I. INTRODUCTION
Author: Hiket Eltutar, Mustafa Igneciler
Pages: 12 to 16
Creation Date: 1958/01/01
I. INTRODUCTION
When cases of opium purchased from different districts of this country are opened for inspection the opium is sometimes found to be covered with mould.
It has been observed that the extent of mould is always greater on opium with a high moisture content. Mould is considered to be due to contamination of the opium while in the producer's possession. When the opium arrives in our warehouse in a contaminated condition it is observed that as a result of the process during which it is manipulated and pressed into the required export shape, mould spreads only over surfaces in contact with air.
Fungi are closely associated with moisture. The speed and extent of their growth depend on dampness and it would therefore be possible to avoid mould by maintaining a dry atmosphere permanently in the warehouse and preventing humidity. From a technical standpoint it would not, however, be convenient to eliminate humidity altogether in the warehouse; besides, this would not be possible, in our opinion, in a building situated near the sea.
We therefore contemplated using fungicides to protect the opium from mould. Part of the research undertaken for this purpose was completed in the Soil Products Office's laboratory; isolation of fungi was carried out in the Veterinary Bacteriological Institute at Pendik; identification was made in the Botanical Institute at the Istanbul University Faculty of Science (Prof. Dr. Heilbron); experiments on isolation of fungi, culture and tests on application of various preparations were performed under the supervision of Dr. Ibrahim Karaca, professor, the Chair of Plant Protection, Faculty of Agriculture of Ankara University.
II. MATERIALS AND METHOD
A . Materials
Opium used in the tests was obtained from various regions of Turkey and supplied by the Soil Products Office.
The following table shows the regions from which samples were obtained in order to study the action of fungus in relation to morphine content and morphine percentage.
One and a half kilos of each of the above samples drawn on 5 to 7 March 1958 were ground four times in the mincing machine and made homogeneous.
Origin |
Morphine percentage |
Serial numbers of cases |
Erbaa |
11. - |
5823 |
Yalvac |
11. - |
5378 |
Bolvadin |
11.50 | 3883 |
Emet |
11.50 | 992 |
Zile |
11.75 | 5441 |
Tavsanli |
12. - |
1443 |
Civril |
12. - |
5963 |
Bozkir |
12.25 | 1553 |
Amasya |
13.75 | 683 |
S. Karaagac |
12. - |
5072 |
The sample used in tests for fungicides was of the standard export type weighing 2 kilos. The preparations used in experiments were sold on the open market and were supplied by manufacturers to the Faculty of Agriculture and the Institute of Agricultural Control, Ankara.
Name |
Manufacturer |
Group according to active substance |
Agresan GN |
I.C.I. |
Organic mercury (Tolymercuriacetate) Mercury 1% |
Anticarie SD |
Saint-Denis |
Hexachlorbenzene |
Arasan |
Du Pont |
Tetramethythiuramdisulphide |
Ceresan T |
Bayer |
EthylmercurychlorideC 2N 5Hg C1 |
Cuprevit Ob 21 |
Bayer |
50 % Copper |
Dithane |
|
|
Fermate |
Du Pont |
Dimethyldithiocarbamate de Fer |
Netzschwefel |
Schering |
Sulphur 90% |
Ortocide 50 |
Standard Oil Company U.S.A. |
Combined Nitrogen and Chlorin |
Perenox |
I.C.I. |
Copper 50% |
Solbar |
Bayer |
Bariumpolysulphide |
Surmesan |
Koruma |
|
Sulphur powder |
Keciborlu |
Sulphur |
Verdesan |
I.C.I. |
Organic mercury |
B. Method
The method used for determination of the action of fungus over morphine in opium was the lime-water method.
