h f hence of Z i n c Oxide
S. B . Scelvinl THE NEW JERSEY ZlNC COMPANY (OF PA.), PALMERTON, PA.
F
UNGI discolor and possibly disintegrate painted surfaces, and thus offset the two primary purposes for the painting of either wood or metal-protection and beautification. The extent of this fungal growth depends primarily on the type of environment and the nature of the paint, I n regions where atmospheric conditions provide an optimum of warmth and humidity, fungal growth on paints is most extensive. I n addition, the degree of fungal damage is determined greatly by the inherent qualities of the paint itself, such as the t,ype of vehicle, t'he hardness and smoothness of the surface, and especially the fungistatic or fungicidal properties of the pigment. Much research has been conducted to determine the most suitable ingredients for rendering paints mildew-resistant. HONever, this work has been approached mainly from a practical point of view-namely, the preparation of a paint, its application to the substratum, the subsequent exposure of the painted surface to molds under conditions suitable for fungal growth, and a qualitative examination for damage by molds (3, 6, 6). Much of this work has shown zinc oxide to be useful in rendering paints mold-resistant (6, 14, 16, 1 6 ) , but no attempt has been made t o investigate how this inhibition occurs-that is, whether the prevention of fungal growth is due to the fungistatic properties of the zinc oxide, to the hardening of the paint surface, or to some other less probable cause. Therefore, although previous work has shown that "zinc oxide pigments . . . tend to prevent mildew" (h), the method and extent of mold inhibition by zinc oxide requires further investigation. The fungi employed, unless otherwise stated, were either isolated directly from a molded paint surface or obtained from other investigators who had previously removed t,he fungi from a mildewed paint, These fungi include Aspelgillus niger, Phoma sp., Aspergillus jlavus, Penicillium sp., Demati.urn sp., Aspergillus sp., and Cladosporium herbarum. INFLUENCE OF ZINC OXIDE ON GROTVTM OF PAINT ,MOLD
A s a preliminary phase the value of the vehicle as a nutrient, was investigated in order to determine whether fungi will grow on a vehicle if no inhibitory pigment is present. The vehicles tested were raw linseed oil, alkali-refined linseed oil, heat-polymerized linseed oil, linseed-tung-soybean vehicle, heat-polymerized refined soybean oil, and heat-polymerized fish oil, in addition to a lead, manganese, or cobalt drier. A small drop of the vehicle was suspended from a cover slip in a van Tieghem cell and inoculated with fungi spores. Each of the vehicles was exposed in triplicate to twelve molds common to paints, with one triplicate set containing no water and the other having two to three drops of distilled water a t the bottom of the van Tieghem cell. When no water was present in the cell and the air surrounding the oil droplet was therefore dry, no spore germination was apparent. However, when the air was well saturated with moisture, the extent of germination varied greatly, 1
Fungal spores were sown on paint yehicle constituents and incubated. Varying degrees of growth were observed, with raw linseed oil providing the most luxurious development. This demonstrates that, for mildew resistance, paint films have to depend upon pigments or other ingredients. The effect of nine types of zinc oxide on the growth of common fungi was studied. The inhibition of fungal growth was found to be a direct function of the surface area of the zinc oxide; a fine-particle-size zinc oxide was particularly effective. Although zinc oxide is able to prevent the growth of mycelium or the germination of spores, it is unable to kill the spores and to prevent their germination after removal from the medium and exposure to a more favorable environment. Therefore, zinc oxide cannot be regarded as possessing fungicidal properties; its action is fungistatic. Respiration studies indicate that the zinc ion affects the carbohydrate metabolism of the fungus and that this property may l e basically responsible for the fungistatic effect preriously described.
