Insecticidal Efficiency of Saturated Petroleum Fractions ‘J
INFLUENCE OF MOLECULAR WEIGHT AND STRUCTURAL CONSTITUTIOA G. W. PEARCE AND P. ,J. CHAPAMAN Yew York State Agricultural Experiment Station, Geneva, A’. Y .
D. E. H. FREAR Pennsylvania State College, State College, P u .
A
‘AST two bade factors appear to be involved in the insecticidal properties of petroleum fractions employed for controlling insect and mite pests on fruit trees: structural cornposition and molecular weight of the hydrocarbons present in such products. The influence of structural Character on insecticidal efficiency has been broadly rstablished in that paraffinic oils have been found superior to naphthenic oils. The conception that “paraffinicity” closely parallels control efficiency would appear t o be established in the case
Two series of narrow boiling petroleum fractions were prepared by fractional distillation of two technical white oils of commercial origin. The oils selected for fractionation represent distinctly paraffinic and naphthenic typee whose viscosities fall within the spra) oil range. The fractions obtained were tested for their insecticidal efficiency using eggs of the oriental fruit moth, Grupholitha molesta Busck, as test material. Correlations between insecticidal efficiency and various properties of the fractions, such as molecular weight, viscosity, density, refractive index, aniline point, and structural character were observed. It is evident from these correlations that conventional spray oils may contain a high proportion of hydrocarbons which are inactive insecticidally. Use of the data as a basis for establishing the properties of an ideal composition as well as their use as standards for a relative evaluation of oils as insecticides is indicated.
of the results. It appetlrr justified on the grounds that iiarlier findings (8, 19) indicated that the aromatic and other constituents removed by extensive acid refining or its equivalent probabl! contribute little to the insecticidal value of the oils It should not be inferred that thP question of thr roxicity of the aromatic, constituents is considered sett,led, but, the advantage 01 eliminating them for the p r w r n t is considerable.
Two water-white saturated petroleum oils were selected a8 base materials from which Iiim-ow boiling fractions could be prepared. These oils were chosen from a group on which both in. secticidal and physical property data were already available The naphthenic oil selected has been reported on by Pearce et al (19) under the code number 41-D3; the paraffinic oil was included in the report by Chapman et a2. (8) as oil 42-M20. The oils were commercially available and represent examples of the so-called technical white oils. The products were water-white and odorless and w x e no doubt obtained by exhaustive acid treatment 01 its equivalent. Compounds of nitrogen, sulfur, and oxygen a> well as arom:ttics and other unsaturates occurring in less refined oils can be assumed as not, being present in any significant amounts. These base or “original” oils were separated into fairly Iitlrrow boiling fractions in two special vacuum distillation columns designed and constructed by M.R. Cannon of thc Pennsylvania State College. Only about 50% of paraffinic oil 42-M20 was distilled in column 1 (25 theoretical plates) ; the largo pressure drop under vacuum conditions prevented completion of the distillation without producing flooding conditions. For this reason a second column (column 2) having fewer theoretical plates was used t u fractionate the 42-1120 residues from column 1 and all of oil 41-D3 (naphthenic). To obtnin sufficient quantities of the fractions for all biological tests anticipated it was necessary to replicate the fractionations The corresponding fractions from each distillation were combined, using refractive index as an indication of constancy of composition. The average deviation from the mean for corresponding fractions obtained viith column 1 was +=0.0001; with column 2, *0.0002. I n the case of the paraffinic oil, 42-1M20, eighteen fractions were obtained, each representing 4.3% by volume of the oviginal oil. Six replicate distillations were carried out in column 1 and five in column 2, yielding 450 and 375 ml. of each composite fraction, respectively. Fractions from column 1 are numbered 1 t o 12; those from column 2, 1’1 t o P8. All physical propertics and biological data reported were obtained on the composite fractions. The napht,henic nil, 41-D3, WRB fractionated into eighteen
of the following species:
Fruit tree leafroller (eggs), Archips argyrospila (Klk.) (19, 20) Codling moth (eggs), Carpocapsa pornonella (I,.) (8) Oriental fruit moth (eggs), Grapholitha molesta (Busck) ( 8 ) Eye-spotted bud moth (eggs), Spilonata ocellana (D. and S.) (8)
Grape-berry moth (eggs), Polychrosis viteana (Clem.) (24) Apple red bug (eggs), Lygidea rnendaz (Reut.) (11) European red mite (all stages), Paratetranychus pilosus (C. and
F.) (10)
Yan Jose scale (nymphs), Aspidiofus perniciosus (Comst.) ( 9 ) California red scale, Aonzdiella aurantii (3Iask.) ( 4 ) Scurfy scale (eggs), Chzonaspis furfura (Fitch) ( 5 ) Parlatoria chinensis (Marl.) (5) The difference in susceptibility between paraffinic and naphthenic oils is not always of the same order for the above species, but in most cases it is marked and of practical significance. It is not to be inferred that naphthenic oils are ineffective but only that they must be used at higher dosages. The present study was undertaken because the precise relation between the molecular weight of petroleum hydrocarbons and their insecticidal efficiency has not been determined. PREPARATION OF EXPERIMEhTAL PETROLEUhI FRACTIONS
The isolation of hydrocarbon fractions of suitable characteristics from crude petroleums was not undertaken in this study. tnstead, the experimental materials were obtained through the fractional distillation of commercially available oils representing distinctly naphthenic and paraffinic types. Only saturated oils mere utilized. Limiting the study to saturated compositions has greatly simplified the preparative work and interpretation 284
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1948
fractions, each 5% by volume of. the original oil. Eight replicate fractionations were carried out m column 2, yielding 650 ml. of each composite fraction, numbered 1 t o 18. Again all data reported apply to the composite fractions. CHEMICAL AKD PHYSICAL CHARACTERS O F FRACTIONS
Because the petroleum hydrocarbon compositions under study here are of high complexity, it has been necessary t o resort to a determination of a variety of physical properties in order to characterizc them from a constitutional viewpoint. In recent years new interprefktions of such properties in terms of structural constitution have become available. These have been taken advantage of whenever possible. The physical property data obtained on the mother oils and their fractions are presented along with constitutional interpretations in the following sections.
