Sodium Orthophenylphenate Research Articles

Influence of selected bactericides on biofilm formation and viability of Xanthomonas citri subsp. citri

Citrus Bacterial Canker (CBC) is a serious disease that affects production of almost all commercial citrus cultivars in subtropical citrus growing regions worldwide. The causal agent, Xanthomonas citri subsp. citri (Xcc), is classified as a quarantine pathogen in European Union (EU) which necessitates treatment of citrus fruit before entry into the EU marketing zone. In this study, the effectiveness of selected bactericides for leaf and fruit disinfection was evaluated. Bacterial aggregation and viability were assayed on abiotic or biotic surfaces in vitro and in planta after treatments with NaCl, CuSO4, NaClO, sodium orthophenylphenate and two hydrogen peroxide based compounds. Most of the bactericides at higher concentrations reduced biofilm formation and Xcc viability compared to the non-treated control, but did not completely prevent or remove biofilms or eradicate Xcc from either biotic or abiotic surfaces. Some of the bactericides at sub-lethal concentrations increased aggregates in which Xcc was viable. Based on these results, we conclude that exposure to the bactericides is variable depending on biofilm formation and that plant and fruit disinfection will not only require treatment with an effective bactericide to kill the bacterium but also include the biofilm disruption.

Re-evaluation of Developmental and Reproductive Toxicity of Ortho- Phenylphenol (OPP) and Sodium Ortho-Phenylphenate (SOPP)

Background: Due to widespread usage, the general public including pregnant women is routinely exposed to the fungicides, Ortho-Phenylphenol (OPP) and Sodium Ortho‑Phenylphenate (SOPP), from many sources. Previous data reviews concluded no effects on development or reproduction in animal studies but upon re-analysis we realize that alternative interpretation may exist. Methods: Developmental and reproductive effects were assessed from studies performed in rats, mice, and rabbits. We identified the most sensitive endpoint(s) for OPP or SOPP as they related to fetal developmental or reproductive toxicity. For quantifying the potential health risk associated with the exposure to OPP or SOPP, we used the lowest dose that caused no developmental or reproductive toxicity or benchmark dose analysis. Results: Developmental effects in OPP-treated rats and mice were decreased fetal body weight, increased incidences in delayed skeletal ossification and post‑implantation loss. In addition, fetal mice exposed to SOPP exhibited malformation. Similar to rats and mice, post‑implantation loss was the developmental effect noted in OPPtreated rabbits. Except for the rats, maternal toxicity appeared to be minimal (mice) or not observed (rabbits) at the lowest dose where developmental effects occurred (mice: cleft plate; rabbits: resorptions). We did not find evidence of OPP affecting reproductive functions but significant deviations from FIFRA Guidelines in these studies may prevent adequate assessment of the reproductive toxicity especially the effects on fertility and mating. Conclusions: The revised data analysis suggests that OPP and SOPP induce fetal toxicity in the absence of maternal effects. Our re-evaluation would be useful in the formulation of a current or updated regulatory strategy for the developmental toxicity of OPP and SOPP.

MRNA from selected genes is useful for specific detection and quantification of viable Xanthomonas citri subsp. citri

The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post‐treatment (0, 3, 24 and 48 h) and analysed by quantitative real‐time reverse transcription PCR (Q‐RT‐PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms (gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445‐bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445‐bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed.

INTEGRATED APPROACHES TO POSTHARVEST DISEASE MANAGEMENT IN CALIFORNIA CITRUS PACKINGHOUSES

INTEGRATED APPROACHES TO POSTHARVEST DISEASE MANAGEMENT IN CALIFORNIA CITRUS PACKINGHOUSES

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.

