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IV. Methods for Characterizing Phosphorus in Organic Wastes

IV. Methods for Characterizing Phosphorus in Organic Wastes

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CHARACTERIZATION OF P IN ORGANIC WASTES



17



Table VI

EVect of Method of Determination on Water Extractable Phosphorus in Poultry Littera

Water soluble P



Manure from diet

Normal corn (NORC)

High available P corn (HAP)

NORC0.1% NPP ỵ phytased

HAPC0.1% NPP ỵ phytasee

NORC0.2% NPP ỵ phytasef

HAPC0.2% NPP ỵ phytaseg

LSD0.05



Colorimetryb

(mg kgÀ1)



ICP‐OESc

(mg kgÀ1)



Overestimation

with ICP‐OES (%)



2394

2349

2157

1235

937

418

351



2486

2525

2395

1414

1305

541

235



4

7

10

13

28

23



a



Adapted from Sims et al. (2000).

Extraction of ‘‘as‐is’’ sample at 1:10 ratio (for 1 h), filtration through Whatman #4 and 0.45 mm

filters, P measured colorimetrically.

c

Extraction of ‘‘as‐is’’ sample at 1:10 ratio (for 1 h), filtration through Whatman #4 and 0.45 mm

filters, P measured by ICP‐OES.

d

Normal corn with 0.1% less nonphytate P (NPP) and phytase.

e

High available P corn with 0.1% less NPP and phytase.

f

Normal corn with 0.2% less NPP and phytase.

g

High available P corn with 0.2% less NPP and phytase.

Phytase was added at 650 U kgÀ1.

b



dilute acids) as sample acidification can result in hydrolysis of acid labile

organic compounds, such as sugar phosphates and monophosphate esters

(McKelvie et al., 1995), and cause inflated inorganic P values. On the other

hand, ICP measures both inorganic and all organic P. For example, in poultry

litter generated from modified diets, Sims et al. (2000) observed 4–28% higher

WEP measured by the ICP than the molybdate method (Table VI).

Another approach to rapidly analyze total P in manures is near‐infrared

reflectance spectroscopy (NIRS). The principle behind NIRS and information on its application in agricultural and food products can be found in

Burns and Cziurczak (1992) and Williams and Norris (2001). Although total

C and N concentrations have been successfully analyzed in manures by

NIRS, there have been mixed results when quantifying the total P content

in manures with NIRS: some workers obtained good correlation between the

total P measured with NIRS and total chemical analyses (Malley et al., 2002)

while others did not (Reeves, 2001; Reeves and Van Kessel, 2000). If this

method can be standardized to determine total P in wastes, it has the

potential to become a robust, cost‐eVective means for use in routine analyses

by manure testing laboratories and in situ field monitoring.



18



G. S. TOOR ET AL.



B. WATER EXTRACTABLE PHOSPHORUS

Water extractable P is that fraction of total P, which is extracted by

shaking a known amount of waste with water. There has been a growing

interest in the use of WEP as an indicator to assess the relative proportion of

dissolved P in organic wastes that could be subject to loss on land application. Studies have shown that WEP can range from <10 to as high as 75% of

total P in biosolids (Brandt et al., 2004; Huang and Shenker, 2004), dairy,

poultry, and swine manures (Angel et al., 2005; Applegate et al., 2003;

Baxter et al., 2003; Dou et al., 2002; Kleinman et al., 2005; Maguire et al.,

2003; Toor et al., 2005c). A positive correlation between WEP in wastes and

dissolved P in surface runoV and leachate has been reported (Kleinman

et al., 2002; McDowell and Sharpley, 2001), suggesting its applicability for

environmental purposes. In addition, it has also been proposed that WEP or

other dilute extractants can be used to provide an in situ management tool to

diagnose excessive P feeding and to assess the relative P availability in waste.

This use of WEP testing as a fecal P indicator would improve optimization

of P eYciency in the confined animal operations (Dou et al., 2002).

Water extractable P consists of organic and inorganic forms of P. Inorganic P is largely composed of dissolved inorganic P or dissolved P minerals.