I
I
1
I
I
289
as 10" F. from those shown. The initial boiling points exhibited some overlapping between consecutive fractions and were not always reproducible in duplicate determinations. Because of the unreliable nature of the initial boiling points, these data are omitted in the plots given in the figures except for the original oils. In general, the curves pass through the experimental points very well, although some idealization was necessary. The effectiveness of the two columns employed in fractionating oil 42-M20 is shown graphically in Figure 1. Fractions 1 to 12, inclusive, were obtained from column 1, and the distillation curves are seen to be flat, indicating fair separation. On the other hand, fractions P1 to P8 from column 2 show considerably wider boiling range and consequent marked overlapping between consecutive fractions. The curves in Figure 2 for the fractions of oil 41-D3 obtained from column 2 are similar. The comparative distillation characteristics of the original oils are shown in Figure 2. The naphthenic oil (41-D3) consists throughout of lower boiling hydrocarbons than the paraflinic oil (42-M20). In terms of viscosity the naphthenic oil is substantially "heavier," having a Saybolt viscosi*y of about 85 seconds a t 100' F. as compared to 69 seconds for the paraffin oil. A pertinent summary of the boiling point data is given in Table I. The boiling points at 10, 60, and 90% distilled were taken from the originals of Figures 1 and 2. The values for the point of 50% distilled a t 760 mm. were obtained by extrapolation on the Brown-Coats chart (6). Extrapolation to other pressure8 with such charts introduces some error if made over a wide range as in this instance-Le., 1 to 760 mm. However, approximate information on the boiling points at 760 mm. of the fractions is useful for comparative purposes, since most boiling range data on
4401 420
380 360 LVV
0
10
20
30
40
50
60
70
80
90
lo0
PER CENT DISTILLED Figure 1. Boiling P o i n t Curves for Oil 42-M20a n d Fractions a t 1 Mm. of Mercury
340
v,
w' 320
Ei
0 300
DISTILLATIONCHARACTERISTICS. Boiling point range has frequently been employed as a specification for spray oils, In general, the method used for determining this characteristic t that of the A.S.T.M. ( 8 ) or a modification (I$). These procedures stipulate that the distillation be conducted at atmospheric pressures. Since many of the fractions dealt with here boil well above the cracking temperature, it was deemed preferable to obtain boiling point data at reduced pressures. M. R. Fenske kindly suppIied specifications and operating details of an apparatus suitable for this purpose. Determination of the boiling point range of the original oils and their fractions was carried out with the Fenske apparatus a t pressures ranging from 0.8 to 2.5 mm. of mercury absolute. A record of the temperature and pressure was taken at the start of distillation and a t 10% intervals up t o and including the point of 90% distilled. The original data extrapolated to 1 mm. of mercury absolute by means of the Brown-Coats chart (6) were used to prepare the distillation curves shown in Figures 1 and 2. The curves should be considered as representing only the relative distillation characteristics of the oils The true boiling points (bubble points) may be as much
I
w
a
280
3
5
260
CL
g,240 3
W
t-
220 200 180
140 0
10
20
30
40
50
60
70
80
90
PER CENT DISTILLED Figure 2. Boiling Point Curves for Oil 4LD3 a n d Fractions a n d Oil 42-M20at 1 M m . of Mercury
100
INDUSTRIAL AND ENGINEERING CHEMISTRY
286
TABLE I. SUMMARY OF BOILING POIXT DATA Fraction
Cumulative yo of Mother Oil
Per Cent 10% 50% 90% B.P. a t 1 Min., O F.
B.P.
Range, 10090%.
60% B.P. 760 hIm., F.
146 43
705 560 605 625 640 650
E'.
Oil 42-hI20 and Fractions 42-M20 1 2 3 4 5 6 7 8 9 10 11
12 P1 P2 P3 P4 P5 P6 P7 P8 41-D3 1 2 3 4 5 6 7 8 9 10
11 12 13 14
1.5 16 17 18
i:3 8.6 12.9 17.2 21.5 25.8 30.1 34.4 38.7 43.0 47.3 51.6 55.9 60.2 64.5 68.8 73.1 77.4 81.7 80.0
5 10 15 20 25 30 35 40 45 50 65 60 65 70 75 80 85
80
16 11
13 18 12
ia
8 10
Oil 41-D3 and Fiactiona 317 423 224 234 166 193 245 186 210 259 207 229 223 241 273 283 235 251 291 246 262 302 258 274 318 274 292 330 288 302 338 294 309 343 298 313 356 807 323 373 818 837 382 330 347 394 843 362 404 854 373 384 412 368 395 420 379
660
670 680
9 9 9 38 39 38 30 40 43 42 42
690 695 705 715 735 740 745 750 760 765 775 785
199
660
54 50 48 46 44 44 41 44 45 49 55 52 51 50 44 41
640 650 655 670 685 695 715 730 740 755
68 60
605 525 550 565 580 500 605 630
spray oils are given a t this pressure a t present. Moreover, it was necessary to have this information in the case of some fractions for calculation of molecular weights. The paraffinic and naphthenic mother oils would be classed as medium and heavy, respectively, under the California system ( 1 % )of classification of spray oils. A summary of the physical property PfiysIcAL PROPERTIES. data on the original oils and their fractions is presented in Table 11, with the insecticidal evaluations expressed as the minimum effective dosage (M.E.D.) required to produce a 95% kill of oriental fruit moth eggs under test conditions described belbw. It was not feasible to evaluate all the fractions insecticidally in the present study. However, physical property data on all fractions are given because it is plmned t,o submit insecticidal data on all fractions eventually. The viscosity values were obtained by Method B, A.S.T.M. Saybolt values mere obtained from conversion tables (63). Kinematic viscosity index values \Yere calculated with the aid of tables (22). Viscosity index is not normally applied to oils of this viscosity range but is given here as an indication of the relative difference in constitution of the two series of fractions. Density values were determined pycnometrically and are corrected for the buoyancy effect of air. The values are accurate to *0.0001 unit. A.P.I. gravity was calculated with the aid of tables (%6). Refractive index was determined with an Abbe refractometer. The values are correct to about *0.0001 unit. Specific refraction was calculated by the Lorentz-Lorenz equation. As a check on the saturated charact'er of the fractions specific dispersion values were determined. The values obtained were essentially the same for all fractions-Le., about 99 x 100-4, using the Gladstone-Dale equation and dispersion values obtained with an Abbe refractometer. Since the specific dispersion values showed all materials to be substantially saturated, no further check on saturation was made. Aniline points were determined by the A.S.T.M. method ( 2 ) . (8) Designation D445-39T.
