Availability, movement, and retention of copper in selected Florida and Vietnamese soils

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Title:
Availability, movement, and retention of copper in selected Florida and Vietnamese soils
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xiii, 130 leaves : ill. ; 28 cm.
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English
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Ho, Van Lam, 1943-
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Subjects / Keywords:
Soils -- Copper content   ( lcsh )
Soils -- Florida   ( lcsh )
Soils -- Vietnam   ( lcsh )
Soil Science thesis Ph. D
Dissertations, Academic -- Soil Science -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 120-128.
Statement of Responsibility:
by Ho Van Lam.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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Full Text












AVAILABIL ITY. A.ND RKTENTEON OF COPPER
Jt SELECTED FLORIDA AND VIETNAMESE SOILS









By

HO VAN TAM


A DISSEPRATION PRESENTED TO THE RAD'ATE CO,"CLfT, OF
THE UNIVERSITY F FLG RIDA
IN PARTIAL FULF"'LII.NT WF THU RE ll;i. :I S X'R THE
i... ... -, OF DOCTOR UOF FL iLO-IOPHY

















P1 ,v. i f O:' LJ.ORIO.


i- /6



















DED CATION


This Dissertation is dedicated to
the author's mother who passed away
before her only son attained the
academic goals that she had
encouraged him to seek.
















ACKNOIkl- -'r2 FC,;-i.NTS


The author wishes to express his sincere appreciation to Dr.

N. Gammon, Jr., Chairman of the Sapervisory Committee, for his valuable

counsel, guidance, c.ud assistance cdring the entire course of this

investigation, and in preparation of this manuscript.

Sincere thanks ire also expressed to D-s. J.G.A. Fiskell, R.C.

Stoufer, T.L. Yuan,and B.C. Volk for their recomnini.datiouns, and con-

structive help in gathering research information and man~.icript review.

My appreciation is given to Dr. M.E. M:!rvel, whc initiated the adminis-

trative work and was instrtuienLal in providing the opportunity fo the

author to attend the University of r'lorida amd whIo served on the Super-

visory Committee until resu- in: -.n overseas assirgnment. Also iy

expressed gratitude is extended to Dr. L.W. Zelazny, who participated

as a member of che Supervisory Commiittee until th' last ph.-se of the

author's research program. It is regretted that Das. Mlrvel ald Zelazny

in their well-deserved positions A-ere prevented fronm being able to

actively review the final dissertation.

The author wouldd like to thank Drs. C.F. E'no and D.F. !lothwell,

Chairman and Gr:aduate Coordinator o Saoil Scienc-e Depart rcnt, rLespec-

tively for their aJi-mnistrative assistance; the iFo-d Foundation and the

Agency for international Developmwnnt (AID) for :'h;ir finarcj.-ti sppor:.

To Dr. H.L. Breland and his staff. who gave va.'able hel with

n:icroe]..ement nnalyss; -to Mr. Harr' Johison, 'r:. A.D. Wair, L nr Mr.









Nguyen Gia Hai for their assistance on sazr:ple collections, routine plant

and soil analyses; to Mrs. Adele Koehler for her excellence in typing

and proofing the final copy of this dissertation, the author is most

grateful.

The author would also like to gratefully recognize his dearly

beloved wife, Phuong, who through t:e 3onec y years when the author was

away from hocne, has taken care of his daughter. To his father and his

sisters for their continuous encouragement which guided him to the

present achievement, the author i.c deeply incebted.
















TABLE OF CONTENTS


ACKNOWLEDGEMENTS .

LIST OF TABLES .

LIST OF FIGURES .

ABSTRACT .

INTRODUCTION .

LITERATURE REVIEW .

Physiological Function

Mechanisms of Cu Moveme
Plant Roots .

Convection .

Diffusion .

Root Interception

Factors Controlling Cu

Total Cu Reserve a

Soil Acidity (pH)

Crystalline and Am


of Cu in Plants .

n. in Soil Prior to Uptake by









Availability in the Soil. .

nd Supply . .



orphous Clay Materials. .
orphous Clay Materials. .


Soil Organic Matter . .

Complexation of Soil Cu2+ with Synthetic Chelating
Agents . . .

Chelated Cu to Total Cu Ratio in the Soil Solution.

Copper Absorption by Plants . .

Interaction Between Cu and Other Elements .
4+ 2+
Measurement of Cu- by Cu -Selective Electrode .


Page

iii

viii

x

xi

1

4

4


. .









TABLE OF CONTENTS
(continued)


MATERIALS AND METHODS. . .

Field Experiments . .

Apopka Fine Sand (HawthorneOrchard). .

Fuquay Fine Sand (Earleton Orchard). .

Greenhouse Experiments. . .

Plant Tissue Sampling, Preparation, and Analyses.

Sampling and Preparation . .

Analyses . . .

Soil Sampling, Preparation, and Analyses. .

Sampling and Preparation . .

Analyses . . .

Statistical Analyses. . .

RESULTS AND DISCUSSION . .

Field Experiments . .

Response of Pecan Trees to Cu Application. .

Distribution of Applied Cu in Surface Layers
Fuquay Fine Sand . .

Availability of Applied Cu to Plants .

Greenhouse Trial. . .

Laboratory Study. . .

Solution Studies . .

Saturated Soil Solution Extract Studies. .

SUMMARY AND CONCLUSIONS. . .


APPENDICES


I Synthetic chelating agents which have been
complexation (Norvell, 1972) .


used for Cu


Page

28

28

29

29

30

32

32

33

34

34

34

41

42

42

45


. . . 110








TABLE OF CONTENTS
(continued)


Page

II Determination coefficients (R 2) of linear curves in
Fig. 2. . . .. 112

III Linear regression analyses of curves in Fig. 8 and
Fig. 9. . .. 113

IV Linear regression analyses of curves in Fig. 12 and
Fig. 13 . ... .... .. 114

V Linear regression analyses of curves in Fig. 15 .... .115

VI Effect of supporting electrolyte on chromatograms of
H20 extract from Fuquay fine sand . ... .116

VII Areas of chromatographical peaks shown in Appendix VI 118

VIII Selected list of chromophoric groups (Boltz, 1966). 119

LITERATURE CITED. . . 120

BIOGRAPHICAL SKETCH . . .. 129















LIST OF TABLES


Table Page

1. Estimated amounts of micronutrients supplied by three
mechanisms to corn roots growing in a heavily fertilized
silt loam soil at pH 6.8 (Barber, 1966) . 8

2. The occurrence of Cu in rocks and soils (Krauskopf, 1972) .. 8

3. General chemical properties of humic and fulvic acid
(Stevenson and Ardakani, 1972). . ... 14

4. Basic fertilization applied to Vietnamese soils ...... 31

5. Some properties of the soils selected for investigation 43

6. Levels of Cu in soil at Hawthorne and Earleton orchards one
year after CuSO4 application. . ... 44

7. Copper levels in leaves of different pecan cultivars in
Hawthorneorchard in the first year after CuSO4 application,
average of all treatments . . .. 46

8. Effect of cultivar and treatment on Cu levels in pecan leaves
in Hawthorne orchard . . ... 47

9. Copper levels in pecan leaves in Earleton orchard .. .50

10. Growth responses of pecans to CuS04 application ...... 52

11. Analysis of variance for growth responses of pecans .... .53

12. Distribution of Cu and other nutrients in 0-15 cm of the soil
profile in the Earleton orchard . .... 56

13. Copper in 0-15 cm of the soil profile of the Earleton orchard
27.5 months after CuSO4 application . .... 58

14. Copper levels in blades of Pensacola bahiagrass (Paspalum
notatum, Flugge) as influenced by CuS04 application in the
Earleton orchard. . . .. 60

15. Correlation coefficients between Cu levels in Pensacola
bahiagrass and various soil extracts. . ... 61

16. Level of Cu in selected Vietnamese soils with and without
CuS04 applications. . . .. ... 63


viii








LIST OF TABLES
(continued)


Table Page

17. Copper (Cu) content and dry weight (DW) of corn grown in
Vietnamese soils . . 64

18. Chelating ability of EDTA and DTPA. . ... 69

19- Formation constants for EDTA and DTPA (Norvell, 1972) 70

20. Chelated Cu to total added Cu2+ ratio (CTR), at different
organic matter and soil Cu levels in Apopka fine sand 72

21. Quantity of Cu2+ completed by soil extracts from Apopka fine
sand . .. . 84

22. Chelated Cu to total added Cu2+ ratio (CTR), at different
organic matter and soil Cu levels in Fuquay fine sand 85

23. Quantity of Cu2+ completed by soil extracts from Fuquay
fine sand . . . 93

24. Chelated Cu to total added Cu2+ ratio (CTR) in selected
Vietnamese soils . . .. 95

25. Quantity of Cu2+ completed by soil extracts from Vietnamese
soils . . . 98

26. Effect of supporting electrolyte on completing ability of
H20 extract from Fuquay fine sand . .. 102

27. Summary of Cu2+ availability in H20 extracts from selected
soils ...... . . 103

28. Approximate wavelength corresponding to maximum and minimum
absorbance in the range from 250 to 300 nm of two chelating
agents and H20 extracts from selected Florida and Vietnamese
soils . . . 106















LIST OF FIGURES


Figure Page

1. Comparison of Cu2+ chelating abilities of 11 chelating
agents in soil solution (Norvell, 1972). ... 21

2. Effect of Cu2+ addition on 0.01 M CaSO4 and two chelating
agents . . ... 68

3. Chromatograms of some organic compounds. .. 74

4. Chromatograms of 0.01 M CaS04 extracts from Apopka
fine sand. . . . 75

5. Chromatograms of H20 extracts from Apopka fine sand. 76

6. Effect of K2S04 on H20 extracts of Florida soils .... 77

7. Effect of K2S04 on H20 extracts of Vietnamese soils. .. 78

8. Effect of Cu2+ Addition on 0.01 M CaSO4 extracts of Apopka
fine sand. . . .. 81

9. Effect of Cu2+ addition on H20 extracts of Apopka fine
sand . . . .. .82

10. Chromatograms of 0.01 M CaSO4 extracts of Fuquay fine
sand . . . 87

11. Chromatograms of H20 extracts of Fuquay fine sand .... 89

12. Effect of Cu2+ addition on 0.01 M CaS04 extracts of
Fuquay fine sand . . ... 91

13. Effect of Cu2+ addition on H20 extracts of Fuquay fine
sand . . . .. 92

14. Chromatograms of H20 extracts from Vietnamese soils. 97

15. Effect of Cu2+ addition on H20 extracts from Vietnamese
soils . . . 99















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



AVAILABILITY, MOVEMENT, AND RETENTION OF COPPER
IN SELECTED FLORIDA AND VIETNAMESE SOILS

By

Ho Van Lam

June, 1976

Chairman: Dr. Nathan Gammon, Jr.
Major Department: Soil Science

The response of pecans (Carya illinoensis, L.) grown in two

Florida soils and of corn (Zea mays, L.) grown in selected Vietnamese

soils to copper (Cu) application was observed. The Cu content of

saturated soil solution extracts, the nature of the organic matter (OM)

2+
in these extracts, and their ability to complex additional Cu were

also investigated.

The Cu treatments for pecans were 0, 1, and 3 ppm in a Hawthorne

orchard (Apopka fine sand) and 0 and 3 ppm in an Earleton orchard

(Fuquay fine sand). Parameters measured were Cu levels in leaves and

growth (tree circumference at 120-cm height). Data on both parameters

were collected yearly. Levels of Cu in leaves from pecans grown at the

3-ppm Cu rate on Fuquay fine sand were significantly higher than the

check during the first year of the experiment but not in later years.

The 3-ppm Cu treatment on the Apopka fine sand produced a steady in-

crease in the Cu levels in the leaves but it was not significant until









the third year. Similarly, the analysis of variance (ANOVA) for tree

growth showed steadily increasing F values in successive years but the

5% level of statistical significance was not achieved. Evidently, a

rather long period is required for surface applied Cu to reach the

active rooting zone of pecans. The downward movement of Cu in the soil

profile was very slow. In Fuquay fine sand, Cu moved ca. 7.5 cm after

27.5 months. It was determined that the Cu applied to the Fuquay fine

sand remained available for root uptake since the Cu level in Pensacola

bahiagrass (Paspalum notatum, Flugge) under pecan trees fertilized with

3 ppm Cu was increased significantly.

Significant differences were observed in the Cu levels in leaves of

different pecan cultivars.

In a greenhouse experiment to test the response of corn to Cu appli-

cations to Vietnamese soils, there was a trend for increased Cu in the

tissue with increased Cu applications which was significant for Ninh-Chu

and Eakmat soils, but not for Trang-Bang, Thu-Duc and Dalat soils.

Total dry weight production did not indicate a consistent response to Cu

application.

Copper in saturated soil solution extracts was very low and the
2+ 2+
ionic Cu measured by Cu -selective electrode, provided such a low

electrode potential that it could not be detected accurately. Saturated

soil solution extracts removed from 1 to 5% of the soil OM. Water

extracts contained more OM than did 0.01 M CaSO4 extracts. By liquid

chromatography, it was found that most of OM in the extracts was small

size compounds, characteristic of fulvic and simple organic acids. The
2+
Cu2+ complexation by organic ligands in soil extracts depended upon

their nature and quantity.








2+
Chelated Cu to total added Cu2 ratio (CTR) of soil extracts was

determined. It was low in CaSO4 extracts and higher in H20 extracts.

The CTR of CaSO extracts ranged from 16.1 to 39.6% in Apopka fine sand

and from 54.7 to 85.3% in Fuquay fine sand. For H20 extracts, CTR

ranged from 60.7 to 92.1% and from 91.8 to 94.0% for Apopka fine sand

and Fuquay fine sand, respectively. The CTR values for Vietnamese

soils, were 69.5 to 82.7% for Ultisols and was about 90% for the Oxisol.

