the estimation of surface residues of benomyl on treated bananas

Pestic. Sci. 1974, 5, 135-145

The Estimation of Surface Residues of Benomyl on Treated Bananas

John Cox, Lawrence Donegan and John A. Pinegar

Tropical Products Institute, 56-62 Grays Inn Road, London WCI X 8L U (Manuscript received 23 May 1973 and accepted 14 September 1973)

A simple rinsing technique for the extraction of benomyl residues present on the skin of bananas, a microbiological assay and a U.V. spectrophotometric assay for its estimation are described. Use of these in a persistence study with benomyl on bananas showed good recovery by both methods. Preliminary work on plantation- treated bananas revealed that the benomyl was persisting unchanged and not decomposing to any appreciable extent during ripening and storage of the fruit.

1. Introduction

Application studies using the fungicide benomyl, methyl 1-(buty1carbamoyl)benzimidazol-2-ylcarbamate, on bananas to control crown rot disease required residue determinations to check dosage rates and persistence. The fluorimetric methods of Pease et aZ.‘r2 were tried but were found to give erratic results for blanks and crop extracts. These methods involve slow, laborious extraction and “clean-up’’ processes and corrections have to be applied for variations in quenching of the fluorescence. Attention was therefore directed to the estimation of surface residues which could be extracted by a simple rinsing procedure and estimated without lengthy “clean-up” to obtain a method suitable for monitoring the efficiency of application methods.

A bioassay was developed in the early stages of the study. At a later date a U.V.

spectrophotometric method (“u.v. assay”) was developed. The validity of these methods for estimating surface residues was checked in a persistence study in 1971-72 using bananas accurately dosed with Benlatea wettable powder (50 % benomyl) at 0.6 mg/kg ( i t . a dosage typical of that received by bananas when dipped in Benlate suspension containing 200 mg benomyl/litre, the level currently recommended by the manu- facturers for field use).

2. Analytical considerations

The estimation of benomyl per se is complicated by the ease with which it decomposes when in solution to yield methyl benzimidazol-2-ylcarbamate (MBC; carbendazimb), which is also fungitoxic. Clemons and Sisler4 noted this during development of the chromatogram in their bio-autograph technique. A rapid conversion in aqueous media and in plants has also been r e p ~ r t e d . ~ Mestres, Tourte and Campo6 noted changes in the

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135

136 J. Cox, L. Donegan and J. A. Pinegar

U.V. spectrum of benomyl with time in various solvents. It seems likely, therefore, that decomposition of benomyl may occur to some extent in the Benlate dip or spray liquor-if this is allowed to stand for several hours prior to application-and in, or on, the crop itself. For convenience the term “benomyl” is used in this paper to mean aggregate benomyl and MBC in terms of original benomyl content.

In preliminary studies with Penicillium citrinum we found benomyl and MBC to be equally toxic on a molar basis to the assay organism and thought this might be due to the rapid decomposition of benomyl in the assay medium so that the antifungal activity observed was due to MBC rather than benomyl. This organism was selected because it showed a suitable sensitivity; the large plate assay method was more convenient and more precise than a bioautographic procedure.

have recently described U.V. spectrophotometric methods for estimating “benomyl” in fruits, bovine fluids or soils which involve solvent extraction and solvent/acid or alkali partitions for “clean-up”. Those of Kirklands, also involve high speed liquid chromatography which permits estimation of “benomyl” and its metabolites, i.e. methyl 4-hydroxybenzimidazol-2-ylcarbamate, methyl 5-hydroxybenzimidazol-2-ylcarbamate and 2-aminobenzimidazole (2-AB). None of the methods referred to so far differentiate between benomyl and MBC, the principal decomposition product. Some ensure complete conversion of the benomyl to MBC by adequate acid treatment at some stage, others either assume it persists in or on the crop as MBC or assume adequate conversion during a brief acid extraction.

