities; submission to OMB for review and approval; comment request; national pollutant discharge elimination system (NPDES) compliance assessment/certification information (renewal), EPA ICR Number 1427.07, OMB Control Number 2040-0110. May 24. Vol. 69, no. 100. Fethiere, R., R.D. Miles, and R.H. Harms. 1990. Influence of synthetic sodium aluminosilicate on laying hens fed different phosphorus levels. Poult. Sci. 69:2195-2208. Field, A.C. and D. Purves. 1964. The intake of soil by grazing sheep. Proc. Nutr. Soc. 23:24-25. GmezAlonso,C. P. Menndez-Rodrguez, M.J. Virgs-Soriano, J.L. Fernndez-Martn, M.T. Ferndez-Coto, and J.B. Cannata-Anda. 1996. Aluminum-induced osteogensis in osteopenic rats with normal renal functions. Calcif. Tissue Int. 64:534-541. Harris, W.D. and P. Popat. 1954. Determination of phosphorus content of lipids. Amer. Oil Chem. Soc. J 31:124-126. Haustein, G.K., T.C. Daniel, D.M. Miller, P.A. Moore, Jr., and R.W. McNew. 2000. Aluminum-containing residuals influence high-phosphorus soils and runoff water quality. J. Environ. Qual. 29:1954-1959. Healy W.B. 1967. Ingestion of soil by sheep. Proc. New Zealand Soc. Anim. Prod. 27:109-115. Healy W.B. 1968. Ingestion of soil by dairy cows. New Zealand J. Agr. Res. 11:487-490. Huff, W.E., P.A. Moore Jr., J.M. Balog, G.R. Bayyari, and N.C. Rath. 1996. Evaluation of toxicity of aluminum in younger broiler chickens. Poult. Sci. 75:1359-1365. Ippolito, J.A., K.A. Barbarick, D.M. Heil, J.P. Chandler and E.F. Redente. 2003. Phosphorus retention mechanism of water treatment residuals. J. Environ. Qual. 32:1857-1864. Kleinman, P.J.A., A.N. Sharpley, B.G. Moyer, and G.F. Elwinger. 2002. Effect of mineral and manure phosphorus sources on runoff phosphorus. J Environ. Qual. 31:2026-2033. Lanyon, L.S. 1994. Dairy manure and plant nutrient management issues affecting water quality and the dairy industry. J. Dairy Sci. 77:1999-2007. Lorentzen, A. 2004. Environmental group says Iowa not enforcing laws. The Associated Press State and Local Wire, May 20.

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60 Mann, R.A., and B.S. Roberts. 2000. Smiths and Roberts Business Law. 11th Ed. West Legal Studies in Business, Thomson Learning, Cincinnati, OH. pp. 997-1002. Martin, L.C., A. J. Clifford, and A.D. Tillman. 1969. Studies on sodium bentonite in ruminant diets containing urea. J. Anim. Sci. 29:777-778. McDowell, L.R. 2003. Minerals in Animal and Human Nutrition. 2nd Ed. Elsevier Sci., Amsterdam. McDowell, L.R. 1997. Minerals for Grazing Ruminants in Tropical Regions. 3rd Ed. Bull. animal science department University of Florida, Gainesville. Meakim, D.W., E.A. Ott, R.L. Asquith and J.P. Feaster. 1981. Estimation of mineral content of the equine third metacarpal by radiographic photometry. J. Anim. Sci. 53:1019-1026. Meyer, D. 2000. Dairying and the environment. J. Dairy Sci. 83:1419-1427. Miles, P.H., N.S. Wilkinson, and L.R. McDowell. 2001. Analysis of mineral for animal nutrition research 3rd ed. University of Florida, Gainesville, FL. Miller W.J. 1983. Phosphorus-ruminant-nutritional requirements, biochemistry and metabolism. National Feed Ingredient Association's Mineral Ingredient Handbook. NFIA, West Des Moines, Iowa. pp.1-14. NRC (National Research Council)S. 1985. Nutrient Requirements of Domestic Animals. Nutrient Requirements of Sheep, 5th Ed. Natl. Acad. Sci. Washington DC. Novak, J.M. and D.W. Watts. 2004. Increasing the phosphorus sorption capacity of southeastern coastal plain soils using water treatment residuals. Soil Sci. 169:206-214. National Research Council. 2000. Watershed Management for Potable Water Supply. Natl. Acad. Press. Washington, DC. OConnor, G.A., H.A. Elliott, and P. Lu. 2002. Characterizing water treatment residuals for P retention. Soil Crop Sci. Soc. Florida Proc. p67-73. Ott, E.A., L.A. Lawrence, and C. Ice. 1987. Use of the image analyzer for radiographic photometric estimation of bone mineral content. Proc. 10th Equine Nutr. Physiol. Sym. Colorado State University. June 11-13. Penn, C.J. and J.T. Sims. 2002. Phosphorus forms in biosolids amended soils, and losses in runoff; effects of water treatment processes. J. Environ. Qual. 31:1349-1361. Powers, W.J. 2003. Keeping science in environmental regulations: role of the animal scientist. J. Dairy Sci. 86:1045-1051.