Vol. 40, No. 2
MOLECVLAR WEIGHTS. The precise determination of the average molecular w i g h t s of petroleum fractions is difficult. For the present study highly accurate values arc not essential. Accordingly it was dccidcd to calculate molecular weights fron: correlations with certain physical properties. Scvcral correlations are available for this purpose. Because of the wide range in molccular size of thc fractions it has been necessary to employ a t least two relations in order to obtain a value for every fraction. As a matter of interest and comparison four correlations for ca1culating molecular ITeight have been employed: e
1. Viscosity at 100" F.-viscosity
a t 210' F.--.molecular
reight. 2. Viscosity a t 100" F.--specific gravity a t 60"/60" F molecular weight. 3. Viscosity at 210" F.--specific gravity a t 60"/60" F molecular wcight. 4. 50y0 boiling point--density at 20°/.1' C.-moleculaT weight. Data and graphs for these correlations supplied by Hirschler Bi summary of the values obtained is presented in Table 111. The data obtained on the paraffinic oil, 42-M20, and its fractions using correlations 1, 2, and 3 are consistent. I n the ring analysip calculations and corrclations between molecular weight arid insecticidal efficiency, given below, the values obtained with correlations 2 and 4 were employed for the paraffinic series The molecular weights obtained for the naphthcnic series shou considerable variation among the several methods of calculation I n order to obtain values showing uniform increments in molecular weight for this series the data obtained with correlations 1 and 4 have been selectcd for use in the ring analysis and correlations with the biological data. HYDROCARBON RINGANALYSIS.I n order to characterize further the materials undcr study so-called "ring analysis" method.. have been applied. Of the several notable methods proposed in the literature the Waterman (65) and the Lipkin-Kurtz (1.4) methods vere most readily applicable to the data a t hand Since the fractions are essentially saturated hydrocarbon compositions, both methods should provide reasonably good estimates of the percentage of naphthene rings and paraffin chaine. The results obtained nith both the Itraterman and the Lipkin-Kurtz methods are given in Table IV. The molecular weight valuep used in the calculations and insecticidal evaluations are included. I n applying the Lipkin-Kurtz technique, density cocfficientff were obtained from the molecular weight us. density coefficient correlation given by Lipkin and Kurtz (15). The results obtained for the per cent naphthcne rings by the two methods show R substantial difference. The deviation i s of the same order and direction as that experienced by Schiossler el al. ( 2 1 ) in applying these methods to compounds and mixture8 of k n o m composition. Regardless of which method gives values nearest the actual, both shorn the same small changcs in composition within each series as w l l as the marked difference between each two. In the paraffinic series there is a trend tom-ards fewer rings among the lighter fractions, rrhereas the naphthenic series shows an opposite trend, tho lighter fractions having a higher percentage of rings. The most significant point for the prescnt study is that the data indicate that the percentage composition within each series is essentially constant q-hilr molecular weight varies. (13) and Mills et al. ( 1 6 ) have been used for all calculations.
INSECTIClDAL EVALUATION OF PETROLEUM FRACTIONS
TESTINSECT.Previous experience had shown (8) that eggti of thrcc economically important oil-susceptible species of insects rcspond almost identically to petroleum oil treatments: codlingmoth, eye-spotted bud moth, and oriental fruit moth. Summer spray programs havc included petroleum oils as an aid in controlling thcsc pcsts, the oils acting primarily as ovicides. Of the three species, oriental fruit moth was the most readily availahlv
'
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1948
287
TABLE11. PHYSICAL PROPERTIES AND MINIMUM EFFECTIVBDOSAGE VALUES
looo P. Fraction
Centistokes
Viscosity Centistokes
69.27 39.55 43.10 45.84 47.96 50.45 52.58 55.06 57.81 60.48 63.34 66.38 69.57 114.5 82.62 85.92 89.36 94.52 98.47 106.9 115.9 131.2 201.5
3.09 1.48 1.74 1.91 2.05 2.17 2.31 2.43 2.57 2.69 2.82 2.93 3.07 4.49 3.53 3.67 3.78 3.92 4.07 4.28 4.55 4.90 6.51
84.78 16.92 41-D3 39.10 4.00 1 42.04 4.92 2 44.67 5.74 3 47.45 6.61 4 50.25 7.47 5 53.73 8.51 6 57.88 9.73 7 63.24 11.26 8 68.56 12.72 9 75.94 14.67 10 82.70 16.40 11 92.15 18.72 12 104.8 21.72 13 121.5 25.60 14 142.2 30.30 15 171.3 36.80 16 213.2 46.05 17 287.1 62.10 18 649.6 140.6 Res. Not est,ablinIied,8ee Figure 3. b Not wtablished, see Figure 4.
3.28 1.41 1.59 1.74 1.90 2.02 2.18 2.35 2.57 2.74 2.98 3.17 3.44 3.73 4.11 4.51 5.04 6.70 6.74 10.66
42-M20 1 2 3 4 5 6 7 8 9 10 11 12 k t res. P1 P2 P3 P4 P5 P6 P7 P8 2nd res.
12.19 4.14 5.25 6.10 6.77 7.53 8.17 8.90 9.71 10.48 11.29 12.13 32.99 23.98 16.38 17.20 18.04 19.29 20.23 22.20 24.30 27.80 43.51
Refractive Index,
210° F. Saybolt eec.
Kineamti’ sac.
36.54
... ...
33 :Ol 33.44 33.96 34.39 34.86 35.25 35.67 36.03 36.47 40.95 37.95 38.37 38.71 39.13 39.59 40.27 41.14 42.28 47.46
ViRcosity Index
De-iW,
dszz
,,&:A& 60° F.
Oil 42-M20 and Fraationa 36.7 110.2 40.7 40.3 39.6 10i:o 89.0 38.4 99.6 105.7 38.3 104.0 38.0 37.7 105.0 37.4 104.0 37.1 104.8 101.6 37.0 104.6 36.7 111.2 34.4 104.8 35.3 110.3 35.6 111.0 35.4 35.3 109.3 35.2 114.0 110.7 34.8 34.5 113.5 34.0 110.2 109.6 32.6
... ...
lzloo
SPecifia Refraction, ,200
Aniline point 0
0.3282 0.3299 0.3300 0 . 3 2 ~ 0,3294 0.3290 0.3291 0.3289 0.3289 0.3287 0.3285 0.3286 0.3283 0.3269 0.3268 0.3278 0.3278 0.3275 0.3276 0.3272 0,3272 0.3267 0.3262
c.’
103.8 89.5 93.8 95.3 96.5 97.3 98.6 99.6 101.5 101 .a 102.0 102.6 103.6 108.3 103.8 105.4 106.3
106.6
107.5 108.0 109.0 109.5 113.0
M.E.D.,
Me./
100 sq. cm. 2.9
Not est. 0
Nd’eat.o Nolest.0
...
6.0 8.6 3.3 3.0 2.1 2.1
i:b 1.6 ...
1.6
i:i
1.6 1.6 2.1
Oil 41.-D3 and Fractions 37.15
... ...