Characterization of fungicide-resistant isolates of Penicillium digitatum collected in California

Isolates of Penicillium digitatum, cause of citrus green mould, were collected in California from infected fruit from packing houses or groves. The fungicide sensitivity of 166 isolates to imazalil (IMZ), thiabendazole (TBZ), sodium ortho-phenylphenate (SOPP), and pyrimethanil (PYR) were determined. All of these fungicides except PYR were in use in packing houses. None were used in groves. Isolates resistant to IMZ, TBZ, and SOPP occurred within packing houses but not in groves. Many were simultaneously resistant to two or more fungicides. Resistance to PYR was found only in three isolates from relatively isolated groves in northern California. The EC 50 levels of IMZ and SOPP among resistant isolates varied, while those resistant to TBZ were primarily of one level. The colony colour, lesion expansion rate and days to sporulate on infected lemons, and the magnitude of sporulation were determined for many isolates. Some minor alterations in colony colour and a slightly reduced lesion size occurred among fungicide-resistant isolates, particularly those resistant to more than one fungicide. Lemons were inoculated with a mixture of conidia from one sensitive and one resistant isolate in equal portions, and then conidia were collected one week later from lesions. The resistant isolates were all resistant to IMZ and some were also resistant to SOPP and TBZ. The proportion of IMZ-sensitive and -resistant conidia was determined and comprised the inoculum to initiate a subsequent decay cycle. A total of 28 pairs of sensitive and resistant isolates were evaluated over four cycles. IMZ-resistant conidia declined rapidly in 26 pairs; few or no IMZ-resistant conidia were present after four cycles. In two pairs the resistant conidia persisted over four cycles with little decline, which suggests that in the absence of IMZ use some resistant isolates may persist for long periods. All of the fungicides would effectively control green mould on fruit arriving from groves with incipient infections, because sensitive isolates predominate there, however, control of infections initiated within packing houses, where resistant isolates predominate, remains a difficult problem. The recently introduced PYR controls resistant isolates that now occur in packing houses, but resistance to this fungicide, which was detected in three isolates from locations where PYR had not been used, indicates it must be used with good resistance management practices.

Micronuclei and cell proliferation as early biological markers of ortho-phenylphenol-induced changes in the bladder of male F344 rats

ortho-Phenylphenol (OPP) and its sodium salt, sodium ortho-phenylphenate (SOPP), are widely used fungicides and antibacterial agents known to cause tumors in the bladders of male F344 rats. Previous studies in our laboratory have shown that micronuclei and cell proliferation were induced in the bladders of treated rats by a high dose of OPP. In our present studies, we investigated the relationship in dose response between these two biomarkers and previously reported tumor formation in the bladders of male F344 rats. Significant non-linear increases in micronuclei (MN) and BrdU-labeling were seen in the bladder cells of rats treated with the 8000 and 12,500 ppm doses of OPP and at 20,000 ppm SOPP. CREST anti-kinetochore staining showed that the micronuclei originated from both chromosomal loss and breakage. In addition, increases in MN were detected in the bladder but not in the bone marrow, underscoring the value of assessing genotoxicity in the target organ. In summary, these studies clearly show that at high doses, OPP and SOPP are genotoxic to the rat bladder. These results also indicate that micronucleus formation and cell proliferation can detect early OPP-induced changes in the rat bladder and may be useful as biomarkers for bladder carcinogens.

Lack of Enhanced Epithelial Cell Proliferation in the Urinary Bladder of Heterozygous p53 Knockout Mice Given Sodium Ortho-phenylphenate or Uracil

In the present study, we investigated epithelial cell proliferation in heterozygous p53 knockout (p53+/-) mice after administration of two urinary bladder non-genotoxic carcinogens, in comparison with that in wild-type littermates (p53+/+). Mice at 10-weeks of age were given 2% sodium ortho-phenylphenate (Na-OPP) or 2.5% uracil in the basal diet for 4 weeks. Uracil evoked a marked elevation of epithelial cell proliferation and development of papillary or diffuse epithelial hyperplasia associated with calculus formation in p53+/- as well as p53+/+ mice. Administration of Na-OPP caused alkalization of the urine in both. Neither Na-OPP nor uracil induced a higher epithelial cell proliferative response in p53+/- mice as compared with p53+/+ mice. While we previously reported p53+/- mice to be highly sensitive to a genotoxic urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine, the present results suggest that p53+/- mice may not have a high susceptibility to induction of urinary bladder tumors by non-genotoxic carcinogens.