The quantity of Cu2+ needed to complete the complexation in indi-

vidual soil extracts was computed and compared to synthetic chelating

2+ 2+ 3+
agents such as EDTA and DTPA. The presence of Ca2+, Mg2+, or Al in

soil extracts reduced the quantity of Cu2+ completed by the soil ex-

tract.


--J .1 .a
















INTRODUCTION


Copper (Cu) deficiency in crops has been known in Florida for many

years. It was observed that an economic crop could not be grown in the

Everglades peat soils without application of Cu fertilizers (Allison

et al., 1927). An abnormal condition of growth in Florida citrus, known

as "exanthema" or "die-back" was fully overcome by application of Cu to

both organic soils (Allison, 1931) and mineral soils (Fudge, 1936).

This provides additional evidence of the early need for Cu on many

Florida soils.

Ranges of 63.9-128 ppm Cu for carrot, 79.8-176 ppm Cu for onion or

spinach, and 102-236 ppm Cu for cauliflower or lettuce were optimum for

these five crops on acid sphagnum peat soil (McKay et al., 1966). Al-

though levels of applied Cu were high, they found that Cu content in plant

tissues ranged from 8.1 to 10.9 ppm, 10.0-10.8 ppm, 19.0-26.8 ppm, 4.7-

6.3 ppm, and 6.6-7.8 ppm for carrot, onion, spinach, cauliflower, and

lettuce, respectively. The need for Cu on some of the sandy soils was

also established (Locascio et al., 1964; Locascio and Fiskell, 1966;

Robertson et al., 1973; Navarro, 1975). Locascio and Fiskell (1966) have

observed 10-fold increases in yield of watermelon (from 270 to 2,410

fruits per acre) by applying 2.5 lb/A Cu in the fertilizer. Robertson

et al. (1973) found that the highest yields of soybean (1,767 kg/ha)

occurred when Cu contents of leaves and seeds were 3 and 9 ppm, respec-

tively. An increase in Cu applications from 0 to 8.96 kg/ha increased the








total yields of cucumber f1.om 3.59 to 15.22 Con/ha (Navarro, 1975).

Copper deficiency has not been reported o coimuTercial pecan

orchards, probably because Cu-containing sprays such as Bordeaux mixture

were used for leaf diseases arid scab in the past. A normal range of 21

to 28 ppm Cu in pecan leaves was reported by Alben and Hammer (1939).

However, a recent survey showed that where Bordeaux sprays had not been

used, Cu levels in pecan leaves could be as low as 6.2 ppm although no

Cu efficiency symptoms have been observed (Gammon and Lam, 1974). In

view of the declining use of Bordeaux sprays, there is a need to estab-

lish with greater certainty the Cu requirement of pecans.

In Vietnam, the need for Cu has been studied very little. However,

some Vietnamese soils are very sandy and in this respect, very similar

to Florida soils, while others are very fine-textured. The warm climate

and high rainfall along with low pH of many Vietnamese soils should

enhance the leaching of Cu from these soils. Thus, there is good reason

to suspect the need for Cu in at least some of these soils.

The availability of Cu to a plant depends upon many soil character-

istics such as piT, quantity and quality of soil organic matter (0,M),

nature of the clay mineral, forms of Cu present, rainfall and related

climatic factors. Hodgson et al. (1965, 1966) reported that more than

98% of Cu in solution was in an organic cor.plexed form, s,.:.:cting that

21-
in neutral soils, very small quantities of rree or equated Cu are

available for adsorption reactions. The importance of soil organic

matter in rfaintainin- adeqi.ate Cu levels in the soil solution For plant

growth is emph.Asized by thi-s 'lseiva:ion.

Our precsnt inves station was designed to study response of pecans

to Cu applications on two flcrid- soils an.n Lt: L~ response of -:or, to Cu






-3-


applications on five Vietnamese soils. An evaluation of Cu availability

in these soils was limited to crop responses and to Cu in H20 and CaSO4

soil solution extracts. The complexation ability of organic ligands

found in these extracts was studied and compared to that of two syn-

thetic chelating agents, EDTA and DTPA.



















h'" iolo :Jc-' Fuinc-itn of Cu in Plints


Coppr is aa esser -ial element in plant growth. The roles of Cu

ini plant metabolism are numerous, varied, and complex. Piochanical

research has estrblLsbd that Cu i3 the prosthetic group in several

,-,etalloprotein e )jPes such as, cy:ochrome oxidase, polyphcooo oxidase

(tyrosi.ase), ascoc-ic acid oxidase, lactase, and butyryl Co--A deiy'lro-

gens. (Arnon and Stout, 19 9; Kpilin and Mann, 1939. UDawL4on, 1956;

Gilbert, 1957; Mayer, Andersoni and Bohning, 1966; Sauchelli, 1969;

Tisdale and Nelson, 1971; Epstein, 1972). Arnon (1.949) re.ort:d that

poyphe;-.o1 oxidase is localized in the chloc pasts, ::hus co.m.rng

that Cu is necessary -in Dhotosynthe..is. Other workers reported he

associacion of Cu with many enzymes Iivvolved in ttih eectcro7n transport

syvotcm aind the Kreb cycle (Evons and Sorge'. 1966"). Sslisbhury and Ross

(1969) stated that Cu is a necessary cotrmpocenrt of p t yain. Furtrhor-

more, they believe that Ca may be a part of nitrate red~I' r. and per-

forms a c.a: aly.c role in nitr'.g'cna fixation.



Mechain.imsi of Cu Mov-menti in SoEl Pri.'.-' to Upcal,., v by Pl1-tt Root'


1. k oinson (1972) described I) ,n.: uppiy of aic conutricn: t (Cu, Zo,

Fe, Alo, .-ind Co) to roots by combhicios of three p-rocesss: coCvecrion,

di ffusion, ani r-.,o i:.t'rceptioM,.


LITF i.lF'.rEVIFW








Convection


Convection is the movemuni of water in soils, carrying those ions

that are freely mobile in soil solution. Movement may occur as a result

of a suction gradient, generated at the root surface by transpiration,

or as a gravitational gradient when water moves downward in the profile.

The percentage of nutrient requirement which can be satisfied by

convection, according to Corey and Schulte (1973), depends on:

1) the plant requirements for the nutrient

2) the concentration of the nutrient in the soil solution

3) the amount of water transpired per unit weight of tissue

4) the effective volume of water, moving in response to potential

gradient, which comes in contact with the root surface.

The contribution of the last process is difficult to determine so

that estimates of the nutrient contribution from convection are usually

based on the concentration of the nutrient and the amount of water trans-

pired per unit weight of tissue (Barber, 1966). Such estimates showed

that Cu was supplied almost entirely by convection (Table 1).


Diffusion


Diffusion of ions nay be caused by their concentration gradient.

This gradient is developed as the result oC removal of ions from the

soil solution in close pcoxinity to a root and/or by addition of fresh

water to the soil solution, as from rainfall. The following equation

illustrates the factors which axe important in determining the rate at

which a soluble nutrient will diffuse to a root surface (Corey and

Schulte, 1973):


dq/dt = DAi(CI C,)/L
1








where


dq/dt

D



A

P



C and C2


represents the rate of diffusion to the root surface

is the diffusion coefficient of the nutrient species in

water

represents total absorbing surface of a plant root

is the fraction of the soil volume occupied by water (it

also includes a tortuosity factor)

are the concentrations of the soluble nutrient at a distance

L from the root surface and at the root surface, respec-

tively.


Ellis et al. (1970) found that the D value of Cu2+ in soil is

affected by the type of clay as well as accompanying anion. They re-

ported that montmorillonite at a concentration of 0.5 meq/g had a D
-6 m2 -1
value of approximately 1 x 10 m sec while kaolinite at the same

-6 2 -1
concentration had a value of 5.2 x 10 cm sec The D values were

greater with CuCi2 than Cu(N03)2, CuSO4, and Cu(OAc)2 in the mentioned

order. Values of D for other clys aR: various concentr.ations were also

reported. These slow rate: of diffusion of Cu indeed restrict their

ability to supply this nutrient to plant rcots.


Root Intercertion


Roots in growing thro-.:h soil, c;t:,i1 sh contact with the mineral

surface; nutrients acquired in this manner are attributed to root inter-

ception (a modification of Jenny's contact exchange concept). The uptake

of nutrients by such contact is ncc deponr ii.en on the processes of con-

vection or diffusion in the usual sein3e, alt;ho>,sh water ~ns b: present

(Passioura, 1968).








An estimate of the possible contribution of root interception to

satisfy the nutrient requirement of a plant can be tradee on the basis of

the following assumptions (Barber, 1966):

1) The maximum amount of nutrient intercepted is presumed to be

"available" in the soil volume occupied by the roots.

2) Roots occupy an average of 1% of total soil volume.

3) About 50% of the total soil. volume is composed of pores; there-

fore, the roots occupy about 2% of the total pore space.

Table 1 presented the amounts of micronutrients supplied by the

above three mechanisms on the basis of these assumptions. The accuracy

of the estimates based on such assumptions is questionable in view of

the fact that the combined amount of the nutrients supposedly supplied

by the interception and convection mechanisms as in the case of Cu and

Zn far exceeded the amount of the total uptake. Nevertheless, the data

show that the convection mechanism contributes a major part of nutrients

of plant requirements.

In soil, all these three mechanisms may be influenced by the ex-

change capacity, pH of the soil, solubility of some minerals, and the

activities of crystalline and amorphous clay materials and OM components.


.Fctors Controlling Cu Availability in the Soil


Total Cu Besep.ve and Supply

Reserve


Krauskopf (1972) reported the Cu contents in soils and in rocks,

Table 2. In nature, Cu occurs chiefly as simple and/or complex sulfide

:minera.s. By far, the most abundant of Cu mineraals is chalcopyrite,


-7-









Table 1. Estimated amounts of ricronutrients supplied by chree mech-
anisms to corn roots growing in a heavily fertilized silt loam
soil at pH 6.8 (Porl;2r, 1966).



Total Amolni. supplied by
Element ....
uptake
uptake Root interception Convection Diffusion


----------- ------ kg/ha ----------- .......


0.16

0.23

0.23

0.80


9) Cl

0.10

0.10

0.10


0.35

0.53

0.05

0.53


0.08

0.17


*
Diffusion is assumed to provide the plant
fied by convection and interception.


requirements not satis-


Table 2. The occurrence of Cu in rocks and soils (Krauskopf, 1972).



Igneous rocks Sedimentary rocks
Crust ------- ----------------------Soils
Granite Basalt Limestone Sandstone Shale

---- -------------------. ppm ---------------


100 4 30


45 10-80









CuFeS2. Several other sulfide minerals may accompany chalcopyrite.

Basic carbonates such as malachite, Cu2(OH)2 CO3 or azurite,

Cu3(OH)2(CO3)2 and the hydrous silicate chrysocolla (CuSi03.2H20) may

be found in an oxidized environment. If anions are scarce, CuO can

exist, although it is not a common mineral. Under reducing conditions,

sulfides were found to be mostly compounded as Cu2S rather than CuS.

From calculated values for standard free energies of formation, Garrels

and Christ (1965) reported the reduction of Cu2+ to Cu took place when

standard redox potential at pH 7.0 reached -153 mv.

Whatever might be the form of Cu in primary rocks, it dissolves in

water during weathering primarily as Cu2+, as long as the solution re-

mains slightly acidic and in the oxidized state. This ionic form is

found in many stable complex ions and molecules as well as organic

complexes.

If Cu is present in solution as a cuprous complex, or if a Cu2+

solution moves into a reducing environment, Cu20 or even Cu metal may

precipitate when the solution becomes alkaline. Generally, however,

enough H2S is present in a reducing environment to precipitate Cu as

CuS or Cu2S, both of which are so very insoluble that they can form even

under moderately acidic conditions. Reactions of this sort leading to

the precipitation of definite compounds are probably not common in soil

formation, and almost certainly are less effective than adsorption as

a general mechanism for removing Cu from solution, a characteristic that

can be correlated with the tendency of Cu atoms to form strong covalent

bonds (Northmore, 1959).


Copper fertilizers


Copper fertilizers are available in both organic and inorganic






-10-


forms. Common sources of Cu for fertilization consist of CuSO 45H20,

CuO, Cu-EDTA, Cu lignin-sulfonate, and Cu polyflavonoids (Murphy and

Walsh, 1972). Rate of application varies and depends on both crops and

methods of application. Generally, 2.2 7.0 kg/ha of inorganic com-

pound has been used if broadcast or 1.1 4.5 kg/ha if banded. Organic

compounds required smaller rates, 0.8 2.4 kg/ha and 0.2 0.8 kg/ha

for broadcasting and banding, respectively.

In most cases, single applications of Cu to soils persist and

produce good responses of crop yield. Smith, Rasmussen, and Hrnciar

(1962) have shown that the mobility of Cu in the soil is relatively

slight, thus explaining its persistence. Reith (1968) pointed out that

residual effects of Cu treatments are long lasting and under field con-

ditions, may be adequate for at least 8 years, depending on the magni-

tude of the application and the soil type. In agreement with Reith,

Berger (1965) pointed out the persistence of Cu in the soil and sug-

gested a halt to Cu treatment after a few years to avoid accumulations

of toxic levels.



Soil Acidity (pH)


Lindsay (1972) in his excellent review of inorganic phase equili-

brium of micronutrients in soil, expressed the solubility of soil Cu by

the equation:


Cu2+ soil Cu-soil + 2H (2)



The log K value for this equilibrium was found to be -3.2 (Norvell and

Lindsay, 1969). This relationship can be expressed as





-11-


(Cu2) = 1032 (H+) (3)


2-
At high pH, because of the increasing concentration of CO and

OH in the soil solution, Cu is likely to precipitate (Fried and Broes-

hart, 1967). As a consequence, excessive liming causes the formation

of Cu(OH)2 and CuCO3 which are not readily available to plants (Purvis

and Davidson, 1948; Menzel and Jackson, 1950; Adam and Pearson, 1967).
2+ +
Copper in the exchange sites may exist as Cu2+ or as CuOH depend-

ing upon the pH of the soil solution (Truog and Bower, 1940). In mildly

acidic systems, however, hydrolysis of copper is relatively unimportant.