2.1. Preliminary studies It was found that such assumptions were not justified in the case of Benlate-treated

bananas, however, as preliminary studies, in which ethyl acetate rinses from Benlate treated bananas were analysed by both the bioassay and a proposed U.V. technique, gave discordant results. The U.V. technique involved extraction of the ethyl acetate rinse with hydrochloric acid followed by spectrophotometric examination. The bioassay method did not involve an acid extraction step. The results obtained were in the main lower than the corresponding bioassay results by up to 25 %. Higher results were obtained if the time of shaking the acid-ethyl acetate mixture was extended from 1 to 60 min or if the ethyl acetate extracts were allowed to stand for 5 days prior to analysis. The partition coefficient of MBC in a hydrochloric acid (0.1 N): ethyl acetate system had been found to be 50 : I in favour of the acid phase. The partition coefficient for benomyl in such a system cannot be determined accurately because decomposition to MBC will occur, the extent of which will depend apparently upon the conditions and length of time since the solution was prepared; it is likely to be lower than the partition coefficient for MBC since the former is a less basic compound. The disparity in results from the U.V. and bioassays indicated that benomyl was present in the extracts and that conversion to MBC was occurring during the extraction procedure. Since, in this particular study the rinsing was conducted two weeks after application of the commercial wettable powder, then some benomyl per se was evidently persisting, despite the ease of breakdown to MBC referred to earlier, probably because the water in the suspension evaporates rapidly leaving the fungicide in a dry state on the waxy, neutral surface of the banana skin.

Several

Estimation of benomyl residues on bananas 137

A hot acid treatment was, therefore, incorporated into the spectrophotometric procedure to ensure complete conversion of the benomyl residue to MBC; and a better agreement with bio-assay results was obtained.

2.2. Changes in solutions and suspensions of benomyl Solutions of benomyl (20 pg/ml) in ethyl acetate, and ethyl acetate saturated with 0.1 N-hydrochloric acid were studied over the 250-300 nm wavelength range using a recording spectrophotometer to estimate the rate of conversion to MBC. The intensity

Wavelength ( n m )

Figure 1. Ultraviolet spectra of benomyl in ethyl acetate. Concentration 20 pg/ml. Cell path length 10 rnm. . . . -, Scanned 1 rnin after dissolution and dilution; - - - -, scanned 16 min after dissolution and dilution; --, scanned 31 min after dissolution and dilution; -, scanned 61 min after dissolution and dilution.

of the major peak at 293 nm and a minor peak at 260 nm decreased with time, at approximately the same rate in each solution, to yield a spectrum practically identical with that of MBC (Figure 1). The reaction showed first order kinetics over a 45 min study period. At room temperature (21 to 24 "C) the half-life, calculated from the rate of decrease of the 293 nm peak with time, was found to be approx. 25 min; a more carefully controlled study at 30 "C showed the half-life in redistilled ethyl acetate to be 18 min.

Bananas experimentally dipped in Benlate suspensions abroad and subjected to the normal commercial transport and ripening procedures were rinsed with ethyl acetate and the U.V. spectrum of the rinses studied over measured time intervals. The spectra showed the same changes with time as the pure benomyl solutions and approximate calculations revealed that the residue was present mainly as benomyl with possibly up


to 10% present as MBC. A Benlate suspension in distilled water was prepared containing 400 pg benomyl/ml and was stored overnight at 30 “C.

Aliquots were withdrawn and diluted with 0.1 N-hydrochloric acid to 10 pg/ml and the U.V. spectrum recorded over measured time intervals. The spectra again showed that little conversion to MBC had occurred possibly because of the low solubility of benomyl (approx. 1 mg/litre). The stability of aqueous Benlate suspension prepared in the field would, of course, depend upon the acidity of the water employed for dilution.