For example, Toor et al. (2005c) observed a significant correlation between

WEP and dicalcium phosphate (r ¼ 0.85) in poultry litters generated from

modified diets. Information about the exact nature of organic P species in

water extracts of wastes is limited at this time, but it is likely that WEP

contains a mixture of organic P species such as labile monoester P and

diester P (McKelvie et al., 1995).

When comparing WEP results from diVerent literature sources using

manure and biosolids, the factors discussed in the following section, such

as waste to solution ratio, length of shaking, fresh or dry samples, centrifugation or filtration, and method of analysis (discussed in total P section),

should be kept in mind as they exert a major influence on measured WEP.

Waste to solution ratio can aVect the amount of WEP in organic wastes.

Various researchers have used diVerent waste to water extraction ratios and

have referred to this fraction as WEP. This has made comparisons between

diVerent studies rather diYcult. For example, Toor and Sims (unpublished

data) observed an increase in concentrations of WEP (inorganic and organic) in dairy feces with increase in manure to solution ratio from 1:10 to 1:400

(Fig. 1). Standardization of WEP methodology has occurred (Kleinman

et al., 2002, 2005; Wolf et al., 2005) and many researchers now use a waste

to solution ratio of 1:100.

Length of shaking waste with extracting solution can also aVect the

amount of WEP extracted. However, the temporal eVect is primarily dependent upon the waste to solution ratio. At a large waste to solution ratio (e.g.,



CHARACTERIZATION OF P IN ORGANIC WASTES



19



Figure 1 EVect of extraction ratio on water soluble phosphorus in two dairy feces samples

collected from Mid‐Atlantic United States.



>1:100), more P may be released into the solution after a longer shaking

time. The chemical composition of the wastes is the principal factor aVecting

the release of P. If there is a higher pool of soluble P in the waste then the

release of P may continue at longer extraction times.

The amount of WEP in organic wastes can also be aVected by drying

the samples. Ajiboye et al. (2004) compared WEP in fresh and oven‐dried

(105  C) organic wastes (dairy, hog manures) and noted an increase in WEP

(Fig. 2). They attributed this increase to hydrolysis of water extractable

organic P to inorganic P in hog manures, and both transformation of

NaHCO3 inorganic P and hydrolysis of NaHCO3 organic P to water extractable inorganic P in dairy manures. In general, drying samples can kill

microorganisms resulting in the release of cellular P, or, can cause desorption of P held on the surfaces of organic and inorganic colloids, thus,

increasing the WEP of the materials.

After extractions, separation of waste and water suspension is accomplished via either filtration or centrifugation. It is assumed that filtration will

remove the P associated with particulates, whereas centrifugation will not

easily remove this particulate fraction. Sims et al. (2000) found that WEP

was 62–78% higher in centrifuged (no filtration) poultry litter extracts than

those filtered through 0.45‐mm filter paper (Table VII).

Although there remains some heterogeneity in WEP measurement introduced from sample processing (fresh or dry samples) and analysis (waste to

solution ratio, length of shaking, centrifugation, or filtration), WEP is an



20



G. S. TOOR ET AL.



Figure 2 Transformation of phosphorus fractions with oven‐drying of the amendments.

(A) Hog manure collected from an agitated storage lagoon, (B) hog manure from a sow barn

(C) hog manure from a nursery barn, and (D) manure from a dairy barn. The whiskers represent

standard errors of the means of three replicates. The terms H2O–Pi and H2O–Po are water‐

extractable inorganic and organic P, NaHCO3–Pi and NaHCO3–Po are NaHCO3–extractable

inorganic and organic P, NaOH–Pi and NaOH–Po are NaOH–extractable inorganic and

organic P; and HCl–P is HCl–extractable P. After Ajiboye et al. (2004).