At pH 6.0,the Cu2+/CuOH ratio is approximately 100:1 and this ratio

increased 10-fold when pH of solution decreased one unit (DeMumbrum and

Jackson, 1957). It can be inferred from the observation of Krauskopf

(1972) that in the pH range at which most economic crops are grown, Cu
2+
in soils is probably present chiefly as the adsorbed Cu2+ or in soil

solution as the ion and various complexes.


Crystalline and Amorphous Clay Materials


Effect of clay type on Cu diffusion coefficient has already been

discussed (Ellis et al., 1970).

Copper as well as other cationic micronutrients such as Zn, Fe,

and Mn can be held near the negatively charged soil surface by electro-

static attraction. They can also enter into specific adsorption pro-

cesses through covalent bonding on the clay surface (Ellis and Knezek,

1972). Isomorphic substitution in the crystal lattice of layer silicates

may also be possible. Hodgson (1963) suggested that solid-state dif-

fusion into the relatively open crystal structure of clay minerals could






-12-


account for adsorption of micronutrients by these minerals. Heydermann

(1959) found that Cu2+ is adsorbed appreciably even by quartz, and of

course, much more strongly by clays. The adsorption capacity of dif-

ferent clay minerals increases in the order kaolinite < illite < mont-

morillonite. Adsorption increases as the pH rises. Ferric hydroxide

2+
is also an adsorbent for Cu2+ provided the pH is above the isoelectric

point of Cu(OH)2 (Hem, 1960).

DeMumbrum and Jackson (1956a) observed that Ca-montmorillonite and

Ca-peat could accumulate Cu or Zn from very dilute, neutral solutions

2+
either in the presence or absence of excess Ca2. They felt that mont-

morillonite and peat had specific exchange sites for Cu as well as Zn.

In another paper (1956b),they reported that Cu or Zn saturation de-

creased the intensity of the 2.8-W infrared absorption band of the

hydroxyl ions in montmorillonite, vermiculite, and kaolinite, indicating

a reaction or bonding with the octahedral OH in layer silicates. This
H
bond could be visualized as a Cu-O-Al or Cu-6-Al in positions where

access by the Cu or Zn ions is possible because of silica net openings,

crystal defects, or at broken edges.



Soil Organic Matter


In a recent review, Stevenson and Ardakani (1972) classified or-

ganic compounds in soil that form stable complexes with metallic ions

into two main groups:

1) biochemicals of the types known to occur in living organisms; and

2) a series of complex polymers formed by secondary synthesis

reactions.

Included in the first group are the organic acids, polyphenols,





-13-


amino acids, peptides, proteins, and polysaccharides. The second group

includes humic and fulvic acids.

The organic acids most effective in forming stable chelate com-

plexes with metallic ions are those of the di- and tricarboxylic hydroxy

types such as citric, tartaric, and malic acid. Sugar acids such as

gluconic, glucuronic, and galacturonic acids may also be important as

solubilizers of mineral matter.

In classical terminology, humic acid (HA) is the material extracted

from soil by alkaline solutions and precipitated upon acidification;

fulvic acid (FA) is the material soluble both in alkali and acid. Num-

erous attempts have been made to devise structural formulas for HA and

FA but as Dubach and Mehta (1963) pointed out, no two humus molecules

may have the precisely identical structure. Contemporary investigators

(Stevenson and Ardakani, 1972) favor a type of HA consisting of micelles

of a polymeric nature, the basic structure of which is an aromatic ring

of the di- or trihydroxyphenol type bridged by -0-, -NH-, -N=, -S-, and

other groups. These compounds contain both free OH groups and the

double linkages of quinones. The overall chemical properties of HA and

FA were reported by Scheffer and Ulrich (1960) and summarized by Steven-

son and Ardakani (1972) as presented in Table 3. The latter workers

reported that in the natural state, HA and FA are intimately bound to

clay, to one another, and to other organic constituents. A variety of

intermolecular bonding forces are involved, including H-bonding, ester

linkages, van der Waals forces, and salt linkages. Dubach and Mehta

(1963) pointed out that even after extraction, it is difficult to free

humic substances from inorganic components such as cations and clay or

organic impurities such as carbohydrate and proteins.







-14-


Table 3. General chemical properties of humic and fulvic acid (Stevenson
and Ardakani, 1972).



Fulvic acid Humic acid

Crenic Apocrenic Brown Humic Gray humic
acid acid acid acid
(Light yellow) (Yellow-brown) (Dark brown) (Gray-black)


------ -------- increase in degree of polymerization -----------------

2,000? -------------- increase in molecular weight ----------+ 3000,000?

45 ----------------- increase in carbon content, % ---------------- 62

48 ----------------- decrease in oxygen content, % ----------------o 30

1,400 --------- decrease in exchange acidity, meq/100 g ----------- 500





_1>-


Soil OM forms complexes witn metals by coagulation, peptization,

ion-exchange, surface adsorpticu, end chelation (Mortensen, 1963).

Schnitzer and Skinner (1965) reported that the stable organometal-

lic complex is obtained from the formation of electrostatic and/or

covalent bonding between the metallic ions and the ligands.


Coagulation peptization


Ong and Bisque (1968), in a study of the coagulation of humic col-

loids by metallic ions, reported that the stability of metal-organic

matter association is due to the Fuoss effect or coiling of the linear

polymers chain as coulombic repulsive forces are reduced by electrostatic

binding.

The coagulation of HA depends upon the pH and the ionic strength

(p) of the solutions (Schnitzer and Khan, 1972). In the absence of

salts, virtually complete peptization occurred at pH 3.0; an increase in

i raised the pH of peptization to pH 4.5- 5.0. Peptization usually

occurs at a somewhat higher pH than coagulation, possibly because of

association of HA particles by hydrogen-bonding (Schnitzer and DeLong,

1955).

Trivalent ions were more effective in coagulating HA than were di-

valent ones. The order of increasing effectiveness of metal ions for
n2+ 2+ 2+ 2+ 2+ 2+ 3+
coagulating HA was Mn < Co < Ni < Zn < Cu < Fe < Al (Khan,

1969).

Wright and Schnitzer (1963) found that the capacity of a number of

metals to coagulate FA at pH 3.5 and 7 decreased in the following order:
3+ 3+ 2+ = Mg2+
Al > Fe > Ca Mg

Hodgson (1963) noticed that organic soils are among those most

commonly deficient in Cu. This occurs because their total Cu content





-16-


is frequently low and their capacity to fix Cu is high.

In a later work, however, Hodgson et al. (1966) found that organic

completing increased the total concentration of Cu in the soil solution

by a factor of about 100. Gupta (1971) demonstrated that the addition

of OM to soil increases exchangeable Cu. The concept of chelation or

complexation therefore, replaces the concept of coagulation and provides

a second way to interpret metal-OM interactions.


Chelation complexation


Geering and Hodgson (1969) calculated the metal to ligand ratio

from the increase in the proportion of metal completed as the concen-

tration of ligand increased and reported that completing agents in soil

solution form 1:1 complexes with Cu. When an equilibrium between the

metal-ligand complex and the dissociated metal and ligand is established,

they estimated a value of 5.5 0.1 for the log10 of formation constant

KML .

There are various factors which contribute to the stability of a

metal chelate. Corvin (1950) cited the importance of atomic radius on

the formation of complexes. Radius ratio of electron-rich group/metal

2+ 2+
ion governs the coordination number of Cu2+. Commonly, Cu2 coordinates
2+
with four ligands. In some cases, coordination of Cu2+ with two ligands

was observed, but very rarely with three or five. Lehman (1963) re-

ported that the number of rings formed by one molecule of chelating

agent with metallic ion, the size of the chelate rings, and the nature

of the donor atoms are of prime importance. He also found that pH of

the solution plays an important role in chelation reactions.

Rashid (1971) extracted HA from marine sediments and fractionated





-17-


these on Sephadex gels into different molecular weight (MW) fractions.

He determined the ability of a number of di- and trivalent metallic ions

to complex with each of these HA fractions at pH 7.0. He found that

under his experimental conditions and on MW basis, the lowest MW frac-

tions completed two to six times more metals than did the high MW

fractions, and that the amounts of divalent metals completed were three

to four times higher than those of trivalent metals. He also reported

that one-third of the total cation exchange capacity of HA exists in the

non-chelating form indicating that not all acidic groups in the HA par-

ticipated in metal completing.

In a spectrophotometric investigation of the FA fraction from peat,

Mitchell (1967) reported at least one Cu chelate was observed. He

suggested that to complex Cu, both -SH and -COOH groups are required

although the -NH2 group may also be involved. He further found that

the optimum acidity.for complex formation with HA is pH 2.5 to 3.5 and

with FA, pH 6.0. Broadbent and Bradford (1952) used the technique of

functional group methylation with diazomethane and dimethyl sulfate to

show that carboxyl and phenolic groups attached to heterocyclic com-

pounds in soil OM extracts are the important functional groups for Cu

bonding. In a later work, Broadbent (1957) used soil as an ion exchange

column for elutriation of OM that was Cu-saturated or Ca-saturated.

Four elutriation peaks were obtained with the Cu-saturated column while

only 2 peaks were obtained with the Ca-saturated one. He concluded that

carboxyl groups completed both Cu and Ca, but that other functional

groups reacted selectively with Cu to the exclusion of Ca.

Lewis and Broadbent (1961a) used a series-of phenolic and carboxylic

acids as model compounds to show that Cu was adsorbed as CuOH+ by





-18-


carboxyl groups whereas Cu2+ was completed by the phenolic groups.

Results of their subsequent research (1965b; indicated that carboxyl

and phenolic groups were important in the organic completing of Cu in

soils, but noted that the forms of Cu bound were not as clearly differ-

entiated as in the model compounds. Varying acidity of the carboxyl

and phenolic functional groups in the heterogeneous soil system was

suggested as a factor preventing results similar to those obtained for

model compounds. Similar research by Schnitzer (1969) confirmed such a

conclusion.

Wei (1959), indicated that Cu was preferentially adsorbed by OM

until its exchange capacity was satisfied and then by clay minerals.

Davies et al. (1969) reported that the bonding strength of Cu by HA

increased as applied Cu content decreased.

Himes and Barber (1957) noticed that where more than one chelating

agent is present, the one which forms the most stable complex will

chelate the most metallic ion until its chelation capacity is reached.

On the other hand, a cation must satisfy two requirements enabling it

to combine appreciably with a chelating ligand. First, an adequate

quantity of the cation must be present in solution or in equilibrium

with the soil solution to permit formation of a significant concentra-

tion of metal chelate. Secondly, the chelate of this cation must

possess sufficient stability to exist in equilibrium with the concen-

tration of the cation and free ligand in soil solution (Norvell, 1972).

Gamble et al. (1970) proposed two mechanisms to account for the

Cu 2+-fulvic acid equilibrium:





-19-


O OH 0 OH
/ 000
C 1 1
either C C- + C u j + (4)



0 OH 0 0
C (5)

or *0 + Cu C (5)




In equation 4, the reacting carboxyl is ortho to a reacting phenolic OH

group. In equation 5, two carboxyl groups are adjacent for Cu complex-

ing. The reactions produce a proton which reduces pH of the soil

solution. Schnitzer and Khan (1972) remarked that it is difficult to

decide at this time whether metal humates and fulvates are simple or

chelate complexes. The formal distinction between simple and chelate

complex is often arbitrary, and it is difficult to differentiate between

the two, especially when the same kind of donor groups are involved.

What is important, however, is that humic substances have the capacity

of binding substantial amounts of metals and thus exert considerable

control over the supply and availability of nutrient elements to plants.

Stevenson and Ardakani (1972) have clarified some of the conflict-

ing views on roles of OM regarding availability of micronutrients.

According to these workers, metals in the soil that occur in insoluble

combinations with OM are largely those that are bound to components of

the HA fraction while the metals found in soluble complexes are mainly

those associated with FA and/or with individual biochemical molecules

such as organic acids.





-20-


2+
Complexation of Soil Cu+ with Synthetic Chelatig Agents


Norvell (1972) reported that in calcareous soils the stability of

the Cu-chelates decreases in the order Cu-DTPA > Cu-HEDTA=Cu-CDTA >

Cu-EDTA, Cu-EDDKA > CU--EGTA =Cu-NTA Cu-CIT >> Cu-P207 = Cu-P 30 10

Cu-OX.

In more acidic soils, he noticed that the stability of all the

Cu-chelates is reduced because of the decline in free ligand concentra-

tions. The relative stability of different Cu-chelates also changes

with pH so that at slightly acid pH values their stability follows the

order of: Cu-HEDTA > Cu-EGTA Cu-DTPA Cu-EDTA = Cu-NTA = Cu-CDTA >

Cu-EDDHA > Cu-P 0 = Cu-P 01= Cu-OX.

-4
By comparison, he estimated that at 10-4 M concentrations, the

three least effective chelating agents, P20 P3010, and OX, should have
9+
no significant influence on complexation of Cu2 in soil solutions. The
2+
effect of CIT on complexation of Cu2+ is not as clear. DTPA, HEDTA,

CDTA, EDTA, EGTA, EDDHA, and NTA would increase complexation of Cu2+

well above levels characteristic of natural Cu-complexes in soil solu-

tion. However, the magnitude of the increase differs greatly among

these chelating agents (Fig. 1).


Chelated Cu to Total Cu Ratio in the Soil Solution


The dominance of organic completed form of Cu in soil solution and

the very high ratio of chelated Cu to total Cu in the soil solution have

already been discussed (Hodgson et al., 1965 and 1966). Metal complex-

ing is higher in soil solutions from surface horizons than from sub-

surface horizon.

Hodgson et al. (1965) based their studies with soil complexation







-21-


C4
o 'C 0 1


C4 co cv O 0
P- I


+n3 01. n D 31V13H3 JO OIlVH0 E)O1
TC


4 0_


CO
rd-







-l4

-41
l-4








0
r-d


4-,
T-4



O)
r-l



8-H














'O
00M
urw
(dC~
41r -4
















0 0tr
U D





-22-


on eight assumptions of which the most important were:

1) The soil solution, when removed from its native environment,

gives a good representation of the completing found under

natural conditions.