Orthophenylphenol and phenylhydroquinone residues in citrus fruit and processed citrus products after postharvest fungicidal treatments with sodium orthophenylphenate in California and Florida.

Sodium orthophenylphenate (SOPP) has been used extensively for >40 years to control postharvest diseases of citrus fruits. Studies of the metabolism of [(14)C]SOPP have identified orthophenylphenol (OPP) as the major metabolite with phenylhydroquinone (PHQ) as a minor metabolite. The whole-fruit tolerance in the United States for OPP is 10 ppm. This study was conducted to quantify terminal OPP and PHQ residues in whole Navel oranges, grapefruit, and lemons following SOPP applications at maximum application rates and following commercial application and fruit storage practices. OPP and PHQ residues also were determined in products processed from treated Navel oranges. OPP residues in lemons, Navel oranges, and grapefruit treated with SOPP using foamer wash and shipping wax applications remained below the 10 ppm tolerance, and PHQ residues were all < or =0.439 ppm. PHQ residues in whole fruit increased with time in commercial storage. OPP residues in all Navel orange matrices except oil remained relatively stable with time in commercial storage; residues in oil declined substantially while in storage.

Dietary effects of ortho-phenylphenol and sodium ortho-phenylphenate on rat urothelium.

Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are pesticides used commercially in the food industry that have been shown to be carcinogenic to rat urothelium. Dietary administration of 1.25% OPP or 2.0% NaOPP caused increased incidences of urothelial hyperplasia and eventually caused tumors in male F344 rats, with NaOPP apparently having a more potent effect. In other studies, various sodium salts such as saccharin and ascorbate enhanced bladder carcinogenesis, although the acid forms of these salts did not. In studies with high dietary doses of these sodium salts, an amorphous precipitate was produced in the urine; precipitate formation was pH dependent. In previous experiments in which high doses of OPP were fed for up to 17 weeks, severe hyperplasia of the urothelium was produced, but without the formation of an urinary amorphous precipitate, calculi, or abnormal microcrystalluria. In addition, we found no evidence of OPP-DNA adduct formation in the urothelium. The present study was conducted to determine if feeding NaOPP * 4 H(2)0 to male F344 rats as 2.0% of the diet resulted in the formation of an amorphous precipitate in the urine, and if NaOPP caused an increased mineral concentration in the urine and/or kidneys. NaOPP administration produced a higher urinary pH than did OPP fed as 1.25% of the diet. Neither amorphous precipitate nor other solids were observed in the urine of the OPP or NaOPP-treated rats, and urinary calcium concentrations in the treated groups were similar to control. OPP and NaOPP had similar proliferative effects on rat urothelium after 10 weeks of treatment by light microscopy, scanning electron microscopy (SEM), and bromodeoxyuridine (BrdU) labeling indices. The results of this study indicate that formation of abnormal urinary solids is not part of the mechanism by which OPP or NaOPP exert their effects on the rat bladder epithelium.

Mycological evaluation of chemically-treated unshelled peanuts.