depending on the type of vehicle, drier, and species of fungus involved. The results summarized in Table I, indicate that raw linseed oil is an excellent medium for the growth of paint molds, whereas the other six substances vary in fungistatic influence, depending on the fungus. I n those instances where germination and subsequent growth were extensive (Figure 1) the hyphae contained large numbers of conspicuous oil globules, an indication that the mycelium can absorb some of the ingredients of the raw oil. I n contrast, when growth was limited to a few mycelial strands, the hyphae contained no visible oil globules but only the normal translucent protoplasm (Figure 1). The conclusion may be drawn that germination of mold spores can occur in the common paint vehicles when exposed to a relatively moist atmosphere. The extent of this growth depends on the type of vehicle, as well as on the quality of the mold present. The mildew resistance imparted by the pigment, therefore, is of great value in the preservation of the paint. Nine different types of zinc oxide were tested to determine their influence on mold growth (Table 11). To determine the most suitable concentration of zinc oxide, a 2 cu. mm. block of agar containing the mycelium of A . niger was placed on 20 cc. of Czapek-Dox nutrient agar (in a Petri dish) to which had been added some U.S.P. zinc oxide. The composition of Czapek-Dox medium is Water, 00. KaNOa, grams KzHPOp, gram .\IgSOA, gram
1000 3.0 1.0 0 5
KC1, gram FeS04, gram Sucrose, grams Agar, grams
0.5 0 01 30 0 20 0
The plates, whose U.S.P. zinc oxide contents varied from 0.025 to S O . O ~ o ,were incubated a t 28" C. after inoculation, and t h e growth of the subsequently developing mycelia was measured. The diameter of the colonies a t the time the control reached 9 8 mm. (220 hours after inoculation) follows: % Zinc
Diam. of Diam. of % Zinc Oxide Colony, II.Im.a Oxide Colony, Mm.0 0.025 58.3 5 0 9.3 0.25 53.0 10.0 7.0 0.5 52.0 20.0 6 3 2.5 20.0 4 Averages of three separate experiments.
% &inc Oxide 30.0 50.0 80 0
Diani. of Colons, A1m.a 5 0 3 5 3 0
Percentages of U.S.P. zinc oxide in the nutrient agar were plotted against the diameter of the mycelium in each medium (Figure 2). It became evident that: A very low percentage of U.S.P. zinc oxide in the culture medium (up to about 0.5y0)had little inhibitory influence on the growth of the fungus; percentages from 0.5 to 5.0 had a markedly increasing effect; and percentages from above 5.0 to 80 were strikingly inhibitory and
At present in the U. S. Navy.
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April, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
331
growth-inhibiting power of the zinc oxide and its specific surface. differed only slightly. However, even the high percentages of However, the exact mechanism whereby zinc oxide particles with U.S.P. zinc oxide in the medium permitted some growth of the greater surface area produce a more marked inhibition of growth mold and, therefore, were not completely fungistatic. has not been studied and, therefore, is still a matter of conjecture. Experiments were conducted t o determine the difference in the inhibitory qualities of the zinc oxides. Cladosporium herbarum, INFLUENCE O F ZINC OXIDE ON SPORE GERMINATION Penicillium sp., Dematium sp., Aspergillus sp., and Aspergillus I n the growth inhibition experiments just described, where t h e niger were grown in standard Petri plates on Czapek-Dox nutridifference in the diameter of the mycelial masses or in the weight ent agar containing 0.5% of the various zinc oxides. The inocuof the fungal mats gave a n index to the inhibitory value of t h e lum consisted of a 5-mm. cube of agar containing the active zinc oxide, the quantitative measurement of the response of t h e mycelium of the fungus; the subsequent growth of the fungus fungus to the growth inhibiting substance was “direct”. To was regulated in a n incubator a t 25” C. The diameters of the verify the relative inhibitory effects of the several zinc oxides, colonies 280 hours after inoculation are listed in Table 111. another series of experiments was conducted in which the inAnalysis of these results indicates t h a t the fine, specially prehibitory values were measured by a method involving a “quantal pared zinc oxide is the most fungistatic of those tested. There response” (12). The fungal parts studied are separated into two is a n inverse relation between particle size and fungistatic power groups, depending on what type of observable response to t h e (Figure 3). Phosphorulation of the zinc oxide somewhat ininhibitory substance is elicited; more specifically, spores exposed fluences its growth-inhibiting properties, and a zinc oxide with t o the zinc oxide are classified as germinated or nongerminated, acicular particles does not differ significantly from related noduand thus their response is measured. lar oxides in