2) Adsorption of cationic micronutrients onto particulate matter

is not significant.

3) Changes in cation concentration in the soil solution by addi-

tion of a radioactive isotope does not measurably alter the

degree of completing in the soil solution.

4) Dilution of the soil solution during the assay does not change

results appreciably.




Copper Absorption by Plants


2+
Copper is absorbed by plants as the Cu2, and may be absorbed as

a salt of an organic complex such as EDTA (Tisdale and Nelson, 1971).

When chelated nutrient reaches the root surface, the plant may be

able to liberate the nutrient ion from the chelating agent and absorb

the nutrient (Hodgson, 1968; Chaney etal., 1972). If, after absorption

of the micronutrient by the plant, the chelating agent does not combine
2+ 2+
with other ions, e.g. Ca2 and Mg at the root surface, it is postu-

lated that the chelate may build up to such a concentration that the

chelating agent and the plant compete for the nutrient (Hodgson, 1968).

Bowen (1969) reported that both Cu and Zn absorption by sugarcane

leaf tissue were characteristic of an active process. Uptake of Cu and

3- -
Zn was reduced by low temperature, 2-3-dinitrophenol, N3-, CN and
3-
As03- and was completely inhibited by amytal (5-ethyl-5-(3-methylbutyl)-

barbituric acid, Na salt) and nembutal (5-ethyl-5-(2-pentyl)-barbituric





-23-


acid, Na salt). Absorption was found to be a steady-state process over

a period of at least two hours. The absorption rate of both Cu and Zn

increased with concentrations up to about 10-4 M where the mechanism

became completely saturated.



Interaction Between Cu and Other Elements


Hiatt et al. (1963) reported that Al3+ at concentrations as low as

0.1 ppm markedly reduced total Cu uptake of wheat roots. This inhibi-

tion is caused by the competition between these two ions for adsorption

sites associated with the roots.

On the other hand, when the Cu content in plants reached its opti-

muip level, the addition of Cu became toxic to plants (Locascio and

Fiskell, 1966). Fiskell and Leonard (1967) recorded that soluble Cu

in excess of 0.1 ppm is toxic to citrus roots. Increased rates of

applied Cu resulted in a significant decrease in B, Fe, K and P content,

whereas Ca, Mn, and Mg were not significantly affected.

One of the earliest studies relating to Cu-P interactions was

conducted in Florida by Forsee and Allison (1944). They reported that

Cu contents of leaves and fruit juice of citrus was decreased as the

level of applied superphosphate was increased. The observation that

excessive application of P induced Cu deficiency in many crops has been

confirmed by many workers (Ervin, 1945; Bingham and Martin, 1956;

Bingham and Garber, 1960; Spencer, 1960).

Locascio et al. (1968) provided additional information related to

Cu-P interaction. They reported that the nature of the P source also

contributed to this interaction. Phosphorus from diammonium phosphate

depressed Cu uptake more than that from either concentrated






-24-


superphosphate or ordinary superphosphate. The mechanism of P-induced

Cu deficiency is not unique. Jamison (1944) reported that when P was

present in large amounts, it would fix Cu, so that Cu became less

available to plants. The idea of P-induced Cu fixation was disputed

by other workers. DeKock et al. (1971) studied the Cu-P interaction

in oats. They reported that aggravation of Cu deficiency with the appli-

cation of P was due to increasing growth of plants and therefore induced

a higher demand for Cu. They also found that N application could en-

hance Cu deficiency.

Fiskell and Westgate (1955) noted that iron chlorosis of crops grown

on sandy soils of Florida was usually a danger signal warning that Cu

was too high and a decrease of P 0 /Cu ratio had taken place. In the

Sanford area, soils having only one pound of available Cu per acre

exhibited Cu-induced iron chlorosis in several crops. Excessively

available Cu in the soil resulted in a stubby root system and the root

surface which was poisoned by excess Cu had reduced ability to absorb

other nutrients. Moore et al. (1957) observed that growth of lettuce

at any one level of Cu was influenced by the Fe supply. The toxic

effects of Cu at high levels of supply were decreased by additions of

Fe, but the adverse effect of high Cu was never completely overcome by

Fe additions.

The antagonism between Cu and Mo was also reported. MacKay et al.

(1966) found that a mutual antagonism existed between Cu and Mo for five

crops. Application of Cu aggravated Mo deficiency in spinach and cauli-

flower, and apparently the application of Mo enhanced Cu deficiency in

carrot, spinach and lettuce. Giordano et al. (1966) reported evidence

indicating that Cu interfered with the role of Mo in the enzymatic

reduction of NO3 in tomato plants.





-z'b-


2+ 2+
Measurement of Cu~ by Cu -Selective Electrode


The use of specific ion electrodes in the study of the soil-water-

plant system is not an entirely new experimental approach. Carlson and

Keeney (1971) cited the works in this field of study of pioneer scien-

tists dated back in the 1940's.



2+
At the present time, Cu -selective electrode is considered as a

useful analytical tool such as in the determination of Cu2 activity

(Orion Research, 1971).

An especially attractive fact is that Cu -selective electrode
2+
measures only the activity of unassociated Cu2+, therefore it can be

used directly and elegantly in the study of Cu complexation. Rechnitz

(1969) proposed measurements of Cu2+ activity under noncomplexing

solution conditions and then again in the presence of the completing

agent. From these measurements and knowledge of the initial concentra-

tions of the reagents involved, the formation constant of the resulting

complex could be obtained. He also remarked that situations involving

a series of complexes, additional equilibria, kinetic complications,

etc., can also be handled through proper design of experiments and use

of appropriate computational techniques.

Carlson and Keeney (1971) reported that the potential of the elec-
2+
trochemical cell created by insertion of Cu 2-selective electrode and a

reference electrode into a sample solution is the sum of a number of

individual potentials:


Ecell E + EM + E (6)


where

E is a constant which includes the internal potential of the







-26-


two electrodes and the asymmetry potential of the membrane

EM is the potential across the membrane

E is the liquid junction potential.


The Nernst equation relates the potential across the membrane to the

2+
activity of Cu2+

A
RT s
E = 2.3 log (7)
1

where

2.3 RT/2F is Nernst factor (29.58 my at 250C)

2+
A is Cu activity in the sample solution
s

A. is Cu2+ activity of the CuS/Ag S membrane (electrode
membrane).


Since A. is constant, its contribution to the membrane potential can be
1
combined with E0 so that


0' RT
cell +2.3 log As + E (8)



As long as EJ does not change as the electrodes are moved from sample

to sample, the change in cell potential will depend only on changes of
2+
Cu2 activity.

2+ 2+
The limit of detection of Cu activity by Cu -selective elec-
-7 -2 -17
trode is ca. 107 M (about 10-2 ppm) in unbuffered and ca. 101 M in

buffered solutions (Carlson and Keeney, 1971). Electrode malfunction
occurs with Ag+ and Hg23+ 2+
occurs with Ag and Hg; and Fe must be held below one tenth of Cu2

concentration (Durst, 1969; Orion Research, 1971). The latter is easily

accomplished by adjusting the sample pH to above 4. In some cases, Cl

and Br interfere with electrode operation. Interference is dependent





-27-


on their levels relative to the level of Cu21- in the sample, and occurs

only if (Orion Research, 1971):


(Cu2+ (C- )2 > 1,6 x 10-6

2+ -2 -12
(Cu )(Br)2 > 1.3 x 101


Because of the fact that activity coefficient of Cu2+ depends on total

ionic strength of the medium (Orion Research, 1971), the maintenance of

a constant ionic strength is necessary if comparable results are to be

obtained. Ionic strength of the sample solution can be controlled by

using supporting electrolyte (Blaede] and Dinwiddie, 1974).
















MATERIALS AND METHODS


Field Experiments


Two young Florida pecan orchards were chosen as experiment sites.

One orchard was planted on a Fuquay fine sand (Arenic Plinthic Paleudult)

in Earleton immediately adjacent to an old pecan orchard. The old

pecan orchard had been sprayed with Bordeaux mixture in 1960 and 1967.

Copper treatments prior to 1950 are unknown. The second was planted on

an Apopka fine sand (Grossarenic Paleudult) in Hawthorne that had been

in sporadic cultivation for more than 75 years. At least once in the

past 20 years, a complete micronutrient frit (including Cu) had been

applied to the area.

Yearly background fertilization was not the same in the two or-

chards. At Earleton, rate of yearly fertilization during the last 5

years varied from 300 pounds to 500 pounds per acre of a fertilizer in

which, percentage of each component was changed from year to year but

averaged 70 lb N, 16 lb P205, and 50 lb K20 per acre annually. The

orchard was last limed (2000 lb/A of dolomite) in November 1965.

In the Hawthorneorchard, 300 lb/A of 15-4-15 fertilizer was broad-

cast every spring and 150 lb/A NH NO every July. In addition, about

4 lb/tree of NH4NO3, depending upon tree size, were applied to the

pecans in February 1974. This orchard had also been limed with 1 ton/A

of dolomite in November 1965.

The effect of Cu fertilization on these two orchards was observed


-28-






-29-


over a period of three growing seasons, from March 1973 to December

1975.


Apopka Fine Sand (Hawthorne Orchard)


Pecan trees (Carya illinoensis L.) in this orchard were grouped

into 7 blocks. Each block consisted of 6 trees of the same kind, either

grafted cultivars or open pollenated seedlings of a cultivar. Tree

circumferences which ranged from 20 to 50 cm were measured at a

120-cm height with a flexible steel tape prior to grouping the trees

for the experiment. These measurements were used to divide each block

of 6 trees into two groups of three of approximately the same size.

Copper was applied as CuSO 45H2 0 (25.45% Cu) to the soil surface

2
around the tree covering an area of about 12.6 m at rates to provide

the equivalent of 0, 1, and 3 ppm per 15 cm depth of soil. The small

amount of Cu applied was first mixed with soil in order to insure a more

even distribution spread over the soil surface. Copper application was

made on March 3, 1973.

Tree growth was determined by remeasuring the circumference of each

tree during the winters of 1973, 1974, and 1975 at 120-cm height when

pecan trees were dormant. Leaf and soil samples were collected for

laboratory analyses, details for which are described later.


Fuquay Fine Sand (Earleton Orchard)


Although the experiments in both orchards were designed in a simi-

lar manner, only two rates of Cu, 0 and 3 ppm, were used in the Earleton

orchard and the trees were grouped into four 6-tree blocks. The trees

of each block were about the same size, but they were not necessarily






-30-


of the same cultivar. All trees were grafted and ranged in size from

25 to 65 cm in circumference at 120-cm height. The culture of the two

orchards was also different. The Hawthorn orchard was disked regularly

to control weeds while the Earleton orchard was planted to Pensacola

bahiagrass (Paspalum notatum, Flugge) and mixed clovers that were grazed

by cattle or mowed without ever disturbing the soil.



Greenhouse Experiments


Five different soil types from Vietnam were used in this study.

Pots containing 800 g soil were given a uniform basic fertilization,

Table 4. Macronutrients (N, P, K) in powdery or crystalline forms were

mixed thoroughly with soils by using a revolving drum. Micronutrients

were applied to the soil surface in the form of a solution prior to

planting.

Copper was applied at 3 rates: 0, 0.3, and 1.8 ppm (0, 0.67, and

4.04 kg/ha Cu or 0, 2.63, and 15.78 kg/ha CuSO4 5H20) by applying Cu

solution to the soil surface.

The soils studied and the number of replications (limited by the

quantities of soil available) were as follows:

1) Ninh-Chu sand (Typic Psammaquent), 6 replications;

2) Trang-Bang sandy loam (Aeric Paleaquult), 3 replications;

3) Thu-Duc loamy fine sand (Typic Paleustult), 2 replications;

4) Dalat clay (Aquic Haplohumox), 2 replications; and

5) Eakmat clay (Typic Haplustox), 2 replications.

Onion (Allium sp.), a crop known to have a high Cu requirement

(Anon., 1966), was planted in all pots but was discontinued because of

fungal diseases. The soils were then permitted to dry before replanting











Table 4. Basic fertilization applied to Vietnamese soils.


Material Rate (Elemental basis)

Source Flement Field* Pot


kg/ha ppm

NH4NO3 N 135 36.5

(NH4)2HPO4 N 23.5
} 135
(NH4)2HPO4 P 26.0

K2SO4 K 135 50.0
MnS04*H20 Mn 0.94 0.4

FeSO 47H20 Fe 0.65 0.4

ZnSO4*7H20 Zn 0.65 0.3

H3BO3 B 0.26 0.1


Florida Cooperation Extension Service, Institute of Food and
Agricultural Sciences, University of Florida, Circular 176B.


-31-





-32-


to corn (Zea mays, L.). Two seeds, of 'Pioneer 3369A', pretreated with

captain and malathion, were placed in each pot. Four days after germina-

tion all pots were tninned to one plant each. Two weeks after seeding,

20 ml of a nutrient solution containing the equivalent of 53 ppm N,

26 ppm P, 49 ppm K, 50 ppm Ca, 30 ppm Mg, and 40 ppm S was added to

each pot.



Plant Tissue Sampling, Preparation, and Analyses


Sampling and Preparation

1. Pecan


Pecan leaf samples were collected from the two experiments in July

of 1973, 1974, and 1975. Each foliar sample consisted of about 100

mature and healthy leaflets selected from the median leaflet and the

median leaf on each twig.

The freshly picked leaves were dried at 650C for a period of 7

days, then ground in a stainless steel mill using a 60-mesh stainless

steel screen. Finely ground samples were stored in capped glass jars

in the laboratory prior to analysis.


2. Grass


Pensacola bahiagrass was sampled from beneath the trees at the

Earleton orchard, in the zone where either 0 or 3 ppm Cu had been

applied. The grass was in leafy vegetative stage, 20 to 25 cma tall at

the time of harvest. It was selectively cut just above the soil sur-

face, contamination with other plant species being carefully avoided.