...
32:90 33.48 34.10 34.86 35.41 36.19 36.79 37.67 38.55 89.72 41.01 42.73 44.85 48.14 61.64
48.4
... ... ... ... 57.8
54.0 48.6 48.5 41.5 40.8 37.8 40.6 36.7 38.6 41.0 50.4 54.7 52.5 49.1
0.8798 0.8756 0.8765 0.8757 0,8742 0.8736 0.8735 0.8740 0.8747 0.8767 0.8765 0.8772 0.8778 0.8788 0.8795 0.8808 0.8824 0.8844 0.8878 0.8962
Consequently, eggs of this species were employed for all insecticidal tests in this study. TESTINO PROCEDURE. Adults were permitted t o oviposit their eggs on thc foliage of small potted quince trees which had been trained to a single stem or shoot. Usually three trees were grown in each pot. The moths were caged around one of the shoots for a period of 48 hours in a greenhouse. Treatments were then applied to all the trecs in the pot, including the infested shoot and the two uninfested ones. The procedure adopted for applying the petroleum fractions to the quince foliage is the result of several years’ experience. I n earlier work (8)a standard orchard power sprayer was used, but it waa not possible to cmploy such equipment for the small quantities of test materials prepared for the present study. Accordingly, after preliminary tests a quart-size paint spray gun was adopted for application of the test materials. The materials were emulsified by use of a hand-operated homogenizer and then diluted to the dcsircd strength for testing. Blood albumin (18)a t the rate of 2 dunces per 100 gallons of finished spray was used as the emulsifying agent. The test emulsions prepared in this manner were transfcrred t o the spray gun and applied as follows : The pot containing the three quince shoots was placed on a small electric turntable set a t 2 r.p.m. The test emulsion was applied with the paint spray gun by directing the spray always downward. An air pressure of 25 pounds per square inch was used for operation of the gun. The total exposure of the foliage to the spray varied somewhat with the size of the trees. Normally an exposure of about 40 seconds was required for thorough wetting of all foliage. Care was taken t o treat only the upper surfaces.
28.7 29.5 29.4 29.5 29.8 29.9 29.9 20.8 29.7 29.5 29.4 29.2 29.1 28.9 28.8 28.6 28.2 27.9 27.2 25.7
91.4 68.5 72.3 75.6 78.3 80.7 82.6 84.5 86.2 87.8 90.2 90.8 92.3 93.8 95.6 96.9 98.4
N0i’abt.b
101.8 106.6
2.6
100.0
5.0 Not est. b
...
N6d;st.b
..... . ...
18.8
...
6.8 7.7 2.9 2.6 1.9 2.3
...
The treated material was placed outdoors until hatching was complete in an untreated control lot, usually 7 to 10 days. A record was then taken of the number of hatched and unhatched eggs with the aid of a binocular microscope. Per cent control efficiency of each treatment was calculated by means of the Abbott equation ( I ) :
yo control efficiency where A
6
-x 100
=A
% hatch in untreated and B = % hatch in treated
This computation corrects for any egg mortality from natural causes, which may be high in certain species of insects. The hatch of eggs in all untreated plots averaged 97.45 =t 0.50%. This value was used as A in the above equation. Since the natural mortality in the controls was very low, the values for per cent control efficiency obtained are practically equivalent to per cent kill or mortality. The efficiency of insecticides is commonly based on the concentration of the active materials in the spray and not on the quantity actually deposited on the plant surface. Previous studies (7,19)have clearly shown that in the case of oil sprays a rather precise and reproducible relation exists between the amount of oil deposited and the kill obtained of the insects involved. On the other hand, the relation between concentration in the spray and kill is subject t o considerable variation because many factors can affect the amount of oil deposited by a given concentration. In view of this it was elected to compare all materials on the basis of the relation of oil deposit to kill.
288
INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLE III.
Fraation 42-M20
1 2
3 4
5 6 7
8 9 10 11 12 1st res. P1 P2 P3 P4 P5 P6 P7 P8 2nd res.
MOLECULAR
WEIGHT
DATAO N
50% b.p. a t 760 mm.
Viscosityiw
Viscosityiw
Viscositym
US.
US.
US.
us.
viscosityzin 328
gravity 329
gravity 329
density 335 237 267 276 288 296
... ... ... ... .
I
.
...
282 292 305 313 317 324 385 343 353 358 367 372 378 389 397 445
...
267 276 281 288 294 300 306 312 317 324 330 388 345 353 358 367 373 382 390 402 447
TABLEIV.
iii
280 290 297 302 309 315 320 329 389 350 358 363 368 375 383 391 403 647
Moleoulai Weight
42-M20 1 3 4
5 6
7
8 9
LO
11 12 tat res. P1 P2
P7
P4 P5 P6 P7 P8
2nd r e i
1
4 5 6 7 8
9 10 11 12 13 14 15 16 17 18
Kea.
%
307 198 208 220 228 237 246 253 266 275 286 296 309 314 330 340 357 366 387 430
%
naphthenic rinm
paraffinic chains
30.1
69 9 69 3 73.1, 72.5 71.1 70 3 70 4 70 5 70 5 70 2 69 8 70 2 69 9 69 1 66 8 68 7 69 1 69 2 70 0 69 4 69 2 68 6 68 2
30.7 26.5 27.5 28.9 29.7 29.6 29.6 29.5 29.8 30.2 29.8 30.1 30.9 33.2 31.3 30.9 30.8 30.0 30.6 30.8 31.4 31.8
2
2 3
AXD
41-D3
fi0.R
78.8 76.5 72.7 70.4
68.1 66.5 65.0 63.0 62.3 61.5 60.4 58,s
58.8 57.3 56.9 56.2 56.9 56.9 58.8
AND
THEIR~ u c n o r s
Viscosityiao US.
li'ractiou 41-D3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
16 17 18 Kea.
Viscosity~w US,
viscositym 307
Viscosityn~o
gravity 310
gravity so9
.".
... .~~ ...
,
247 266 275 286 296 309 314 330 340 357 366 387 430
259 267 275 284 292 303 311 320 333 348 362 379 396 427 490
.
50%F-
a t 760 mm. US.
WS.
I
254 260 269 281 287 297 308 320 330 343 3 57 372 388 408 471
densit> 290 198 208 220 228 237 246 253 266 274 282 288 292 304 318 .
I
. I
."