2.3. Ethyl acetate rinse Attempts to determine total “benomyl” residues in banana skins and pulps spectrophotometrically have proved unsuccessful to date because of difficulties in removing from the extracts those impurities which absorb at similar wavelengths to MBC. It was found that surface deposits of “benomyl” removed from whole bananas by a

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simple rinsing with ethyl acetate (Section 3.1) could be converted into MBC, extracted into hydrochloric acid and determined spectrophotometrically without further “clean- up”, and this method was adopted because of its speed and simplicity. A differential analysis for the fungicide and its breakdown products is desirable especially in view of the finding of unchanged benomyl residues on bananas but such an analysis would of necessity be complex and tedious. The U.V. spectrum of MBC in hydrochloric acid shows two sharp peaks of maximum absorption at 282 and 275 nm (Figure 2). The ratio of these absorbances, measured from a basepoint at 310 nm, is constant at 1.20 over the analytical concentration range and can be used to detect interference from other U.V. absorbing constituents if present in the hydrochloric acid extract.

A third peak, at 223 nm, in the spectrum was found to vary in intensity, depending on the history of the extract, indicating interference by other constituents. A solution of MBC (6.59 pg/ml), equivalent to original benomyl (10 pglml), in 0.1 N-hydrochloric acid has an absorbance of 0.64 at 282 nm measured in a 10 mm cell which provides a suitable analytical sensitivity for a monitoring analysis of this type.


3. Experimental

3.1. Extraction of samples The whole banana is weighed and it is then suspended by forceps vertically over a small filter funnel inserted in a suitable flask. Pure ethyl acetate (50 ml) is allowed to run slowly (10 to 20 ml/min) from a pipette on to the fruit care being taken to see that all sides of the fruit are rinsed thoroughly. The extract is concentrated to approx. 5 ml in a rotary evaporator or vacuum still.

3.1.1. Dilution for bioassay For the bioassay the concentrate is cooled and transferred to a measuring cylinder or volumetric flask, made up to 10 ml with ethyl acetate and mixed. A portion of this is diluted two-fold as it is desirable that samples are assayed at at least two different dilutions ; the degree of concentration and dilution should be adjusted to yield solutions containing 1 to 8 pg “benomyl”/ml.

3.1.2. Dilution for U.U. assay For the U.V. assay hydrochloric acid (0.1 N, 0.2 ml) is added to the concentrate which is mixed and heated to 75 ”C for 10 to 15 min with occasional shaking. (As ethyl acetate boils at 77 “C a controlled bath is necessary.) The mixture is cooled, transferred to a cylinder and made up to 10 ml.

3.2. Bioassay 3.2.1. Preparation of standard solution A stock solution containing pure benomyl(1 mgjml) in ethyl acetate is prepared. From this, standard solutions containing 8,4,2 and 1 yg/ml are prepared by dilution in ethyl acetate.

3.2.2. Preparation of assay medium Oxoid Potato Dextrose Agar (PDA) (3.9% w/v) is prepared according to the manu- facturer’s directions. The medium is filled out in 250 ml amounts and autoclaved at 103 kN/m2 (15 Ib/in2) for 15 min.

3.2.3. Preparation of inoculum, Penicillium citrinum M140 The test organism is grown at 27 “C on Malt Agar in Petri dishes until there is visible evidence of conidiospore formation when the conidiospores are harvested. Distilled water (a few ml) is added to the plate together with a few sterile glass beads and the plate is agitated. The resulting spore suspension is then transferred to an empty sterile universal bottle and then further diluted with distilled water until the density of the suspension approximates to Brown’s Opacity Tube No. 2.

3.2.4. Preparation of assay plates Plates are prepared from assay medium that has been melted and held at 50 “C in the water bath. Each bottle of assay medium (250 ml) is inoculated aseptically with 2.5 ml (i.e. 1 % v/v) of the P. citrinum MI40 conidiospore suspension and thoroughly mixed.


The inoculated agar is aseptically poured into a level, sterile 12 in x 12 in assay plate. The plate is tilted at first, if necessary, to ensure that an even depth of agar covers the whole plate. I t is desirable to prepare the plates on the day on which the assay is to be carried out, as plates which have been refrigerated do not give such sensi- tive results as freshly poured ones. When the agar is set and the plates are thoroughly cool, holes are punched with a cork borer (8 mm diam.) and the agar discs are removed (a pen nib, bent to form a hook is convenient for this). Sixty-four wells are punched in each 12 in x 12 in assay plate. One drop of molten PDA is then added to each well to seal the bottom.

