Die Nahrung 36 (1992) 5 , 485-489
Mycotoxic flora and mycotoxins in smoke-dried fish from Sierra Leone FELIXTINA E. JONSYN and G. P. LAHAI Department of Biological Sciences, Njala University College, University of Sierra Leone, P. M. Bag, Freetown, Sierra Leone
Summary Examination of 20 samples of smoke-dried fish of the Ethmolosa sp. commonly called “Bonga”, from homes and markets in Njala (Sierra Leone) revealed the presence of 4 Aspergilli species: A.j?avus Links ex Fries, A . ochraceus Wilhelm, A . tamarii Kita and A. niger van Tieghem. Fresh or properly preserved smoke-dried fish showed no signs of fungal contamination. Mouldy fish extracts contained varying amounts of aflatoxins B,, G,, G, and ochratoxin A. Isolates of A.jlavus grown on yeast extract sucrose (YES) medium, produced considerable amounts of aflatoxin B, and G , and trace amounts of G,. O n YES medium A . ochraceus produced large amounts of ochratoxin A but no penicillic acid.
Zusammenfassung Mykotoxinbildende Flora und Mykotoxine in rauchgetrocknetem Fisch aus Sierra Leone Die Prufung von 20 Proben rauchgetrockneter Fische (Ethmolosa sp., gewohnlich als ,,Bonga“ bezeichnet) von Familien und Markten in Njala (Sierra Leone) zeigen das Vorkommen von 4 Aspergilli-Species: A.jlavus Links ex Fries, A . ochraceus Wilhelm, A. tamarii Kita und A . niger van Tieghem. Frische oder richtig praservierte rauchgetrocknete Fische weisen keine Anzeichen einer Pilzkontamination auf. Mit Pilzen infizierte Fischextrakte enthalten variierende Mengen an Aflatoxinen B,, G,, G2 und Ochratoxin A. Isolate von A.flavus, die auf Hefeextrakt-Sucrose(YES)-Medium wachsen, bilden betrachtliche Mengen an Aflatoxin B, und G, sowie sehr geringe Mengen an Aflatoxin G,. A. ochraceus produziert auf YES-Medium groI3e Mengen an Ochratoxin A, aber keine Penicillinsaure.
Introduction In Sierra Leone fish accounts for two thirds (67%) of animal protein consumption and 14% of the total protein intake [9]. It is used as an ingredient in the preparation of weaning food for young children. To ensure easy and long storage, low transportation costs and special flavours, fish is generally smoke-dried. However, poor storage facilities result in deterioration of the product. Evidence of spoilage is provided by the presence of maggots, insects and fungi in stored fish. Literature search has shown that only a few studies with respect to toxigenic fungi on fish or fish products have been carried out. In Nigeria, NWOKOLO et al. [6] demonstrated high levels of aflatoxin in fungi-contaminated fish. A related study on salt-dried fish in Sri Lanka [2] revealed the predominance of Aspergilli species on this product. However, the strain of A.fluuus isolated was nonaflatoxigenic. This study was therefore carried out to determine the mycoflora of smoke-dried fish from Sierra Leone and the presence/absence of known mycotoxins.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
0027-769X/92/0509-0484$3.50+ .25/0
486
Die Nahrung 36 (1992) 5
Materials and methods Collection of samples Fish samples used in this study were the common “Bonga” (Ethmolosa sp.) which is available all year round, relatively cheap, its susceptibility to fungi is inapparent (no evidence of fungal attack can be seen until the scales are removed) and it is very popular due to its taste with both rich and poor. Fish samples obtained from the local market and homes in the study area were placed in sterile bags and transported to the laboratory.
Isolation of mycojlora Dried scales were removed by hand and samples showing visible fungal growth or ones with poor appearance were selected. Selected samples were broken by hand into tiny pieces and 1 g lots were plated onto malt extract agar (MEA) (Oxoid) and incubated at room temperature for 8 days. A total of 6 replicates was made. Spores from each colony on MEA were further monocultured in potato dextrose agar and pure colonies sent to the Commonwealth Mycological Institute (CMI), Kew, UK, for identification.