fungistatic powers. Leaded zinc oxides are among Much experimentation has been carried on t o determine t h e the poorest fungistatically, and their fungistatic effect correbest methods for measuring the response of a n organism t o an sponds t o their particle size and actual zinc oxide content. inhibitory agent by this method of quantal response ( 7 , 8, 10These results were further substantiated by dry weight deterIS)-for example, recorded definite standards t o be met in t h e minations. Known and equal quantities of A. niger spores were laboratory assay of toxic agents. The purpose of these standards sown in Erlenmeyer flasks; each contained 50 cc. of Czapek-Dox is t o permit accurate comparison of many toxic agents by main, medium and 0.5% of one type of zinc oxide, and was incubated taining the environment constant under rather complex, b u t for 2 weeks a t 25” C. The flasks were then steam-sterilized, and arbitrary, conditions. the mycelial mats washed in hot water and 5y0 acetic acid (to reFor the following experiments, the main purpose of which was move adhering zinc oxide particles), dried a t 102’ C., and to establish and compare the inhibitory properties of several types weighed. The results. Table IV indicate the same general trend as did the diameter measurements in a standard Petri plate. To determine whether there was any correlaFigure 1. Photomicrographs of Extensive Mycelial Growth Containing tion between the rate of growth of A. niger in a Oil Globules (above) and Limited Growth Containing No Oil (below) zinc oxide medium and the amphoteric properties of the zinc oxides, the fungus was grown in 50 cc. of the Czapek-Dpx medium containing zinc oxide, and hydrogen-ion concentration was measured at the time of inoculation and after 2-week growth at 25’ C. The results (Table IV) seem particularly striking in that, with two excebtions, the changes in hydrogen-ion concentration were of the same order of magnitude; also, the control showed one of the smallest increases in acidity, and zinc oxides C and D, which exhibited the greatest inhibitory influence, seemed t o exert the least buffering action on the nutrient solution. The obvious conclusion is that the greater the fungistatic effect of a zinc oxide, the lower is its buffeting power. The author is a t a loss to explain this conclusion. The most striking results are those indicating the relation between particle size of oxide and inhibition of growth. Three experimental zinc oxides were used:
-
Zinc Oxide No. KH-1021 KH-959 KH-958
Particle Size Grama per Sq. meters sq. meter per gram 0.505 1.98 1.21 0.826 1.19 0.84
Dematium sp., Phoma sp., Cadophora richardsiae, and A. niger were grown in agar media containing 0.5, 1.0, 3, and 6% of the three zinc oxides in the manner described above. Analysis of the diameters of the colonies when the control reached 90 mm. (Table V) seems t o show t h a t the extent of growth inhibition is related t o the particle size of the zinc oxide (Figure 4). I n fact, there is a direct relation between the
INDUSTRIAL AND ENGINEERING CHEMISTRY
338 OF TABLE I. GROWTH
iilkaliRefined Linseed Oil
Raw Linseed Oil
Culture A . niger A . repens Phorna sp. Cladosporium SP. Torula SP. A. tevreus Cadophora richardsiae Dematium sp. Penicillium sp. A . echinulatus Aspergillus sp. = extensive mycelial growth; growth.
TABLE 11. Zinc Oxide A Particle size, Microns Range 0.4-2.0 Average Total S (as SO;) Total P b as (PbO)
25” C.
HeatPolymerized Linseed Oil
HeatPolymerized Linseedand Refined TungSoybean Soybean Oils Oil
HzO
+++ +++ ++++ ++ +++ +++++ +++ - +++ + ++’$ ++ +++-k +++ ++ +++ +++ +++ +++ +++ ++ ++ +++++ ++ ++ +++ ++ ++ ++ ++ +++ +++ +++ + =++mc)derate +grctwth; ++- = slight+growth;+- -
+++
because fungal spores are exposed to a toxic substance and the percentage of spore germination measured without the spores having been subsequently removed from the poisonous compound, washed, and sown in a nutrient medium t o bring about germination. The “killing” or fungicidal character of the compound is not being determined, but rather the power of the toxicant to prevent the transition of the spore from a resting or “static” condition to a n active one. Because a spore does not germinate in the presence of a given compound is no indication that the spore has been killed, but only t h a t it has been rendered temporarily inactive and is still viable. Therefore, the germination experiments involve the laboratory assay, not of fungicidal, but of fungistatic action. T o denote a compound which inhibits the growth of a fungus or prevents the normal germination of its spores, the word “fungistate” is suggested. This term is t o be distinguished from “fungicide”, which characterizes a compound having the power of killing a fungus, either in spore or vegetative form.
CULTURESa ON PAINT VEHICLES AF’BER 21-DAY INCUBATION AT
0.8 0.2 0.2
++ ++++ +++ + +++
= no
CHARACTERISTICS OF ZINC OXIDES
B