3.2.5. Plating-out Both sample and standard dilutions are added to the plate with an automatic 50 p1 dispensing pipette. Each sample and standard dilution is replicated four times, a Latin Square or a quasi-Latin Square design being used to ensure randomisation. The plates are then incubated at 27 “C for approx. 24 h.

3.2.6. Calculation of results The diameters of the zones of inhibition are measured with vernier calipers or optical magnifier. The average zone diameters of the standard dilutions are plotted on the linear axis against concentration on semi-log graph paper. The concentration of each sample dilution is then read from this standard graph.

3.3. U.V. assay 3.3.1. Reagents Standard MBC solutions. A solution of MBC [13.2 pg/ml; i.e. equivalent to benomyl (20 pglml)], is prepared in ethyl acetate saturated with 0.1 N-hydrochloric acid by appropriate dilution of a stock solution of pure MBC in ethyl acetate (0.66 mglml). If necessary this can be prepared from a stock solution of pure benomyl in ethyl acetate (1 mglml). This solution (2 ml) is treated with hydrochloric acid (0.1 N, 2.0 ml) and ethyl acetate (50 ml), the mixture is heated to 75 “C for 15 min with occasional vigorous shaking or is agitated on a rotary evaporator. The mixture is cooled and made up to 100 ml with ethyl acetate.

Freshly prepared each day 1 Ethyl acetate reagent: Ethyl acetate

saturated with 0.1 N-hydrochloric acid Hydrochloric acid reagent : Hydrochloric

acid (0.1 N) saturated with ethyl acetate

3.3.2. Preparation of standard curve Aliquots (2.5, 5.0 and 7.5 ml) of the standard MBC solution are diluted to 10 ml with the ethyl acetate in test tubes fitted with ground glass stoppers to yield solutions containing “benomyl” 5, 10 and 15 pg/ml respectively. Hydrochloric acid reagent (10.0 ml) is added to each tube, the tubes stoppered, shaken vigorously for 1 to 2 min and allowed to settle until the lower layers are clear.

The upper ethyl acetate layers are removed by suction and portions of the acid layers are transferred to 10 mm silica cells. These solutions are scanned, preferably on a


recording U.V. spectrophotometer, in the 250 to 310 nm range using the hydrochloric acid reagent as reference.

The absorbances for each solution are measured at the peak maxima at 282 and 275 nm from the baseline at 310 nm. The absorbances obtained at the 282 maximum are plotted against the “benomyl” concentrations of 5, 10 and 15 pg/ml and should yield a straight line. Alternatively the slope, concentration in pg/ml divided by absorbance, can be calculated and used in computing the final assay results. This should be near 15.6 pg “benomyl” ml-1 unit absorbance-’. The ratio, absorbance at 282 nm divided by absorbance at 275, is calculated and should give a constant value near 1.2. Instead of a standard curve a duplicate standard can be run, if preferred, at the 10 pg/ml level with each set of assays and the results calculated from its slope which should show little day- to-day variation.

If a recording spectrophotometer is not available then the absorbances should be measured at the wavelengths given above (making the necessary instrument adjust- ments between each wavelength reading). It should be noted that the peak at 282 nm is very sharp and should there be slight differences in instrument wavelength calibration, e.g. due to temperature changes in the room, then the maximum may appear slightly displaced.

On a non-recording spectrophotometer the location of this maximum should be checked, noted and the instrument carefully set at this wavelength reading for the analysis, as a difference of i- I nm can have a significant effect on the absorbance measurement. The acid solutions are stable and show no changes in absorbance at 275 and 282 nm for several hours.

3.3.3. Estimation

Hydrochloric acid reagent (10.0 ml) is added to rzch treated extract (from Section 3.1.2), and the procedure followed as in Section 3.3.2 and the “benomyl” content determined from the standard curve of absorbance at 282 nm. The residue on the banana is calculated from the equation:

Surface residue “benomyl” - “benomyl” pg/ml in final extract x 10 - mg/kg banana weight (g)

The ratio of the absorbances from the two wavelength maxima are determined as given in the instructions for the standard curve. If this ratio falls outside the range 1.1 to 1.3 then other U.V. absorbing constituents present in the extract are interfering and the analytical result will be suspect.