CHARACTERIZATION OF P IN ORGANIC WASTES



21



Table VII

EVect of Filtration Versus Centrifugation on Water Extractable Phosphorus Release

from Poultry Littera

Water soluble P

b



Manure from diet

Normal corn (NORC)

High available P corn (HAP)

NORC0.1% NPP ỵ phytased

HAPC0.1% NPP ỵ phytasee

NORC0.2% NPP ỵ phytasef

HAPC0.2% NPP ỵ phytaseg

LSD0.05



Filtration

(mg kg1)



Centrifugation (no

filtration)c (mg kgÀ1)



Overestimation with

centrifugation (%)



2486

2525

2395

1414

1305

541

235



6800

6624

7045

5030

3561

2497

1215



63

62

66

72

63

78



a



Adapted from Sims et al. (2000).

Extraction of ‘‘as‐is’’ sample at 1:10 ratio (for 1 h), filtration through Whatman #4 and 0.45 mm

filters, P measured colorimetrically.

c

Extraction of ‘‘as‐is’’ sample at 1:10 ratio (for 1 h), filtration through Whatman #4 and 0.45 mm

filters, P measured by ICP–AES.

d

Normal corn with 0.1% less nonphytate P (NPP) and phytase.

e

High available P corn with 0.1% less NPP and phytase.

f

Normal corn with 0.2% less NPP and phytase.

g

High available P corn with 0.2% less NPP and phytase.

Phytase was added at 650 U kgÀ1.

b



important part of total P that should be measured in wastes. WEP results are

extremely useful in understanding both the plant available P pool and the

resulting potential influence on water quality if these wastes were to reach

water bodies.



C. PHYSICOCHEMICAL FRACTIONATION

The physicochemical fractionation method is widely used to diVerentiate

P forms in leachate and surface runoV waters (Haygarth and Sharpley,

2000), and this method can also be used to characterize P forms in liquid

wastes such as dairy and swine slurry and wastewater produced from sewage

treatment. Physicochemical fractionation of P in liquid wastes into reactive,

unreactive, dissolved, and particulate forms (Fig. 3) is based on two simple

criteria: (1) size: whether the P is present in the dissolved or particulate

fraction, with the distinction made by filtration through a filter paper (usually 0.45 or 0.2 mm); and (2) digestibility: whether the P is present in a

reactive or unreactive fraction, with the distinction made by digesting the

wastes with some form of acid or oxidizing agent (Haygarth and Sharpley,



G. S. TOOR ET AL.



22



Figure 3



Physicochemical fractionation of phosphorus.



2000). Dissolved reactive P and total reactive P are directly measured (without

digestion) on filtered and unfiltered samples, respectively, by the acid molybdate method. Both total dissolved P and total P fractions are determined on

filtered and unfiltered samples, respectively, after digestion (Ebina et al., 1983;

Rowland and Haygarth, 1997). The other P forms are calculated as:













Total particulate P ¼ total P – total dissolved P

Total unreactive P ¼ total P – total reactive P

Dissolved unreactive P ¼ total dissolved P – dissolved reactive P

Particulate unreactive P ¼ total unreactive P – dissolved unreactive P

Particulate reactive P ¼ total reactive P – dissolved reactive P.



According to this fractionation scheme, there are four categories of P forms

under two main groups. Reactive P is thought to mainly consist of orthophosphate and includes P in dissolved and particulate phases. Unreactive dissolved

and particulate P contains labile and recalcitrant organic P compounds. The

possible composition of P compounds in these physicochemical forms is as

follows:

Dissolved reactive P includes orthophosphate that are soluble in water and

some of the easily hydrolyzable organic P forms such as labile monoesters

(sugar phosphates, mononucleotides) and diesters (DNA, RNA, phospholipids). The organic P species in the fraction are a result of the acid‐mediated

hydrolysis during P analysis by acid molybdate method (McKelvie et al.,

1995).

Particulate reactive P is that fraction of total P that is insoluble in water

but may contain P sorbed on the surfaces of clay, Fe, Al, or Ca oxides and

hydroxides.

