Accepted Manuscript

Quantitative Analsis Of Malachite Green And Leucomalachite Green Residues In

Fish Purchased From The Markets In Malaysia

Penz Penz Kwan, Sanjoy Banerjee, Mohamed Shariff, Nur Ain Syahira Ishak,
Fatimah Md. Yusoff

PII: S0956-7135(18)30194-4

DOI: 10.1016/j.foodcont.2018.04.031

Reference: JFCO 6093

To appear in: Food Control

Received Date: 02 February 2018

Revised Date: 13 April 2018

Accepted Date: 15 April 2018

Please cite this article as: Penz Penz Kwan, Sanjoy Banerjee, Mohamed Shariff, Nur Ain Syahira
Ishak, Fatimah Md. Yusoff, Quantitative Analsis Of Malachite Green And Leucomalachite Green
Residues In Fish Purchased From The Markets In Malaysia, Food Control (2018), doi: 10.1016/j.
foodcont.2018.04.031

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1 QUANTITATIVE ANALYSIS OF MALACHITE GREEN AND LEUCOMALACHITE

2 GREEN RESIDUES IN FISH PURCHASED FROM THE MARKETS IN MALAYSIA

4 Penz Penz Kwana, Sanjoy Banerjeea, Mohamed Shariffa,b*, Nur Ain Syahira Ishakb, Fatimah

5 Md. Yusoffa,c

7 aInstitute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia

8 bFaculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia

9 cDepartment of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM

10 Serdang, Malaysia

11

12 *Corresponding author

13 Mohamed Shariff

14 Phone: +60122839845

15 E-mail: [email protected]

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16 Abstract

17 Malachite green (MG) is still being used as an antiparasitic and antifungal agent in aquaculture.

18 This is due to the fact that many stakeholders are not aware of the potential genotoxic and

19 carcinogenic properties of MG. Since the consumption of fish in Malaysia was recorded to be

20 the highest in Southeast Asia in 2013, it is therefore important to ensure that fish sold in the

21 market are free of MG and its metabolite, leucomalachite green (LMG). In this study, MG and

22 LMG residues in muscle tissue of commonly consumed fish such as red tilapia (Oreochromis

23 hybrid), African catfish (Clarias gariepinus), barramundi (Lates calcarifer), hybrid grouper

24 (Epinephelus fuscoguttatus × Epinephelus lanceolatus) and striped catfish (Pangasius

25 hypophthalmus) were analysed using liquid chromatography tandem mass spectrometry. The

26 fish were purchased from 11 different markets in the state of Selangor, Malaysia. The results

27 showed that the sum residues of MG and LMG in the present study ranged from 0.53 to 4.10

28 µg/kg with the highest residue detected in domestic striped catfish. This study indicates that

29 MG is still being used and aquaculture stakeholders should be made aware of the judicious use

30 of MG for the benefit of public health.

31

32 Keywords: Fish; LC-MS/MS; Leucomalachite green; Malachite green; Residue

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33 1. Introduction

34 Fish is a common source of animal protein for the people. It contains long chain omega-3 fatty

35 acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Erdman, Oria,

36 & Pillsbury, 2011), as well as vitamins and minerals which are beneficial for a healthy diet

37 (Sidhu, 2003). In Malaysia, the per capita consumption of fish was 52.4 kg/year in 2013

38 (DOFM, 2014) being the highest in Southeast Asia. The commonly consumed fish recorded in

39 2016 for freshwater species were catfish, red tilapia, river catfish; and brackish/marine species

40 were sea bass, red snapper and grouper (DOFM, 2017). Given that the market demand and

41 consumption of fish is high in Malaysia, aquaculture plays an important role. To meet the

42 current demand, many farms culture fish in an intensive environment. However, intensive fish

43 farming induces stress and increases fish susceptibility to disease (Baldwin, 2010). In order to

44 prevent the occurrence of diseases, the use of chemicals such as acriflavine, methylene blue,

45 formalin and malachite green (MG) as a prophylactic treatment has become a common practice

46 (Mohamed, Nagaraj, Chua, & Wang, 2000).