No attempt was made to measure grass yield. Technique of sample pre-

paration was the same as that for pecan leaves.






-33-


3. Corn


Eight weeks after planting, the entire above-ground portion of each

plant was collected, washed with distilled water, dried and ground in

the same manner as the pecan leaves and bahiagrass.


Analyses


All plant tissues were redried in an oven at 650C for at least 2

hours before weighing for laboratory analysis (Jones and Steyn, 1973).

Five grams of dried, ground samples placed in 50-ml Pyrex glass beakers,

were ashed in a muffle furnace(Type 1600 Thermolyne) at 475 150C for

8 hours (Issac and Jones, 1972). The ash samples were treated with

20 ml of 5 N HC1 plus 5 ml of concentrated HNO3 and taken to dryness

on an electric hot plate. Excess carbon was removed from samples by

adding 5 ml of 30% H202 and evaporating on the hot plate. This step was

repeated if significant quantities of carbon persisted. Then, 10 ml of

5 N HC1 were added and following evaporation, the residue was dried to

dehydrate silica. The residue was treated with 0.1 N HC1, warmed to

dissolve salts, and filtered through a Nalgene funnel, using No. 42

Whatman paper. Quantity of Cu, Zn, Mn, Fe, Ca, and Mg were determined

on the filtrate by atomic absorption spectroscopy (AA). A model B,

Beckman flame emission spectrophotometer was used to determine K in the

filtrate and P was determined by the ammonium molybdate-ascorbic acid

procedure (Watanabe and Olsen, 1965) using a Model 20, Bausch and Lomb

colorimeter at 660 nm.






-34-


Soil Sampling, Preparation, and Analyses


Sampling and Preparation


In Hawthorneand Earleton orchards, four 15-cm deep soil cores,

taken from the Cu treated zone of individual pecan trees, were mixed

thoroughly and used as a composite sample. Soil samples were taken

from 6 replications at Hawthorneand 4 at Earleton, 1 year after Cu

application.

Twenty-seven and a half months after Cu fertilization (June 18,

1975), the top 15 cm of the soil profile in Earleton orchard were

sampled in 2.5-cm increments. Six replications were collected. Five

months following that (November 11, 1975), samples were again taken to

represent the 0 7.5 and 0 15 cm depths from Earleton and Hawthorne

orchard, respectively. These samples were taken from treatments with

the highest and lowest OM contents based on analysis of earlier sam-

ples. These samples were to be used in Cu complexation studies.

In the greenhouse study with Vietnamese soils, after the corn

harvest, the whole soil in each pot was air dried, screened through a

2-mm aluminum seive and stored in paper bags under laboratory atmos-

pheric conditions for various studies.


Analyses


Preliminary chemical analyses


Soil pH in H O and extractable Ca, Mg, K, and P by 0.7 N NH OAc in

0.45 N HOAc buffered at pH 4.8, were determined by the procedures given

in Bulletin No. 102 Southern Cooperative Series (Page, 1965). Soil OM

was determined by the K2Cr207-H2 )4 wet combustion method (Jackson, 1958).






-35-


Micronutrient analyses


Aqua Regia Dissolution


Micronutrients reported in this dissertation are Cu, Mn, Zn, and

Fe. Value representing the total content of each of these micronu-

trients was obtained from aqua regia dissolution (Gammon, 1976).

Duplicate 2-g samples, treated with 10 ml concentrated HC1 plus

5 ml concentrated HNO3, were brought to dryness on a hot plate. Mineral

salts were dissolved in 0.1 N HC1 and filtered through a Nalgene funnel

and No. 42 Whatman paper. The filtrates were analyzed by AA for Cu, Zn,

Mn, and Fe.


Chelating Agent Extraction


Duplicate 20-g samples and 40 ml EDTA-triethanolamine extractant,

consisting of 0.05 M EDTA, 0.01 M CaCl and 0.23 M triethanolamine (TEA)

buffered at pH 8.5, were placed in a 125-mi Pyrex Erlenmeyer flask and

shaken on an Eberbach shaker at 140 reciprocations per minute with a

stroke length of 4.5 cm for 30 minutes (Lam and Gammon, 1976). The

suspensions were centrifuged at 2000 rpm for 10 minutes and filtered

through No. 42 Whatman paper. Copper was determined on the filtrate

by AA.


Dilute Hydrochloric Acid Extraction

1. Extraction by 0.1 N HC1


Duplicate 2.5-g soil samples and 25 ml 0.1 N HC1 were placed in

50-ml Pyrex Erlenmeyer flasks and shaken for 15 minutes. The extracts

after filtration were analyzed for Cu by AA. The results represented

the amount of Cu held by OM (Fiskell, 1965).








2. Extraction by 1.0 N HC1


The use of this IIC1 concentration ii the extraction was an attempt

to obtain a better correlat4or between the data of soil and plant analy-

ses than had been observed with more dilute acid extracts.


Saturation Extraction


Six replicates of 100-g soil samples were placed in plastic 0.2-i

filter units (Nalgene). Deionized water or 0.01 M CaSO4 solution was

added to produce a saturated condition. The unit was capped to prevent

evaporation and left overnight. Suction (ca. 62 cm Hg) was then applied

to remove ca. 10 ml of soil solution. Additional 10 ml of H20 or 0.01 M

CaSO4 was added and the suction reapplied. This step was repeated until

a total volume of soil extracts which was slightly less than the original

volume added to the soil sample was collected (Gammon, 1976). The soil

solutions extracted from the same soil were combined and mixed thoroughly

in order to have a sample large enough for the following determinations:
2+
1) Copper titrimetric analysis, using Cu -selective electrode;

2) Total cation concentration by conductivity bridge;

3) Organic matter in the soil solution by carbon analyzer;

4) Fraction size separation by gel permeation chromatography; and

5) Total Cu in the soil solution by AA.


Copper Titrimetric Analysis

1. Apparatus

2+
A commercial Cu -selective electrode (Model 94-29, Orion Research)

and a double junction reference electrode (Model 90-20, Orion Research)

were used in these determinations. The electrode leads were connected


-3b-






-37-


to a digital pH meter (Model 701, Orion Research). All electrode poten-

tials were read at room temperature. Reproducible stirring was achieved

by using a heat insulated magnetic stirrer and Teflon-covered bar.


2. Reagents


Copper solutions were prepared by successive 10-fold dilutions,

2+
beginning with a primary standard 1000 ppm Cu2+ solution (Matheson

2 -1
Coleman and Bell). Solutions ranged in concentrations from 102 to 10
2+
ppm Cu2+ were stored in new 100-ml Nalgene plastic volumetric flasks to

prevent the loss of Cu2+ through adsorption on the inner walls of con-

tainers (Blaedel and Dinwiddie, 1974).

In order to maintain a constant ionic strength in the working range

2+
of the standard curve, a series of Cu2+ standards in the supporting

electrolyte were prepared by mixing 10 ml Cu solution of various con-

centrations and 90 ml 0.01 M CaSO4 in 100-ml plastic Nalgene volumetric

flasks to provide a Cu concentration range of 102 to 10-2 ppm.


3. Procedure

Preparation of standard curve


A 25-ml aliquot of each standard solution was placed in a 100-ml

plastic Nalgene beaker, which was put on the heat insulated magnetic

stirrer and the electrode potential was measured. The plot of electrode

potentials of standards vs. log10 of standard concentrations was used

as a standard curve. This curve was redetermined daily prior to the

titration of unknown samples.


Solution studies


The effects of two chelating agents (EDTA and DTPA) and 0.01 M





-38-


CaSO4 (a soil extractant) on the activity of Cu2+ were studied.

-5 -5
A 25-mi aliquot of 5 x 10 M EDTA or 5 x 10 M DTPA plus 1 ml of

0.5 N K2SO4 (a supporting electrolyte) was placed in a 100-ml plastic
2+
Nalgene beaker and the potential for Cu -selective electrode read,
2+
followed by a titration with 100 ppm Cu2+ solution without the support-

ing electrolyte until the increase of electrode potential indicated a

recovery of added Cu2+ from the standard curve, identical to that

observed in the presence of the electrolyte alone.

No supporting electrolyte was necessary in the 0.01 M CaSO, solu-
2+
tion because added Cu(NO3)2 from 100 ppm C2+ standard was too low to

cause a significant change in the ionic strength of the sample solution.

Between titrations, the electrodes were cleaned by rinsing with a

stream of distilled water from a squeeze bottle, followed by immersion

in 0.025 M H2S04, which was stirred until the electrode response reached

a constant value, a value which was determined daily. The electrodes

were then removed from the acid, rinsed with distilled water, blotted

dry with a tissue and then immersed in the solution to be measured.

When not in use, the electrodes were stored dry in air as specified by

the manufacturer.

When the electrode potential of 0.025 M H2SO did not reach con-
2+
stant value, the Cu -selective electrode surface was wet polished by

hand with a piece of abrasive plastic ca. 2.5 x 2.5 cm (Orion Research)

for 30 seconds. This was repeated, if necessary, until a constant value

was obtained.

The pH of the solutions before and after titration were checked by

using a combination pH electrode (Fisher, Model Microprobe No. 13-639-

92) connected to a Beckman Zeromatic pH meter.





-39-


Saturated soil solution extract studies


A 25-ml aliquot of the saturated soil solution extract plus 1 ml

0.5 N K SO was utilized for potential reading and titration, using

the same technique as described above.

Total Cu2+ added was plotted against Cu2+ remaining in the soil extract

by using linear regression analysis. Each curve consisted of two por-

tions: the top portion of the curve showed the normal response to Cu2+

not completed while the lower portion showed the effect of completing

agentss. The breakpoint of the curve was obtained by extrapolating

its two portions. Points near the breakpoint were omitted since this

portion is curvilinear. The percentage of Cu2+ completed from total
2+ 2+
added Cu2+ at the breakpoint was termed chelated Cu2+ to total added
2+
Cu2+ ratio (CTR).

The complexation ability of the soil extract could be compared with

that of EDTA or DTPA by use of the equation:


L x Cu
Complexation ability of the soil extract, mole = S (9)
CuL
L


where


L is the quantity of synthetic chelating agent in 25 ml solu-

tion, in mole
Cu is the quantity of Cu2+ completed by L, in mole
Cu is the quantity of Cu2+ completed by ligands in 25 m sole
2+
Cu is the quantity of Cu completed by ligands in 25 ml soil
S
extract, in mole.


Total Cation Concentration in the Soil Extract


Total cation concentration in the soil extract was calculated from





-40-


electrical conductivity (EC) of soluble salts in the sample solution

which, in turn, was calculated from cell resistances (Bower and Wilcox,

1965) obtained by use of a conductivity bridge (Beckman, Model RC 16B2):


0.0014118 x R
EC, mho/cm (at 250C) = std
R
ext


(10)


(11)


Total cation concentration, meq/1 = 10 x EC, mmho/cm
where


0.0014118




Rstd



R
ext


is the EC of the standard 0.01 N KC1 solution in mho/cm

at 250C

is the cell resistance when the conductivity cell fille

with standard 0.01 N KC1 solution

is the cell resistance when the conductivity cell fillet

with sample solution.


d


d


Organic Matter Measurement


Duplicate 75-mi aliquots of saturation extract were brought to

dryness. Dry weights of the residues were recorded prior to the C

measurement which was determined by dry combustion method and performed

on a high-temperature induction furnace (Lezo, Model 100) using iron

and tin accelerators (Allison et al., 1965). The %OM was calculated

from the equation:


%OM = %C x 1.724


(12)


Fractionation by Gel Permeation Chromatography


Chromatograms of the soil extracts were obtained from a liquid

chromatograph (Water Associates, Model ALC-202) under the following





-41-


operating conditions:

1) Length of column : 122 cm.

2) Stationary phase : Porasil EX (75- 125 j).

3) Mobile phase : dionized, degased water.

4) Flow rate : 0.4 ml per minute.

5) Detector : UV absorption detector. The UV detector was de-

signed for operation at one of two constant wavelengths: 254

or 280 nm. All chromatograms reported in this study were

obtained at the 280 nm setting.


Total Cu in the Soil Extract


The total Cu was obtained directly from the saturation extract by

use of an atomic absorption spectrophotometer (Model 503, Perkin Elmer).



Statistical Analyses


Data collected from the field experiments and greenhouse trial

were subjected to statistical analyses by analysis of variance (ANOVA)

and Duncan's multiple range test (DMRT).

All statistical analyses were executed under the Statistical

Analysis System (Barr and Goodnight, 1972). Linear regression analysis

which was used in the titration study was performed by using a program-

mable hand calculator (Model 25, Hewlett-Packard), following the pro-

cedure outlined by the manufacturer (Hewlett-Packard, 1975).
















RESULTS AND DISCUSSION


Field Experiments


Fuquay fine sand contained more extractable nutrients than did

Apopka fine sand. The nutrient contents of both soils were within the

moderate range for Florida soils (Table 5). In soil samples collected

under individual trees, the Fuquay fine sand OM content in the top

15-cm surface horizon ranged from 2.07 to 3.14% averaging 2.36%. The

top 7.5 cm of the soil is considerably higher in OM content and ranged

2.60 to 4.30% as compared to a range of 1.29 to 2.06% of the next 7.5 cm

layer. This accumulation of OM in the surface is in direct contrast to

the Apopka fine sand which was disked regularly and hence the 0 15 cm

horizon was of uniform OM content, ranging from 0.92 to 1.97%, averag-

ing 1.39%. The significance of these OM differences will be discussed

later.

The extractable Cu values of soil samples taken 12 months after

CuSO4 treatment are given in Table 6. The values would appear to be

adequate based on the minimum level for sensitive crops of 0.2 ppm for

DTPA-TEA extraction at pH 7.3 established by Follett and Lindsay (1970).