Extraction and identification of toxins 5 g mouldy fish were put in a 100 ml ERLENMEYER flask and 20 ml chloroform-HCI (99 : 1) was added. The flask was allowed to stand for 2 h and then the contents emptied into a porcelain mortar and a slurry was made using the pestle. The extract was filtered through a Whatman No. 4 filter paper. The filtrate was collected and allowed to evaporate to approximately 5 to 6 ml (overnight in a fume cupboard). Various concentrations of this filtrate and 5 pl standard solutions of aflatoxin B,, B,, G,, G2,ochratoxin A and penicillic acid were spotted on precoated TLC plates (Merck 5553). Plates were developed in toluene/ethyl acetate/formic acid (6 : 3 : 1) for 1 h and later viewed under long and short range UV light.
Testing the toxin production potential of isolates A spatula full of one week old spores of each isolate was used for individual innoculation of 50 ml yeast extract (4%) sucrose (10%) (YES) medium in a 250 ml ERLENMEYER flask. Incubation for 3 wks. at room temperature then followed.
Extraction and chromatography of toxins Using a sterile pipette, 10 ml sample was withdrawn from the YES-medium culture, put into a test tube and 10 ml chloroform-HC1 (99: I ) was added. The test tubes were shaken vigorously for 10 min and centrifuged at 2000 rpm. The lower chloroform layer was removed and put into a 50 ml beaker. The extraction was repeated twice. Chloroform layers were pooled, allowed to evaporate (as above) and TLC analysis carried out as already described. This extraction and TLC analysis of samples from the Y ES-medium was performed weekly for a period of 3 weeks.
Confirmatory tests To test the authenticity of the aflatoxions, two methods were used: 1 . The developed TLC plates were allowed to dry and later sprayed with aqueous sulphuric acid [7]. 2. Using the method of COKER et al. [3], derivative formation of aflatoxin and trifluoroacetic acid (TFA) was determined. Spray treatment of the developed TLC plate with p-anisaldehyde [8] and exposure of TLC plates to ammonia fumes [ I ] were the two methods used to confirm the presence of ochratoxin A.
JONSYN/LAHAI: Mycotoxins in smoke-dried fish
487
Results Mycoflora of “Bonga” Out of the 20 fish samples examined, 14 appeared mouldy. From these mouldy samples, 13 fungal isolates were made. Eleven (84%) were Aspergilli sp. These included known toxigenic strains like A.flavus (36%) and A . ochraceus (27%); A . tamarii (29%) and A. niger (8%). Syncephalastrum racemosum Cohn ex Schroeter was also identified.
Mycotoxins detected Thin layer chromatography of 6 mouldy fish extracts revealed the presence of (2 to 5) fluorescent spots under UV light (Table 1). From these fluorescent compounds aflatoxins B,, GI, G , and ochratoxin A were positively identified and confirmed through the appearance of yellow fluorescent spots of standard aflatoxins and aflatoxin containing extracts, after spray treatment with sulphuric acid and the “blue cap” at Rf 0.1 of these standards and samples after trifluoroacetic acid derivatization. Ochratoxin was also Table 1 Thin layer chromatography of mouldy fish extracts Sample No.
Number of spots on TLC
1
4
2
5
Colour under UV
Rf. value
Identity of toxin*
OTA* AFB1* AFG,* OTA AFB, AFG AFG, AFG, AFB AFG,
Before spray
After spray
blue-green green blue green blue-green green blue green green
blue blue pink blue
0.72 0.49 0.29 0.23
blue blue pink blue blue
0.72 0.48 0.28 0.24 0.20
3
2
blue-green green
blue blue
0.13 0.23
4
3
blue-green blue green
blue pink blue
0.72 0.28 0.23
5
4
blue-green blue green green
blue pink blue blue
0.73 0.29 0.24 0.19
6
4
blue-green green blue green
blue blue pink blue
0.72 0.47 0.29 0.23
7 (control)
2
blue-green white
blue -
0.72 0.32
*
,
,
-
AFB, AFG, AFG, OTA AFB, AFG, -
OTA = ochratoxin A; AFB, = aflatoxin B,; AFG, = aflatoxin G I ; AFGz = aflatoxin G,
Die Nahrung 36 (1992) 5
488
confirmed by its bright green fluorescent spot turning blue after spray treatment with p-anisaldehyde and also turning bright blue after exposure to ammonia fumes. Using the comparison of fluorescence technique of the standards and sample extracts, it was established that the amount of mycotoxins present in mouldy fish varied from trace to moderately high (Table 2). .____
Sample No.