In the present work, the effect of propionic acid (ammonium salt) at 3000 mg/kg of unshelled peanuts (PA1) and at 5000 mg/kg (PA2), grapefruit seed extract at 5000 mg/kg (GF1) and 10,000 mg/kg (GF2), sodium orthophenylphenate at 2500 mg/kg (SOP1) and at 5000 mg/kg (SOP2) and thiabendazole at 1000 mg/kg (TBZ1) and at 5000 mg/kg (TBZ2) was studied for controlling total and potentially aflatoxigenic fungi in unshelled peanuts (UP). Samples of sound mature UP were moistened by adding water and kept refrigerated till they reached ca 16% moisture. The samples were then sprayed with the chemical solutions and incubated at 30 +/- 2 degrees C for 28 days. Control samples were sprayed with water. An evaluation of total and aflatoxigenic fungi was made, in pods of UP and in kernels obtained aseptically, before and at 7, 14, 21 and 28 days of incubation, by serial dilution in culture media Dichloran Rose Bengal Chloramphenicol (total fungi count) and in Aspergillus flavus parasiticus Agar (potentially aflatoxigenic count). In relation to the period and conditions of this experiment the overall best treatment was PA2, when the lowest average value of total and aflatoxigenic fungi were obtained in UP and were maintained in its kernels. Although SOP2 treatment could control fungal contamination in pods, it was not effective in controlling contamination through the kernels. The other treatments were ineffective.

Laboratory evaluation of chemical control of aflatoxin production in unshelled peanuts (Arachis hypogaea L.).

Propionic acid (ammonium salt) at 3000 mg/kg (PA1) and 5000 mg/kg (PA2) of unshelled peanuts (UP); grapefruit seed extract at 5000 mg/kg (GF1) and 10,000 mg/kg (GF2); sodium orthophenylphenate at 2500 mg/kg (SOP1) and 5000 mg/kg (SOP2); thiabendazole 1000 mg/kg (TBZ1) and 5000 mg/kg (TBZ2) were studied in the laboratory, to verify their efficiency in controlling fungal growth and aflatoxin (AF) production on moist UP (16-18% moisture content). Moist UP were put into polyethylene bags with cotton plugs and incubated at 30 +/- 2 degrees C for 28 days. Treatments were considered efficient when the AF content (B1 + G1) remained under 30 micrograms/kg. PA1 treatment was efficient till 14 days of incubation and PA2 during the whole incubation period (28 days). All other treatments were not efficient, showing AF contents from 150 to 108,333 micrograms/kg during the incubation periods. Propionic acid, used as ammonium propionate, at 5000 mg/kg shows promise in controlling aflatoxin production when applied to moist unshelled peanuts.

Post-harvest control of aflatoxin production in in-shell moist peanuts with sodium ortho-phenylphenate: III. Storage tests

The present experiment aimed to evaluate the effect of sodium ortho-phenylphenate (SOP) application to in-shell moist peanuts for the control of aflatoxin production. Previous studies showed the need to improve the SOP solution distribution on peanut pods to evaluate the product. Thus, in this experiment the place of the spray system was the bag filler pipe of the pre-cleaning machine in the warehouse. In the 1989 rainy season two lots of 120 bags of in-shell moist peanuts were sprayed with 0.5 and 1% SOP solutions and aflatoxin production was not controlled. In the dry season of 1989 and in the rainy season of 1990, in-shell moist peanuts were sprayed with 5% SOP solution. The coverage of pods with the solution was efficient, allowing a uniform distribution of SOP solution on the pods. The results showed that only the 5.0% concentration of SOP solution utilized controlled the external fungal growth when a naked eye observation was made, however did not control aflatoxin production when applied to in-shell moist peanuts, probably due to the internal presence of Aspergillus flavus and because the fungicide could not penetrate inside to reach the kernels.

Sodium Lignin Sulfonate and Sodium Orthophenylphenate Interaction on Browning of Pear Fruits

Immersion of Anjou pears (Pyrus communis L. cv. Beurre d Anjou) in sodium lignin sulfonate (SLS), a flotation agent used in hydraulic handling of pears, did not cause injury leading to skin browning. Immersion of cut pear slices in SLS discolors pear fruit flesh, but the discoloration derived from SLS pigments does not intensify with time. When the fungicide sodium orthophenylphenate (SOPP) was combined with SLS, necrotic skin mottling occurred with increased immersion times and temperatures. A white precipitate in the SLS SOPP solution accompanied phytotoxicity of pear skin tissue. Acidification of alkaline SOPP solutions (pH 11.3) with 0.01 N HCl down to pH 10 produced mild skin necrosis. Both acid (0.01 N HC1) and alkaline (0.01 n KOH) solutions of SOPP and SLS-SOPP combinations caused browning of pear flesh.