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61 Rengel, Z. 2004. Aluminum cycling in the soil-plant-animal-human continuum. BioMetals 17:669-689. Rosa, V., P.R. Henry, and C.B. Ammerman. 1982. Interrelationship of dietary phosphorus, aluminum and iron on performance and tissue mineral composition in lambs. J. Anim. Sci. 55:1231-1240. Rotz, C.A., A.N Sharpley, L.D. Satter, W.J Gburek, and M.A. Sanderson. 2002. Production and feeding strategies for phosphorus management on dairy farms. J. Dairy Sci. 85:31423153. Smith, D.R., P.A. Moore Jr, C.V. Maxwell, B.E. Haggar, and T.C. Daniel. 2004. Reducing phosphorus runoff from swine manure with dietary phytase and aluminum chloride. J Environ. Qual. 33:1048-1054. Soon, Y.K. and T.E. Bates. 1982. Extractability and solubility of phosphate in soils amended with chemically treated sewage sludges. Soil Sci. 134:89-96. Tomas, F.M., and M. Somers. 1974. Phosphorus homeostasis in sheep. I. Effect of ligation of parotid salivary ducts. Aust. J. Agric. Res. 25:475-483. Underwood, E.J. and N.F. Suttle. 1999. The Mineral Nutrition of Livestock. 3rd Ed. Midlothian, Wallingford, UK. US Environmental Protection Agency (USEPA). 1997. Animal Waste Disposal Issues: Office of Inspections, Washington. DC. US Environmental Protection Agency (USEPA). 2003. Ecological soil screening level for an interim final report. OERR, Washington DC. Valdivia, R. 1977. Effect of dietary aluminum on phosphorus utilization by ruminants. Ph.D. Dissertation, University of Florida, Gainesville. Valdivia, R., C.B. Ammerman, P.R. Henry, J.P. Feaster, and C.J. Wilcox. 1982. Effect of dietary aluminum and phosphorus on performance, phosphorus utilization and tissue mineral composition in sheep. J. Anim. Sci. 55:402410. Valdivia, R., C.B. Ammerman, C.J. Wilcox, and P.R. Henry. 1978. Effects of dietary aluminum on animal performance and tissue mineral levels in growing steers. J. Anim. Sci.47:1351-1360. Water Resources. 2005. The official government website of Greensboro NC; water treatment process. Greensboro, NC, http://www.greensboro-nc.gov/water/ supply/treatment.htm. Accessed: May 17, 2005. Williams, S.N., L.A. Lawrence, L.R. McDowell, and N.S. Wilkinson. 1991a. Criteria of evaluate bone mineral in cattle: II. Noninvasive techniques. J. Anim. Sci. 69: 1243-1254.

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62 Williams, S.N., L.R. McDowell, A.C. Warnick, L.A. Lawrence, and N.S. Wilkinson. 1992. Influence of dietary phosphorus level on growth and reproduction of growing beef heifers. Int. J. Anim.. Sci. 7:137-142. Williams, S.N., L.R. McDowell, A.C. Warnick, L.A. Lawrence, and N.S. Wilkinson. 1990. Dietary phosphorus concentrations related to breaking load and chemical bone properties in heifers. J. Dairy Sci. 73:1100-1106. Williams, S.N., L.R. McDowell, A.C. Warnick, N.S. Wilkinson, and L.A. Lawrence. 1991b. Phosphorus concentrations in blood, milk, feces, bone and select fluids and tissues of growing heifers as affected by dietary phosphorus. Liv. Res. for Rural Dev. 3:67-79. Williams, S.N., L.R. McDowell, A.C. Warnick, N.S. Wilkinson, and L.A. Lawrence. 1991c. Criteria of evaluate bone mineral in cattle: I. Effect of dietary phosphorus on chemical, physical, and mechanical properties. J. Anim. Sci. 69:1232-1242. Whetter, P.A., and D.E. Ullrey. 1978. Improved fluorometric method for determination of selenium. J Assoc. Off. Anal. Chem. 4:927-930. Zafar, T.A., D. Teegarden, C. Ashendel, M.A. Dunn, and C.M. Weaver. 2004. Aluminum negatively impacts calcium utilization and bone calcium-deficient rats. Nutr. Res. 24:243-259.

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63 BIOGRAPHICAL SKETCH Rachel Van Alstyne was born in Roches ter, NY, on March 4, 1979, to Fred and Andrea Van Alstyne. She was raised in the city of Rochester until the birth of her brother, Timothy. At age seven, Rachel and her family moved to the suburbs of Rochester, to the town of Fairport, NY. From the age of 16, she maintained se veral jobs providing enough monetary solidity to attain the educa tion she desired after high sc hool graduation. Rachel has always had an incorrigible desire to be near animals. With the exception of her employment at Bruggers Bagels as a shift mana ger, in all of her jobs she was able to surround herself with animals and/or wildlife. Rachels employment pursuits led her from veterinary hospitals to the Seneca Park Zoo in Rochester, where she worked as an animal care attendant better known as a zoo keeper." Rachel found it to be difficult during her first year as an undergraduate, since she did not have the agricultural background many of her classmates did, but she persevered, and maintained a near perfect GPA, proving that she was cut out for this lifestyle. She desired enhancement in the field of animal care, and she began her pursuit to be a veterinarian. During her junior year, Rachel decided that ve terinary college was not on the path for which she was best suited. She graduated from Cornell University with her bachelor's degree in animal science in 2002. Upon graduati on, she was undecided as to which field she desired to pursue in gradua te school, and began work. In 2003 Rachel applied and was accepted to a master's program at the University of Florida with Dr. Lee

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64 McDowell. After diligent days and evenings working in both the lab and barn, through hurricanes, lost power, sick lambs, and inad equate staffing she was able to receive her degree in 2005. While at the Univ ersity of Florida she obtai ned a second master's degree, concurrently, in management, from the Wa rrington College of Business. Unlike many students, Rachel was unable to take even one semester to herself without enrollment in classes. Her second degree in management from the college was both simulating and cumbersome, but she was able to finish bot h degrees within a pe riod of two years.