Use of EDTA-TEA pH 8.5 extractant is apparently a stronger extractant

for Cu as it extracted about twice as much Cu as the DTPA-TEA at pH 7.3

(Lam and Gammon, 1976). However, a 10-fold difference between extract-

ants would be necessary if the values in Table 6 were to indicate an

inadequate Cu level by the Follett and Lindsay (1970) standards. The


-42-







-43-


n o
un


co

0


-4
C C







I u
i-4


o *.o
N c0,
r-(
i-4



m m









0 o\o

o -1
o m

N
o


+--
CO)
U) r-1

-4 0
Q C n en-4 a
o
Ni CM M 0
CO 0 ~o
3 Qn
CO

0 W
o in >

on> L(n rn ,in
U') Lf4N- L'


00 0



o
o o


o-4

o N









N '.0


CN 0 0





r-
i4 CN 0



i-4


C,, 00 CM
un eon 'D
'.0~


N l

cn r-


Nl N1 Lr'

Lt( Lt4 U'


4-I

0 4-1 r- 0 ,.


40 0 3






ca Eno
co ro CO m




oo on
*I *l-l l l .-
> -1 a i






CO


U I o 04

O C -4 -1 6
ZC H0 S
T H-i QS
r t- E- Qd Mr


-i




co



4-i
C,




,04

0)











co
c
0

4-1
a













4.1
(0
()
0























0
a






























4-1
C,

















c4
C

11
4-

























vl *4


00 (N

r- 'o


' 00 0

cn o



N-,-

C") 0


00 -It I'D

CN CN C0


-i C 0n

O1 4- 0


0
u


(0 4-1
)4 Ca





o e















0

t4-
CH

p-

















0
cn






-44-


Table 6. Levels of Cu in soil at Hawthorne and Earleton orchards one
year after CuSO4 application.



Soil extractant
Treatment
Treatment Aqua regia 0.1 N HC1 1.0 N HC1 EDTA-TEA


--------------------------------- Cu, ppm---------------------------



Apopka fine sand

0 1.7b 1.0c 2.3b 1.5c

1 3.4a 1.4b 2.4b 2.5b

3 3.8a 2.9a 3.6a 3.la



Fuquay fine sand

0 3.7b 1.3b 2.3b 1.3b

3 6.3a 2.5a 3.6a 4.2a

*
Each value is the average of 6 determinations, Hawthorne orchard.
4-
Values in column for each location not followed by the same
letter are significantly different at 5% level according to
DMRT.

Each value is the average of 4 determinations, Earleton orchard.






-45-


data show the Cu level increased following Cu treatments and also tend

to confirm, within the variation error, that most of the Cu added is

present in the 0 15 cm soil depth.


Response of Pecan Trees to Cu Application


Response of pecan trees to Cu treatments was examined by using two

parameters: Cu level in leaves and tree growth.


Copper levels in leaves

Hawthorne Orchard


No significant response to treatment was found 1 year after Cu

application. There was evidence that Cu levels in the leaves varied

with the cultivar (Table 7). Grafted 'Kernodle' had consistently higher

levels of Cu in the leaves throughout the 3-year experiment, whereas

'President' seedlings were usually the lowest. However in subsequent

years, there was a marked increase in the Cu level of President seed-

lings grown on soils receiving 3 ppm Cu (Table 8). The delayed response

to soil application of Cu is shown by the increasing level of leaf Cu

with time. Only during the third year, did the overall Cu level in the

leaves of trees receiving 3 ppm Cu on the soil become significantly

higher than that of the first year.


Earleton Orchard


'Desirable' grafts made up 58% of the trees in this experiment.

The remaining trees consisted of 7 different cultivars. No attempt was

made to group the trees by cultivar. Only 0-and 3-ppm treatments were

used, because of the limited numb.,r of trees available at this site.






-46-


Table 7. Copper levels in leaves of different pecan cultivars in
Hawthorne orchard in the first year after CuSO4 application,
average of all treatments.



Cultivar Cu in leaves


ppm

Kernodle graft 10.3a'

Desirable graft 8.7ab

Moreland graft 9.2a

Desirable seedling 9.4a

Cape Feare seedling 8.7ab

Moreland seedling 8.6ab

President seedling 7.3b

*
Each value is the average of 6 determinations.

Values not followed by the same letter are significantly
different at 5% level according to DMRT.





-47-


Table 8. Effect of cultivar and treatment on Cu levels in pecan leaves
in Hawthorne orchard.




Year sampled
Cultivar
1973 1974 1975


--------------- Cu, ppm ----------------


Kernodle graft

Desirable graft

Moreland graft

Desirable seedling

Cape Feare seedling

Moreland seedling

President seedling

Overall mean


Control

10.8a1

8.8a

9.3a

10.8a

8.0a

7.8a

7.0a

8.9B


Kernodle graft

Desirable graft

Moreland graft

Desirable seedling

Cape Feare seedling

Moreland seedling

President seedling

Overall mean


1 ppm Cu

10.0a

8.0a

9.0a

9.3a

9.3a

8.5a

7.5a

8.8B


application

9.5abcd

8.3abcd

8.8abcd

9.0abcd

9.5abcd

7.8bcd

7.5cd

8.6B


9.5abcd

9.8abcd

9.8abcd

9.8abcd

10.Oabc

7.5cd

7.3d

9.1B


9.5ab

8.8ab

9.3ab

9.0ab

9.3ab

9.8ab

8.0b

9.1B


10.0ab

9.3b

8.0b

8.0b

8.5b

8.0b

7.3b

8.4B











Table 8 (Continued)


Year sampled
Cultivar ---
1973 1974 1975

---------------- Cu, ppm ---------------------



3 ppm Cu application

Kernodle graft 10.3a 10.8a lO.Oab

Desirable graft 9.3a 9.0abcd 14.8a

Moreland graft 9.3a 10.5a 9.0ab

Desirable seedling 8.3a 9.0abcd 8.5b

Cape Feare seedling 8.8a 9.8abcd 10.Cab

Moreland seedling 9.5a 9.3abcd 10.8ab

President seedling 7.3a 10.3ab 12.Sab

Overall mean 8.9B 9.8AB 10.8A


Each value is the average of 2 determinations.

Values in column not followed by the same letter are significantly
different at 5% level according to DMRT.
+
TOverall means not followed by the same letter are significantly
different at 5% level according to DMRT.








Reported Cu levels in the leaves of this orchard are shown in Table 9.

The significant difference in leaf Cu attributed to treatment in the

first year after Cu application may have been a response to favorable

moisture conditions which promote' root growth near the soil surface.

The following 2 springs were unusually dry and no significant difference

was observed. A similar response to soil moisture has influenced Mg

levels in pecan leaves (Gam:oni et a.. 1960';. Although the level of Cu

in the leaves increased in the succeeding year and the average Cu level

in the third year was signiflcancly higher than that of the two previous

years, this increase was attributed to factors influencing growth other

than the Cu applications since the Cu level in the leaves without Cu

treatment was also increasing.

In general, the Cu level in the leaves from both orchards, even

3 years after Cu fertilization, was low when compared to the normal

range (21 28 ppm) reported by Alben and Hammer (1939). Broadcast Cu

on the soil surface appeared not to reach the active root zone under

most conditions and as a consequence there was little increase in the

levels of Cu in the pecan leaves.


Growth response


The growth response was evaluated by percentage increase in trunk

circumference (IC) measured at approximately 120-cm height which was

measured annually during the dormant season. The IC value was computed

as follows:


th
IC Circumference at n year Circumference before Cu application
Circumference before Cu application
(13)

This parameter was considered to be indicative of tree growth with a
















Table 9. Copper levels in pecan leaves in Earleton orchard.



Year sampled
Treatment
1973 1974 1975

----------------------------- Cu, ppm-----------------------------

0 6.2b' 6.9a 7.4a

3 7.la 7.2a 7.9a

Mean 6.6B0 7.OB 7.7A



Each value is average of 12 determinations
4-
Average values in column not followed by th same letter are
significantly different at 5% level according to DMRT.

'Average values in line not followed by the same letter are
significantly different at 5% level according to DMRT.





-51-


correction for the differences in tree size at the time the experiment

was initiated.

The IC values are reported in Table 10. Although there is some

indication of responses to the Cu fertilization, the increase of IC is

not significant. Differences in rate of growth are also noted for dif-

ferent cultivars and probably are the result of general vigor of the

cultivar, leaf disease resistance, and size of nut crop set. A lower

N supply and larger nut crops (Table 10) could well be factors slowing

growth in the Earleton orchard.

The ANOVA for the growth response is presented in Table 11. None

of the treatments reached the 5% level of significant difference but

there is a consistent increase in the probability of tree response to

Cu treatment in both orchards from the first to the third year of the

experiment. This trend, plus the fact that the Cu level in'the leaves

from soil receiving 3 ppm Cu (Table 8) was significantly higher in the

third year, posed two questions:

1) How much time was required for Cu to move from the soil surface

to the active root zone?

2) Was the applied Cu readily available for plant absorption?

The following sections will provide information on the data col-

lected to help answer these questions.


Distribution of Applied Cu in Surface Layers of Fuquay Fine Sand


The distribution of Cu in Fuquay fine sand (Earleton orchard) 27.5

months after CuSO4 application was studied, using soil samples taken in

2.5-cm increments from the surface to a depth of 15.0 cm. This kind of

study could not be made at Hawthorne because the soil was regularly





-52-


Table 10. Growth responses of pecans to CuSO4 application.



Cu Increase in trunk circumference (IC)
Cultivar Treatment
First year Second year Third year


ppm ----- -------- % ------------------


Hawthorne orchard

All 0 60 110 140

All 1 70 130 170

All 3 60 120 180


Kernodle graft All 70 140 190

Desirable graft All 70 140 190

Moreland graft All 70 140 170

Desirable seedling All 40 70 100

Cape Feare seedling All 70 140 190

Moreland seedling All 50 110 140

President seedling All 60 120 160


Earleton orchard

All 0 30 40 70

All 3 20 50 80






-53-


Table 11. Analysis of variance for growth responses of pecans.



Source of Sum of Mean F
df Prob > F
variation squares square value


Hawthorne Orchard

First year

0.021 0.010

0.981 0.047


0.205

0.981


0.034

0.047


Second year

0.062 0.031

1.427 0.068


0.812

1.427


0.135

0.068


Third year

0.176

0.140


0.291

0.140


Treatment

Residual


Cultivar

Residual





Treatment

Residual


Cultivar

Residual


0.22





0.73







0.46





1.99


0.80





0.63







0.65





0.11


Treatment

Residual


Cultivar

Residual


0.352

2.936


1.747

2.936


1.26





2.08


0.30





0.10














Table 11 (Continued)


Source of Sum of Mean F
squares squParrob > F
variation squares square value


Earleton orchard

First year

0.003 0.003

0.102 0,005


0.005

0.107






0.052

1.002


Second year

0.005

0.005



Third year

0.052

0.046


0.65








1.05








1.15


0.56








0.33








0.30


Treatment

Residual






Treatment

Residual






Treatment

Residual


-------






-55-


disked to a depth of 10 to 15 cm in order to control weeds. At Earleton,

the soil was not disturbed and weed and grass growth was controlled by

grazing or regular mowing. The data are presented in Table 12.

In all cases, Cu in the top 5.0 cm accounted for about 60% of Cu

content of the first 15.0 cm of the soil profile and the top 7.5 cm

accounted for approximately 80% of this Cu. Although the level of Cu

in each 2.5 cm layer was higher in the treated than in the untreated

soils, the differences were statistically significant only to a depth

of 7.5 cm (Table 13). It is evident that most of the applied Cu has

remained very near the soil surface and hence it would remain largely

unavailable to tree roots. In the top 7.5 cm of the surface soil, only

old pecan roots, which did not bear any root hairs were found. Such

roots would not absorb nutrients.

Distribution of other nutrients in this soil is also of interest.

The distribution of Zn is similar to that of Cu although Zn is more

mobile than Cu (Hawkes and Webb, 1962). This response is attributed to

the small quantities that have been applied from time to time in the

mixed fertilizer (Table 12). The accumulation of Mn near or in the soil

surface may be related to the slightly higher soil pH and to the reduc-

4+ 2+
tion of Mn4 to Mn when the soil becomes saturated with water. Under

such conditions, Mn2+ may be carried to the surface by evaporation and

reoxidized to insoluble Mn4+ (Amir and Gammon, 1976). Other elements

applied as fertilizer Ca, Mg, K, and P have accumulated in the soil

surface to some extent but these retentions are probably more closely

linked to the higher OM and the ability of this material to retain ions

by its properties of exchange capacity. Annual leaf drops will also

contribute to the accumulation of nutrients at the soil surface.












Table 12. Distribution of Cu and other nutrients in 0-15 cm of the soil
profile in the Earleton orchard.




