Tracing the Calcium, Phosphorus, and Iron from a Limed and Unlimed Lateritic Soilto the Grass and to the Animal Blood1

JUAN A. BoNNET2

DATA are presented on the effect of lime applica-tion on the percentage composition of available

calcium, phosphorus, iron, magnesium, and manga-nese in a lateritic soil from- Puerto Rico, and on thepercentage composition of these total minerals in thegrass growing in this soil. Data are also presented onthe effect of the unlimed and limed grass on the cal-cium and inorganic phosphorus in the blood serumand on the iron and hemoglobin in the blood of femalegoats.

EXPERIMENTAL PROCEDUREEighteen plots, each with an area of 0.4 acre, were selected

in a field of "Fajardo clay" at the Experiment Station farmin Rio Piedras, Puerto Rico. Fajardo clay is a lateritic soil ofthe humid region of Puerto Rico, acid in reaction, with alevel or gently sloping relief, derived from old, high alluvialmaterial and from outwash fans of adjacent shale hills. Halfof the randomized plots were limed to pH 6.5. The amount oflimestone varied from 8 to 10 tons per acre.

The field was planted in the middle of July 1943 with amixture of Para grass, Panicum purpurascens, and Caribgrass, • Eriachloa polystachya, the former known as "Malo-jillo" and the latter as "Malojilla", two valuable pasture andsoilage grasses in the humid lowlands of Puerto Rico.

Five consecutive grass crops were harvested lip to October1945; of which the third and fifth were fertilized with am-monium sulfate at the rate of 500 pounds per acre.

Ten virgin female goats were randomized in their pensand given the parasite treatment of 12 grams of phenothiazineper os. The goat experiment was divided into three periods,as follows: Pre-feeding, October 19 to November 14, 1944;pre-ges'tation, November 15, 1944, to January 15, 1945; ges-tation, January 16 to July 15, 1945.

Separate composites of the unlimed and limed grass were,cut daily in strips from each corresponding plot and ground.Eight pounds of the chopped unlimed and limed grass werefed, respectively, to each of the corresponding group of fivegoats. The feeding box in each pen avoided the contaminationof the grass with urine and excrement. The amount of residualgrass left daily by each animal was also weighed. A com-posite sample from the grass was taken .daily for moistureanalysis. A record was therefore kept of the amount of greenand dry grass consumed by each animal. Each goat was alsoweighed on three consecutive days about the middle and theend of each month, respectively. Rainwater was supplieddaily. Tp induce breeding, at approximately the same time,each animal was given, on January 29, 1945, S rngms ofdiethyl stylbestrol per os.

METHODS OF ANALYSIS3

Soils.—Three composite samples of the soil- were takenfrom each plot, the first previous to the lime application; thesecond and third, 15 and 23 months, respectively, after thelime anplication. Each soil sample was analyzed for exchange-

able calcium, magnesium, and manganese and for so-calledavailable phosphorus and iron. The pH values were alsodetermined.' Exchangeable bases were run by Peech's (5)* method

extracting with N neutral ammonium acetate solution. A .Coleman spectrophotometer was used to read the transmit-tances corresponding to the colored solutions of magnesiumand manganese.

The so-called available phosphorus and iron in the soilwere extracted with Morgan's extracting solution of Nsodium acetate buffered at pH 4.8 with acetic acid as follows:12.5 grams of air-dried soil and 25 ml of extracting solutionwere placed in a test-tube, 6 inches long and I inch in diameterand the tube stoppered and shaken horizontally for 2 minutesin a reciprocating shaker at a speed of about 120 shakingcycles per minute.

Available phosphorus and iron were determined in aKlett-Summerson photocolorimeter, the former as per Wolff's(7) procedure and the latter by the method of Saywell andCunningham as described by Parks, et al. (4).

Grass.—Composite samples of the standing grass from theunlimed and limed plots were taken, 14, 17, and 32 monthsafter liming, respectively.

The minerals were determined by the procedure of Parks,et al. (4), omitting the dithizone extraction. A Coleman spec-trophotometer was used to read the transmittances cor-responding to the colored solutions of manganese, magnesium,phosphorus, and iron.

Blood.—Blood samples were .taken from each goat at thebeginning of the pre-feeding period, i month after the begin-ning of the pre-gestation period, and thereafter, every middleof the month. About 10 ml of blood were drawn from eachanimal by a direct puncture of the jugular vein, 2 ml for thehematological test and 8 ml for the chemical test. The 2-mlblood portion was poured into a' lo-ml test-tube containingo.i ml of a mixed solution of 6% ammonium oxalate and 4%potassium oxalate, previously evaporated .to dryness.