1 2 3 4 5 6
Table 2 Aflatoxin/ochratoxin levels in mouldy fish
Amount of Bl
GI
G2
OTA
++ +++ + ++
+ ++ + ++ + +
-
+ +- +
f
-
-
-
+ -
++
+ + = equivalent to 10 p1 standard + = equivalent to 5 pI standard = equivalent to trace amount -
=
not detected
Growing the isolates in YES medium served two purposes: 1. It confirmed the existence of toxigenic fungi on fish, as mycotoxins isolated from this medium corresponded with those found on mouldy fish extracts. 2. The levels and exact time of production of these mycotoxins could be monitored. In this study it was seen that A . ochraceus produced ochratoxin as early as the 1st week of incubation, whereas aflatoxins were only detected on the 2nd week of incubation. Judging from the intensity of the fluorescent spots on TLC, there was a daily increase in mycotoxin production by these strains of fungi. This continued until the end of the incubation period (3 weeks) (Table 3). Table 3 Detection of mycotoxins and rate of production by Aspergilli isolates grown of YES medium Number of spots on TLC
Toxin detected
A . ,flaws
4
A . ochraceus
2
AFB, AFG, AFG, OTA PEA
Fungus
Incubation days 3
7
-
-
k -
+-
15
21
++ + k ++ -
+++ ++ k
+- + +
AFB, = aflatoxin B,; AFG, = aflatoxin G I ; AFG, = aflatoxin G,; OTA = ochratoxin A; PEA = penicillic acid + + + = equivalent to 15 to 20 p1 of standard + + = equivalent to I0 pI of standard + = equivalent to 5 p1 of standard = nCJt detected
JONSYN/LAHAI: Mycotoxins in smoke-dried fish
489
Discussion In Nigeria, NWOKOLO et al. [6]have shown that the heaviest source of aflatoxin exposure in semi-savanah and forest areas is dried fish. Levels of 400 - 800 ppb were recorded in these regions. This study has revealed not only the presence of aflatoxins in smoked-dried fish but also of ochratooxin A. The existence of ochratoxin A, a powerful nephrotoxin, is not well recognized in Africa. This toxin, as well as aflatoxin is very stable and it can survive cooking temperatures [4] but shows no significant reduction after processing [5] compared with aflatoxin. Since smoked-dried fish is an important and popular part of the diet of Sierra Leoneans, it constitutes a possible source of repeated aflatoxin and ochratoxin exposure, both of which are well recognized as potential causes of serious health hazards. In the light of the above, new and improved methods of extracting and determining the exact amount of these mycotoxins in smoked-dried fish are under consideration. There is need also to investigate the mycoflora of other preserved fish and sea products from Sierra Leone in order to determine whether they contribute to mycotoxin exposure. References [I] Association of Official Analytical Chemist, Official Methods of Analysis: Natural Poisons. p. 258. Ed. by W. HORWITZ, 12th Ed., 1975. [2] ATAPATTU, R. and U. SAMARAJEEWA, Mycopathologia 111, 55 - 59 (1990). [3] COKER,R. D., B. 0. JONES and M. J. NAGLER,Mycotoxin Training Manual. London Tropical Development and Research Institute, 1984. [4] JONSYN,F. E., The toxicity of various mycotoxins to bacteria: An examination of five toxigenic strains of fungi. MSc. Thesis. University of Muenster, West Germany, 1979. [5] JONSYN,F. E., Mircen Journal 5, 457-462 (1989). [6] NWOKOLO, C., and P. OKONKWO, Transaction of the Royal Society of Tropical Medicine and Hygiene 72, 329-332 (1978). [7] PRZYBYLSKI, W., J. Assoc. Off. Anal. Chem. 58, 163 (1975). [8] SCOTT,P. M., J. W. LAWRENCE and W. VAN WALBEEK, Appl. Microbiol. 20, 839-842 (1970). [9] TEUTSCHER, F., F A 0 J. Food Nutrit. 12 (2), 2-10 (1986). FELIXTINA E. JONSYN,Department of Biological Sciences, Njala University College, University of Sierra Leone, P.M. Bag. Freetown, Sierra Leone Received October 28, 1991
Mycotoxic flora and mycotoxins in smoke-dried fish from Sierra Leone FELIXTINA E. JONSYN and G. P. LAHAI Department of Biological Sciences, Njala University College, University of Sierra Leone, P. M. Bag, Freetown, Sierra Leone
Summary Examination of 20 samples of smoke-dried fish of the Ethmolosa sp. commonly called “Bonga”, from homes and markets in Njala (Sierra Leone) revealed the presence of 4 Aspergilli species: A.j?avus Links ex Fries, A . ochraceus Wilhelm, A . tamarii Kita and A. niger van Tieghem. Fresh or properly preserved smoke-dried fish showed no signs of fungal contamination. Mouldy fish extracts contained varying amounts of aflatoxins B,, G,, G, and ochratoxin A. Isolates of A.jlavus grown on yeast extract sucrose (YES) medium, produced considerable amounts of aflatoxin B, and G , and trace amounts of G,. O n YES medium A . ochraceus produced large amounts of ochratoxin A but no penicillic acid.
