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

1.1 - Background

According to EU legislation (Regulation (EU) 2017/625, which from 2023 is supplemented with Regulation(EU) 2022/1644, Regulation (EU) 2022/1646, Regulation (EU) 2022/931 and Regulation (EU) 2022/932), 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

A5: Beta-agonists

A6: Prohibited substances

Group B Veterinary drugs and contaminants:

B1: Antibacterial agents

B2a: Anthelmintics

B2c: Carbamates and pyrethroids

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 Authority (NSFA), without prior notification to the farmers. Samples were taken at all stages of farming in order to represent farmed fish during production. Substances monitored in Group A include growth promoters like steroids and stilbenes, and unauthorized drugs. Unauthorized drugs considered most relevant for aquaculture are chloramphenicol, nitrofurans, metronidazole and dyes. Since the use of the dyes malachite green, crystal violet and brilliant green is not allowed for food producing species (Commission Regulation (EU) No. 37/2010), they are considered Group A substances and hence monitored in samples throughout the production chain. However, according to Regulation (EU) 2017/625, these dyes belong to the group B3e. Thus, in order to fulfill criteria for group B sampling, 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 (Commission Decision 2003/181/EC; CRL, 2007; European Commission, 2003), and European reference laboratories (EU-RLs) (Commission Decision 2003/181/EC; CRL, 2007; European Commission, 2003). Table 1 gives an overview of MRPLs of relevant compounds.

1.3 - Group B, veterinary drugs

In order to protect public health, current EU legislation (Commission Regulation (EU) No. 37/2010) provisions the assignment of Maximum Residue Limits (MRLs) for all legally applied pharmacologically active substances in products intended for human consumption. An MRL denotes the highest permitted residual concentration of a legally applied veterinary drug and is evaluated for each substance and each food product individually. Consumption of food with drug residues below the MRL should not pose a health risk to the consumer. For fish, the MRLs 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.

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 (Commission Regulation (EC) No. 1881/2006) has set a Maximum limit (ML) for some of the contaminants in fish, while for others, such as pesticides, polycyclic aromatic hydrocarbons (PAH), perfluorocarbons (PFC) and brominated flame retardants (BFR), maximum limits have not been established.

2.1 - Sampling

Samples were taken on fish farms or slaughterhouses, by official inspectors from the NFSA, in all fish-producing regions in Norway. The sampling plan was randomised according to season and region. In 2022, the monitoring program included Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), turbot (Scophthalmus maximus), Atlantic halibut (Hippoglossus hippoglossus), Arctic char (Salvelinus alpinus), Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor) .

Samples were transported to the Institute of Marine Research (IMR) in a frozen state. For most analyses, the Norwegian quality cut (NQC) was used (Johnsen et al., 2011). However, both NQC and individual liver samples were collected for analysis of antibiotics. 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 was 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 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 was analysed by chemical methods. The maximum residue limits for veterinary drugs are set for muscle and skin in natural proportions (Commission Regulation (EU) No. 37/2010). Therefore, according to the analytical protocol, any detection of drug residues in the muscle or liver was 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 1). A summary of the analytical methods and their limit of detection (LOD) or limit of quantification (LOQ) is shown in Table 1. 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.

3.1 - Substances with anabolic effects and unauthorized substances

In 2022, a total of 1074 pooled fillet samples (5 fish/sample) were tested for residues of illegal substances, including stilbenes (142 pooled samples), steroids (141 pooled samples), beta agonists (142 pooled samples) and unauthorized veterinary drugs (649 pooled samples). The samples were mainly taken from Atlantic salmon, but also samples from rainbow trout, Atlantic cod, brown trout and Arctic char were analysed. With regards to illegal substances, the samples are monitored for and evaluated towards presence. No residues of unauthorized compounds were detected in any of the samples. The individual substances included in the monitoring of these substance groups, analytical methods, and legal action limits are listed in Table 1, Materials and Methods.

3.2 - Veterinary drugs

Samples analysed for veterinary drugs were collected from fish at processing plants, representing fish ready for human consumption. The maximum residue limit (MRL) for veterinary drugs is defined for muscle and skin in natural proportions (Commission Regulation (EU) No. 37/2010). Therefore, according to the analytical protocol, any detection of drug residues in a sample of 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

Antibacterial agents were monitored through a combination of a three-plate bioassay and chemical methods. The broad groups a) quinolones, b) amphenicols and tetracyclines and c) sulfonamides were screened in livers of 1640 fish (Table 3). A total of 128 pooled fillet samples, representing 640 fish were analysed by chemical methods (Table 4). No residues were detected in any of the samples analysed.

3.2.2 - Group B2a, Anthelmintics

The residues of anthelmintics, such as anti-sea-lice agents (Table 5) and agents for treatment of endoparasites (Table 6) were monitored in a total of 520 pooled fillet samples, representing 2600 fish.

Residues of the anti-sea lice agent emamectin were detected in two out of 124 analysed samples, at concentrations of 3.9 and 7.8 µg/kg. The concentrations were below the MRL of 100 µg/kg (EU 37/2010). Residues of lufenuron were found in two out of 113 analysed samples. The concentrations measured were 1.2 and 1.5 µg/kg and were below the MRL of 1350 µg/kg. Residues of imidacloprid were detected in two out of 118 samples analysed. The concentrations were 10 and 13 µg/kg, and were below the MRL of 600 µg/kg. Residues of other agents in this group were not detected in any of the samples.

3.2.3 - Group B2c, Carbamates and pyrethroids

In 2022, carbamates and pyrethroid substances were monitored in 197 samples, representing 985 fish (Table 7).

Cypermethrin was detected in 41 out of 178 pooled fillet samples of Atlantic salmon, 6 out of 15 samples of rainbow trout, and both samples of Arctic char analysed. Residues of deltamethrin were found in two samples of salmon and one sample of Arctic char.