Soil pH 0.1 N HC1 extractable
Soildepth (H0) Cu Zn Mn Fe Ca g K P
depth (HCu) OM Zn Mn Fe Ca Mg K P


ppm ----- -------------


4.12

3.40

2.55

2.00

1.50

1.46


4.72

3.45

2.53

2.11

1.65

1.43


Control

34.6

29.6

16.8

7.8

4.1

6.0


33.3

29.8

16.7

7.8

5.1

5.3


3 ppm Cu application

6.6 33.8 37.4

6.1 25.8 26.4

3.8 13.4 16.0

2.0 8.3 8.2

1.3 4.9 5.3

0.8 5.3 4.3


39.3

48.2

64.3

72.4

68.6

55.8


33.7

43.6

55.1

51.1

43.2

35.9


998

818

1146

411

291

266


1089

914

672

456

398

213


179

96

51

37

28

27


134

90

53

33

21

16


Each value represents the average from 6 profiles.


cm




0- 2.5

2.5-5.0

5.0-7.5

7.5-10.0

10.0-12.5

12.5-15.0


0- 2.5

2.5-5.0

5.0-7.5

7.5-10.0

10.0-12.5

12.5-15.0


5.9

5.8

5.7

5.7

5.7

5.7


5.9

5.8

5.8

5.7

5.7

5.7


---------------------














Table 12 (Extended)


1.0 N HC1 extractable

Cu Zn Mn Fe Ca Mg K P


NH OAc extractable

Ca Mg K P


------------------------------ ppm -------


2.5 35.3 54.6 268

1.7 27.2 43.5 278

1.2 16.3 21.0 297

0.5 6.2 10.3 304

0.2 4.0 7.4 320

0.1 3.5 7.8 321




11.1 31.7 49.6 256

9.3 22.8 34.7 265

5.3 11.3 15.7 283

2.8 6.2 10.9 296

1.6 3.2 7.7 312

1.1 3.7 7.0 315


Control

939 104 150 118 586 141 150 5.4

789 75 84 109 462 97 86 4.3

505 45 50 90 346 54 52 3.6

399 30 35 77 248 35 35 4.2

284 20 32 68 188 26 29 3.9

263 18 30 51 153 22 27 2.9


3 ppm Cu application

1082 132 174 99 708 174 181 5.6

882 81 84 87 549 91 87 3.3

665 53 47 73 406 56 47 3.2

490 33 34 60 302 38 36 3.4

314 22 29 38 203 26 31 2.4

210 17 30 24 146 21 31 1.6


---_~--------_I--_-


---- ----- -------------------------- -- --


ppm




-Jo-


Table 13. Copper in 0-15 cm of the soil profile of the Earleton orchard
27.5 months after CuSO4 application.*


Soil depth


Aqua regia


Method of extraction

0.1 N HC1 1.0 N HC1


cm


----------------------- Cu, ppm ----------------------


0-2.5

2.5-5.0

5.0-7.5

7.5-10.0

10.0-12.5

12.5-15.0

Overall mean


0-2.5

2.5-5.0

5.0-7.5

7.5-10.0

10.0-12.5

12.5-15.0

Overall mean


13.3a

11.Oa

6.3b

3.8bc

2.7c

2.3c

6.6


3 ppm Cu application

6.6a

6. la

3.8b

2.Oc

1.3cd

0.8cd

3.4


11. la

9.3a

5.3b

2.8c

1.6c

1.lc

5.2


10.3a

7.7b

4.3c

2.2cd

1.ld

0.8d

4.4


Each value is the average of 6 determinations.

Values in column not followed by the same letter are significantly
different at 5% level according to DMRT.


EDTA-TEA


3.3bci

2.8c

1. c

1.2c

0.8c

0.7c

1.8


Control

1.4cd

1.3cd

0.8cd

0,4cd

0.ld

0.ld

0.7


2.5c

1.7c

1.2c

0.5c

0.2c

0.lc

1.0


2.7cd

1.8cd

1.2d

0.5d

0.5d

0.5d

1.2


_.:_______ __ __~_~I_


------`-- -' `-~ ------- --






-59-


Availability of Applied Cu to Plants


Samples of Pensacola bahiagrass were taken in the Earleton orchard

under pecan trees fertilized with 0 and 3 ppm Cu to determine if in

fact the applied Cu was available for root uptake. Analysis of the

grass for Cu (Table 14) showed that the applied Cu was available and

increased the total Cu in plant tissue. Although this grass is capable

of rooting to great depth, most of the roots are concentrated near the

soil surface, where most of the applied Cu was found. This is taken as

additional evidence that the lack of a significant response in the pecan

trees was because of the strong retention of Cu in the surface soil

above the main tree rooting zone. The apparent response to 3-ppm Cu in

the Hawthorneorchard during the third season may have been accentuated

by physical movement of Cu to the rooting zone from the disking opera-

tion.

Data from the bahiagrass analysis were used to examine the correla-

tion between the Cu content of the grass and the different methods of

soil extraction (Table 15). All methods of soil extraction proved to

be significantly correlated with the Cu level in the bahiagrass at 0 -

2.5 cm and 2.5 5.0 cm depth. The HC1 extractions seemed to be slightly

better correlated than aqua regia and EDTA-TEA pH 8.5. These correla-

tions provide more evidence that the grass roots were actively taking up

Cu from the surface soil, an area not likely to be utilized by the pecan

tree roots.



Greenhouse Trial


In studying plant response to a nutrient application, a field

experiment would be preferred to a greenhouse trial because many





J&U-


Table 14.


Copper levels in blades of Pensacola bahiagrass (Paspalum
notatum, Flugge) as influenced by CuS04 application in the
Earleton orchard.


Cu Cu level
Treatment in grass*


ppm ppm

0 5.2bt

3 6.9a


Each value is the average of 6 determinations.

'Values not followed by the same letter are significantly dif-
ferent at 5% level according to DMRT.
















Table 15. Correlation coefficients between Cu levels in Pensacola
bahiagrass and various soil extracts.



Soil Method of soil extraction
depth
Aqua regia 0.1 N HCI 1.0 N HCI EDTA-TEA


cm --------------------- r value---------------

0-2.5 0.73** 0.81** 0.80** 0.73**

2.5-5.0 0.68** 0.77** 0.76** 0.68**

5.0-7.5 0.59* 0.55NS 0.49NS 0.50NS


** Significant at the 1% level.

* Significant at the 5% level.

NS Non-significant.


-tJ.-






-62-


artifacts would be involved in the latter study. The limited quantity

of Vietnamese soils restricted even the extent of the greenhouse trials.

The data obtained from these soils is indicative rather than conclusive.

Chemical analysis of the five soils utilized in this experiment was

reported in Table 5. Ninh-Chu soil (Typic Psammaquent) was alkaline,

very low in OM, low Cu and Zn. Three of the other soils were acidic,

variable in OM, and low in micronutrients (Cu, Zn, Mn). Eakmat soil

(Typic Haplustox) had a medium level of OM and contained considerably

more Cu (Table 16), but this level was well within the normal Cu range

found in soils (Fiskell, 1965).

Responses of corn growing in these Vietnamese soils to different

levels of applied Cu are reported in Table 17. Copper content of corn

grown in untreated soils was consistently lower than the normal range

of 7 20 ppm, reported by Jones and Eck (1973) for whole corn plants

at 3- to 4-leaf stage. An increase in plant Cu in response to Cu

application was observed; however, this was statistically significant

only for the Ninh-Chu soil. Because of the high variation among indi-

vidual treatments the number of replications for the other soils was

insufficient for statistical significance. Although the Eakmat soil

had a sizable Cu reserve, there was an increase in tissue Cu and dry

weight production from the Cu treatments. Evidently the small amount

of added Cu was much more available to the plant than the large reserve

of soil Cu. Only corn grown in Trang-Bang soil exhibited normal vege-

tative growth. Corn grown in the four other soils developed nutrient

deficiency symptoms which definitely influenced plant development.

Phosphate deficiency was observed on the plants grown in Ninh-Chu and

Eakmat soils, Ca deficiency appeared in plants on Dalat soil and N




TwJ--


Table 16. Level of Cu in selected Vietnamese soils with and without
CuSO4 applications.



Extractant
Treatment ----------
Aqua regia 0.1 N HC1 1.0 N HC1 EDTA-TEA

------------------------------ Cu, ppm--------------------------------


1. Oc
2. b
3.5a


1.O0b
2. 0ab
2.5a


1. 0b
1.Ob
2.0a


9.5b
1l.Oab
12.5a


53.5a
56.5a
57.5a


Ninh-Chu
0.6b
0.5b
0.8a


Trapg-Bang
0.8b
0.8b
l.a


Thu-Duc
0.5b
0.5b
1.5a


Dalat
1.5b
1.8b
2.8a


Eakmat
5.3a
5.0a
5.8a


1. lb
1.Ob
2.6a




1.Oc
1.5b
2.5a




0.5b
0.5b
2.0a




2.5b
2.8b
4.3a




7.3b
7.0b
8.8a


0.5b
0.5b
2.0a




1.5b
1.5b
2.5a




0.5b
0.5b
1.5a




1.5c
2.5b
3.5a




5.0a
5.0a
5.5a


Each value is the average of 7 determinations.

Values in column for each location not followed by the same letter
are significantly different at 5% level, according to DMRT.

Each value is the average of 3 determinations.

Each value is the average of 2 determinations.


0
0.3
1.8


0
0.3
1.8


0
0.3
1.8


0
0.3
1.8

















0
4+

d

1





it














U
(d















H-4





4-CA
tS
























H-4
Ud
3





Ql
r
n



i

+-
6C







tJ
oc


0 0
* C
-4 -:1


Br


II g
U




0
0











0
1
c




5-i

*U
C


'V
{ 3
U (f
0)
Mu
Hc


CI a
U C,


0
CO
N
60 cY
r-


( *




Su'
00






S .0






Gc
M0


C r-


-a
4i
cu


4-1




4u
oc




























0 0 0









oJ
$l


r-4
































0 0 0
0u

















tv0 0
C.







co

01







00


*IC+-+
u 41
























0 0 0
0 0 0 0
4) a a o) 4







o) o 0









*dl r rl 00
Uc


o a01-4


U U U r-l U

*K i- +i- un


Uo


U 0 0
C M
IJ ao






-65-


deficiency on the Thu-Duc soil. These hunger signs were corrected by

using supplemental nutrition solutions, but as Epstein (1972) pointed

out, even after the correction, plants still did not resume full normal

growth. No deficiency symptom was observed on corn grown in Trang-Bang

soil, but the limited soil volume in the pots was inadequate for an

8-week corn plant. Because of this problem, pot size rather than Cu

become the critical factor limiting growth on this soil. The relation-

ship between Cu content in the whole plant and dry-weight did not show

any particular pattern. Corn grown in Trang-Bang soil produced the most

dry-weight but Cu content was among the lowest. Correlation coefficient

(data not presented) of Cu content and plant size was very low even for

Cu treatments on the same soil.

Unusual fertility problems and small volume of soil available for

each plant probably masked any possible Cu limitations in these soils.




Laboratory Study


The Cu content of the soil solution is of direct importance in Cu

absorption by plants. This section deals with Cu2+ in the saturated soil

solution extracts and the completing of Cu2+ by these extracts. The

complexation ability of the extracts will be compared to those of syn-

thetic chelating agents, EDTA and DTPA. Since the total Cu in the soil

solution is reported to be more than 98% completed (Hodgson et al., 1965),

knowledge of some of the factors which influence complexation should be

beneficial in determining the most efficient manner in which to supply

crops with supplemental Cu. The purpose of this study was to approximate

the natural condition of the soil solution in the field, therefore very

simple extractant solutions, H20 and 0.01 M CaSO were used.
4'





-66-


Solution Studies


Synthetic chelating agents for soil extraction in micronutrient

analyses as well as for supplying micronutrients to plant roots have

come into general use in commercial agriculture. Two of the agents in

common use are EDTA and DTPA. Since these agents have been successfully

used to transport micronutrients to plant roots in the soil, they were

selected for preliminary study. The CaSO4 solution used in the satu-

rated soil solution extracts was selected as the third material to study

2+
for possible interaction with Cu2

The effects of CaSO4 and two chelating agents, EDTA and DTPA, on
2+
the activity of Cu2+ are shown in Fig. 2. As expected, 0.01 M CaSO

did not reduce Cu2+ activity.

In order to avoid the possible effect of dipositive ions on the

chelating agents (EDTA and DTPA), 0.5 N K SO4 was used to provide the

supporting electrolyte instead of 0.01 M CaSO With EDTA, the addition

of Cu2+ did not significantly increase the electrode potential to indi-

cate the presence of Cu2+ until Cu2+ had been added in the quantities

shown in Fig. 2 and Table 18. Beyond this point, the electrode poten-

tial increased directly with the amount of Cu2+ indicating no chelating
2+
of additional Cu2. However, with DTPA, the electrode potential did

not increase directly with Cu2+ additions until the breakpoints on the

curves were reached indicating that more than one functional group was

involved in the chelation reaction and that the efficiency was not as

great as EDTA. These differences were due to the differences in the

two chelates. Norvell (1972), Table 19, reported up to three chelation

reactions of DTPA with Cu as well as chelation of Ca in acidic medium.

Although the formation constant of CuL is high, the presence of two






























.cr)

S 0 0
-'0 0 '- 0 0 t
COO vC cc 0




0) Cc 00 c 0







r- r- --I rHHr
00 0 it00 00
Su r S u S l3










eo 00 0 e T0





000 000 co
U-l 04r C)








HHH HNH 0
+++ +++ +
a )n) n Le ) 0l


















C14
C; in Lr LI Ji Ln l iL n









II I I I I IC
Nm000 0000 m0











U4


c-l ~*
Tl

fc





-68-


wdd 'pajnseuw+ no
*&


^.
*^t.



e\
^.


*'s.















Table 18. Chelating ability of EDTA and DTPA.


Quantity of metal
Chelating at breakpoint Vol. CTR Initial
Vol. CTRpH
agent Al Ca Mg Cu pH


--- ---------108 mole -------- ml %


EDTA

125 0 0 0 121 26.7 100 4.85

125 18 0 0 76 28.5 100 4.50

125 0 18 0 116 32.7 100 4.60

125 0 0 16 115 30.7 100 4.75


DTPA

125 0 0 0 210 27.4 96 4.15

125 18 0 0 203 29.3 95 4.00

125 0 20 0 200 33.3 94 4.10

125 0 0 17 193 31.2 94 4.10


Volume of solution at breakpoint.





-70-


Table 19. Formation constants for EDTA and DTPA (Norvell, 1972).



Log of formation constant
Reaction
EDTA DTPA


19.7

3.3


Cu + L CuLt

CuL + H CuHL

CuL + Cu Cu2L



Al + L AlL

AlL + H + A1HL



Ca + L CaL

CaL + H CaHL

CaL + Ca Ca2L



Mg + L MgL


22.6

5.2

6.2


17.6


20.3

4.7


11.5

3.5


11.9

6.6

2.7


9.6


10.4


All values were adjusted to an ionic
the DeBye-Huckel equation.

'L Ligand, EDTA or DTPA.


strength of 0.01 M using






-71-


other chelates (CuHL and Cu L) with low formation constants resulted in

an equilibrium which provided a CTR < 100%. In case of EDTA, the forma-

tion constant of CuHL was too low to influence the CTR provided by the

first chelation reaction (Table 19).