Zusammenfassung Mykotoxinbildende Flora und Mykotoxine in rauchgetrocknetem Fisch aus Sierra Leone Die Prufung von 20 Proben rauchgetrockneter Fische (Ethmolosa sp., gewohnlich als ,,Bonga“ bezeichnet) von Familien und Markten in Njala (Sierra Leone) zeigen das Vorkommen von 4 Aspergilli-Species: A.jlavus Links ex Fries, A . ochraceus Wilhelm, A. tamarii Kita und A . niger van Tieghem. Frische oder richtig praservierte rauchgetrocknete Fische weisen keine Anzeichen einer Pilzkontamination auf. Mit Pilzen infizierte Fischextrakte enthalten variierende Mengen an Aflatoxinen B,, G,, G2 und Ochratoxin A. Isolate von A.flavus, die auf Hefeextrakt-Sucrose(YES)-Medium wachsen, bilden betrachtliche Mengen an Aflatoxin B, und G, sowie sehr geringe Mengen an Aflatoxin G,. A. ochraceus produziert auf YES-Medium groI3e Mengen an Ochratoxin A, aber keine Penicillinsaure.
Introduction In Sierra Leone fish accounts for two thirds (67%) of animal protein consumption and 14% of the total protein intake [9]. It is used as an ingredient in the preparation of weaning food for young children. To ensure easy and long storage, low transportation costs and special flavours, fish is generally smoke-dried. However, poor storage facilities result in deterioration of the product. Evidence of spoilage is provided by the presence of maggots, insects and fungi in stored fish. Literature search has shown that only a few studies with respect to toxigenic fungi on fish or fish products have been carried out. In Nigeria, NWOKOLO et al. [6] demonstrated high levels of aflatoxin in fungi-contaminated fish. A related study on salt-dried fish in Sri Lanka [2] revealed the predominance of Aspergilli species on this product. However, the strain of A.fluuus isolated was nonaflatoxigenic. This study was therefore carried out to determine the mycoflora of smoke-dried fish from Sierra Leone and the presence/absence of known mycotoxins.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
0027-769X/92/0509-0484$3.50+ .25/0
486
Die Nahrung 36 (1992) 5
Materials and methods Collection of samples Fish samples used in this study were the common “Bonga” (Ethmolosa sp.) which is available all year round, relatively cheap, its susceptibility to fungi is inapparent (no evidence of fungal attack can be seen until the scales are removed) and it is very popular due to its taste with both rich and poor. Fish samples obtained from the local market and homes in the study area were placed in sterile bags and transported to the laboratory.
Isolation of mycojlora Dried scales were removed by hand and samples showing visible fungal growth or ones with poor appearance were selected. Selected samples were broken by hand into tiny pieces and 1 g lots were plated onto malt extract agar (MEA) (Oxoid) and incubated at room temperature for 8 days. A total of 6 replicates was made. Spores from each colony on MEA were further monocultured in potato dextrose agar and pure colonies sent to the Commonwealth Mycological Institute (CMI), Kew, UK, for identification.