Curing to prevent chilling injury during cold disinfestation and to improve the external and internal quality of lemons

In 1988, 2 experiments were undertaken to test the effect of curing on the external and internal quality of lemons. The experiments were designed to determine the optimum postharvest handling regime for protecting lemons from chilling injury during the cold disinfestation process that is necessary for entry into markets such as Japan. In experiment 1, a mixture of Eureka and Lisbon lemons was cured for 0, 2,4, 8, 16, or 32 days at ambient temperature, then cold disinfested at 1�C for 16 days and stored for 4, 8, or 16 weeks at 10�C. In experiment 2, a mixture of Eureka and Lisbon lemons was treated with either thiabendazole (TBZ) (500 mg a.i./L) or sodium ortho-phenyl phenate (SOPP) (200 mg a.i./L) by bulk dipping before processing, or by an in-line spray during processing. The fruit was then cured for 0, 2, 4, 8, 16, or 32 days, cold disinfested as above, and stored at 10�C for 28 days. Curing for 16 days reduced chilling injury symptoms in experiment 1 but increased oleocellosis in experiment 2. Curing for 16 days also reduced the incidence of fungal breakdown caused by Penicillium spp. and Geotrichum candidum Lk. ex. Pers. When used as a bulk dip for pretreating lemons prior to curing, TBZ was more effective at controlling fungal breakdown than when used as an in-line spray, and more effective than SOPP used as a bulk dip or in-line spray. Red spotting of the rind was also reduced by bulk dipping in TBZ. Curing for 4 or more days produced increases in extractable juice. Development of yellow colour occurred during curing for 16-32 days and was enhanced by storage at 10�C. Curing for at least 4 days, when used in conjunction with a bulk dip of TBZ (500 mg a.i./L), can be used to protect lemons against fungal breakdown and to increase juice. Curing for 16-32 days can be used to develop even yellow coloration of the fruit and to decrease chilling injury.

Influence of Fungicide and Chemical Salt Dip Treatments on Crater Rot Caused byRhizoctonia carotaein Long-Term Storage

Nine fungicides and two chemical salts were tested for their ability to reduce mycelial growth of Rhizoctonia carotae on V8 agar. Among the fungicides, only sodium orthophenylphenate (SOPP, Dowcide A) and mancozeb (Dithane M-45) completely inhibited growth and were fungicidal. Benomyl, chlorothalonil, and iprodione also inhibited mycelial growth but were fungistatic. The fungicides captan, dicloran, thiabendazole, and thiophanate methyl reduced growth of the fungus by 4−85% of that in unamended agar. Two chemical salts, potassium carbonate (K 2 CO 3 ) and sodium bicarbonate (NaHCO 3 ), at 0.1 M totally inhibited growth of R. carotae and were fungicidal (.)

Metabolism of phenylhydroquinone by prostaglandin (H) synthase: possible implications in o-phenylphenol carcinogenesis

o-Phenylphenol (OPP) and its sodium salt sodium ortho-phenylphenate (NaOPP) are broad spectrum fungicides and antibacterial agents. Both are urinary bladder and renal carcinogens in the Fischer 344 rat. OPP is converted by mixed-function oxidases in the liver to phenylhydroquinone (PHQ). Since appreciable amounts of prostaglandin (H) synthase (PGS) are found in rat bladder and kidney-medullary papilla, the target sites of OPP- and NaOPP-induced tumors, we hypothesized that a secondary PGS-mediated activation of PHQ to phenylbenzoquinone (PBQ) may occur in the bladder and kidney. We have studied the metabolism of PHQ by PGS in the presence of arachidonic acid and hydrogen peroxide as co-factors. These studies showed that PHQ is indeed metabolized to a product having identical spectral and electrochemical properties to PBQ. The disappearance of PHQ with time was stoichiometric to the formation of PBQ. Less than 10% of PHQ was converted to PBQ in the absence of enzyme, indicating that auto-oxidation may play only a minor role in the conversion of PHQ to PBQ. Similar results were obtained when PGS was replaced with either myeloperoxidase or horseradish peroxidase and hydrogen peroxide as co-factor. These studies suggest that the peroxidative metabolism of PHQ by PGS to the reactive PBQ could play an important role in OPP-induced urinary bladder and kidney carcinogenesis in rats.