Dissolved unreactive P is thought to primarily contain organic P compounds,

however, this fraction can also contain some inorganic compounds, such as



CHARACTERIZATION OF P IN ORGANIC WASTES



23



Table VIII

Composition of Dissolved Unreactive Phosphorus in Marine Environmentsa

Dissolved unreactive

P compound class

Monophosphate esters



Composition (%)



Phospholipids



10–100

55–77

23–5

10–100

ATP <1

DNA/RNA <5

3–11



Phosphonates

Polyphosphates



5–10

0–50



Nucleotides and nucleic acids



a



Method of analysis

Enzymatic assays

Modified UV oxidation

Persulfate‐modified UV

Enzymatic assays

Firefly bioluminescence

Multiple methods

Cross flow filtration and Polymyxin

B treatment

31

P‐NMR

Acid reflux–UV oxidation



Adapted from Benitez‐Nelson (2000).



polyphosphates, which are not detected with the acid molybdate method (Ron

Vaz et al., 1993; Thomson‐Bulldis and Karl, 1998). Relatively less information

is available about the nature of organic P forms in this pool, whereas some data

are available on the nature of organic P compounds in marine waters and

sediments. Benitez‐Nelson (2000) using various methods such as enzymatic

assays, ultraviolent (UV) oxidation, cross flow filtration, and 31P‐NMR distinguished various classes of organic P compounds in marine waters and sediments into monophosphate esters, nucleotides and nucleic acid, phospholipids,

phosphonates, and polyphosphates (Table VIII).

Particulate unreactive P is a fraction in which the nature of the P compounds is relatively unknown. However, it may contain P sorbed on mineral–

humic acid complexes. Toor et al. (2005b) speculated that particulate unreactive P in dairy eZuent may originate from coatings of soil particles (e.g., clay,

Ca, Fe, Al), eaten by dairy cows during grazing in animal rumen, by inositol

hexaphosphate.

Adequate knowledge about P speciation in wastes, gained by physicochemical fractionation, can provide invaluable information about the potential bioavailability of these P forms if they reach water bodies. For example,

dissolved reactive P is known to be immediately available to aquatic biota,

while dissolved unreactive P may become available over a short period of time

(Whitton et al., 1991). On the other hand, the recalcitrant nature of particulate reactive and unreactive P in the environment means that these fractions

have a low bioaccessibility and might not constitute a direct short‐term threat

to surface water quality. Therefore, physicochemical fractionation can provide a rapid and low cost means to assess the relative bioavailability of

diVerent forms of P in organic wastes.



24



G. S. TOOR ET AL.



D. SEQUENTIAL PHOSPHORUS FRACTIONATION

Sequential chemical P fractionation is primarily used for solid materials

but can also be used for liquid wastes after some form of drying (e.g., air‐,

oven‐, freeze‐drying). The earliest work on P fractionation in manures using

chemical extractants was documented by Funatsu (1908) and Tsuda (1909).

They sequentially extracted P forms in herring guano as: (i) acid soluble

(inorganic P, phytic acid type material), (ii) alcohol–ether soluble (phospholipids), and (iii) residual (nucleic acid type material). Other workers later

used this methodology to separate P forms in poultry manure, farmyard

manure, and cow manure (McAuliVe and Peech, 1949). They found that

organic and inorganic P was present in equal proportions. This fractionation scheme was later adopted by Peperzak et al. (1959) and later Barnett

(1994a,b) modified this method, as illustrated in Fig. 4, and analyzed a large

number of dairy, poultry, and hog manures (Table IX). However, this

method is not widely used by many researchers because it provides limited

information about organic P forms, does not distinguish between inorganic

P forms, and is time consuming.

The other sequential chemical fractionation method, which has been

more popular among researchers, is largely adopted from the fractionation

method suggested for soils by Hedley et al. (1982b). The Hedley fractionation scheme employs acid or alkaline reagents (mild to strong), where

material (soil, waste) is sequentially extracted with H2O or resin, NaHCO3,

NaOH, HCl, or H2SO4 and then the P extracted by each extractant is

analyzed for inorganic P and total P (Fig. 5). The diVerence between the

total P and inorganic P in each extractant is assumed to be organic P.