.. sample of the treated foliage is extracted with petroleum ethrr and the amount of color in the extract is then determined in a photoelectric colorimeter. From a knowledge of the concentration of dye in the oil applied, the amount deposited can lar calculated. The data obtained are expressed in terms of milligrams of oil per 100 sq. cm. of leaf surface. Using the procedure outlined, all fractions tested were partiall? saturated with Oil Red-0 before application to the quince foliage (Oil Red-0 a t the concentrations used was found to have no toxicity to the test eggs.) A sample consisting of fifty 0.875-inch disks was taken from the center portion of the leaves for oil deposit analysis as soon as the spray was dry.
RING ANALYSISDATAAND MINM.IUM EFFECTIVEDOMGE
Lipkin-Kurtz Analysis
41-D3
42-1120
Molecular Weights (Naphthenic Series)
Only the upper surfaces of the quince leaves were treated, in order to avoid complications due to the pronounced difference in the amount of oil deposited on the upper and lower surfaces of the foliage (8). By confining the spray to the upper surface and taking mortality records on the same surface, these complications have been eliminated. Preliminary work indicated that many fractions would have to be tested a t extremely low concentrations in order to evaluate them properly. This necessitated the development of a special method for determining the amount of oil deposited (17). The procedure consists in partial saturation of the oil with an oilsoluble dye before application t o the foliage. A represent,ativr
Fraction
OILS
Molecular Weights (Paraffinic Series)
I
Vol. 40, No. 2
39.2 21.2 23.5 27.3 29.6 31.9 33.5 35.0 37.0 87.7 38.5 39.6 41.2 41.2 42.7 43.1 43.8 43.1 43.1
41.2
Waterman Analysis Rings in naphthenia paraffinic PQr rings chains mole C,H%* Oil 42-M20 and Fraations
x
I _
I
%
1.3
-4.6
0.9 0.9 0.9
t0.2 f0.2 f0.2 0.0
1.0 1.1 1.1 1.1 1.1
1.2
1.2 1.2
1.3
1.6 1.6 1.4 1.4 1.5 1.5 1.5
1.5 1.7 1.9
Oil 41-D3 and 'Fractions 47.7 52.3 67.6 32.4 65.0 35.0 61.7 38.3 58.6 41.4 56.4 43.6 55.0 45.0 53.1 46.9 51.4 48.6 50.5 49.6 48.8 51.2 47.5 52.5 45.9 54.1 46.7 53.3 45.0 55.0 44.4 55.6 44.6 55.4 43.8 56 2 44.3 59.7 , 55.1 44.9
2.5
2.1 2.1 2.2 a.2 2.2 2.2 2.2 2.2 2.3 2.3 2.4 2.4 2.5 2 5 2.6 2.7 2.7 3.0 3.3
-0.1
-0.2 -0.2 -0.2 -0.4 -0.4 -0.4
-0.E --I . z ,2 -0.8 --0 .8 -1.0 -1.0 -1.0
,o
--1.4
---I , 8
.-3.0 -2.2 --a, 2 --2,4
.-2.4
-2.4 -2.4 -2.4 .-2,4 -2.6 ,-2,6 -2.8 .-2,8 -3 0 --3.0 --3 .7 -3.4 -3 4 -4.0 ,-4,6
No. of carbon atoms
h5.E.I) hfg./ 100 sq. c m >
23.6 16.9 19.1 19.7 20 1 20 6 21 0 21 4 21 9 22 3 22 7 23 2 23 6 27 8 24 7 25 3 25 6 26 3 26 7 27.3 27.9 28.8 32.0
2.9 N o t eat
Nodest
Ndt'kt 5:0 3.R 3.3 3.0 2.1 2.1
1:n 1.6 1:6
i:fi
1.6 1.6 2.1
22 1 14 3 15 0
si
0 16 5 17 1 17.7 18 2 I9 2 19 8 20 6 21.3 22 3 22 6 2.7 8 24 5 25 7 20 4 27 9 31 .O
6:P
7.7 2.9 2.6 1.P
2.3
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1948
289
- - - - - - - - - - - - - - - - - - - - - - - --
----- - - - -----
1
0
F 75 -
Z
W
0
a w
a
is0 -
-
-
M 20-3 VIS.46 M.L.D.-NOT EST
C
M.E.D.-S
-
-
M 20-PI VIS.83 M.E.D.-1.6
-
M20-P7
VIS.116 M.E.D.-I.6
I
I 1
I
> C 0
I 5z w
u
LL
LL
W
-I
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0 0
Figure 3.
OIL DEPOSIT -MG PER 100 SQ. CENTIMETERS Dosage-Control EfficiencyCurves for Oil 42-M20 and Fractions (Paraffinic Series)
obtained over most of A compilation of the average - deposits the range of concentrations included in this study is presented in Table V. Inspection of the average deposit obtained a t the several concentrations shows that the relation between them is linear. The average per cent deviation of individual treatments given in the last column indicates that considerable variation in the amount deposited by B given concentration may be expected. This probably represents the extreme condition, since the average deposits‘are based on data taken over a 3-year period. It is believed deposition rate can be controlled easily within the variations shown, even under orchard conditions. While this varia-‘ tion is not too large for practical considerations, it is far too large for experimental comparisons of the efficiency of oils. For this reason only the present study is based on oil deposit us. mortality rather than oil concentration in the spray us. mortality. MINIMUMEFFECTIVEDOSAQEOF FRACTIONS. Insect toxicologists have employed various points on the dosage-mortality curve to compare inaecticides. The point of 60% mortality (median lethal dose) is often used, for example. In the present
work a point much closer to 100% mortality-Le., 95%-has been selected as experience has shown this point to be more reliable and significant in the case of oil sprays. h addition it representr a degree of kill frequently aimed a t in commercial orchard spraying. Accordingly, the efficiency ratings of the fractions tested are expressed in terms of the dosage necessary for a control efficiency of 95%. These ratings are given the term “minimum
TABLEV. RELATION BETWEEN OILDEPOSITAND CONCENTRATION IN SPRAY UNDER EXPERIMENTAL CONDITIONS Av. Deposition.
% Oil in Spray 0.10 0.20 0.40 0.60 0.80 1.00
KO. of Treatments 19 32 22 11 7 9
’
Mg./ 100 Sq. Cm. Leaf Gurface 0.67 1.33 2.43 3.75 5.50 6.73
Av. Deviation
of Each Treatment
from Mean, % 30.4 30.9 23.4 22.3 18.9 14.6
Vol. 40, No. 2 .