Czapek-Agar medium was used for reproduction of fungus. According to (1), components of the above medium are as follows:
Water |
1,000 cc |
NaNo 3 |
3 g |
K 2HPo 4 |
1 g |
Mg.So 47H 2O |
0.5 g |
KC1 |
0.5 g |
FeSO 47H 2O |
0.01 g |
Sugar |
30 g |
Agar |
15 g |
Inoculation of Aspergillus was effected in Czapek medium poured in Petri containers and the product was distributed by quantities of 25 mg for each Petri box. Then the Petri boxes were placed in a thermostatically controlled container at 30°C and were left to allow reproduction of fungus.
With regard to tests on opium, the opium was cut in shapes of cakes 0.5 cm square, so that they could be held in Petri boxes. These cakes were softened in water; then fungus was inoculated into them. Thereafter, the opium cakes were laid in the powder product and were completely covered with the powder and entirely hidden. Then they were placed in Petri boxes containing damp blotting paper. The Petri boxes were left in the thermostatically controlled container at 30°C. or at room temperature of 25-27°C.
III. VARIETIES OF FUNGI GROWING ON NATURAL OR MANIPULATED OPIUM
In this country, four kinds of disease are known to afflict poppy plants (Papaver somniferum L.) owing to such factors as climatic and soil conditions. Among these are cryptogamic parasites. However, available publications do not show the kinds of fungi attacking opium poppy. It has been observed here that opium in warehouses sometimes grows mouldy; mould factors always showing the same etiological and symptomatological properties were isolated at the Veterinary Bacteriological Institute at Pendik and were identified by Prof. Dr. Heilbron.
One of the two kinds of fungus thus identified was indicated as Aspergillus Niger. According to (1), it appears that this fungus belongs to the series Aspergillus Niger A. Awameri Nakazawa. The second kind was identified as Aspergillus minimus Wehmer. A. Minimus species are not widespread, and are found on rare occasions on dry leaves (2).
Class |
Ascomycetes |
Order |
Plectascimene |
Family |
Aspergillacea |
Species |
Aspergillus |
According to (3) No. 3 Aspergillus species include 119 kinds. Up to the present only Aspergillus Niger has been found in Turkey. Now As. Minimus has been discovered in opium and only two varieties out of 119 have been determined in this country.
Both varieties have been found in raw opium purchased from different regions, and fungi growing on it are probably due to contamination either in the fields or while it is in the producer's possession.
IV. ACTION OF FUNGUS ON MORPHINE
In order to ascertain whether fungus in opium has any action on the morphine content, samples of 1.5 kg each were drawn from opium produced by regions mentioned in part II of this article and mixed four or five times in the small mincing machine.
When results of analysis showed that the opium had been made thoroughly homogeneous, the samples were divided into two parts and placed in glass jars of one litre each.
The lids of ten jars were closed; the other ten were left open, so that the opium remained in contact with air. Moisture and morphine content were determined on the same day.
Origin |
Moisture percentage |
Morphine percentage |
Erbaa |
16.42 | 14.51 |
Yalvac |
12.02 | 12.68 |
Bolvadin |
16.81 | 13.41 |
Emet |
13.16 | 12.96 |
Zile |
15.47 | 12.18 |
Tavsanli |
16.87 | 13.82 |
Civril |
13.99 | 13.28 |
Bozkir |
12.95 | 13.13 |
Amasya |
17.55 | 14.69 |
S. Karaagac |
15.48 | 12.80 |
No mouldiness appeared on opium contained in the jars which had been left open; the samples in closed jars were gradually covered with mould, which spread completely over the opium within thirty days. The samples were tested at times, and their morphine content checked.
Whenever more than one analysis was required, this was repeated three times, but no error beyond the permitted margin was found. In order to compare the findings, results of analyses were calculated on dry product basis.