Hematological test.—A o.i-ml portion of the oxalatedblood was used for the red blood cell and white blood cellcounts. A o.7-ml portion of this blood was used for hemato-

. crits and O.I ml for hemoglobin. For the determination ofhemoglobin, o.i ml of oxalated blood was diluted to 20 mlwith 0.1% sodium carbonate solution and the transmittanceof the colored solution was read in a Coleman spectropho-tometer, using filter PC-4 at a wave length of 540 milli-microns. The amount of hemoglobin was calculated by refer-ence to a standard transmitttance-concentration curve pre-pared from cow's blood whose hemoglobin content was de-termined by the. Van Slyke's method (2).

Chemical test:—Iron was determined in a 0.5 ml portion ofoxalated blood bv the Wong's (8) modified method and the.transmittance of the colored solution was read in the Colemanspectrophotometer with filter PC-4 at a1 wave length of 480millimicrons.

The nonoxalated blood was centrifuged immediately afterbeing drawn for 5 minutes at 2,800 r.p.m. in an internationalclinical centrifuge. This was found necessary, to avoid hemo-lysis, since the red blood cells of goat's blood are quite fragile

'Contribution from the Soils Department, Agricultural Experiment Station, University of Puerto Rico, Rio .Piedras, P. R.2Head, Soils Department. The writer wishes to express his appreciation to Mr. Alfonso Riera, Assistant Chemist of the Soils

Departnjent, and to the following members of the Animal Husbandry Department of the Agricultural Experiment Station of theUniversity of Puerto Rico for their cooperation in the animal experiment: Dr. F. E. Armstrong, Associate Veterinarian; Mr. LuisRivera Brenes, Assistant Animal Husbandman; Mr. V. Quinones, Assistant Chemist; and Mr. R. Orlandi, Research Assistant,who determined the blood count.• 3A complete description of the methods of analyses and transmittance concentration curves will be published later in the.

Journal of Agriculture, University of Puerto Rico.4Figures in parenthesis refer to "Literature Cited", p. 297.

295

296 SOIL SCIENCE SOCIETY PROCEEDINGS 1946

and minute, 4.1 microns in diameter. The fibrin sealing theplasma was loosened carefully with a wooden rod, and theplasma was poured down or centrifuged again if necessary.

Calcium in blood serum was determined by the method ofRoe and Kahn (6), using a Klett-Summerson photoelectriccolorimeter with red filter 66 at a wave length range from640 to 700 millimicrons.

Inorganic phosphorus in blood serum was determined by"the method explained by Levinson and MacFate (3), butusing the Coleman spectrophotometer with filter PC-4 anda wave length of 600 millimicrons.

PRESENTATION AND DISCUSSION, OF DATAThe'increase of available calcium and phosphorus

and the decrease of available iron in the soil due toliming was highly significant, 15 and 23 months,respectively, after the lime was applied to the soil(Table i). The decrease of available manganese inthe soil, due to liming, was highly significant 15months after liming and significant 23 months afterliming. The difference between the available mag-nesium content of the limed and unlimed soil was notsignificant.

• The increase of calcium and the decrease of man-ganese in the grass due to liming was highly signifi-cant for the second and third crops, while the increaseof calcium was significant for the fourth crop andthat of manganese was not significant (Table 2). Theincrease of phosphorus in the grass due to liming washighly significant for the second and fourth crops butwas not significant for the third crop. There was nosignificant'change in the iron content of the grass dueto liming in the three crops and in the magnesiumcontent of the second and fourth crops. However, theincrease of magnesium in the grass crop due to lim-ing was highly significant for the third crop.

The increase in the grass yield due to liming wassignificant for the first and third crops (Table 3) ;however, the difference between the respective yieldsof the unlimed and limed soil, for the second, fourth,and fifth crop and for the total of five crops was notsignificant. -

There were no significant differences in the pre-gestation period between the hemoglobin and iron inthe blood, and the calcium and phosphorus in the

'serum (Table 4). of the goats fed with unlimed andlimed grass.TABLE I.—The pH values and exchangeable calcium, magnesi-

um, manganese, and available phosphorus and iron in un-limed and limed lateritic Fajardo clay, dry basis.

TABLE 2.—Total calcium, magnesium, manganese, phosphorusand iron in three crops of Para-Carib( grass grown in un-

limed and limed lateritic Fajardo clay, air-dry basis.

Treat- .ment pH

Parts per million

Ca Mg Mn P Fe

15 Months After LimingUnlimed . . . 4.6

6.1849

6.8111 80172

. 423 13

61172

23 Months After LimingUnlimed . . .Limed. . . . .

4-46.3

9925,351

156156

,295

21-56

4512

Treatment

Unlimed. .