47

48 Malachite green is one of the commonly used antifungal and antiparasitic agents in aquaculture.

49 However, due to its toxic effects and potential carcinogenesis, this chemical is not permitted to

50 be used in any animal or food for human consumption in the US (Leonard, 2011) and the EU

51 (EFSA, 2017). Fish exposed to MG treatment may undergo immunodepression. Studies on

52 common carp Cyprinus carpio treated with MG (3 mg/dm3 for 1 h, 4 times every 2 days)

53 revealed that the fish red blood cells and mean corpuscular haemoglobin concentration

54 decreased significantly, whereas the mean cell volume, haemoglobin level and percentage of

55 abnormal erythrocytes increased (Witeska, Kondera, & Belniak, 2013). In another study, using

56 proteomic analysis of primary cultured peripheral blood mononuclear cells of Asian catfish,

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57 Pangasianodon hypophthalmus exposed to MG at 1 and 10 ppb for 48 h revealed toxicity in

58 the mitochondrion (Pierrard et al., 2012). In a research conducted on the physiological

59 processes of zebrafish (Danio rerio), it was reported that the percentage of spawning pairs

60 dropped after 5-days exposure to 0.065 ppm MG (White, Davies, & Henry, 2012).

61

62 Apart from fish, the toxicity of MG and LMG has also been shown in mice. Toxicity in mice

63 can be closely related to humans as it is the most commonly used animal to model human

64 disease (Uhl & Warner, 2015). Thus, the toxicity of MG and LMG indicated in mice should be

65 of concern to humans. Studies on mice that were exposed to MG (27, 91, 272, 543 mg/kg b.

66 wt. for 7, 14, 21, 28 days) exhibited DNA damage in liver, biochemical disturbances in the

67 major glucolytic-gluconeogenic pathways, hepatic marker enzymes, and depleted glutathione

68 (Donya et al., 2012). In addition, Liu and Li (2014) found that MG showed deleterious dose-

69 related effects on mouse embryo exposed to 10, 100 and 1000 ng/mL MG for 72 h. Werth

70 (1958) as cited in Culp and Beland (1996), reported an increased number of tumours in Wistar

71 rats fed with MG at 20 to 33% of the lethal dose for a long period .

72

73 Given that MG is a drug without maximum residue limits (MRLs) (Rahkonen & Koski, 2002),

74 ‘zero tolerance’ provisions were established in the EU where no measurable levels of MG and

75 LMG residues are permitted in food or animal feed (Heberer, 2011), as both compounds are

76 not registered as veterinary drug with food producing animals (Heberer, 2009). The zero

77 tolerances were introduced to give consumers the greatest possible protection from substances

78 with possible potential hazard according to the hazard prevention principles and precautionary

79 measures (Heberer, 2011). Despite being restricted, MG is still being used in many countries

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80 (Srivastava, Sinha, & Roy, 2004), due to its low cost, effectiveness and lack of alternatives

81 (Alderman, 1985).

82

83 In the fish tissue, MG is absorbed and metabolised into leucomalachite green (LMG) which

84 persist for a long period in the fatty tissues due to its lipophilic nature (Fallah & Barani, 2014).

85 According to Anadón, Martínez-Larrañaga, Ares, Castellano, & Martínez (2017), lipophilic

86 chemicals can be stored and accumulated over time in the human fatty tissues and can be

87 mobilized into the milk during lactation. In addition, the researchers also stated that lipophilic

88 chemicals have the capacity to pass through the placental barrier and into the fetal blood stream.

89 Since LMG is able to accumulate in the fat tissue, skin and gonad of fish (Jiang, Xie, & Liang,

90 2009), there is a potential health hazard to the consumers due to the accumulation of this toxic

91 chemical in the long run. At present, although the consumption of fish is high in this country,

92 little information is available on the residue levels of MG and LMG in fish sold in the markets.

93 Hence, the aim of this study was to screen commonly consumed fish sold in the markets in

94 Selangor, Malaysia for residues of MG and LMG.

95

96 2. Materials and methods

97 2.1. Sample collection

98 Five species of fish from different domestic markets were sampled to analyse residues of MG

99 and LMG. Locally produced red tilapia (Oreochromis hybrid), African catfish (Clarias

100 gariepinus), hybrid grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus),

101 barramundi (Lates calcarifer) and domestic striped catfish (Pangasius hypophthalmus) as well

102 as imported frozen striped catfish fillet were purchased in triplicates from 11 different markets

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103 located around densely populated areas in the state of Selangor. The fish samples were

104 immediately placed in an icebox and transported to the laboratory to be kept at -20 °C.

105

106 2.2. Chemicals and reagents

107 All chemicals used were of HPLC or analytical grade unless stated otherwise. Acetonitrile,

108 methanol and formic acid were obtained from Merck (Germany). Hydroxylamine

109 hydrochloride was obtained from Sigma-Aldrich (USA) and d6-LMG standard was purchased

110 from Witega (Germany). Fresh ultrapure water was obtained using a Milli-Q system (Millipore,

111 Bedford, USA).

112

113 2.3. Sample preparation for LC-MS/MS analysis

114 The level of MG and LMG residues were studied in the fish muscle tissue using a sensitive and

115 reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method. This method

116 was adapted from Roudaut, Delepine, Bessiral, & Sanders (2005) with some modifications. In

117 brief, homogenized fish muscle (2 g ± 0.005 g) was carefully weighed (GR-200, AND, Japan)

118 into a 50 mL conical tube (Eppendorf, Germany). Subsequently, 600 µL ultrapure water was

119 added. Then, 2 mL of 5 g/L hydroxylamine HCl in ultrapure water was added and the mixture

120 was vigorously vortexed (VTX-3000L, Uzusio, Japan) for 30 sec and was mixed using an

121 orbital shaker (Protech, Malaysia) at 100 rpm for 10 min. Then, 8 mL of acetonitrile was added

122 to the mixture, vortexed for 30 sec and mixed using the shaker for 10 min at 100 rpm. The

123 mixture was then centrifuged at 7122 ×g for 10 min to separate the supernatant and the

124 precipitate. An aliquot of the supernatant was taken and filtered using 0.45 µm syringe filter

125 (Pall Corporation, New York) into a sterile amber glass vial. The internal standard (d6-LMG)

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126 was added with a final concentration of 20 µg/L. Then 20 µL of final solution was injected into

127 TSQ Quantum UltraTM triple stage quadrupole (QqQ) (Thermo Scientific, CA, USA).

128

129 The analysis was performed using Accela autosampler and pump UPLC (Thermo Scientific,

130 USA), with Hypersil GOLD 50 × 2.1 mm, 1.9 µm column (Thermo Scientific, USA). The flow

131 rate of the chromatography was set at 100 µL/min. whereas for the mass spectrometry, TSQ

132 Quantum was used with heated electrospray ionization (H-ESI) at 4003 V. The vaporizer and

133 capillary temperature were 200 °C and 350 °C respectively, and the total run time for each

134 sample was 15 min.

135

136 2.4. Statistical analysis

137 The data were analysed for statistical significance between different amount of MG and LMG

138 residue found in fish by analysis of variance (one-way ANOVA) and independent sample t-

139 test. For the analysis of variance, post hoc comparison was conducted using Tukey test. The

140 correlation was also performed between fish species and markets. The significance of the

141 results was ascertained at p<0.05.

142

143 3. Results and discussion

144 Table 1 shows residues of MG and LMG from five different fish species purchased from

145 different markets. Residues of MG and LMG were found in 16 out of 37 samples (43.2%)

146 (Table 2), with sum residues ranging from 0.53 to 4.10 µg/kg (Table 1). The mean

147 concentration of sum residues of MG and LMG was highest in domestic striped catfish,

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148 whereas the red tilapia had the lowest residues. From all the samples examined, 8.1% showed

149 above 2 µg/kg sum residues of MG and LMG which exceeded the minimum required

150 performance limits (MRPLs) of the Commision Decision (2004/25/EC), whereas in 54.1% it

151 was undetectable (Fig. 1). There were no correlation between the residues of MG and LMG

152 detected in different fish species and the source of markets they were purchased from.

153

154 Table 2 shows that there is a higher probability of purchasing barramundi fish (60%) with

155 residues of MG and LMG, followed by hybrid grouper (50%), domestic striped catfish (50%),

156 imported frozen striped catfish fillet (42.9%), African catfish (25%) and red tilapia (20%). The

157 different residue levels could be attributed to the hardiness of the fish species in terms of their

158 tolerance to stress and susceptibility to infectious agents. Thus, there is a lower probability of

159 purchasing fish with MG and LMG residues in hardy fish such as tilapia and catfish as

160 compared to barramundi and hybrid grouper. Tilapia (El-Sayed, 2006) and catfish (Davis,

161 Griffin, & Gray, 2002; El-Sayed, 2006) have higher resistance to stress and disease thus

162 requiring less frequent prophylactic treatments.

163

164 The present findings corroborated with the fact that MG is still being used by fish farmers in

165 different countries. In the EU (RASFF, 2017) and US (USDA, 2015), food is monitored

166 through a surveillance program for the presence of veterinary drug residues of possible health

167 concern such as MG and LMG. Data obtained from Rapid Alert System for Food and Feed

168 (RASFF) of the European Commission from 2005 to 2017, showed MG and LMG residues in

169 fish and fish products (Table 3). The recent data obtained from RASFF showed MG and LMG

170 residues detected in frozen pangasius fillet, climbing perch, mud goby fish, red tail barb, eel

171 and other fish tissues from different countries (Table 3). Apart from this, in Slovenia, 7 out of

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172 33 fish samples from the market and farms contained residues of MG (Bajc, Doganoc, &

173 Gačnik, 2007). In India, Tripathi, Khanna, & Das (2007) showed that foodstuffs from the rural

174 area contained more prevalent residues of MG than those from the urban markets. In Croatia,

175 18.1% of farmed fish had residues of MG (Bilandžić, Varenina, Kolanović, Oraić, & Zrnčić,

176 2012); and in China, Fu, Zheng, Qiu, Wang, & Fang (2013) reported 56.1% of freshwater fish

177 samples contained residues of MG ranging between 0.5 and 148 µg/kg.

178

179 In the present study, it is evident that the total amount of MG and LMG residues detected were

180 lower compared to other reported studies mentioned above. This may be due to several possible

181 reasons such as the warm climate in this tropical country as Bajc et al. (2011) reported that

182 temperature of the water can influence the persistence of MG and LMG residues in fish. In

183 warm water, fish has a higher metabolism rate that may accelerate the elimination of MG and

184 LMG residues from the fish. Jiang et al. (2009) reported that the persistence of MG and LMG

185 is also dependent on the fat composition of the fish tissue. This means that fish with higher

186 content of lipid will take longer elimination time for MG and LMG. In addition, the persistence

187 of MG is affected by the initial MG concentration and the growth rate of the fish (Sudova,

188 Machova, Svobodova, & Vesely, 2007). Another possible reason for the low amount of MG

189 and LMG detected in the present study could be due to the fact that accumulation of MG in the

190 fish was from an indirect source such as feed and/or contaminated water bodies.

191

192 Several studies reported that on an average, the residues of LMG were considerably higher than

193 MG in the fish tissues (Bajc et al., 2011; Bergwerff, Kuiper, & Scherpenisse, 2004; Tan et al.,

194 2011). This is because the lipophilic properties of LMG causes it to persist longer than MG

195 (Máchová et al., 1996). However the residues of MG in the present study, were by far higher

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196 than LMG (p<0.05) in most of the samples. Poe and Wilson (1983) reported that LMG can be

197 slowly oxidised back to MG during prolonged storage. The consistent lower levels of LMG

198 compared to MG in most samples from the present study was probably due to the storage

199 method of fish. Generally fish are highly perishable and have a relatively short shelf life

200 (Regenstein & Regenstein, 1991). In some local markets, stalls open at pre-dawn hours and

201 close in the evening and it is common to use ice flakes on the fish, but is not practiced in some

202 cases. Leaving fish exposed to ambient temperature will accelerate the microbiological and

203 enzymatic spoilage of the fish (Nor Hasyimah, James Noik, Teh, Lee, & Pearline Ng, 2011).

204 Fish in temperate regions have psychrotrophic (cold-tolerant) bacteria, whereas fish in the

205 tropical regions usually have non-psychrotrophic (mesophilic) spoilage bacteria (FAO, 1994)

206 which accelerates decomposition in the absence of ice. Lipids can be oxidised to peroxides,

207 aldehydes, ketones and lower aliphatic acids (Wąsowicz et al., 2004) and the speed of this

208 oxidation process is dependent on the surrounding temperature. Thus the warm temperature

209 may have influenced the oxidation of LMG back to MG resulting in higher levels of MG found

210 in the present study.

211

212 Although the residues of MG and LMG in fish from this study were found to be low, however,

213 in the long run, there could be possible adverse effect to the consumers. Mitrowska et al. (2007)

214 studied the stability of MG and LMG in carp muscle exposed to different cooking techniques

215 and found no reduction of LMG after boiling and baking for 15 min. Furthermore, human

216 intestinal microflora is capable of degrading MG to LMG (Henderson, Schmitt, Heinze, &

217 Cerniglia, 1997) which is more toxic (Culp et al., 1999), hence posing consumers to possible

218 health risk.

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219 4. Conclusion

220 Results of this study showed that residues of MG and its metabolite LMG were detected in

221 several fish species from the domestic markets. This could be due to the fact that many

222 stakeholders are not aware of the potential genotoxic and carcinogenic properties of MG. In

223 addition, the farmers, due to their ignorance, may not be adhering to the guidelines on the

224 judicious use of chemicals. The presence of such chemical in food for human consumption

225 should be monitored closely due to its health risk. This study serves as baseline information

226 for the government agencies which monitor and regulate the aquaculture industry. Therefore,

227 proper surveillance program should be enforced to avoid negative impact to the nation’s

228 aquaculture industry. This will ensure that food is free of harmful chemical residues and is safe

229 for human consumption.

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230 Acknowledgement

231 This study was funded by Fundamental Research Grant Scheme (FRGS), Malaysia, project

232 number 07-01-14-1487FR. The first author received a scholarship from MyBrain, KPT,

233 Malaysia for her PhD study. Appreciation also goes to the Faculty of Food Science and

234 Technology, Universiti Putra Malaysia for the use of LC-MS/MS.

235

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365

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60 54.1

Percentage (%) 50

40

30
18.9 18.9
20

10 5.4
2.7
0.0
0
n.d. >0 - 1 >1 - 2 >2 - 3 >3 - 4 >4 - 5
Concentration (µg/L)

Figure 1: Percentage of detection in fish with different range of total malachite green
(MG) and leucomalachite green (LMG) concentrations
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Highlights

 MG and LMG residues were detected in fish purchased from Malaysian markets

 Red tilapia, African catfish, barramundi, grouper, striped catfish were tested

 Fish samples had up to 4.10 µg/kg residues of MG and LMG

 Sensitive and reliable LC-MS/MS method for MG and LMG quantitation was used
1 Table 1: Malachite green (MG) and leucomalachite green (LMG) residues in muscles of different fish available in the market

Market Fish MG (µg/kg) LMG (µg/kg) MG + LMG (µg/kg)

1 Frozen striped catfish fillet (Import) n.d. n.d -
Striped catfish (Domestic) 1.77 ± 0.03c 0.05 ± 0.00j 1.82d
Striped catfish (Domestic) 0.41 ± 0.00m 0.31 ± 0.01f 0.72jk
Tilapia n.d. n.d. -
Barramundi 0.55 ± 0.02l 0.11 ± 0.01h 0.66kl
Hybrid grouper 0.74 ± 0.00ij n.d. 0.74j
2 Striped catfish (Domestic) n.d. n.d. -
Tilapia 0.44 ± 0.01m 0.09 ± 0.00hi 0.53m
African catfish 0.61 ± 0.01kl 0.11 ± 0.00h 0.72jk
Barramundi 0.92 ± 0.02g 0.36 ± 0.02e 1.28f
Hybrid grouper n.d. n.d. -
3 Striped catfish (Domestic) n.d. n.d. -
Tilapia n.d. n.d. -
African catfish n.d. n.d. -
Barramundi 1.12 ± 0.01f 0.06 ± 0.00ij 1.18g
4 Striped catfish (Domestic) n.d. n.d. -
Tilapia 0.33 ± 0.02n 0.30 ± 0.00f 0.63l
African catfish n.d. n.d. -
Barramundi n.d. n.d. -
Hybrid grouper n.d. n.d. -
5 Striped catfish (Domestic) 0.67 ± 0.02jk 0.23 ± 0.00g 0.90i
Tilapia n.d. n.d. -
African catfish n.d. n.d. -
Barramundi n.d. n.d. -
Hybrid grouper 0.55 ± 0.01l 0.52 ± 0.00c 1.07h
 6 Frozen striped catfish fillet (Import) 1.64 ± 0.00d 0.53 ± 0.02c 2.17*c
Striped catfish (Domestic) 3.51 ± 0.00a 0.59 ± 0.00b 4.10*a
7 Frozen striped catfish fillet (Import) n.d. n.d. -
Striped catfish (Domestic) n.d. n.d. -
8 Frozen striped catfish fillet (Import) n.d. n.d. -
Striped catfish (Domestic) n.d. n.d. -
9 Frozen striped catfish fillet (Import) n.d. n.d. -
Striped catfish (Domestic) n.d. n.d. -
10 Frozen striped catfish fillet (Import) 1.52 ± 0.01e 0.21 ± 0.00g 1.72e
Striped catfish (Domestic) 2.16 ± 0.03b 0.35 ± 0.00e 2.51*b
11 Frozen striped catfish fillet (Import) 0.84 ± 0.02h 0.88 ± 0.00a 1.72e
Striped catfish (Domestic) 0.76 ± 0.03i 0.43 ± 0.01d 1.19g

2 Values (mean ± standard error) with different superscript letters in the same column are significantly different (p<0.05); n.d.: not detected; *
3 indicates that value exceeds the MRPLs of 2 µg/kg set by the European Commission.
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4 Table 2: Percentage of fish detected with residues of malachite green (MG) and
5 leucomalachite green (LMG) according to different species

Type of fish Total number Fish with Percentage

of samples residues (%)
Barramundi 5 3 60.0
Hybrid grouper 4 2 50.0
Striped catfish (Domestic) 12 6 50.0
Frozen striped catfish fillet (Import) 7 3 42.9
African catfish 4 1 25.0
Red tilapia 5 1 20.0
Total 37 16 43.2

6 Results showed are a pool of 3 replicates for each sample.

Table 3: Residues of malachite green (MG) and leucomalachite green (LMG) found in fish and fish product (RASFF, 2017)

Date of Notifying country Fish or fish product Country of Substance Amount

case origin (µg/kg)
1 28/9/2017 Belgium Frozen catfish (Clarias spp.) Vietnam LMG -
2 19/5/2017 Switzerland Frozen whole catfish (Clarias macrocephalus) Vietnam LMG 29
3 6/12/2016 Netherlands Cultivated trout Denmark LMG 4.96
4 12/8/2016 Netherlands Cultivated trout Denmark LMG 5.9
5 4/3/2016 France Frozen shrimps (Penaeus monodon) Bangladesh MG 1.6
LMG 0.91
6 4/3/2015 United Kingdom Frozen fish Vietnam LMG 39
7 16/6/2014 United Kingdom Frozen red tail tin foil barb (Puntius spp.) Vietnam MG -
LMG -
8 23/4/2014 Germany Mud goby fish (Pseudapocryptes lanceolatus) Vietnam via the MG 6.26
Netherlands
9 22/4/2014 Germany Frozen Red Cheek Barbs (Puntius orphoides) Vietnam MG 4.6
10 17/4/2014 United Kingdom Frozen yellow catfish Vietnam MG 0.9
LMG 3.1
11 14/1/2014 Germany Frozen Climbing perch (Anabas testudineus) Vietnam via the LMG 18.9
Netherlands
12 20/12/2013 Poland Chilled carps Poland, with raw MG 9.21
material from the LMG 243, 78
Czech Republic
13 8/11/2013 Switzerland Walking catfish chunk (Clarias batrachus) Vietnam via the MG 36
Netherlands
14 1/7/2013 Czech Republic Chilled trout Poland LMG 6.66
15 24/1/2012 Austria Chilled trout Germany LMG 62.4
16 6/1/2012 Germany Frozen trout Turkey LMG 3.48
17 21/12/2011 United Kingdom Frozen catfish chunks Vietnam MG 1.6
LMG 3.9
18 9/12/2011 Germany Raw and frozen king prawn tails India via Belgium LMG 22.4
19 4/11/2011 Czech Republic Live trouts Poland LMG 1.08
20 1/11/2011 Czech Republic Live trouts Austria LMG 1.24
21 13/10/2011 United Kingdom Frozen gutted catfish Vietnam LMG 6.2
22 15/6/2011 Germany Cultured caviar (Huso dauricus) China LMG 4.39
23 21/4/2011 Germany Trout roe Denmark LMG 2.5
24 26/11/2010 Germany Trouts (Salmo trutta) Germany MG 3.3
25 8/10/2010 United Kingdom Frozen tilapia steaks China LMG 20
26 1/9/2010 France Frozen cooked shrimp tails Thailand LMG 3.3
27 16/3/2010 Poland Frozen pangasius fillet Vietnam MG -