Proliferative response of renal pelvic epithelium in rats to oral administration of ortho-phenylphenol, sodium ortho-phenylphenate and diphenyl

Changes in DNA synthesis levels and morphology as observed by scanning electron microscopy (SEM) and light microscopy in rat renal papilla and pelvis following oral administration of ortho-phenylphenol (OPP), sodium ortho-phenylphenate (OPP-Na) and diphenyl, were investigated in F344 rats. OPP and OPP-Na treatment for 4 weeks was associated with elevated DNA synthesis in both renal papilla and pelvis, in addition to distinct morphological cell surface alterations. Sequential light microscope observation revealed induction of renal papillary necrosis from week 4, followed by regeneration hyperplasia at weeks 16 and 24, but no changes in the renal pelvis in the OPP case. OPP-Na not only similarly affected the renal papilla, but also brought about development of hyperplasia in the renal pelvis. No proliferative response of the kidney was apparent in rats fed diphenyl. The present study indicated that the proliferative responses in the renal pelvic epithelium following OPP-Na are similar to those induced by this chemical in the urinary bladder, and that in both cases they are indicative of promoting or carcinogenic potential.

Synergistic effect of fungicides on resistant strains of Penicillium italicum and Penicillium digitatum

In vitro and in vivo tests were carried out to study the inhibitory effect of mixtures of sodium orthophenylphenate, methyl-1-(butyl-carbamoyl)-2-benzimidazole (‘Benomyl’) and sodium orthophenylphenate, 2-(4-thiazolil)-benzimidazole (‘Thiabendazole’) on growth of strains of Penicillium italicum and P. digitatum resistant to sodium orthophenylphenate, ‘Benomyl’ and ‘Thiabendazole’. ‘In vitro’ tests showed a synergistic effect of the mixtures; 10 ppm of sodium orthophenylphenate and 10 ppm of the benzimidazolic compound in most cases resulted in complete growth inhibition. Navel oranges inoculated with P. italicum spores showed no damage after 7 days of incubation at 25°C and 80–90% relative humidity when treated with a mixture of 100 ppm of sodium orthophenylphenate and 100 ppm of Thiabendazole.

Comparative Effects of Applying Imazalil By Dipping or By Incorporation Into the Plastic Film On Decay Control, Distribution and Persistence of This Fungicide in Shamouti Oranges Individually Seal-Packaged

Seal-packaging presents another practical way of providing fungicides to individual fruits by incorporating the fungicide in the packaging film. Imazalil-containing film prevented the development of Penicillium digitatum and significantly inhibited that of Diplodia natalensis and Alternaria citri in petri dishes; this indicates that imazalil exerts its action through the vapor phase. Film containing imazalil (at concentration of 2000μg/g) markedly reduced the decay of Shamouti oranges inoculated with P. digitatum if the sealing was done immediately following the inoculation, and only slightly if sealing was delayed 24 h. Disinfection with sodium orthophenylphenate combined with sealing in imazalil-containing film provided further reduction in decay, even if the film was applied 24 h after inoculation. The effectiveness of fungi cides, whether applied to the fruit directly or in the film, was not affected by sealing. Distribution of imazalil among the film, flavedo and albedo was monitored through out the storage period of sealed fruit. The film could serve as a reservoir releasing the fungicide gradually, so that its antifungal activity may replace that previously provid ed by waxing without the risk of excessive toxic residues.