For soils, H2O or resin extractable P is thought to be composed of

dissolved inorganic P, whereas NaHCO3 and NaOH extractable fractions

may be a mixture of amorphous and crystalline Al and Fe phosphates, and

some physically and chemically protected organic P. The relatively stable

fractions extracted with acids (HCl or H2SO4) are assumed to be Ca bound

phosphates. Use of this approach often assumes that similar forms exist in

wastes although the matrices of manures are based on organic residues while

soils are dominated by mineral phases. In addition, soils contain more Al

and Fe than manures, while manures have greater concentrations of Ca and

Mg than soils.

Leinweber et al. (1997) and Sharpley and Moyer (2000) and Dou et al.

(2000) used the Hedley fractionation method to characterize P in a range of

dairy, poultry, and swine manures (Table X). Dou et al. (2000) reported that

H2O, NaHCO3, NaOH, and HCl removed 67, 13, 5, and 5% P for dairy

manures and 50, 20, 5, and 25% P for poultry manures, respectively. The

major amount of P was extracted from manures with H2O and NaHCO3.

This is in contrast with soils, where most of the P was removed with NaOH



CHARACTERIZATION OF P IN ORGANIC WASTES



Figure 4



25



Manure phosphorus fractionation scheme. Adapted from Barnett (1994a).



and HCl extractions (He et al., 2003a; Sharpley, 1996; Tiessen et al., 1984).

Therefore, it is inappropriate to apply soil fractionation procedures on

manures, as it is not clear exactly what forms of P are being removed by

the diVerent fractionation steps. However, with precise knowledge of

extracted P forms available via spectroscopic methods, as discussed in the

following section, it should be possible to accurately develop and interpret

waste fractionation procedures.



26



Table IX

Fractionation of Phosphorus Forms in Various Manures According to the Fractionation Scheme of Barnett (1994b) as Shown in Fig. 4a



Manure type

Dairy (15 herds)



Total P (g kgÀ1)

9 (6–16)b



18 (13–23)



Layer (11 flocks)



24 (16–30)



Hogs (16 herds)



29 (20–40)



a



Adapted from Barnett (1994b).

Range of values.

c

Nucleic acid type material.

d

Inositol hexaphosphate or phytic acid type material.

e

Phospholipids.

b



Range (%)



CoeYcient of

variation (%)



63.2

27.7

7.8

1.4

34.8

11

53.4

0.9

49.3

17.3

33.2

0.6

54.7

15.2

29.7

0.4



55.3–71.2

18.1–35.8

2.4–13.9

1.1–1.8

21.4–58.4

1.5–16.6

26.2–75.5

0.4–1.3

39.8–70.0

6.6–31.8

24.7–44.4

0.4–0.8

42.2–76.6

9.2–26.9

13.7–45.3

0.3–0.5



8.1

18.4

46.7

19.6

31.1

45.7

24.4

33.4

21.4

41.2

20.2

19.5

20.3

36

41.6

15.8



G. S. TOOR ET AL.



Broiler (13 flocks)



Inorganic P

Residual Pc

Acid soluble organic Pd

Lipid Pe

Inorganic P

Residual P

Acid soluble organic P

Lipid P

Inorganic P

Residual P

Acid soluble organic P

Lipid P

Inorganic P

Residual P

Acid soluble organic P

Lipid P



Percentage of total P



CHARACTERIZATION OF P IN ORGANIC WASTES



27



Figure 5 Sequential waste phosphorus fractionation scheme. Adapted from Hedley et al.

(1982b) and Tiessen et al. (1984).



Case Study: Identification of Phosphorus Compounds in

Cattle, Broiler, and Swine Manure

Turner and Leytem (2004) first sequentially extracted P forms from

broiler, cattle, and swine manures and then performed solution state NMR

analysis on these extracts. Their results indicated that H2O extraction primarily removed orthophosphate (>89% of total P) in swine manure and

broiler litter, while the H2O extract of cattle manure contained most of the P

as organic forms (monoesters: 37%, DNA: 20%) and only 42% as orthophosphate (Table XI). Orthophosphate was 82–98% in NaHCO3 extracts of

cattle and swine manure, whereas broiler litter NaHCO3 extracts contained



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