95
--
- - - --
- -
C O R R ELATIONS BETWEEN CONSTlTUlION AND INSECTICIDAL EFFICIENCY
75 03-1 VIS39 M E.D -NOT E ST
hf 0 L E C U L A H WEIOHTAND VIS COSITY. The ring 25analysis data submitted are con, 1 sidered satisfactory evidence that within each series of _ _ _ _ 95 fractions, structural constitution 75 broadly speaking, is tD3-4 D3-13 D 3-17 Z e s s e n t i a l l y conVIS 47 VIS 105 VIS 213 d 50 0stant. Therefore, 0 M ELI-NOT E S T M E D.-77 MED-23 plots of the miniY d 25 2 5 -mum effective dosn Y age values against __ molecular weight and viscosity valuee for each series are valid. Such graphs are shown in Figure 5 (data fromTables VlS.63 111 and IV). D3-14 4 1 D 3 R E S M E D -NOT EST The curves apVIS. 122 VIS.650 1 ______i pear to be hyperbolic and, in general, the experimental points fit well, especially in the case of the paraffinic fraction8 (P curves). The naphthenic fractions (N curves) show more deviation, but it is believed the curveB drawn represent the true behavior of these fractions as near as may be ascertained from the O I L DEPOSIT-MG. PER 100 SQ. CENTIMETERS data. Figure 4. Dosage-Control Efficiency Curves for Oil 4LD3 and Fractions (Naphthenic Series) A number of important observations may be made on the data appearing in Figure 5. First, insecticidal effectiveness effective dosage" (M.E.D.) and represent the milligrams of oil becomes constant above certain minimum niolecular weights and per 100 sq. cm. of leaf surface required to give a control efficiency viscosities for each series. For the naphthenic serics the critical (kill) of 957, against eggs of the oriental fruit moth. molecular weight is about 350 and the critical viscosity aboul The values given in the last column of Tables 111and IV were 140 seconds. The critical values for the paraffinic series are 340 arrived a t from the dosage-control efficiency curves shown in for molecular weight and 80 seconds for viscosity. Below the Figures 3 and 4. The horizontal dotted line in each graph rcpcritical points in each series insecticidal efficiency falls rapidly to resents 95% control efficiency and the point of intersection with immeasurable values. the dosage-mortality curve is taken as the minimum effective The comparativc eficiency between the two series of fractione dosage value. Fraction number, Saybolt viscosity a t 100' F., $ perhaps best illustrated by the compilation in Table VI. The and minimum effective dosage value are included in the body of values for naphthenic and paraffinic compositions a t 10-second each graph. The tabular data from which these curves were intcrvals of viscosity are shown, derived from the viscosity graph prepared are omitted to preserve space. The data required in Figure 5. A paraffinic hydrocarbon composition having B over two hundred treatments, involving about a quarter of a viscosity of 60 seconds shows a high degree of effectiveness-i.e., million eggs. The tests were conducted during the late summer a minimum effective dosage of 3.2, whereas a naphthetiic commonths of 1943, 1944, and 1945. The dosage-control efficiency position of the same viscosity is totally ineffective, tho minimum Curves shown usually involve all data from a t least two years' effective dosage being apparently infinite. The data illustrate tests and in the case of the mother oils all the data from throe years further that a naphthenic composition having a viscosity of arc employed. The reproducibility of the curves is estimated to about 100 seconds ie necessary before measurable values me be a b u t =t5%. -
50
I
5 I
-
,
I
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I
February 1948
spparent. The critical viscosity levels at the points of maximum effectiveness are underlined in the table. Thus, in addition t o being more efficient in the range of maximum effectiveness for both series, the paraffinic compositions are highly effective a t considerably lower viscosities than in the case of naphthenic oils. This may be of significance in relation to plant tolerance. The evidence in the literature as well as current practice indicates that within limits fruit trees show increasing tolerance towards oils as the viscosity is reduced. ANILINE POINT, REFRACTIVE INDEX, AND DENSITY.Examination of the physical properties and minimum effective dosage values of the fractions shown in Table V indicates that if the various properties are plotted against the minimum effective dosage values, curves similar to those in Figure 5 are obtained. To illustrate this point Figure 6 has been prepared. The three properties selected are ones frequently employed for characterization of petroleum products. The curves are essentially of the same nature as those shown in Figure 5. This might be expected, since the change in aniline point, refractive index, and density within each series is simply a reflection of the change in molecular weight. The data represented by the curves, then, can be considered additional evidence that within each series chemical composition, broadly speaking, is constant. This evidence is not entirely independent of the ring analysis data because the latter represent special interpretations of refractive index, density, and molecular weight data. However, aniline point is independent of the ring analysis data. Plots of other properties against minimum effective dosage values which will give the same type of correlations shown in Figures 5 and 6 are 50% boiling point and number of carbon atoms. Again the change in these characters within each series IS proportional t o change in molecular weight and, thus, curves Rimilar to those in Figure 5 and 6 are to be expected. The difference in chemical composition between the two seriea of fractions is well illustrated by the large horizontal spread between the N and P curves in Figure 6 and also the viscosity graph in Figure 5. On the other hand the spread in molecular weight between the two series (Figure 5) is comparatively much smaller. This is an illustration of the well-known fact that the physical properties involved are frequently more affected by changes in structural composition than by changes in molecular weight. DILUTION EFFECT
It is plain from the data submitted in Figures 3 and 4 that the lighter fractions in each series have little or no value insecticidally for the species involved. Thus, about the first eleven fractions of oil 41-D3 are more or less ineffective. Since each fraction rcpregents 5% of the mother oil, 55% of the latter may be considered inactive. The effect of the presence of the inactive portion on the minimum effective dosage values of the original oil is approximately linear, as is suggested by the following considerations. I f the minimum effective dosage value of oil 41-D3-namely, 5.0
TABLEVI.
COMPARISON O F EFFICIENCY O F PAR.4FFINIC AND NAPHTHENIC COMPOSITIONS AT VARIOUSVISCOSITIES
Saybolt
Viwxity
at 100' F.
60 60
70 80 90 100 110 120 130 140 150
d
-
291
INDUSTRIAL AND ENGINEERING CHEMISTRY
M.E.D.
(Parqffinio Series) Curve P
i:a
1.9 1.6 1.6
1.6 1.6 1.6
1.6 1.6 1.6
M.E.D. (Nrtpbthenio Series) Curve N
... ... ...
...
6:i 4.1 3.0 2.6 2.3 2.3
P
-
8
8-
V
'a 0
-
6w -
a
6 4-
I _ I Q 2W
s I 0'
50
I
1
100
SAYBOLT VISCOSITY
5
-
4
8-
'5
6-
0
I I50
I
AT 100 DEGREES
1 200
F.
0
a
J I I
d.
w
2
.