Extraction and identification of toxins 5 g mouldy fish were put in a 100 ml ERLENMEYER flask and 20 ml chloroform-HCI (99 : 1) was added. The flask was allowed to stand for 2 h and then the contents emptied into a porcelain mortar and a slurry was made using the pestle. The extract was filtered through a Whatman No. 4 filter paper. The filtrate was collected and allowed to evaporate to approximately 5 to 6 ml (overnight in a fume cupboard). Various concentrations of this filtrate and 5 pl standard solutions of aflatoxin B,, B,, G,, G2,ochratoxin A and penicillic acid were spotted on precoated TLC plates (Merck 5553). Plates were developed in toluene/ethyl acetate/formic acid (6 : 3 : 1) for 1 h and later viewed under long and short range UV light.
Testing the toxin production potential of isolates A spatula full of one week old spores of each isolate was used for individual innoculation of 50 ml yeast extract (4%) sucrose (10%) (YES) medium in a 250 ml ERLENMEYER flask. Incubation for 3 wks. at room temperature then followed.
Extraction and chromatography of toxins Using a sterile pipette, 10 ml sample was withdrawn from the YES-medium culture, put into a test tube and 10 ml chloroform-HC1 (99: I ) was added. The test tubes were shaken vigorously for 10 min and centrifuged at 2000 rpm. The lower chloroform layer was removed and put into a 50 ml beaker. The extraction was repeated twice. Chloroform layers were pooled, allowed to evaporate (as above) and TLC analysis carried out as already described. This extraction and TLC analysis of samples from the Y ES-medium was performed weekly for a period of 3 weeks.
Confirmatory tests To test the authenticity of the aflatoxions, two methods were used: 1 . The developed TLC plates were allowed to dry and later sprayed with aqueous sulphuric acid [7]. 2. Using the method of COKER et al. [3], derivative formation of aflatoxin and trifluoroacetic acid (TFA) was determined. Spray treatment of the developed TLC plate with p-anisaldehyde [8] and exposure of TLC plates to ammonia fumes [ I ] were the two methods used to confirm the presence of ochratoxin A.
JONSYN/LAHAI: Mycotoxins in smoke-dried fish
487
Results Mycoflora of “Bonga” Out of the 20 fish samples examined, 14 appeared mouldy. From these mouldy samples, 13 fungal isolates were made. Eleven (84%) were Aspergilli sp. These included known toxigenic strains like A.flavus (36%) and A . ochraceus (27%); A . tamarii (29%) and A. niger (8%). Syncephalastrum racemosum Cohn ex Schroeter was also identified.
Mycotoxins detected Thin layer chromatography of 6 mouldy fish extracts revealed the presence of (2 to 5) fluorescent spots under UV light (Table 1). From these fluorescent compounds aflatoxins B,, GI, G , and ochratoxin A were positively identified and confirmed through the appearance of yellow fluorescent spots of standard aflatoxins and aflatoxin containing extracts, after spray treatment with sulphuric acid and the “blue cap” at Rf 0.1 of these standards and samples after trifluoroacetic acid derivatization. Ochratoxin was also Table 1 Thin layer chromatography of mouldy fish extracts Sample No.
Number of spots on TLC
1
4
2
5
Colour under UV
Rf. value
Identity of toxin*
OTA* AFB1* AFG,* OTA AFB, AFG AFG, AFG, AFB AFG,
Before spray
After spray
blue-green green blue green blue-green green blue green green
blue blue pink blue
0.72 0.49 0.29 0.23
blue blue pink blue blue
0.72 0.48 0.28 0.24 0.20
3
2
blue-green green
blue blue
0.13 0.23
4
3
blue-green blue green
blue pink blue
0.72 0.28 0.23
5
4
blue-green blue green green
blue pink blue blue
0.73 0.29 0.24 0.19
6
4
blue-green green blue green
blue blue pink blue
0.72 0.47 0.29 0.23
7 (control)
2
blue-green white
blue -
0.72 0.32
*
,
,
-
AFB, AFG, AFG, OTA AFB, AFG, -
OTA = ochratoxin A; AFB, = aflatoxin B,; AFG, = aflatoxin G I ; AFGz = aflatoxin G,
Die Nahrung 36 (1992) 5
488
confirmed by its bright green fluorescent spot turning blue after spray treatment with p-anisaldehyde and also turning bright blue after exposure to ammonia fumes. Using the comparison of fluorescence technique of the standards and sample extracts, it was established that the amount of mycotoxins present in mouldy fish varied from trace to moderately high (Table 2). .____
Sample No.
1 2 3 4 5 6
Table 2 Aflatoxin/ochratoxin levels in mouldy fish
Amount of Bl
GI
G2
OTA
++ +++ + ++
+ ++ + ++ + +
-
+ +- +
f
-
-
-
+ -
++
+ + = equivalent to 10 p1 standard + = equivalent to 5 pI standard = equivalent to trace amount -
=
not detected
Growing the isolates in YES medium served two purposes: 1. It confirmed the existence of toxigenic fungi on fish, as mycotoxins isolated from this medium corresponded with those found on mouldy fish extracts. 2. The levels and exact time of production of these mycotoxins could be monitored. In this study it was seen that A . ochraceus produced ochratoxin as early as the 1st week of incubation, whereas aflatoxins were only detected on the 2nd week of incubation. Judging from the intensity of the fluorescent spots on TLC, there was a daily increase in mycotoxin production by these strains of fungi. This continued until the end of the incubation period (3 weeks) (Table 3). Table 3 Detection of mycotoxins and rate of production by Aspergilli isolates grown of YES medium Number of spots on TLC
Toxin detected
A . ,flaws
4
A . ochraceus
2
AFB, AFG, AFG, OTA PEA
Fungus
Incubation days 3
7
-
-
k -
+-
15
21
++ + k ++ -
+++ ++ k
+- + +
AFB, = aflatoxin B,; AFG, = aflatoxin G I ; AFG, = aflatoxin G,; OTA = ochratoxin A; PEA = penicillic acid + + + = equivalent to 15 to 20 p1 of standard + + = equivalent to I0 pI of standard + = equivalent to 5 p1 of standard = nCJt detected
JONSYN/LAHAI: Mycotoxins in smoke-dried fish
489
Discussion In Nigeria, NWOKOLO et al. [6]have shown that the heaviest source of aflatoxin exposure in semi-savanah and forest areas is dried fish. Levels of 400 - 800 ppb were recorded in these regions. This study has revealed not only the presence of aflatoxins in smoked-dried fish but also of ochratooxin A. The existence of ochratoxin A, a powerful nephrotoxin, is not well recognized in Africa. This toxin, as well as aflatoxin is very stable and it can survive cooking temperatures [4] but shows no significant reduction after processing [5] compared with aflatoxin. Since smoked-dried fish is an important and popular part of the diet of Sierra Leoneans, it constitutes a possible source of repeated aflatoxin and ochratoxin exposure, both of which are well recognized as potential causes of serious health hazards. In the light of the above, new and improved methods of extracting and determining the exact amount of these mycotoxins in smoked-dried fish are under consideration. There is need also to investigate the mycoflora of other preserved fish and sea products from Sierra Leone in order to determine whether they contribute to mycotoxin exposure. References [I] Association of Official Analytical Chemist, Official Methods of Analysis: Natural Poisons. p. 258. Ed. by W. HORWITZ, 12th Ed., 1975. [2] ATAPATTU, R. and U. SAMARAJEEWA, Mycopathologia 111, 55 - 59 (1990). [3] COKER,R. D., B. 0. JONES and M. J. NAGLER,Mycotoxin Training Manual. London Tropical Development and Research Institute, 1984. [4] JONSYN,F. E., The toxicity of various mycotoxins to bacteria: An examination of five toxigenic strains of fungi. MSc. Thesis. University of Muenster, West Germany, 1979. [5] JONSYN,F. E., Mircen Journal 5, 457-462 (1989). [6] NWOKOLO, C., and P. OKONKWO, Transaction of the Royal Society of Tropical Medicine and Hygiene 72, 329-332 (1978). [7] PRZYBYLSKI, W., J. Assoc. Off. Anal. Chem. 58, 163 (1975). [8] SCOTT,P. M., J. W. LAWRENCE and W. VAN WALBEEK, Appl. Microbiol. 20, 839-842 (1970). [9] TEUTSCHER, F., F A 0 J. Food Nutrit. 12 (2), 2-10 (1986). FELIXTINA E. JONSYN,Department of Biological Sciences, Njala University College, University of Sierra Leone, P.M. Bag. Freetown, Sierra Leone Received October 28, 1991
![[Craniotomy in Sierra Leone?].](/assets/img/hold.png)