Dales of analyses |
|||||
---|---|---|---|---|---|
Regions |
9.III.1955 Morphine percentage |
2.V.1955 Morphine percentage |
23.XI.1955 Morphine percentage |
17.II.1958 Morphine percentage |
Difference between first and last tests. Morphine % |
Erbaa |
17.36 | 17.60 | 17.56 | 17.52 | 0.16 |
Yalvac |
14.41 | 14.14 | 13.93 | 14.20 | 0.21 |
Bolvadin |
16.12 | 15.78 | 15.60 | 15.98 | 0.14 |
Emet |
14.86 | 14.96 | 14.53 | 14.60 | 0.26 |
Zile |
14.40 | 14.47 | 14.17 | 14.25 | 0.15 |
Tavsanli |
16.62 | 16.64 | 16.29 | 16.35 | 0.27 |
Civril |
15.44 | 15.05 | 15.53 | 15.45 | 0.01 |
Bozkir |
15.08 | 14.87 | 15.19 | 15.10 | 0.02 |
Amasya |
17.81 | 17.87 | 17.91 | 17.85 | 0.04 |
S. Karaagac |
15.14 | 14.93 | 14.79 | 15.00 | 0.14 |
As will be seen in the above table, differences in morphine contents between the first and last analyses are as follows : Bozkir, Amasya, Civril : 0.01 - 0.04; Zile, Erbaa, S. Karaagac, Boldavin : 0.14 - 0.16; Yalvaç, Emet, Tavsanli : 0.21 - 0.27.
In conclusion of the foregoing it is understood that for the dry product at any rate, opium without mould shows no difference in its morphine content even at the end of a period of three years.
|
Dates of analyses |
Difference between first and last tests | |||
Regions |
9.1955 |
2.V.1955 |
23.XI.1955 |
17.II.1958 |
|
Erbaa |
17.36 | 17.21 | 17.92 | 17.52 | 0.16 |
Yalvac |
14.41 | 13.97 | 14.08 | 14.36 | 0.05 |
Bolvadin |
16.12 | 15.54 | 16.18 | 16.10 | 0.02 |
Emet |
14.86 | 14.17 | 14.84 | 14.86 |
- |
Zile |
14.40 | 13.98 | 13.97 | 14.30 | 0.10 |
Tavsanli |
16.62 | 15.92 | 16.86 | 16.70 | 0.08 |
Civril |
15.44 | 14.56 | 15.07 | 15.33 | 0.11 |
Bozkir |
15.08 | 14.63 | 15.20 | 15.20 | 0.18 |
Amasya |
17.81 | 17.82 | 17.66 | 18.08 | 0.27 |
S. Karaagac |
15.14 | 15.02 | 15.23 | 15.19 | 0.05 |
It will be noted from the table above that morphine content determined on opium produced by Emet was the same on the first and the last analysis; differences between first and last tests for morphine content in opium from Boldavin, Yalvaç, S.Karaagaç, Tavsanli and Zile range between 0.02 and 0.10. As for Givril, Erbaa, Bozkir and Amasya opium, differences between the first and the last analysis for morphine content are from 0.11% to 0.27%.
It may be concluded from these data that mouldy opium in closed jars, as well as mouldless opium in open jars, did not show any considerable difference in morphine content at the end of a period of three years.
V. ELIMINATION OF FUNGI BY MEANS OF FUNGICIDES
Action of fungicides over fungi was at first tested in an artificial medium (Czapek), then in a natural medium (opium); and prolonged studies were made.
A. Tests on the products in an artificial medium
Products obtained from different firms were sprayed at the rate of 25 mg for each Petri box and laid homogeneously on Agar-Agar surface. As will be seen in the following table, some of the products remained inert, whereas others showed long-lasting action.
In the following table, sign ( - ) indicates inertia and (+) indicates beginning of growth of fungus.
This test was repeated four times and the average of days shown in the table was taken.
|
DAYS | |||||||||||
Name of product |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
... |
60 |
Agresan GN |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
Anticarie |
- |
- |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
|
+ |
Arasan |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
|
+ |
Ceresan T |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
|
Dithane |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
|
+ |
Fermate |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
|
+ |
Cupravit |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Netzschwefel |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Orthocide 50 |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
|
+ |
Solbar |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Surmesan |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Pelenox |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Sulphur powder |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Verdesan |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
Control |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
|
+ |
Upon examination of the results it was noted that Aspergillus Awomari appeared within four days after culture. Growth of fungus was not prevented by the products Anticarie, Cupravit, Netzschwefel, Solbar, Surmesan, Perenox and sulphur powder. As for Arasan, Dithane, Fermate and Orthocide : their action ceased at the end of a week, and fungus began to grow.
On the other hand, the products Agrosan GN, Ceresan and Verdesan prevented growth of fungus during sixty days.
Each product was used in equal quantities of 25 mg. Some were spraying products, doses of which did not of course, correspond to powder doses. Should these have proved efficient, we should have contemplated their application to opium. But - as shown in the above table - the results were entirely insufficient. With regard to the powder products, these were tried in lower density (10 mg for each Petri box), and growth of fungus did not start much sooner, but it developed with greater speed later on.
During these experiments, we did not look for the minimum active dose to be applied, but studied potency of products and duration of their action. We therefore did not find it necessary to use lower doses than 10 mg in our researches.
B. Application of fungicides on opium
As started in part II of this article, Aspergillus Niger was inoculated into opium which was cut in the shape of cakes. Then the cakes were completely covered with the product and placed in Petri boxes, where fungus developed in inverse proportion with the product's efficiency.
By comparing the following table with the previous one, it will be noted that the same products gave the same results in both experiments, so that satisfactory action of Agrosan GN, Arasan, Cerasan T., Orthocide 50 and Verdesan was confirmed. Tests were continued with these products.
DAYS |
DAYS | ||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Name of product |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|
Name of product |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|
Agrosan GN |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
* |
Solbar |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Anticarie |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Surmesan |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Ceresan T |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
** |
Sulphur powder |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Cupravit |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
|
Orthocide 50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
*** |
Dithane |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Verdesan |
(Available quantity insufficient) |
|
|
|
|
|
|
|
|
|
|
Fermate |
- |
- . |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Arasan |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
|
Netzschwefel |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Control |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
Perenox |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
|
|
|
|
|
|
|
|
|
|
|
|
|
Observations : * 11 days later in one case. ** 15 days later in one case. *** 5 days later in one case : 10 days later in another case.
Experiments on opium with each one of the above products were repeated five times with the method described and results were as follows :
DAYS | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Name of product |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
Agresan GN |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
2 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
3 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
4 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Arasan |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
2 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
3 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
4 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
Ceresan T. |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
2 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
3 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
4 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Orthocide 50 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
2 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
3 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
4 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
+ |
+ |
+ |
- |
+ |
Verdesan |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
As will be seen from table 7, no fungus appeared on opium after use of Agresan GN, Ceresan T, Verdesan, and no sign of it was observed. However, with the use of Arasan and Orthocide 50, fungus began to grow on the eleventh day and spread over the medium.
The active substance of the above three products, action of which proved efficient, is mercury.
VI. CONCLUSIONS
Fungi observed on raw and manipulated opium are Aspergillus Awomeri Nakazava and Aspergillus minimus Wehmer.
No mould appeared on opium samples preserved in open jars.
Opium samples preserved in closed jars grew progressively mouldy depending on moisture content and were completely covered with mould within a month's time.
No marked difference was observed in three years’ time in morphine content of opium samples, either with or without mould.
Fourteen kinds of product containing various active substances were tested against fungi in artificial and natural media.
Results obtained both in artificial and natural media completely confirmed each other.
Products containing mercury (Agresan GN, Ceresan T. and Verdesan) applied to opium in natural media prevented growth of fungus, thereby giving entirely satisfactory results.
VII. LITERATURE
CHARLES, Thos. 1945, A Manual of the Aspergilli, Baltimore, 1 x + 373
VEHMER, 1899, Aspergillus Minimus, Bot. Control, 80 : 449- 461.
Centraalbureau voor 1947, Sehimmelcultures, List of Cultures. Baarn, Holland 145
MIGULA, W., 1927, Kriptogamen - Flora von Deutschland, Deutsch-österreich und der Schweiz. BD. III. Pilze.
SÖZEN HAYRI, 1950, Analitik Toksokoloji : Istanbul Universit esi.