4-
2-
-
MOLECULAR
WEIGHT
Figure 5. Correlation between Minimum Effective Dosage of Fractions and Viscosity and Molecular Weight Circled points, mother oils N, naphthenic series P, p a r a 5 n i o seriea
mg.-is corrected by 55% inactive constituents, the resulting value is 2.3 mg., which is the same as the average for all fractiom in the range of maximum efficiency. In a similar manner the value for the paraffinic oil, 42-M20, might be corrected for the presence of 45% inactive material. Again the corrected value, 1.6 mg., is equal t o that found for the most efficient portion of t h b oil. While these calculations have been idealized somewhat by judicious selection of the percentage of each oil considered totally ineffective, for practical considerations this is warranted. In effect, then, one may consider that the dilution of the original oils by the ineffective constituents has an approximately additive or linear effect on their respective minimum effective dosages. On the other hand, the effectof the lighter fractions on the physical properties of the original oils is not linear in most instances. This probably accounts in part for the fact that the points for the original oils shown in Figures 5 and 6 are a t some distance from their respective curves in some cases. In view of the foregoing observations it appears that conventional spray oils probably contain various amounts of ineffective hydrocarbons. Some of the very large differences iD efficiency observed between paraffinic and naphthenic oils studied in earlier work will no doubt have to be modified by correction for the dilution effect of inactive constituents. However, the data submitted on the two series of fractions studied support all previous findings that paraffinic oils are generally superior to naphthenic oils. MECHANISM OF KILLING ACTION
A point which appears worth discussion concerns the explanation for the rapid change in insecticidal efficiency of the fractiom over a relatively narrow range in molecular weight. From Table IV and Figure 5 it is observed that in both series of fractions the change from the least effective fractions to the most efficient cor-
INDUSTRIAL AND ENGINEERING CHEMISTRY
292
---
I an ideal oil composition. RS follows:
The properties of such an oil would he
Saybolt viscosityat looo F. Uasulfonated residue (A.S.T.hI.) Density, :so Refractive index, B.P. range a t 1 mm. Hg (90%) 50% b.p. a t 1 mm. Hg iMoleoular weight Pour point Homogeneity
. i ’
8
2 -
0L.--
L
1
1 1.4600
I ,_I_L__L1 14650 1.4700 1,4750 1.4800
.J
r
REFRACTIVE
J
INDEX-N?’
I
.83
a5
~.
--*
DENS T I
-
I
?
Figure 6. Correlations between Minimum Effective Dosage of Fractions and Some Physical Properties Ciroled pointm, mother oils N, naphthenic series P, paraffinio series
responds to a change of about five carbon atoms or 70 units in molecular weight. Similarly, other properties (Table 11) of the fractions change very slowly while insecticidal efficiency proceeds rapidly to a maximum. This suggests that some property of the fractions which has not been evaluated is probably related t o the killing mechanism. The relatively high volatility of the first few fractions from both oils may account in part for their insffectiveness, but unpublished evidence is available which indicates that volatility is not a factor in the case of the higher boiling fractions. However, until quantitative measurements on the amount of oil volatilized per unit of time and the time required for the killing process to be completed are asccrtained, one cannot be certain regarding the role of volatility. It would seem io he a factor in the case of kerosenelike fractions. APPLICATIONS OF DATA
Lt is probably premature to attempt direct application of the
data to current practices in the use of spray oils, because the iesults are restricted t o the response of a single species of insect. On the other hand, one would expect the general pattern of the correlations observed with eggs of the oriental fruit moth t o be approximated by eggs of closely related species such as codling Qoth, eye-spotted bud moth, and grape-berry moth. Within the limitations indicated various applications of the data may be made-for example. they provide a basis for defining
Vol. 40, No. 2
70-80sec. X o t leva than 92% 0.840 (mnx.) 1.484 (min.) 100 50 F. 370’ f 5’ F. 340 z t 5 X o t greater than 30° 8 . h nonblended distillate portion of petroleum
*
The oil described by these properties would presumably b e classified under present convention as a summer or foliage spraj oil. Some unpublished evidence indicates that the oil defined above is also highly efficient as a dormant treatment. At present industry is no doubt unprepared to provide an oil of such close definition except a t great expense. Heretofore, however, such standards have not been available and they are presented here in an attempt to indicate xhat future developments may demand i n the case of petroleum oil sprays. Another possible application of the data concerns the appraisal of more or less highly refined petroleum fractions for their insecticidal value without resorting to direct biological cvaluatiori Such an appraisal could be carried out by use of the viscositr correlation shown in Figure 5. The method would, first, consist of a preliminary separation of the oil into a number of fairlj narroi? boiling fractions by a distillation procrss. A subsequent determination of the viscosity and a ring analysis of each fraction would provide information from which the minimum effective dosage value of the original oil could be predicted from interpolations between the viscosity curves in Figure 5. This pro. cedure would take into account the “dilution effect” of constituents of low molecular weight as well as the influence of structural composition of the constituents. In this manner one could obtain a relative evaluation of oils which should be useful as a guide iu developing improved products or inspection of many of t h e products now on the market. ACKNOWLEDGMENT
The writers wish to express their appreciation and thank8 tu the following for their invaluable advice and assistance: RI. R Fcnske and Rodney Hersh, Petroleum Refining Laboratory, and M. R. Cannon and R. W. Schiessler, School of Chemistry and Physics, all of the Pennsylvania State College; and to A. W Avens and G. E. R. Hervery of the S e w York State Agricul. tural Experiment Station. Thanks are also extended to thr Don, Chemical Company for providing financial aid at the heginning of this investigation. LITERATURE CITED
Abbott, W. S., J . Econ. Enlomol., 18, 265-7 (1925). Am. Soc. Testing Materials “Standards on Petroleum Product? and Lubricants,” 1943. Baker, II., “Report of Control Studies on Parlatoria chinensic (Marl.),” private communication, 1944. Boyce, A. M ., Citrus Experiment Station, Riverside, Calif.. private communication, 1945. Brann, J. L., “Biology and Control of the Scurfy Scale, Chionaspis furfura (Fitch),” Ph.D. thesis, Cornell Univ., 1944. Brown. G . G., and Badger, P. A, ”Brown-Coats Vapor Pressure Chart for Hydrocarbons,” G. G. Brown, Univ. of hfich. Ann Arbor, Mich., 1833. Chapman, P. J., Pearce, G. W.. and AvenA$ A . W.. .I. Ernn Bntomol., 34, 639747 (1941). Ibid., 36, 241-7 (1943). Ibid., 37, 305-7 (1844). Dean, 11. W., N. Y . Agr. Expt. Sta., Bull. 703, 61-5 (1943). Dean, 11. I\’., and Chapman, P. J., Ibid., Bull. 716 (1946). Elinore, J. W,, Calif. Dept. Agr., Div. Chem., Special Pub. 107 (1930).