3.4. Modified U.V. assay

Further work has shown that the U.V. analysis can be simplified with no loss in precision by the omission of the ethyl acetate concentration stage. The extraction efficiency is only slightly reduced by this modified procedure as the partition coefficient of MBC is high.

142

3.4.1. Reagents MBC solution (13.2 pg/ml) in ethyl acetate (prepared as described for the basic method).

J. Cox, L. Donegan and J. A. Pinegar

Ethyl acetate Hydrochloric acid (0. I N)

Hydrochloric acid (5 N)

3 4.2. Preparation of standard curve Aliquots (2.5, 5.0 and 7.5 ml) of the standard MBC solution are diluted to 50 ml with ethyl acetate in 50 ml glass stoppered measuring cylinders. (Actual volume to bottom of stopper is approx. 65 to 70 ml.)

Hydrochloric acid (5 N, 1 .O ml) is added to each cylinder, the cylinders stoppered and shaken gently. Hydrochloric acid (0.1 N, 10.0 ml) is then added and the system shaken vigorously for 1 to 2 min. The contents are allowed to settle until two clear layers are obtained. A “blank” is prepared similarly by omitting the MBC.

The upper ethyl acetate layers are removed by suction and the absorbances of the acid layers determined as previously described (3.3.2). The standard curve is plotted and the ratio of the absorbances at 282 and 275 nm at each concentrationis calculated as previously.

3.4.3. Estimation The whole banana is rinsed with 50 ml ethyl acetate as previously described, all washings being run into a 50 ml glass stoppered measuring cylinder via a funnel.

Hydrochloric acid (5 N, 1 .O ml) is added to the cylinder, the cylinder is stoppered and vigoroujly shaken for 20 to 30 5. The cylinder is then heated to 75 “C for 10 to 15 min with occasional shaking. A “blani;” is prepared in the same way using pure ethyl acetate.

The solution is cooled to i-oom temperature and then hydrochloric acid (0.1 N, 10.0 ml) is added, the cylinder is stoppered and vigorously shaken for 1 to 2 min. The contents are allowed to settle until two clear layers are obtained and the procedure in Section 3.3.2 followed and the “benomyl” residue calculated as previously described. A volume alteration will be observed in the ethyl acetate and acid layers, this being due to their slight mutual miscibilities. KO volume correction is required if the standard curve is prepared as described.

3.5. Persistence of “benomyl” on ripening banana A persistence study was conducted to determine what proportion of applied benomyl remained on the surface of bananas during a three week ripening period to test the validity of using a rinsing procedure for the extraction. The resultant extracts were divided and analysed by both methods, the results are given in Table 1.

Green bananas were accordingly treated with benomyl at a level equivalent to approx. 2 mg/kg on the skin (i.e. 0.6 mg/kg on the whole fruit) and were assayed by the procedures described after 0, 7, 14 and 21 days’ storage. The benomyl was applied by a spotting technique as follows.

A 100 pl pipette was filled with a Benlate suspension containing benomyl(1 mg/ml) and two aliquots of 50 pl each were spotted in the form of 50 to 60 droplets each of


approx. 1 pI at spacings of 2 to 3 mm on to two faces of the banana. Care was taken to keep the droplets at least 2 cm away from either end of the banana to avoid uptake by the fibrous corky tissue at the ends and coalescence was guarded against to prevent run off. Spotting in a warm stream of air helped to prevent this. The Benlate suspension was vigorously mixed and the pipette rinsed with the suspension before each measurement to guard against sedimentation effects.