Limed. . . .

TABLE 3.—unlimed

Sec

Tl

FO

Parts per million

Ca

;ond Crop2,1992,811

iird Crop2,0083,351

urth Crop2,9193,38l

Mg

14 Mont1,5091,638

17 Montl1,8242,212

32 Mont2,1662,088

Mn

hs After I229137

is After L15684

hs After ]243181

P

aiming2,1002,43°

iming2,7493,047

^iming. .2,4502,929

Fe

149158

196'i 60

124121

-Yield in tons per acre of green Para-Carib grass inand 'limed lateritic Fajardo clay, air-dry basis. ,

Number of cropso

Treat-'ment

Unlimed . .Limed. . . .

i, nonitro-

gen ap-plied*

8.98II.OO

2, nonitro-

gen ap-' plied

7478.03

3,nitro-

gen ap-plied*

/9-59

10.33

4, nonitro-

gen ap-plied

8.928.62

5,nitror

gen ap-plied

9.829.81

'Total

• 44-7847-79

*Significant difference between yields.

TABLE 4.—Percentages of, hemoglobin, iron in blood, calcium,and inorganic phosphorus in serum of goats fed with un-

limed and limed grass.

PeriodHemo-globin,grams*

Fe inblood,mgmsf

Ca in'serum,

.mgmst

Pinserum,mgmsf

Unlimed GrassBefore pre-gesta-

tion . . . . . . . . . . .End of pre-gesta-

tion . . . . . . . . . .End of gestation

10.5

IO.I8.2

48.2

•37 TJ 1 ' *35-4

11.4

12-5 '13-8

4-94.04.2

Limed GrassBefore pre-gesta-

tion . . . . . . . . . .End of pre-gesta-

tion . . . . . . . . . .End of gestation

H-510.7

7-9

51-2

39-034-2

.. 10.6

12.2H.I

. '. 5-5

4-25-2

*Grams hemoglobin per 100 ml of blood.tMilligrams iron per. loo ml blood.^Milligrams calcium or phosphorus per 100 ml serum.

The goats were healthy and vigorous at the end ofthe pre-gestation period, but were skinny, bony, andweak at the end of the gestation period. Of the 10goats, 6 aborted, i died, and 2 gave birth to weakkids. This malnutrition was due to a low protein con-tent, around 3.5%, in the grass fed. All of the goafs,irrespective of treatment showed at the end of gesta-

BONNET: TRACING MINERAL CONSTITUENTS OF SOIL 297

tion a reduction in weight, in red and white bloodcells, in hemoglobin, and in blood iron. However, nochange was evidenced in the calcium and inorganicphosphorus of the blood serum. It seems that theanimals were able to replace them from their bones.

The lowest amount of inorganic phosphorus ob-tained in 100 milliliters of the goats' blood serum was4 mgms. This does not point to a deficiency of phos-phorus in the unlimed or limed grass, since accord-ing to Beeson, et al. (i), blood phosphorus levels of3.50 mgms of inorganic phosphorus (P) 'per 100milliliters plasma might be considered as deficient.

The reduction of iron in the blood followed that ofhemoglobin. After calculating the iron combined withhemoglobin and multiplying the hemoglobin by thefactor 3.35, as proposed by Wong (8.) on-the basisthat hemoglobin contains 0.0335% iron as FC> it wasfound that a considerable part of the iron was notcombined with hemoglobin. The uncombined ironvaried from o.i to 19.9 mgms in 129 blood tests.

When the animal is fed with a grass of low proteincontent, malnutrition symptoms mask the mineral andblood picture of the animal. It is of importance, there-fore, to supplement the> protein diet with an extraamount of a free-mineral protein. Urea, c. p., is ourchoice for the next trial.

SUMMARYLivestock in the arid area of Puerto Rico is, in

general, stronger and more healthy and vigorous thanthat in the humid area. The mineral content of the soilis of much concern.

A field experiment on lateritic.Fajardo clay wasstarted in humid Puerto Rico to study the mineralchanges involved from the soil to the grass to theanimal blood. The study was initiated with calcium.

Data for the unlimed and limed soil are reported,including pH, exchangeable calcium, magnesium, andmanganese, and available phosphorus and iron.

Data of the above total minerals and of grass yieldare also reported for three crops of a mixture ofPara grass, Panicum purpurascens, and Carib grass,Eriochloa polystachya, unlimed and limed.

.Data are also presented on calcium and phosphorusin the blood serum and hemoglobin and iron in theblood of goats before and after the pre-gestation andafter the gestation periods.