3+ 2+ 2+ 2+
Addition of Al Ca or Mg reduced the quantity of Cu neces-

sary to saturate the chelating agents. This tends to disagree with the

work of Pfeiffer and Schmitz as cited by Martell and Calvin (1952).

These workers reported that the stability of chelates of EDTA with Cu2+

and other metallic ions was greatly dependent on the nature of the

2+ 2+
remaining positive ions in the solution (e.g. Ca Mg ). However,

2+ 2+
they showed that the interference of Ca2+ was not the result of Cu2

displacement but caused a weakening of the chelates.

Under the conditions of the experiment, additional metallic ions

did not change the CTR of EDTA, but that of DTPA slightly decreased

(Table 18). Perhaps, the concentration of metallic ions used in this

study was too low to affect the CTR for EDTA.


Saturated Soil Solution Extract Studies

Apopka fine sand


Samples were selected from the 0, 1, and 3 ppm Cu treatments to

represent sites of the highest and the lowest soil OM contents at each

treatment level.

It was not necessary to add supporting electrolyte to CaSO4 extracts,

Table 20. The 0.5 N K SO was used to raise the total ionic strength of

H20 extracts and avoid the possible effect of dipositive ions (from

CaSO4, if it were used) on ligands in the saturated soil solution ex-

tracts. The total cation concentration in the soil extract which was

calculated from electrical conductivity, was used as an indication of






-72-


C: C N -| CN C- N
o 0 0 0 0 0
0 0 0 0 0 0



0 0

* i


o o



4 K o" r-4
C U I
C)U 0 T 0u







Li U, m C) C o o c 0 C) r=a
-H C; C; wC
u C l 0 44




*- u I m o co 0 r co





o ) L V ) L)
U I 0 )
















-H \ m 0
1 0 0 8c 0 0 0 U











4J ca-
blj O- 4.l .
' E I u "O O ,







4+ )) C) C) 0) *C
* C 0 wu C C
u 00 4 Lf4.4 4 Cu
SH

V (


c u co5
Cuo U D
44 U H- 4O
? q 0 0 0Cu
aU -H 00
0J 44 .E- .BI v- C)


0o z


Cm z u -m

U ca 0 H 0- 0 0u 41 -4.4
ca 0 u cc
u U = U u 0
U u O O O O O C




1 vl U a^4 o 1
L a)H 0 0 0 0 0 r-4 M
E 00


o 0 0 0 0 00 '>-I .



0 u t, -


C) *-Iu 0 '. 0' -4 r- 0 u
v-I 0 0 *
Hl r l r I








the total ionic strength. At the present time, there is no available

technique for direct measurement of the total ionic strength of soil

extract.

Approximately 1% of the soil OM was removed in the saturated soil

solution extraction. The extraction with 0.01 M CaSO, released a very

small quantity of OM to the extract probably because of the coagulation

effect of Ca2+ which would precipitate a portion of water-soluble OM.

The Cu treatments further reduced the OM in the soil extract. The OM

in the water extracts was higher than that in CaSO4 and was not influ-

enced by Cu treatment, Table 20. The extracted OM was examined by the

gel permeation chromatography technique to establish the approximate

fraction sizes of these compounds. In this procedure, the longer the

retention time, the smaller the fraction. A comparison of chromato-

grams of known compounds (Fig. 3) to those of the soil extracts (Fig. 4,

5) indicated that the major fraction of OM in CaSO4 extracts was of

smaller size than that of H-bonded, agglomerated EDTA and DTPA (Fig. 4).

Organic compounds from water extracts contained fractions of larger size.

The dominant components had a retention time of 8 to 11 minutes (Fig. 5)

indicating a fraction size in the range of agglomerated EDTA and DTPA.

The difference in fraction size of organic compounds from CaSO4 extracts
2-
and water extracts is probably caused by the effect of the SO4 present

in the CaSO4 extracts. In aqueous solution, organic compounds tend to

link together by H-bonding and/or H20 bridges, and in gel permeation

chromatography, would have the appearance of larger molecules. However,
2-
in the presence of SO4 these H-bonding and H20 bridges are broken,

producing smaller size components (Fig. 6, 7) which was eluted at the end

of the selective permeation. The organic compounds in the H20 extracts,
2-
in the presence of SO4 from the supporting electrolyte (K2SO4) were

also less influenced by H-gonding and H20 bridges.






-74-


C)
0
C
0
()
a

0)
o
L.



*o
0
0
a)
rr:





DTPA


ACETONE


0


EDTA


0


L. I I I I I I I I I I
S* 18 16 14t- 12
18 16 14 12 10 8 6 4 18 16 12

Retention time, minutes
1. Acetone 2. H-DTPA 3. H-DTPA in the presence of
0.02 N K2SO4
4. Na-EDTA 5. Na-EDTA in the presence of 0.02 N K SO4
* t
Begin selective permeation. End selective permeation.
Fig. 3. Chromatograms of some organic compounds.





-75-


I I


Retention time, minutes


Control, soil OM = 0.92%
Control, soil OM = 1.97%
Soil treated with 1 ppm Cu
Soil treated with 1 ppm Cu
Soil treated with 3 ppm Cu
Soil treated with 3 ppm Cu


in 1973, soil OM
in 1973, soil OM
in 1973, soil OM
in 1973, soil OM


Begin selective permeation.
Fig. 4. Chromatograms of 0.01 M CaS04 extracts from Apopka fine
sand.


0
CL
0
a


0


a.

o

c
C


= 1.15%
= 1.76%
= 1.09%
= 1.69%


I I I |


I


!






-76-


Retention time, minutes


Control, soil 0OM = 0.92%
Control, soil OM = 1.97%
Soil treated with 1 ppm Cu
Soil treated with 1 ppm Cu
Soil treated with 3 ppm Cu
Soil treated with 3 ppm Cu


in 1973, soil OM = 1.15%
in 1973, soil OM = 1.76%
in 1973, soil OM = 1.09%
in 1973, soil OM = 1.69%


Begin selective permeation.
Fig. 5. Chromatograms of H 0 extracts from Apopka fine sand.
2








































Zn


--


Az 4
-4 4 -







4 4
A A 't .4 .4 4

-4 4 4 1 4~~1
4~1111 .4 44 A'4 4 -
Ao *,: 4444





ca*^ 4 :


U )^ ^ r 4 *



co
_ ____ ___ 1 44
'CT
vn \ ^<


c ^

I* I


- (0
4)
'0~
3
^C


E
c1l



0- 0

0

U-
CC








0 0

co
uu

XX

0 0


C C


esuodsoJ jopiooej


-/I-


4





i













DALAT SOIL


O 1 I TRANG-BANG SOIL
O
CL
1.1





0






THU-DUC SOIL



S_' rb
........ .. __




I I I I I I I
24 20 16 12 8 4 0

Retention time, minutes

a H20 extract in the presence of 0.02 N K2S04
b H20 extract alone
-k
Begin selective permeation.
Fig. 7. Effect of K2SO4 on 1120 extracts of Vietnamese soils.





-79-


The chromatographic patterns of H20 extracts of control samples

were identical. Note the stronger instrument response in sample #2

which had the highest OM content, Fig. 5. Extracts from Cu treated

soils provided a more distinct peak with a retention time of about 6.5

minutes. Evidently, most organic compounds extracted by both extrac-

tants were of relative low molecular size. Such materials would be

expected to be simple organic acids which could be found in the fulvic

acid fraction of soil OM.

Total Cu in saturated soil solution extracts was very low and un-

complexed Cu2+ was not detectable by Cu +-selective electrode under the

described operating conditions. This made the comparison of the two Cu

values impossible. The CTR for CaSO4 extracts ranged from 16.1 to

39.6% while those for H20 extracts ranged from 60.7 to 92.1% (Table 19).

This could be expected from the competition between Ca and Cu for com-

plexation with OM. Total content of OM in soil extracts seemed to have

little effect on the CTR which again reflects the type of OM in the

extract. The three samples which provided the highest CTR (samples #1,

2, and 6) had about the same chromatographic pattern (Fig. 5) and indi-

cated a higher fraction size than those in the other 3 extracts.
2+
Effects of Cu2+ additions on electrode potential of soil extracts

are depicted in Fig. 8 and 9. Slopes of the curves from CaSO4 extracts,

prior to breakpoints were very steep indicating that the complexation

reaction was not predominant. Since the extracts were dilute solutions

(Table 20), ions present in the extract might be considered as indepen-

dent of one another in the solution (ion interactions were not signifi-

cant) (Hunt, 1963). The hydrolysis of Cu2+ was negligible in the acidic

medium. Taking these two factors into consideration, the quantity of
































Control, soil OM =
Control, soil OM =
Soil treated with
Soil treated with
Soil treated with
Soil treated with


0.92%
1.97%
Sppm Cu in 1973, soil OM = 1.15%
Sppm Cu in 1973, soil OM = 1.76%
3 ppm Cu in 1973, soil OM = 1.09%
I ppm Cu in 1973, soil OM = 1.69%


2+
Fig. 8. Effect of Cu addition on 0.01 M CaSO4 extracts of
Apopka fine sand.


Legend:




-81-


5 436 2 1


S/


/ / /
6.00o I / d

//

E r



4,00 /
0/ // /


o / I











Cu added,
I //









//





0 2.00 4.00 6.00 8.00 0. 00
Cu2+ added, ppm


C.oo















II II II

000


000
-4l -r- rql
0 0 0

40 CO 01 0V


ON ONO


0










o c' c
a)Q 0) a c
.4.
1 U1U U










W: ci U) a)





CCu
*> r C.J


V4
0 N&
m 44



\s I-3






-S1
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md I -lrIl no









S0* 0 020
004!
4J 4! 4
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-83-


2+
Cu completed by organic compounds in the soil extracts was reported

in Table 21. The presence of Ca and Mg in CaSO extracts at relatively

high concentrations reduced the quantity of available ligands for Cu.

The effect of Al was small and uniform except for two confirmed varia-

2+
tions. The total quantity of added Cu2+ removed from its ionic state

by the OM was equivalent either to 3 x 10-8 to 89 x 10-8 mole of EDTA

or to 2 x 10-8 to 51 x 10-8 mole of DTPA.

2+ 2+
The response of Cu2+ -selective electrode to the applied Cu2+ in the

H20 extracts had rather flat slopes (Fig. 9) indicating that the water-

extractable OM was highly active in completing Cu when Ca and Mg concen-

trations were in the normal range of these soils. The values of Cu

-8 39x1-
complexation ability of H20 extracts ranged from 22 x 108 to 329 x 108

mole of EDTA or from 13 x 10-8 to 189 x 10-8 mole of DTPA.



Fuquay fine sand


Total OM in Fuquay fine sand was higher than that of Apopka fine

sand, as was the OM in the soil extracts (Table 22). Effects of 0.01 M

CaSO4 and water on the OM in saturation extracts were similar to those

observed for the Apopka fine sand. Chromatographically, CaSO4 extracts

had similar patterns but peak heights were much greater (Fig. 10), indi-

cating that the amount of OM was larger. This was confirmed by the car-

bon analysis. The organic components in the H20 extracts of Fuquay fine

sand were larger in quantity and lower in fraction size than those of

Apopka fine sand (Fig. 11). Retention time in the chromatographic column

of most organic compounds in H20 extracts ranged from 10 to 13 minutes

which corresponded to a molecular size smaller than agglomerated EDTA.

Extracts of samples #1 and #3 (Figi. 11) presented a small peak at about





-84-


Table 21. Quantity of Cu2+ completed by soil extracts from Apopka
fine sand.



Cu Sol Metal in the extract
Cu Soil
Treatment OM A Ca Mg Cut
Al Ca Mg Cu,


m-------------- 108 moles ----------------


0.01 M CaSO extraction
-_ 4


0.92

1.97

1.15

1.76

1.09

1.69


0.92

1.97

1.15

1.76

1.09

1.69


21

21

21

21

43

Trace


4904

4832

4508

4957

4742

4095


Water extraction

21 244

21 291

21 102

21 313

21

21 255


976

1042

791

1215

646

1373




82

111

53

129



102


79

86

33

30

3

28




137

288

70

63

21

318


Micronutrients were not computed.

tTotal Cu2+ added less the Cu2+ detected by ion selective electrode.

'Quantity of cation in the 25 ml aliquot.


ppm







-85-


m0 0 0
S 0 0 0 0 -





c
-4



S t-
0)0 0 0 044

vJ 4Z 01
SC.) 3 0) M N 0 '..o

OD u
w C4 -K4

0 w
SO U 0 0


4 u
Q)U U a
E ca E *e
u C CO N 4 N U








4- 0 0 C 0.
N10 N 0 s I4

C) 0' 0 0 04






ca -4 *
y ou o


- 0. 0 0 0 0 0

S0 0 0

Cu, C. L a q- a)
0 I" .







o o u
S0 0 0 0 r-o
h 0 00 0 r





r. m U x
_ C- C O. ..

o o 3




-W 4 0) N 0
0 4- f


0o 0 a -4 o _c e
0o w q a


41 V W) w
z- 0. C 0 u 4
o 4J 40 If c 0
0 03 03 U~ 03 CC) 4C
uu w0 4

-1 Q) E C) Ml an C' )
0. 0 0 0 0 Cw 0
- 0 0 0 0 4 c i



-4r
CN c w r- a


Htr-4 00 Cu
0 0 c N- N So
JE n 0 0 0 0 l 4.

0 z


-4


41 a 0 0 m -ru
-o e a
Hd

































Legend:

1. Control, soil OM = 2.44%
2. Control, soil OM = 4.30%
3. Soil treated with 3 ppm Cu in 1973, soil OM = 2.77%
4. Soil treated with 3 ppm Cu in 1973, soil OM = 4.21%
Begin selective permeation.
Fig. 10. Chromatograms of 0.01 M CaSO4 extracts of Fuquay fine
sand.




-87-











Retention time,


minutes