The precision of the “spotting” technique was checked by spotting the bottom of a conical flask in the same way. The residue was treated directly with hydrochloric acid (0.1 N, 10 ml), heated to convert the benomyl to MBC then cooled and examined spectrophotometrically. Nine replicates gave a range of 92 to 114 pg/lOO p1 aliquot with a mean of 107.3; S.D. was 6.8 pg and S.E. of the mean was 2.26 pg. The strength of the suspension was also checked by examining duplicate aliquots (5 ml) spectrophotometrically after treatment with hydrochloric acid and suitable dilution. Both duplicates showed the “benomyl” content to be 980 pg/ml. This study showed that the “spotting” technique yielded a slightly higher dosage, 107 pg/lOO p1, than the 98 pg calculated from the strength of the suspension. The difference might be due to the longer drainage time involved in the pipetting method and perhaps a slight sedimentation of the Benlate in the pipette. S.D. of 6.8 pg was larger than hoped for but was considered acceptable in view of the tedious nature of the application method.

TABLE 1. Recovery of “benomyl” from bananas treated with 107 pg benomyl as determined by bioassay and U.V. absorption techniques

Experiment 1 Experiment 2 Number of Method of estimation days after Banana Bioassay U.V. Bioassay U.V. treatment sample recovery % Ratio recovery % Ratio

A B

0 C D Average A B

7 C D Average A B

14 C D Average A B

21 C D Average

104 88 96

108 99

112 94

130 82

104 130 111 105 133 120 97 97

104 119 104

103 88 88

102 95 96 82

113 74 91

107 103 93

115 104 92 89

110 102 98

1.01 1 .oo 1.09 1.06 1.04 1.17 1.15 1.15 1.11 1.14 1.21 1.08 1.13 1.16 1.14 1.05 1.09 0.94 1.17 1.06

102 103 105 93

101 96

105 100 102 101 108 110 108 96

106 89 86

102 88 91

92 89 91 80 88

101 93 82

109 96 97 90

100 87 94 85 85

102 83 89

1.11 1.16 1.16 1.16 1.14 0.95 1.13 1.22 0.94 1.06 1.11 1.22 1.08 1.10 1.13 1.05 1.01 1 .oo 1.06 1.03

Bioassay U.V. assay Mean ~ ratio, 1.10; Rangeofratios,0.94to 1.22;s.~.0.08.


In each of two experiments 16 bananas were treated in this manner. When the applied suspension had dried the treated bananas were placed in an incubator at 15 to 18 “C, care being taken not to damage the treated faces. Untreated (control) bananas were also placed in the incubator. “Benomyl” on the surface of the bananas was estimated, using the rinsing and assay procedures described, after storage intervals of 0,7, 14 and 21 days, four bananas (A, B, C and D) and a control being analysed on each occasion. The results obtained are given in Table 1 expressed as percentage “benomyl” recovered

Bioassay result U.V. assay result

taking the initial dose as 107pglbanana. The ratio was also calculated

and included in the Table for convenience in comparing both methods.

4. Results The recoveries showed no appreciable decrease with time over the 21 days storage and ripening period and the lowest recovery, by the U.V. assay, was 80 %. The overall average recovery was 103% by the bioassay method and 94% by the U.V. assay. The methods

Bioassay result U.V. assay result

were compared by simple statistical consideration of the ratio, . I t is

seen that the mean ratio of 1.1 indicates that the bioassay values are systematically higher since the deviation of 0.1 from the expected ratio of 1 .OO is highly significant in relation to S.E. of the mean. This bias is difficult to explain as preliminary work and subsequent examination (which will be published later) of a number of experimentally treated shipments showed better agreement between the methods. The ratio is not of course affected by the precision of the “spotting” technique or by the storage interval as the assays were conducted on common extracts.

The peak ratios of the absorbances at 282 to 275 nm were also measured in all the U.V. assays” and were found to give an average value of 1.20 with S.D. of +_ 0.017

showing that there was negligible interference with the method from other u.v. absorbing materials in the extracts.

Analysis of control bananas showed, in all cases, apparent “benomyl” contents of less than 0.2 mg/kg by the bioassay and less than 0.1 mg/kg by the U.V. method.

“

5. Discussion The preliminary studies on ethyl acetate rinses from treated bananas had revealed that most of the benomyl on the banana surface was persisting unchanged and it seemed likely that the remainder would be present as MBC, the major breakdown product. The high recoveries obtained in the persistence study would indicate that the rinsing effectively removed surface deposits of MBC if this had formed to any appreciable extent during the storage.

refers to hydroxy derivatives of MBC in bovine fluids and 2-AB in soils but no information is available on the speed of formation of these compounds in crops. 2-AB is formed from MBC by treatment with hot strong alkali and it was thought that little, if any, would develop in benomyl treated bananas in view of the neutral, thick skinned nature of this fruit and the relatively short residence time of the fungicide and its product(s), i.e. 3 to 4 weeks from application to consumption. 2-AB

Recent

Estimation of benomyl residues on ~ ~ M I M S 145

has a similar U.V. spectrum to MBC with two poorly resolved peaks of almost equal absorbance at 274 nm and 279 nm showing, on a molar basis, approximately half the absorbance of the corresponding MBC peaks (275 nm and 282 nm). A small proportion of 2-AB if present in the extracts would not affect the U.V. assay greatly; the ratio of the absorbances at 282 and 275 nm would be slightly reduced. It is not fungitoxic and if it had formed to any appreciable extent in the persistence study the bi0assaylu.v. assay ratio would have been markedly depressed.

Although the methods described here do not measure total benomyl content in bananas, i.e. residues in the skin and in the pulp, they should nevertheless be of value in monitoring commercial shipments of benomyl treated bananas to check on evenness of application and the order of overall contamination. The rinsing and spectrophotometric procedures are simple and rapid and will detect at least 80 ;/o of the total "benomyl" burden on the whole fruit. The validity of the latter analysis can be checked by measuring the ratio of the absorbance of the two maxima at 282 and 275 nm. Further- more the characteristic spectrum of MBC can be readily distinguished from that given by thiabendazole (another fungicide, used on bananas, which is also extracted by ethyl acetate and hydrochloric acid treatments) as the latter shows a broad spread maximum at 303 nm and a secondary maximum at 245 nm.

The bioassay procedure is a longer procedure which is susceptible to interference by other fungicides, if present, but would be of value where a suitable spectrophotometer is not available or in estimating other fungicides of the benomyl type providing a suitable test organism were chosen.

It is worth noting that Bailey et af." studying thiabendazole, a compound of low solubility similar to benomyl, applied to bananas found that, on average, 94 % of the total residue in the whole fruit remained on or in the skin.

The rapid conversion of benomyl to MBC in ethyl acetate and other solvents occurring during the extraction procedure could give rise to misleading information on the persistence of benomyl per se in other crops if not properly considered by the analyst.

Acknowledgements We thank our colleague Dr J. Marriott, for his suggestion that we consider estimating surface residues and also Miss Linda Boss and Mr Peter Terry for their valuable assistance in conducting the many extractions and assays involved in this work. We are also grateful to Geests Industries Limited for supplying bananas and Du Pont Limited for supplying Benlate and pure benomyl.

References 1. Pease, H. L.; Gardiner, J. A. J. ugric. Fd Chem. 1969,17,267. 2. Pease, H. L.; Holt, R. F. J . Ass. off . anulyt. Chem. 1971,54,1399. 3. Erwin, D. C.; Mee, H.; Sims, J. J. Phytopathology 1968,58,528. 4. Clemons, G. P.; Sisler, H. D. Phytoputhology 1969,59, 705. 5. Peterson, C. A.; Edgington, L. V. Phytopathology 1970,60,475. 6. Mestres, R.; Tourte, J.; Campo, M. Truu. Soc. Phurm. Montpellier 1971,31 ( I ) , 49. 7. White, E. R.; Kilgorc, W. W. J. ugric. FdChem. 1972,20, 1230. 8. Kirkland, J. J. J. ugric. Fd C'hetn. 1973, 21, 171. 9, Kirkland, J. J.; Holt, R. F.; Pease, H. L. J. ugric. Fd Chern. 1973, 21, 368.

10. Bailey, D. M.; Cutts, D. F.; Donegan, L.; Phillips, C. A.; Pope, R. J. Fd Technol. 1970,5,89.

the estimation of surface residues of benomyl on treated bananas

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