Both cypermethrin and deltamethrin are synthetic pyrethroid substances, used as pharmaceutical delousing agent applied as bath treatment in aquaculture farm cages, but also as insecticide in large-scale commercial agricultural applications. Residues of cypermethrin and deltamethrin in fish may therefore also originate from transfer via plant-based ingredients in fish feed. 

There are no pesticide MRLs established for cypermethrin or deltamethrin in fish muscle (EFSA, 2015, 2023). The maximum levels of cypermethrin measured were 4.1 µg/kg in salmon, 1.4 µg/kg in rainbow trout, and 2 µg/kg in Arctic char, and were all below the MRL of 50 µg/kg (EU 37/2010), which is established for cypermethrin residues from veterinary drugs. The MRL for deltamethrin used as veterinary drug is established at 10 µg/kg for fin fish. The maximum level of deltamethrin (1.5 µg/kg in Arctic char) detected was below this MRL.

None of the other carbamate or pyrethroid substances included in the monitoring, were detected in any of the samples.

3.2.4 - Group B2d, Sedatives

No residues of isoeugenol or eugenol were found in any of the 50 samples analysed for these sedatives (Table 8).

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 304 pooled samples in 2022. The results are summarised in Tables 9 to 11.

3.3.1.1 - Organochlorine pesticides

For several of the pesticides, the amount present is calculated as a sum including metabolites or transformation products (EU DG SANTE, 2017). The results for these groups of pesticides are presented in Table 9. To calculate the sum of the components, conversion factors (Table A1, Appendix) are used to adjust for different molecular weights (EU DG SANTE, 2017). The sums in Table 9 were calculated according to the upper bound (UB) formula. For DDT and Chlordane levels were calculated as the sums of all measured metabolites, as well as the sums of metabolites according to the legal residue definitions established through Reg. (EC) No 149/2008. When using UB calculations, the numerical value of LOQ is used as a concentration value for each non-quantified analyte. UB thus 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 organochlorine pesticides are summarised in Table 10.

There are currently no MRLs established in fish fillet for any of the listed pesticides (EU, 2014).

3.3.1.2 - Dioxin, dl-PCBs and PCB-6

The levels of dioxin (PCDD+PCDF), dl-PCBs and PCB-6 in farmed fish are shown in Table 11. The data is mainly represented by Atlantic salmon, but in 2022 also samples from rainbow trout, Arctic char, Atlantic cod, turbot and Atlantic halibut were examined. The sums of dioxins, dioxins + dl-PCBs and PCB-6 are calculated as upper bound (Commission Regulation (EU) No. 1259/2011). Accordingly, the numerical LOQ values were used for congeners with levels below LOQ.

The levels of dioxins and dl-PCBs are reported as ng toxic equivalents 2005 (TEQ05)/kg and represent 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 the World Health Organization (WHO), and the toxicity of each congener is expressed relative to the most toxic form of dioxin, which has a TEF value of 1 (Commission Regulation (EU) No. 1259/2011; Van den Berg et al., 2006).

In 2022, dioxin levels found in fish fillet were somewhat lower than in the previous year. For salmon, the median of the sum of dioxins was 0.12 ng TEQ/kg w.w. The maximum value found in salmon (0.34 ng TEQ/kg w.w.) was below the EU maximum level of 3.5 ng TEQ/kg w.w. The median of the sum of all 29 PCDD/F and dl-PCBs was 0.32 ng TEQ/kg w.w for salmon (0.04 ng TEQ/kg w.w. lower than in 2020) and 0.37 ng TEQ/kg w.w for rainbow trout similar to 2021. The highest result for sum dioxin and dl-like PCBs was 0.79 ng TEQ/kg w.w., measured in tubot. All measured values were below the EU maximum level of 6.5 ng TEQ/kg w.w. The median of PCB-6 for salmon was 2.6 μ g/kg w.w and 3.6 in rainbow trout, with maximum concentrations of 5.7 and 5.1 μ g/kg w.w, respectively. For PCB-6, a maximum level is set at 75 μ g/kg w.w. in the EU.

3.3.2 - Group B3b, Organophosphorous compounds

Organophosphorous pesticide residues, chlorpyriphos, chlorpyriphos-methyl and pirimiphos-methyl, were determined in 197 pooled fillet samples, representing fillet of 985 fish (Table 12). No residues of chlorpyriphos or chlorpyriphos-methyl were detected in any of the samples. Pirimiphos-methyl was detected in 5 of 178 samples of Atlantic salmon. The maximum concentration was 0.60 µg pirimiphos-methyl/kg w.w.. There is currently no MRL established for pirimiphos-methyl in fish fillet (EU, 2014). No residues were detected in samples of rainbow trout, Arctic char, Atlantic cod or Atlantic halibut.

3.3.3 - Group B3c, Chemical elements

In 2022, monitoring of the levels of chemical elements, such as arsenic (and inorganic arsenic), total mercury in addition to methylmercury, cadmium, lead included 71 samples of Atlantic salmon, 5 samples of rainbow trout, 2 samples of Atlantic cod, one sample of turbot and one sample of Arctic char (Table 13). Mono-, di- and tributyltin were analyzed in 54 samples of Atlantic salmon, 5 samples of rainbow trout, 2 samples of Atlantic cod and one sample of Arctic char.

The concentrations of total mercury were found below the EU maximum level, which is set at 0.50 mg/kg w.w. for these species. The highest concentrations of total mercury were 0.06 mg/kg w.w. in salmon, and 0.08 mg/kg w.w. in Atlantic cod (Table 13). Mercury was mainly present as methylmercury, which was assessed in 21 samples of Atlantic salmon (Table 14).