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
February 1948 (13)
Hirschler, A. E., papers presented before Div. of Petroleum Chemistry, A.C.S., pp. 153-69, Memphis, Tenn., April 1942; J . Znst. Petroleum, 32, 33-61 (1946).
Lipkin, M. R., and Kurtz, S. S., Jr., papers presented before Div. of Pet.roleum Chemistry, A.C.S., pp. 47-53, Atlantic City, N. J., September 1940. (15) Lipkin, M. R., and Kurtz, S. S., Jr., IND. ENO.CHEM.,ANAL. ED..13,291-5 (1941). (16) Mills, I. W., Hirschler, A. E., and Kurtz, S. S., Jr., papers presented before Div. of Petroleum Chemistry, A.C.S. Meeting in Print, pp. 91-114, September 1945. (17) Pearce, G. W., and Avens, A. W., “A Colorimetric Method of Determining Oil Deposit on Plant Surfaces Treated vith Oil Sprays,” in preparation. (18)Pearce, G. W., and Chapman, P. J., N. Y. Agr. Expt. Sta., Bull.
(22) (23)
(14)
293
Standard Oil Development Co., ‘‘Basic Values for Calculatinp Viscosity Index,” Circ. 30.50 (1938). Standard Oil Development Go., “Conversion Tables for Kine. matic and Saybolt Viscosities,” Supplement to Circ. 30.90A (1938).
(24)
Taschenberg. E. F., “Studies on the Control of the Grape-Berry Moth, Polgchrosb viteana (Clem.),” Ph.D. thesis, Cornell Univ..
(25)
Vlugter, J. C., Waterman, H. I., and van Westen, H. A., J
(26)
Wilhelm, R. M.,“Tag Manual for Inspectors of Petroleum’ 25th ed., Brooklyn, N. Y., C. J. Tagliabue Mfg. Co., 1929
1945.
Znst. Petroleum Tech., 21, 661-76, 701-08 (1935).
698, 15-16 (1942).
(19)Pearce, G. W., Chapman, P. J., and Avens, A. W., Enfomol.,35, 211-20 (1942). (20) Penny, D. D., Ibid., 14,428-33 (1921). 121)
J. Econ.
Sohiessler, R. W., Clarke, D. J., Rowland, C. S., Sloatman, W. S., and Herr, C. H., Petroleum Refiner, 22, 390 (1943); Proc. Am. Petroleum Znst.. 24, Seo. 3, 49-74 (1943).
RECEIYEDFebruary 24, 1947. Abstract of a theeis submitted to the Gradu. ate School of the Pennsylvania State College by G. ITr. Pearce, June 1946, BC partial fulfillment of the requirements for the Ph.D. degree. Journal Paper 698, New York State Agricultural Experiment Station, Geneva, N. Y . Au. thorired for publication as Paper 1360 in the Journal Series of the Penn. sylvania Agricultural Experiment Station.
Protoplasts from Plant Materials Properties of Protoplasts Released by Anaerobic Fermentation with Clostridium roseurn JONATHAN W. WHITE, JR., LEOPOLD WEIL, JOSEPH NAGHSKI, EDWARD S. DELLA MONICA, AND J. J. WILLAMAN Eastern Regional Research Laboratory, Philadelphia 18, Pa.
’I’he 2,000,000 tons of leaf waste incident to the commercial production of vegetable crops contain 50,000 tons of protein and 20 tons of carotene, besides other lipoid constituents. As a preliminary to the recovery of these materials, some method of concentration is desirable. It can be accomplished by release of the protoplasts as a result of a 2-day fermentation of the cell walls. The dried protoplasts contain from 31 to 56% protein, 18 to 27% (ipoids, and 0.04 to 0.2% carotene, representing a two- to aeven fold increase in concentration ovcr the original leaves. The protein of leaf protoplasts was digestible by trypsin. The process is also applicable to fleshy tissue such as carrot roots, sweet potato tubers, and winter squash. Concentrates containing up to 2.2% carotene were preQaredfrom carrots without solvent extraction.
W
HEN cryptostegia leaves (C. grandijoru) were subjected to anaerobic fermentation by Clostridium roseurn as a pre-
treatment in recovery of rubber, the cell walls were digested and the cell contents (protoplasts) liberated (4, 7). The protoplaste remained as discrete entities and could readily be separated from .the bagasse (cuticle, ribs, vascular tissue). Since the fractionation .of the leaves was so clean cut and the recovery of the protoplasts and of their water-insoluble constituents so high, it was deemed advisable to apply the fermentation process to other plant materials as a first step in the preparation of proteins and lipoids. This paper reports the successful preparation of protein, fat, and carotene concentrates from leaves of turnip, lettuce, beet, pass, and broccoli; from whole pea and snap bean vines and alfalfa; from the roots of carrots and sweet potatoes; and from the fruit of winter squash. Because of the special interest of this laboratory in the utilization of the leaf wastes incident to the .m-~ductionand processing of vegetables, the leaves chosen for
study were mostly from this source. The 2,000,000 tons of such fresh waste produced annually in the United States (8) contain 50,000 tons of protein and 20 tons of carotene. Any method for recovering that protein and carotene is intriguing. The preparation of protein concentrates from grass has beeu attempted with some success. A mechanical process patented in England (8) recovered only part of the proteinaccous material Sullivan (9, 10) dissolved the proteins with dilute alkali and reprecipitated them with acid. The crude product contained 58% protein, which was 56% of the’total protein in the grass. Surplus and cull carrots are the predominant source of commer. cia1 carotene, Many present methods for the preparation of carotene necessitate drying the fresh roots in order to make the carotene accessible t o nonaqueous solvent extraction. This drying ip costly and involves considerable loss of carotene by oxidation. Holmes and Leicester (3) have proposed dehydrating the roote with acetone prior to solvent extraction. This method eliminates the oxidative destruction of carotene, but the large quantities of solvent involved make the process expensive. Barnctt (9) developed a method of recovering a carotene concentrate from fresh carrots by mechanical means, This method requires extensive disintegration of the plant tissues. In fact, unless the walls of the individual cells are broken to allow the escape of the carotenecontaining particles, the yield of carotene is low; furthermore, a considerable amount of pulp is incorporated into the concentrate. resulting in much lower carotene”purity. FERMENTATION OF LEAFY VEGETABLE WASTES
Leaf material from eight species of plants was used in thia study to determine the applicability of the fermentation process for the recoverv of protoplasts. The source, degree of maturity, and portions of plants used in the experiments were as follows:
