Monitoring program for pharmaceuticals, illegal substances, and contaminants in farmed fish

— – Annual report for 2018

Sammendrag

This report summarises the monitoring data collected in 2018 on the status of illegal substances, pharmaceuticals and contaminants in Norwegian farmed fish. A total of 13 920 fish were collected. Samples examined for illegal compounds could be collected at all stages of farming and are representative of farmed fish under production. The samples were analysed for substances with anabolic effects or unauthorized substances. No residues of illegal compounds were detected. Samples tested for approved veterinary drugs and contaminants were collected at processing plants, and are representative of Norwegian farmed fish ready for human consumption. Residues of anti sea lice agents were found in four samples, the levels present were below the Maximum Residue Limit (MRL) for all samples. Other veterinary drugs, like antibiotics or drugs used against internal parasites were not found. No environmental contaminants were found above the EU maximum limits.

1 - Introduction

1.1 - Background

According to EU legislation (96/23/EC), all food producing animals should be monitored for certain substances and residues thereof. The following residues or substance groups are monitored in Norwegian farmed fish:

Group A Substances with anabolic effects and unauthorized substances:

A1: Stilbenes, derivatives and their salts and esters

A3: Steroids

A6: Prohibited substances

Group B Veterinary drugs and contaminants:

B1: Antibacterial agents

B2a: Anthelmintics

B2d: Sedatives

B3a: Organochlorine compounds

B3b: Organophosphorus compounds

B3c: Chemical elements

B3d: Mycotoxins

B3e: Dyes

B3f: Others

 

1.2 - Group A, Substances with anabolic effects and unauthorized substances

Fish tested for illegal compounds were collected at the farm by official inspectors from the Norwegian Food Safety Authorities, without prior notification to the farmers. Sampling can be done at all stages of farming and are representative of farmed fish during production. Group A includes growth promoters like steroids and stilbenes, and unauthorized drugs. Unauthorized drugs considered most relevant for aquaculture are chloramphenicol, nitrofurans, metronidazole and dyes. The dyes; malachite green, crystal violet and brilliant green are not allowed to use for food producing species (EU 2010/37), they are therefore considered an A substance and hence sampled throughout the production chain. However, according to directive 96/23 these dyes belong to the group B3e. Therefore, some of the samples assigned to analysis of dyes were also collected at the slaughterhouse.

To ensure harmonized levels for the control of unauthorized substances, the analytical methods should meet a minimum required performance limits (MRPLs) set by the European Union (2002/657/EC), and European reference laboratories (EU-RLs), (CRL 2007). Table. 7.3 gives an overview of MRPLs of relevant compounds.

 


1.3 - Group B, veterinary drugs

In order to protect public health, Maximum Residue Limits (MRLs) have been established. According to current EU legislation (EU 37/2010) each substance is assigned a MRL, which is the highest permitted residual concentration of legally applied pharmacologically active substances in products intended for human consumption. The MRLs for fish are set for muscle and skin in natural proportions. Samples examined for veterinary drugs were collected from fish at processing plants and the samples are representative of fish ready to be placed on the market for human consumption. In order to use a veterinary drug for food producing animals, a maximum residue limit (MRL) has to be evaluated. The MRL is the highest permitted residual concentration of legally applied pharmacologically active substances in animals or animal products intended for human consumption. Consumption of food with drug residues below the MRL should not pose a health risk to the consumer.

 

1.4 - Group B, contaminants

Samples examined for contaminants were collected from fish at processing plants, and are representative of fish ready for human consumption. The EU (EU 1881/2006) has set a Maximum limit (ML) for some of the contaminants in fish, while for others, like the pesticides, PAH, PFC and BFR, maximum limits have not been established.

 

1.5 - Ethoxyquin

Ethoxyquin (EQ) is a synthetic antioxidant, widely used as an additive (E324) in components for animal feed for pets and livestock to preserve product quality and increase shelf live. Because of its high efficacy, EQ has also been widely used by the global fishmeal industry both as a nutritional preservative, but also to avoid oxidation and self-ignition under long-distance transport.

2 - Material and methods

2.1 - Sampling

Samples were taken on fish farms or slaughterhouses, by official inspectors, in all fish-producing regions in Norway. The sampling plan was randomised according to season and region. In 2018, the following fish species were included in the monitoring program: Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), Atlantic halibut (Hippoglossus hippoglossus), Arctic char (Salvelinus alpinus) and Atlantic cod (Gadus morhua).Samples were transported to IMR in a frozen state. For most samples, the Norwegian quality cut (NQC) was used for further analyses (Johnsen 2011). However, for most of the samples collected for analysis of antibiotics, individual livers were also collected. Samples to be used for analyses of substances with anabolic effects or unauthorized substances also included small fish from early life stages and in these cases, the whole fish except head, tail and gut were homogenised. The samples were analysed as pooled samples comprising five fish from the same cage/farm.

 

2.2 - Pre-treatment

Upon arrival at IMR the sample identification were anonymised for the analysts. A back-up sample was stored for all samples. Pooled samples of muscle from five fish from the same cage/farm were homogenised before analyses. Samples of liver were excised from the fish in samples to be screened for residues of antimicrobial agents by the microbiological inhibition zone assay. Liver samples were examined individually, if residues were detected, the back-up sample of muscle would be analysed by chemical methods. The maximum residue limits for veterinary drugs are set for muscle and skin in natural proportions (EU 37/2010). Therefore, according to the analytical protocol, any detection of drug residues in the muscle or liver would be followed by a re-analysis of the back up sample, consisting of muscle and skin in natural proportions, in duplicate.

 

2.3 - Analytical methods

The laboratory routines and most of the analytical methods are accredited in accordance with the standard ISO 17025 (Table 7.3). A summary of the analytical methods and their limit of detection (LOD) or limit of quantification (LOQ) is shown in table 7.3. The LOD is the lowest level at which the method is able to detect the substance, while the LOQ is the lowest level for a reliable quantitative measurement. For all methods, a sample blank and a quality control sample (QC) with a known composition and concentration of target analyte, are included in each series. The methods are regularly verified by participation in inter laboratory proficiency tests, or by analysing certified reference material (CRM), where such exist.

 

2.3.1 - Group A substances

A1, Stilbenes

Stilbenes were extracted by water and acetonitrile. Liquid-liquid extraction was used for sample clean up. The stilbenes were and analysed by LC-MS/MS.

A3, Steroids

Steroids were extracted by water and acetonitrile. Liquid-liquid extraction followed by solid phase extraction was used for sample clean up, before the samples were analysed by LC-MS/MS.

A6, Illegal veterinary drugs

Chloramphenicol

Chloramphenicol was extracted with ethyl acetate. Liquid-liquid extraction was used to purify the extract. The samples were analysed by LC-MS/MS.

Nitrofurans

The nitrofuran metabolites were extracted with aqueous hydrochloric acid and derivatized with nitrobenzaldehyde. Solid phase extraction was used for sample clean up. The analytes were determined by LC-MS/MS.

Metronidazole

Metronidazole and its metabolite hydroxymetronidazole were extracted by ethyl acetate. Solid phase extraction was used for sample clean up. The analytes were determined by LC-MS/MS

Malachite green (MG), crystal violet (CV), brilliant green (BG)

The analytes were extracted with acetonitrile and dichloromethane. Samples clean-up were performed by solid phase extraction. MG, CV, BG and the metabolites leuco malachite green (LMG) and leuco crystal violet (LCV), were determined by LC-MS/MS.

 

2.3.2 - Group B substances

B1, Antibacterial agents (antibiotics)

The presence of antibacterial agents was determined by a three plate microbiological assay or by chemical analysis.

Microbiological assay

For the three-plate microbiological inhibition method, a specific bacterial strain was added to a plate containing growth agar and. Small pieces of liver were placed on the plates before incubation. If the samples contained residues of antibacterial agents, the bacterial growth would be inhibited in a zone around each piece of liver tissue. Thus, a transparent zone with no bacterial growth surrounding the liver sample would indicate a positive sample. Any positive detection had to be verified by chemical analysis of muscle and skin.

Oxolinic acid, flumequine, enrofloxacin, ciprofloxacin and trimethoprim

The analytes were extracted with acetonitrile and water. The analysis was performed by LC-MS/MS.

Oxytetracyclin

The analyte was extracted with acetonitrile. Liquid-liquid extraction was used to purify the extract. Oxytetracyclin was analysed by LC-MS/MS.

Florfenicol

The analyte was extracted with ethyl acetate. Liquid-liquid extraction was used to purify the extract. The samples were analysed by LC-MS/MS.


B2a, Anthelmintics

Diflubenzuron, teflubenzuron, lufenuron and hexaflumuron

The analytes were extracted with acetone. Solid phase extraction was used for sample clean up. The samples were analysed by LC-MS/MS (Samuelsen et al. 2014).

Emamectin

Emamectin was extracted with acetonitrile, and analysed by LC-MS/MS.

Ivermectin

Ivermectin was extracted with organic solvent, and the extract were purified by solid phase extraction. The samples was analysed by LC-MS/MS

Cypermethrin and deltamethrin

Cypermethrin and deltamethrin were extracted by soxhlet extraction. The extracts were purified by gel permeation chromatography. The samples were analysed by GC-MS/MS.

Fenbendazole

Fenbendazole was extracted using methanol and water. Sample clean up was performed by liquid-liquid extraction. The samples were analysed by LC-MS/MS.

Praziquantel

Praziquantel was extracted from the sample by acetone, and analysed by LC-MS/MS.

B2d, Sedatives

Isoeugenol

Isoeugenol is analysed by GC coupled to a flame ionization detector (FID).

B3a, Organochlorine compounds

Dioxins, dl-PCBs, PCB-6 and PBDEs.

This is an adaptation to modern clean-up equipment of the US-EPAs (Environmental Protection Agency) methods No. 1613 and 1668. Separation and quantification were performed by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The method measures all of the 29 compounds on the WHO list: 17 PCDD / PCDF congeners, four non-ortho substituted PCBs: PCB -77, 81, 126 and 169 and eight mono-ortho substituted PCBs: PCB-105, 114, 118, 123, 156, 157, 167 and 189 (Berntssen, Julshamn et al. 2010). The PCBs included in PCB-6, PCBs no. 28, 52, 101, 138, 153 and 180, were analysed by GC-MS/MS. The PBDEs were analysed by GC/MS in a relevant solvent fraction from the EPA clean-up procedure (Pirard, De Pauw et al. 2003).

PCB-6

The six PCBs were extracted by hexane using an accelerated solvent extractor. The extract was purified by sulphuric acid before detection and quantification by GC-MS (Berntssen et al. 2011). The method quantifies the PCBs no. 28, 52, 101, 138, 153 and 180.


Chlorinated pesticides

Pesticides were extracted by organic solvent, and the extract were cleaned-up by column chromatography, before the pesticides were analysed by HRGC-HRMS.

B3b, Organophosphorus compounds

Azamethiphos and dichlorvos

The analytes were extracted with acetonitrile, and analysed by LC-MS/MS.

Chlorpyriphos and Pirimiphos

Chlorpyriphos, chlorpyrifos-methyl, pirimiphos-methyl and pirimiphos-ethyl were extracted by soxhlet extraction. The extracts were purified by gel permeation chromatography. The samples were analysed by GC-MS/MS.

B3c, elements

Lead, mercury, cadmium and arsenic

The sample was decomposed by acid treatment, assisted by heat and high pressure. The metals were analysed by inductively coupled plasma mass spectrometer (ICP-MS) (Julshamn, Maage et al. 2007).

Inorganic Arsenic

Inorganic arsenic was extracted by hydrochloric acid in hydrogen peroxide at 90 °C. Inorganic arsenic includes As (III) and As (V). As (III) was oxidised to As (V) during the extraction. Inorganic arsenic was separated from other arsenic compounds by anionic exchange HPLC, and detected by ICP-MS.

Methylmercury

Methylmercury was extracted by Tetramethylammonium Hydroxide. The pH was adjusted before derivatization and extraction by hexane. The samples were analysed by GC-ICP-MS.

Tributyltin

Tributyltin was extracted by acetic acid/methanol. The pH was adjusted before derivatization and extraction by hexane. The samples were analysed by GC-ICP-MS.

B3d, Mycotoxins

Enniatin and beauvericin

The mycotoxins; beauvericin, enniatin A, enniatin A1, enniatin B and enniatin B1were extracted with acetonitrile and water. Solid phase extraction was used for sample clean up. The mycotoxins were analysed by LC-MS/MS.

B3f, Others

HBCD

HBCD was extracted by a soxhlet apparatus, using a mixture of acetone and hexane. Sulfuric acid was used for purification. The extract was further cleaned up by an alumina column. The HBCD isomers were analysed by LC-MS/MS.


TBBPA

TBBPA was extracted by a soxhlet apparatus using a mix of acetone and hexane. Sulfuric acid was used for purification. O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was used for derivatization. The extract was purified using column chromatography. TBBPA was analyzed by GC-MS using Electron Ionization (EI).

PFC

PFCs were extracted by methanol, the extract was purified by solid phase extraction. PFCs were analysed by LC-MS/MS.

PAH

PAHs were extracted by dichloromethane and cyclohexane using an Accelerated Solvent Extractor (ASE). The extract was purified by solid phase extraction and analysed by GC-MS/MS.

Ethoxyquin

EQ and EQDM were extracted with hexane from pooled muscle samples, after saponification in a mixture of ethanol, NaCl and NaOH. EQ and EQDM were quantified by reversed-phase high-performance liquid chromatography with fluorescence detection, using an external standard curve, as previously described by Bohne et al. (2007), with modifications described by Ørnsrud et al. (2011).

Table 2.1. Number of fish analysed for each substance.

CompoundsFishAtlantic salmonRainbow troutAtlantic halibutArctic charTurbot
A1 Stilbenes
Zeranol, 17alpha-Estradiol, 17alpha-Ethinyl-estradiol, 17beta-Estradiol, beta-Zearalanol, Dienestrol, Diethylstilbestrol, Estriol, Estrone, Hexestrol81576050 5 
A3 Steroids
16-Hydroxystanozolol, 17alpha-Boldenone, 17alpha-Trenbolone, alpha-Nandrolone, Boldenone, Chlor-Testosterone, Epitestosterone, Methyl-Boldenone, Methyltestosterone, Nortestosterone, Stanozolol, Testosterone, Testosterone propionate, Trenbolone, Trenbolone-acetate81576550   
A6 Illegal drugs
Chloramphenicol82077050   
Metronidazole8157703510  
Nitrofuran metabolites (AOZ, AMOZ, AHD, SEM)8257704510  
Malachite green, Crystal violet, Brilliant green*83577545105 
B1 Antibiotics
Florfenicol26025010   
Oxytetracycline100100    
Flumequine, Oxolinic acid, Enrofloxacin, Ciprofloxacin, Trimethoprim36034515   
Quinolones (liver), Tetracyclines (liver), Amphenicols (liver), Sulphonamides (liver)150013651155105
B2 Other veterinary drugs
Emamectin84581040 5 
Cypermethrin, Deltamethrin60057525   
Diflubenzuron, Teflubenzuron, Hexaflumeron, Lufenuron80074055 5 
Ivermectin756010 5 
Praziquantel49545540   
Fenbendazole50455   
Isoeugenol140140    
B3a Organochlorine compounds
Pesticides48544540   
Dioxin and dl-PCBs3503405  5
PCB-6680650205 10 5
B3b, Organophosphorus compounds
Azamethiphos, Dichlorvos2502455   
Chlorpyriphos, Pirimiphos60057525   
B3c Chemical elements
Lead Cadmium Mercury Arsenic43541025   
Inorganic arsenic Methylmercury100955   
Tributyltin26025010   
B3d, Mycotoxins
Beauvericin, Enniatin49546030 5 
B3e, Dyes
Malachite green, Crystal violet, Brilliant green *4604555   
B3f, Others
PBDE3503405  5
HBCD, TBBPA34032020   
PAH35532525 5 
PFC36535015   
Ethoxyquin37533535 5 

Some of the samples collected have been analysed by more than one method. Therefore, the total of fish in this table will be higher than the number of fish collected.*

According to directive 96/23, malachite green, crystal violet and brilliant green belongs to the group B3e. However, these dyes are not allowed to be used for food producing animals, therefore samples analysed for dyes have been collected as both group A samples (illegal drugs) and group B samples (dyes).

3 - Results

3.1 - Substances with anabolic effects and unauthorized substances

A total of 1 085 pooled fillet samples from 5 425 fish, were examined for of illegal substances. The analyzed samples were mainly from Atlantic salmon, but also samples from rainbow trout, and Arctic char were examined. For illegal substances, any presence of the compound will lead to a non-compliant result, regardless of the level.

 

3.1.1 - Stilbenes

The presence of stilbenes was examined in 163 pooled samples. None of the included stilbenes was detected in the samples analysed.

 

3.1.2 - Steroids

The presence of steroids was examined in 163 pooled samples. None of the substances was detected in the samples analysed.

 

3.1.3 - Unauthorized veterinary drugs

Totally 751 pooled samples were analyzed for unauthorized veterinary drugs. No residues of malachite green, crystal violet, brilliant green, chloramphenicol, nitrofurans or metronidazole were detected.

 

3.2 - Veterinary drugs

Samples analysed for veterinary drugs were collected from fish at processing plants and are representative of fish ready for human consumption. The maximum residue limit for veterinary drugs is defined for muscle and skin in natural proportions (EU 37/2010). Therefore, according to the analytical protocol, any detection of drug residues in the muscle or liver would be followed by a re-analysis of the backup sample, consisting of muscle and skin in natural proportions, in duplicate.

 

3.2.1 - Group B1, antibacterial agents

The antibacterial agents were determined by a combination of the three plate bioassay and chemical methods. The broad groups a) quinolones, b) amphenicols and tetracyclines and c) sulphonamides, were measured in livers from 1 500 fish. Florfenicol, (52 pooled samples), oxytetracyclin (20 pooled samples) and flumequin, oxolinic acid, enrofloxacin, ciprofloxacin and trimethoprim (72 pooled samples) were also analysed by chemical methods. No residues were detected in any of the analysed samples. The LOQs of the respective compounds are listed in Table 7.3.

 

3.2.2 - Group B2a anthelmintics

The levels of the anthelmintics; teflubenzuron, diflubenzuron, cypermethrin, deltamethrin, emamectin, ivermectin, praziquantel or fenbendazole were determined in 510 pooled muscle samples representing 2 550 fish. Emamectin was detected in 3 out of 162 pooled samples of Atlantic salmon. The highest concentration of emamectin was found at 5.2 µg/kg. This concentration is below the MRL of 100 µg/kg (EU 37/2010). Residues of the anti sea lice agent lufenuron was found in one sample at a concentration of 12 µg/kg. The MRL for lufenuron is 1350 µg/kg (EU 37/2010). Residues of other agents in this group were not detected in any of the samples. LOQs for the substances are specified in Table. 7.3.

 

3.2.3 - Group B3b. Organophosphorous compounds

No residues of azamethiphos or dichlorvos were detected in the 50 samples analysed for these analytes.

 

3.2.4 - Group B3d. Sedatives

Residue of isoeugenol was not detected in any of the 28 samples analysed for this sedative.

 

3.3 - Contaminants

Samples analysed for contaminants were collected from fish at processing plants, and are representative of fish ready for human consumption.

 

3.3.1 - Group B3a, Organochlorine compounds

The levels of organochlorine compounds were determined in 239 pooled samples. The results are summarised in Table 3.1 to 3.3.

 

3.3.2 - Organochlorine pesticides

For a number of the pesticides, the amount present is calculated as a sum including metabolites or transformation products (SANTE 2015). The results for these groups of pesticides are presented in table 3.1.

Table 3.1. The sum of groups of pesticides (µg/kg w.w.) in fillets of farmed fish

 Atlantic SalmonRainbow trout
SumSamples898
DDTMedian (UB)6.58.8
Max (UB)1312
EndosulfanMedian (UB)0.850.86
Max (UB)3.10.89
Aldrin and dieldrinMedian (UB)1.31.6
Max (UB)3.62.2
ChlordaneMedian (UB)1.01.1
Max (UB)2.51.7
HeptachlorMedian (UB)0.470.50
Max (UB)1.30.67
ToxapheneMedian (UB)2.62.5
Max (UB)9.15.5

To calculate the sum of the components, conversion factors (table 7.4) are used to adjust for different molecular weights (SANTE 2015). The sums in table 3.1. were calculated according to the upper bound (UB) formula. When using UB calculations, the numerical value of LOQ is substituted for analytes with levels below LOQ. UB represents a “worst case scenario”. As an example, all measurements of endosulfan are below LOQ, however, a sum is generated based on the LOQ-values.

The results for the other pesticides are summarised in Table 3.2. The highest level measured was 2.3 µg/kg w.w. of trans-nonachlor and 4.1 µg/kg w.w. hexachlorobenzene.

Table 3.2. Pesticides (µg/kg w.w.) in fillets of farmed fish.

PesticideAtlantic salmonRainbow TroutLOQ
 Samples898 
α-Hexachlorocyclo- hexane#Values120 
MedianLOQLOQ 
Max0.24LOQ0.13-0.60
β-Hexachlorocyclo- hexane#Values32 
MedianLOQLOQ 
Max0.190.260.13-0.60
δ-Hexachlorocyclo- hexane#Values00 
MedianLOQLOQ 
MaxLOQLOQ0.13-0.60
γ-Hexachlorocyclo- hexane#Values00 
MedianLOQLOQ 
MaxLOQLOQ0.13-0.60
Hexachlorobenzene#Values888 
Median1.41.5 
Max4.12.70.06-1.0
Pentachlorobenzene#Values30 
MedianLOQLOQ 
Max0.37LOQ0.30-1.2
Trans-Nonachlor#Values898 
Median0.741.0 
Max2.31.90.13-0.60
Endrin#Values40 
MedianLOQLOQ 
Max0.49LOQ0.15-0.71
Mirex#Values214 
MedianLOQLOQ 
Max0.120.110.05-0.24
Octachlorstyrol#Values827 
Median0.090.10 
Max0.180.200.03-0.12

3.3.3 - Dioxin, dl-PCBs and PCB-6

The levels of dioxin, dl-PCBs and PCB-6 in farmed fish are shown in Table 3.3. The data is mainly represented by Atlantic salmon, but also samples from rainbow trout, Atlantic halibut, and turbot have been examined.

The sums of dioxins, dioxins + dl-PCBs and PCB-6 are calculated as upper bound (EU 1259/2011). Accordingly, the numerical LOQ values were used for congeners with levels below LOQ.

The level of dioxins and dl-PCBs are reported as ng toxic equivalents 2005 (TEQ05)/kg, and represents the sum of 17 different PCDD/F and 12 dl-PCBs where each congener was multiplied by a Toxic Equivalency Factor (TEF). TEF values are determined by WHO, and the toxicity of each congener has been expressed relative to the most toxic form of dioxin, 2,3,7,8-TCDD which has a TEF value of 1(EU 1259/2011).

For salmon, the median of the sum of dioxins was 0.23 ng TEQ/kg w.w. The maximum value of 0.47 ng TEQ/kg w.w. is below the EU maximum limit of 3.5 ng TEQ/kg w.w.

The median of the sum of all 29 PCDD/F and dl-PCBs was 0.58 ng TEQ/kg w.w for salmon. The highest result for salmon was 1.3 ng TEQ/kg w.w. All values were below the EU maximum limit of 6.5 ng TEQ/kg w.w.

The median of PCB-6 for salmon was 4.7 μg/kg w.w. The EUs maximum limit for PCB-6 in fish is 75 μg/kg w.w. and the highest concentration of PCB-6 measured in 2018 was 12 μg/kg w.w. in an Atlantic halibut sample.

Table 3.3 Dioxins, dl-PCBs and PCB-6 in fillets of farmed fish.

 Atlantic SalmonRainbow troutAtlantic halibutTurbotMaximum limit
 Samples681 1 
Sum dioxins (ng TEQ/kg w.w.)Median0.23- - 
Max0.470.24 0.173.5
Sum dioxin + dl-PCBs (ng TEQ/kg w.w.)Median0.58- - 
Max1.30.60 0.656.5
 Samples130411 
PCB-6 (µg/kg w.w.)Median4.76.0-- 
Max118.9125.575

 

3.3.4 - Group B3b. Organophosphorous compounds

The pesticides chlorpyriphos, chlorpyrifos-methyl, pirimiphos-methyl and pirimiphos-ethyl were analysed in totally 120 pooled samples, 115 of the samples were salmon and 5 of the samples were rainbow trout, no residues were found.

 

3.3.5 - Group B3c, Chemical elements

In 2018, the highest measured concentration of total mercury were 0.062 mg/kg w.w. in a salmon sample and 0.027 mg/kg w.w. in a rainbow trout sample (Table 3.4). The EU maximum limit is 0.50 mg/kg w.w. for mercury in the species analysed in this report (EU 1881/2006). Thus, the concentrations measured in all samples are below the maximum limit. In addition to mercury, methylmercury was measured in 20 samples. The result showed that the levels of methylmercury (Table 7.1) were similar to the level of mercury in the same samples.

The concentrations of cadmium in most samples analysed since 2002 have been lower than the LOQ. In 2018, one sample had levels above the LOQ. The highest concentration measured was 0.002 mg/kg w.w. which is well below EUs maximum limit of 0.05 mg/kg w.w. (EU 1881/2006).

Arsenic is determined as “total arsenic”, comprising the sum of all arsenic species. The median level of total arsenic in Atlantic salmon was 0.75 mg/kg w.w., and the highest concentration measured was 2.2 mg/kg w.w. (Table 3.4). None of the samples had concentrations of inorganic arsenic above the LOQ (Table 7.1), indicating that arsenic in fish is present mainly as organo-arsenic compounds of low toxicity (Shiomi 1994). There is currently no EU upper limit for neither total arsenic nor inorganic arsenic in fish fillets.

A quantifiable concentration of lead was detected in one of the 87 samples analysed. The EU maximum level for lead in muscle meat of fish is 0.30 mg/kg w.w. (EU 1881/2006). The highest concentration measured was 0.011 mg/kg w.w. Thus, all samples were well below the limit.

Monobutyltin was found in concentrations above the LOQ in 12 out of 50 samples analyzed, the maximum level was 2 µg/kg w.w.. All samples analysed for dibutyltin were below the LOQ. Tributyltin was detected in 17 of the analysed samples. The highest measured level was 0.3 µg/kg w.w.. There is currently no EU upper limit for tin in fish fillet.

Table 3.4. Chemical elements in fillets of farmed fish

Element Atlantic SalmonRainbow troutArctic CharCodLOQEU- Limit
 N825    
Mercury (mg/kg w.w.)#Values805    
Median0.0200.022    
Max0.0620.027  0.0020.50
Arsenic (mg/kg w.w.)#Values825    
Median0.750.79    
Max2.21.1  0.003n.a.
Cadmium (mg/kg w.w.)#Values10    
Median--    
Max0.002LOQ  0.009-0.0020.050
Lead (mg/kg w.w.)#Values10    
Median--    
Max0.011LOQ  0.005-0.010.30
 N502    
Monobutyltin (µg Sn/kg w.w.)#Values111    
Median0.5-    
Max20.40  0.4-1n.a.
Dibutyltin (µg Sn/kg w.w.)#Values00    
Median--    
MaxLOQLOQ  0.2-0.5n.a.
Tributyltin (µg Sn/kg w.w.)#Values152    
Median0.080.3    
Max0.20.3  0.06-0.09n.a.

 

3.3.6 - Group B3d, Mycotoxins

In 2018, 93 pooled samples were analysed for enniatin A, enniatin A1, enniatin B, enniatin B1 and beauvericin. No residues of these mycotoxins were detected.


3.3.7 - Group B3f, others

The group B3f, others is a group not required for finfish products by the directive 96/23EC, but are deemed relevant for analyses in Norwegian aquaculture by the NSFA and IMR. This group currently consist of brominated flame retardants (BFR), perfluorinated compounds (PFC) and polyaromatic hydrocarbons (PAHs). These are undesirable compounds present in the environment and may affect food safety. In addition, in 2018, levels of the technological feed additive ethoxyquin (EQ) and its main transformation product ethoxyquin dimer (EQDM) were examined.

 

3.3.8 - Brominated flame retardants

PBDE, TBBPA and HBCD are compounds used as flame retardants. The summarised PBDE-7 (28, 47, 99, 100, 153, 154, 183) and PBDE 66, 119 and 138 are shown in Table 3.5. The highest level of PBDE-7 was 0.96 μg/kg w.w. with a median value of 0.46 μg/kg w.w for salmon. Out of 64 pooled samples of Atlantic salmon and 4 pooled samples of rainbow trout, TBBPA was found at a quantifyable level in one sample of salmon (0.05 μg/kg w.w.). HBCD was analysed in 68 samples, the highest level was 1.2 μg/kg w.w. The median concentration of HBCD in salmon was 0.11 μg/kg w.w.. There is currently no EU maximum limit for BFRs in food.

Table 3.5 BFR (µg/kg w.w.) in fillets of farmed fish.

 Atlantic SalmonRainbow troutTurbotLOQ
 Samples6811 
UB-Sum PBDE 7Median0.46-- 
Max0.960.610.40 
PBDE 66#Values6811 
Median0.011-- 
Max0.0350.0100.0110.002-0.01
PBDE 119#Values4111 
Median0.0030-- 
Max0.0140.00270.00270.002-0.01
PBDE 138#Values000 
Median--- 
MaxLOQLOQLOQ0.003-0.02
 Samples6440 
TBBPA#Values10  
Median--  
Max0.05- 0.03-0.14
 Samples6440 
UB-Sum HBCD(α,β,γ)Median0.120.11  
Max1.20.27  

 

3.3.9 - Perfluorinated compounds

A total of 73 samples were analysed for the PFCs. All results were below the LOQ (Table 7.3). EU has no maximum level for PFC in food.


3.3.10 - Polycyclic aromatic hydrocarbons

The results for PAH are summarised in table 3.6. PAH was analysed in 71 samples, of which 65 samples were from salmon, five from rainbow trout and one was Arctic char. There is no maximum limit for PAH in fresh fish (EU 835/2011).

Table 3.6 PAH (µg/kg w.w.) in fillets of farmed fish.

PAHAtlantic salmonRainbow troutArctic charLOQ
 Samples6551 
5-methylchrysene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Benz(a)anthracene#Values5000.09 - 0.13
Max0.2LOQLOQ 
Benzo(a)pyrene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Benzo(b)fluoranthene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Benzo(c)fluorine#Values1000.09 - 0.13
Max0.2LOQLOQ 
Benzo(ghi)perylene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Benzo(j)fluoranthene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Benzo(k)fluoranthene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Chrysene#Values8000.09 - 0.13
Max0.3LOQLOQ 
Cyclopenta(cd)pyrene#Values2000.09 - 0.13
Max0.1LOQLOQ 
Dibenz(ah)anthracene#Values0000.09 - 0.13
MaxLOQLOQLOQ 
Dibenzo(a,e)pyrene#Values0000.44 – 0.66
MaxLOQLOQLOQ 
Dibenzo(a,h)pyrene#Values0000.44 – 0.66
MaxLOQLOQLOQ 
Dibenzo(a,i)pyrene#Values0000.44 – 0.66
MaxLOQLOQLOQ 
Dibenzo(a,l)pyrene#Values0000.44 – 0.66
MaxLOQLOQLOQ 
Indeno(1,2,3,-cd)pyrene#Values0000.09 - 0.13
MaxLOQLOQLOQ 

3.3.11 - Ethoxyquin

EQ and EQDM levels were measured in 74 pooled fillet samples. The samples were mostly taken from Atlantic salmon, but also samples from rainbow trout and Atlantic char were included (Table 3.7 of EQ and EQDM was calculated as upper bound, using the numerical LOQ values for measurements below LOQ.

For salmon samples, the median level of the sum of EQ&EQDM was 0.11 mg/ kg ww. Rainbow trout and trout contained EQ&EQDM at a median concentration of 0.09 mg/kg ww. One sample of Atlantic char was analysed and this sample contained 0.04 mg EQ&EQDM/ kg ww .

The maximum values of EQ and EQDM were 0.01 and 0.34 mg/kg ww, respectively, and were found in salmon.

Table 3.7 Ethoxyquin (mg/kg w.w.) in fillets of farmed fish.

 Atlanti salmonRainbow troutAtlantic charLOQ
 Samples6771 
EQ (mg/kg ww)#Values417- 
Median0.0020.001LOQ 
Max0.010.009LOQ0.001
EQDM (mg/kg ww)#Values6671 
Median0.110.08- 
Max0.340.150.040.005
Sum EQ&EQDM (mg/kg ww) UB     
Median0.110.09- 
Max0.340.150.04 

4 - Discussion

4.1 - Unauthorized substances

No residues of unauthorized substances were detected in any of the analysed samples.

 

4.2 - Veterinary drugs

Most samples reviewed in this report are from fillets of farmed fish. However, as the liver has a central function in the distribution and elimination of veterinary drugs, liver samples were analysed for antibiotics. Even though the bioassay used for the antibacterial agents is less sensitive than the chemical analytical methods, the higher concentrations of antibacterial agents in liver compared to fillet enhance the ability to detect any residues. Moreover, the ability of the bioassay to detect a wider range of antibiotics than the more specific chemical methods renders the method useful for screening purposes. Any positive detection by the inhibition assay is verified by chemical analysis of the corresponding fillet sampled from the same fish. In accordance with previous results from the last years, no residues of antibiotics or endoparasitic agents were detected.

Residues of the anti sea lice agents emamectin and lufenuron were found in three and one sample, respectively. The percentage of positive samples for anti sea lice agents were lower than in 2017. Residues of emamectin have also been detected previously; however, this is the first time residues of lufenuron has been detected in this surveillance. All samples had levels below the MRL.

 

4.3 - Contaminants

Although the level of dioxins and dl-PCBs decreased from 2006 until 2012, reflecting the increased inclusion of vegetable ingredients in the feed, the level appears to have stabilized at approximately 0.5 ng TEQ/kg w.w. in farmed Atlantic salmon. This level has been stable from 2012 up to, and including, 2018.

No environmental contaminants were found above the EU maximum limits (ML) in 2018, for the contaminants where MLs have been implemented. However, the EUs MLs for food are not toxicologically based, but derived from the ALARA (as low as reasonably achievable) principle, with the aim to prevent those commodities with the highest contaminant levels to reach the market.

To evaluate the toxicological relevancy of the different contaminant levels, tolerable intake values are implemented. The Tolerable weekly intake (TWI) estimates the amount per kg body weight (bw) of a potentially harmful chemical that can be ingested per week over a lifetime without appreciable health risk. The TWI is a threshold level set by international risk assessment bodies, such as WHO and JECFA, or EFSA in Europe. The compound group with the strongest impact on restricting the recommended intake of fish is the dioxins and dl-PCBs. The TWI for dioxins and dl-PCBs was re-evaluated by EFSA in 2018 (EFSA 2018), and a new TWI of 2 pg WHO-TEQ/kg bw was established. This TWI is 7-fold lower than the previous TWI set in 2001 by the Scientific Committee of Food (SCF, 2001). Importantly, the new TWI ascertains the risk of dioxins and dl-PCBs, not the combined risk and benefit of fish consumption. Therefore, the NFSA has commissioned a new report from the Norwegian Scientific Committee for Food and Environment (VKM) on the risk and benefit of seafood consumption

 


4.4 - Ethoxyquin

Due to limited data on toxicity of EQ and its metabolites, a precautional maximum residue level (MRL) at the limit of analytical quantification (0.05 mg/kg), is currently applied in the EU (EFSA 2005). The list of products where a MRL has been established includes products of animal origin, but no MRL has yet been defined for fish. Measurements of fillet from Atlantic salmon, rainbow trout and Atlantic char do not show an exceedance of this level for EQ alone. However, EQ is metabolized quickly in salmon and accumulates mainly as EQDM in the edible parts of the fish. The median concentration of the sum EQ&EQDM exceeds the currently set precautional MRL value. Yet, with the measured level, a daily intake of one kg of salmon is still considered acceptable without appreciable health risks based on the currently applied ADI established by JECFA in 2005. A risk assessment of ethoxyquin by EFSA is currently in progress.

5 - Conclusion

No substances with anabolic effect were detected in any of the samples analysed.

None of the veterinary drugs were detected at levels exceeding the MRL established for fish. Emamectin or lufenuron were detected in a total of four samples; but the measured levels were below their respective MRLs.

For contaminants, no samples exceeded the EUs maximum limits, where such limits have been established (sum dioxins, sum dioxins and dl-PCBs, PCB-6, mercury, lead and cadmium).

6 - Advice

The new TWI for dioxins and dl-PCBs of 2 pg WHO-TEQ/kg bw ascertains risk only, and a holistic risk-benefit perspective is not considered. We therefore support the decision by the NFSA to commission a new report from VKM concerning the risk and benefit of fish consumption.

7 - Tables

 

Table 7.1. Inorganic arsenic and methylmercury in fillets of farmed fish

  Atlantic SalmonRainbow troutLOQ
 N191 
Inorganic arsenic (µg/kg w.w.)#Values00 
Median-- 
MaxLOQLOQ2-3
Methyl-mercury (mg Hg/kg w.w.)#Values191 
Median0.020- 
Max0.0600.0190.001

 

 

Table 7.2. PFCs (µg/kg w.w.) in fillets of farmed fish

CompoundAtlantic SalmonRainbow troutMax valueLOQ
PFBA703 1.0
PFBS1.0
PFDA0.2
PFDoDA0.2
PFDS0.2
PFHpA0.2
PFHxA0.5
PFHxS1.0
PFNA0.2
PFOA0.4
PFOS0.2
PFOSA0.5
PFTeDA0.2
PFTrDA0.2
PFUdA0.2

 


Table 7.3. Summery of analytical methods

Group of substancesAnalyte1MethodLOD (µg/kg w.w.)LOQ (µg/kg w.w.)Level of action (µg/kg w.w.)Laboratory
A1 StilbenesDiethylstilbestrolLC-MS/MS1 PresenceEurofins
Dienestrol1
Hexestrol1
β-Estradiol1
α-Estradiol1
Estriol1
Estrone1
Ethinyl estradiol1
A3 Steroidsα-nandrolonLC-MS/MS1 PresenceEurofins
β-nandrolon1
α-trenbolon1
β-trenbolon1
Trenbolone-acetate2
16-Hydroxy stanozolol1
α -Boldenone1
Boldenone1
Chlor-Testosterone (Clostebol)1
Epitestosterone1
Methyl-Boldenone (Dianabol)1
Methyltestosterone1
Nortestosterone/ Nandrolone1
Stanozolol1
Testosterone1
Testosterone-propionate2
A6 Annex IV substancesChloramphenicolLC-MS/MS0.25 Presence (MRPL = 0.3)IMR
MetronidazoleLC-MS/MS0.3 Presence (MRPL = 3.0)
Hydroxy-metronidazole2.0 
Nitrofuran AOZLC-MS/MS0.5 Presence (MRPL =1.0)
Nitrofuran AHD0.6 Presence (MRPL =1.0)
Nitrofuran AMOZ0.4 Presence (MRPL =1.0)
Nitrofuran SEM0.5 Presence (MRPL= 1.0)
B1 Antibacterial Substances Micro-biological methodQuinolones3-plate Screening Method2200 100-600IMR
Tetracyclines200 100
Amphenicols200 1000
Sulfonamides400 100
B1 Antibacterial substances Chemical methodOxolinic acidLC-MS/MS 40100IMR
Flumequine40600
Enrofloxacin10100
Ciprofloxacin10100
Trimethoprim250
OxytetracyclineLC-MS/MS 30100Eurofins
FlorfenicolLC-MS/MS 0.51000IMR
B2a AnthelminticsPraziquantelLC-MS/MS 1n.a.IMR/ Eurofins
FenbendazoleLC-MS/MS 1n.a.
EmamectinLC-MS/MS 2-10100
DiflubenzuronLC-MS/M 1-101000
Teflubenzuron1-50500
Hexaflumuron1-50500
Lufenuron1-501350
IvermectinLC-MS/M2n.a.Eurofins
CypermethrinGC-MS 550
Deltamethrin1010
Isoeugenol3GC-FID 506000
B3a Organo-chlorine compoundsDioxins and dlPCBHRGC-HRMS 0.0001-0.1 ng TEQ/kg6.5 ng TEQ/kgIMR
PCB-6GC-MS GC-MS/MS 0.004 – 0.575
PesticidesHRGC-HRMS 0.003-0.8n.a.Eurofins
B3b Organo-phosphorus compoundsAzametiphosLC-MS/MS 10n.a.Eurofins
Dichlorvos
Chlorpyriphos Chlorpyrifos-methylGC-MS 5n.a.
Pirimiphos-methyl Pirimiphos-ethyl 10n.a.
B3c Chemical elementsLeadICP-MS 0.005- 0.01 mg/kg0.3 mg/kgIMR
Cadmium 0.001- 0.002 mg/kg0.05 mg/kg.
Arsenic 0.003 mg/kgn.a.
Mercury 0.002 mg/kg0.5 mg/kg
Inorganic arsenicLC-ICP-MS 4-6n.a.
MethylmercuryGC-ICP-MS 1n.a.
TributyltinGC-ICP-MS 0.3-0.5n.a.
B3d MycotoxinsBeauvericin, Enniatin A, A1, B and B1LC-MS/MS 10n.a.Eurofins
B3e, dyesMalachite greenLC-MS/MS0.15 Presence (MRPL=2)IMR
Leuco malachite green0.15 
Crystal violet0.30 Presence
Leuco crystal violet0.15 Presence
Brilliant green0.15 Presence
B3f, othersPBDEGC-MS 0.003-0.009n.a.IMR
HBCDLC-MS/MS 0.006-0.01n.a.Eurofins
TBBPAGC-MS 0.03-0.2n.a.Eurofins
PAHGC-MS/MS 0.5-1.0n.a.IMR
PFCLC-MS/MS 0.5-13n.a.IMR
EthoxyquinHPLC-FLD 0.001n.a.IMR
Ethoxyquin dimer0.005n.a.
1 All methods used muscle as sample matrix except for microbiological methods for antibacterial substances (B1), were liver was used 2 Only screening method, positive results have to be confirmed by a chemical method.

 

Table 7.4. Calculation of sums for certain pesticides.

SumSubstances included in the sumConversion factor
DDT (sum of p,p-DDT, o,p-DDT, p,p-DDD, o,p-DDD, p,p-DDE,and o,p-DDE expressed as DDT)op-DDT1
pp-DDT1
op-DDD1.108
pp-DDD1.108
op-DDE1.115
pp-DDE1.115
Endosulfan (sum of alpha- and beta-isomers and endosulfan-sulphate expressed as endosulfan)alpha-endosulfan1
beta-endosulfan1
endosulfan sulphate0.962
Aldrin and dieldrin (Aldrin and dieldrin combined expressed as dieldrin)dieldrin1
aldrin1.044
Chlordane (Sum of cis- and trans-isomers and oxychlordane expressed as chlordane)trans-chlordane1
cis-chlordane1
oxychlordane0.967
Heptachlor (sum of heptachlor and heptachlor epoxide expressed as heptachlor)heptachlor1
trans-heptachlor epoxide0.959
cis-heptachlor epoxide0.959
Toxaphene (sum of toxaphene 26, toxaphene 50 and toxaphene 62)Toxaphene 261
Toxaphene 501
Toxaphene 621

8 - Referances

Berntssen, M. H. G., Julshamn, K., Lundebye, A. K. (2010). Chemical contaminants in aquafeeds and Atlantic salmon (Salmo salar) following the use of traditional- versus alternative feed ingredients. Chemosphere 78: 637-646.

Berntssen, M. H. G., Maage A., Julshamn, K., Oeye, B. E., Lundebye, A. K. (2011). Carry-over of dietary organochlorine pesticides, PCDD/Fs, PCBs, and brominated flame retardants to Atlantic salmon (Salmo salar) fillets. Chemosphere 83: 95-103.

Berdikova Bohne, V. J., Hove, H., & Hamre, K. (2007). Simultaneous quantitative determination of the synthetic antioxidant ethoxyquin and its major metabolite in Atlantic salmon (Salmo salar, L), ethoxyquin dimer, by reversed-phase high-performance liquid chromatography with fluorescence detection. Journal of AOAC International, 90(2), 587-597.

Chan, D., Tarbin, J. A., Stubbings, G., Kay, J., & Sharman, M. (2012). Analysis of incurred crystal violet in Atlantic salmon (Salmo salar L.): comparison between the analysis of crystal violet as an individual parent and leucocrystal violet and as total crystal violet after oxidation with 2, 3-dichloro-5, 6-dicyanobenzoquinone. Food Additives & Contaminants: Part A, 29, 66-72.

CRL (2007). CRL guidance paper (7 december 2007) CRLs view on state of the art analytical methods for national residue control plans.

EFSA (2004) Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission to assess the health risks to consumers associated with exposure to organotins in foodstuffs.

EMA (2013) European public MRL assessment report (EPMAR) for lufenuron (fin fish), EMA/CVMP/651740/2013

EU (1996). Council Directive 96/23/EC on measures to monitor certain substances and residues thereof in live animals and animal products.

EU (2002). 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results

EU (2006). Commission regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs.

EU (2010). Commission Regulation (EU) No. 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin.

EU (2011). Commission regulation (EU) No 835/2011 of 19 August 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for polycyclic aromatic hydrocarbons in foodstuffs.

EU (2011). Commission Regulation (EU) No. 1259/2011 amending Regulation (EC) No. 1881/2006 as regards maximum levels for dioxins, dioxin-like PCBs and non dioxin-like PCBs in foodstuffs.

EU (2014). Commission implementing regulation (EU) No 967/2014 of 12 September 2014 amending Regulation (EU) No 37/2010, as regards the substance ‘lufenuron’

European Commission, Health & Consumer Protection Directorate-General, Scientific Committee on Food (2001).Opinion of the SFC on the risk assessment of dioxins and dioxin-like PCBs in food. [Update based on new scientific information available since the adoption of the the SCF opinion of 22nd November 2000. Adopted 30 May 2001.

European commission directorate-general for health and food safety (2015). Safety of the Food Chain Pesticides and biocides. SANTE/11945/2015.

Hamre, L. A., Lunestad, B. T., et al. (2011). An evaluation of the duration of efficacy of emamectin benzoate in the control of Caligus curtus Muller infestations in Atlantic cod (Gadus morhua). Journal of Fish Diseases 34: 453-457.

Johnsen, C. A., Hagen, Ø., Adler, M., Jönsson, E., Kling, P., Bickerdike, R., Solberg, C., Björnsson, B. T., Bendiksen, E.Å. (2011). "Effects of feed, feeding regime and growth rate on flesh quality, connective tissue and plasma hormones in farmed Atlantic salmon (Salmo salar). Aquaculture 318: 343-354.

Joint FAO/WHO Expert Committee on Food Additives (2015). Summary and Conclusions of the Joint FAO/WHO Expert Committee on Food Additives and Contaminants (JECFA).81st meeting on Veterinary Drug Residues in Food.

Julshamn, K., Maage, A., Norli, H. S., Grobecker, K. H., Jorhem, L., Fecher, P. (2007). Determination of arsenic, cadmium, mercury, and lead by inductively coupled plasma/mass spectrometry in foods after pressure digestion: NMKL1 interlaboratory study. Journal of Aoac International 90: 844-856.

Samuelsen, O. B., Lunestad, B. T., Farestveit, E., Grefsrud, E. S., Hannisdal, R., Holmelid, B., Tjensvoll, T., Agnalt, A. L. (2014). Mortality and deformities in European lobster (Homarus gammarus) juveniles exposed to the anti-parasitic drug teflubenzuron. Aquatic Toxicology 149: 8-15.

Shiomi, K. (1994). Arsenic in marine organisms: chemical forms and toxicological aspects. Advances in environmental science and technology-New York: 261.

VKM (2014). Benefit-risk assessment of fish and fish products in the Norwegian diet – an update.

Scientific Opinion of the Scientific Steering Committee. Norway. ISBN: 978-82-8259-159-1.

Ørnsrud, R., Arukwe, A., Bohne, V., Pavlikova, N., & Lundebye, A. K. (2011). Investigations on the metabolism and potentially adverse effects of ethoxyquin dimer, a major metabolite of the synthetic antioxidant ethoxyquin in salmon muscle. Journal of food protection, 74(9), 1574-1580.