Safety evaluation of the food enzyme phospholipase A2 from the genetically modified Aspergillus niger strain PLA.

The food enzyme phospholipase A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) is produced with the genetically modified Aspergillus niger strain PLA by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of egg and egg products, in the processing of fats and oils by degumming and for the production of modified lecithins (lysolecithin). As residual total organic solids (TOS) are removed in the refined fats and oils during degumming, dietary exposure was calculated only for the remaining two food manufacturing processes. For egg processing, the dietary exposure was estimated to be up to 1.712 mg TOS/kg body weight (bw) per day in European populations. Wet gum can be used to produce lysolecithin with the highest dietary exposure of 1.61 mg TOS/kg bw per day in children at the 95th percentile when used as a food additive. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1350 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated overall dietary exposure, resulted in a margin of exposure of at least 851. A search for the similarity of the amino acid sequence of the food enzyme to those of known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus niger strain PME.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus niger strain PME by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. It is intended to be used in fruit and vegetable processing, for juice production and fruit and vegetable processing for products other than juices. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.095 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the pectinesterase to be used in the toxicological studies, because both production strains are derived from the same recipient strain, the location of the inserts is comparable, no partial inserts were present and the production methods are essentially the same. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,852 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure of at least 19,495. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to pollen allergens, cannot be excluded. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme phospholipase A2 from porcine pancreas.

The food enzyme phospholipase A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) is obtained from porcine pancreas by Sanyo Fine Co., Ltd. It is intended to be used in three food manufacturing processes: egg processing, flavouring production and yeast processing. In the absence of sufficient data provided by the applicant to characterise the source of food enzyme, its production and chemical characterisation, coupled with insufficient information about food manufacturing processes to which the food enzyme is applied, the Panel was unable to assess the safety of the food enzyme.

Safety evaluation of the food enzyme containing endo-polygalacturonase and cellulase from the non-genetically modified Talaromyces cellulolyticus strain NITE BP-03478.

The food enzyme containing endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) and cellulase (4-(1,3;1,4)-ß-d-glucan 4-glucanohydrolase; EC 3.2.1.4) activities is produced with the non-genetically modified Talaromyces cellulolyticus strain NITE BP-03478 by Meiji Seika Pharma Co., Ltd. It is intended to be used in eight food manufacturing processes: baking processes, brewing processes, fruit and vegetable processing for juice production, wine and wine vinegar production, fruit and vegetable processing for products other than juices, fruit and vegetable processing for refined olive oil production, coffee bean demucilation and grain treatment for starch production. Since residual amounts of total organic solids (TOS) are removed during three food processes (refined olive oil production, coffee bean demucilation and grain treatment for starch production), dietary exposure was not calculated for these food processes. For the remaining five food processes, dietary exposure was estimated to be up to 3.193 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 806 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 252. A search for the similarity of the amino acid sequences of the food enzyme to known allergens was made and six matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, especially in individuals sensitised to pollen. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme catalase from the non-genetically modified Aspergillus niger strain CTS 2093.

The food enzyme catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) is produced with the non-genetically modified Aspergillus niger strain CTS 2093 by Shin Nihon Chemical Co., Ltd. It is considered free from viable cells of the production organism. The food enzyme is intended to be used in eight food manufacturing processes: baking processes, cereal-based processes, coffee processing, egg processing, vegetable processing for juice production, processing of tea, herbal and fruit infusions, herring roe processing and milk processing for cheese production. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 3.61 mg TOS/kg body weight (bw) per day in European populations. In addition, it is used in the production of acacia gum with the highest dietary exposure at the 95th percentile of 0.018 mg TOS/kg bw per day in infants, when acacia gum is used as a food additive. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 56 mg TOS/kg bw per day, the mid-dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of 16. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel considered the margin of exposure as insufficient to exclude safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme ß-galactosidase from the non-genetically modified Aspergillus oryzae strain AE-LA.

The food enzyme ß-galactosidase (ß-d-galactoside galactohydrolase; EC 3.2.1.23) is produced with the non-genetically modified Aspergillus oryzae strain AE-LA by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used for lactose hydrolysis in milk processing, production of fermented milk products, whey processing and the manufacture of enzyme-modified dairy ingredients. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 1.651 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,656 mg TOS/kg bw per day, the highest dose tested. This results in a margin of exposure of at least 1,003. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme ß-galactosidase from the non-genetically modified Neobacillus sp. strain AE-LT.

The food enzyme ß-galactosidase (EC 3.2.1.23) is produced with the non-genetically modified Neobacillus sp. strain AE-LT by Amano Enzyme Inc. The strain is not cytotoxic and does not harbour any known virulence factor or antimicrobial resistance gene. The presence of viable cells of the production strain in the food enzyme could not be excluded, but the likelihood of this being a hazard is considered low. The food enzyme is intended to be used for lactose hydrolysis in milk processing and the manufacture of galacto-oligosaccharides (GOS). The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.971 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,223 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 412. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme α-amylase from Cellulosimicrobium funkei strain AE-AMT.

The food enzyme α-amylase (4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with the non-genetically modified Cellulosimicrobium funkei strain AE-AMT by Amano Enzyme Inc. The food enzyme is free from viable cells of the production organism. It is intended to be used in starch processing for maltotriose production. Since residual amounts of total organic solids (TOS) are removed by purification steps applied during starch processing, the estimation of a dietary exposure is considered unnecessary. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose of 230 mg TOS/kg body weight (bw) per day. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and nine matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the native and thermolabile forms of the food enzyme mucorpepsin from Rhizomucor miehei strain MMR 164.

The food enzyme mucorpepsin (aspartic endopeptidase, EC 3.4.23.23) is produced with the non-genetically modified microorganism Rhizomucor miehei strain MMR 164 by Takabio. The enzyme is chemically modified to produce a thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in milk processing for cheese production. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.98 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,320 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,300. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and five matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of a food enzyme containing aspergillopepsin I and II from the Aspergillus niger var. macrosporus strain PTG8398.

The food enzyme with aspergillopepsin I (EC 3.4.23.18) and aspergillopepsin II (EC 3.4.23.19) activities is produced with a non-genetically modified Aspergillus niger var. macrosporus strain PTG8398 by Meiji Seika Pharma Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in wine production. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.14 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 919 mg TOS/kg bw per day, the highest dose tested which, when compared with the estimated dietary exposure, results in a margin of exposure above 6,700. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the thermolabile form of the food enzyme mucorpepsin from Rhizomucor miehei strain MMR 164.

The food enzyme mucorpepsin (aspartic endopeptidase, EC 3.4.23.23) is produced with the non-genetically modified microorganism Rhizomucor miehei strain MMR 164. The enzyme is chemically modified by DuPont Nutrition Biosciences (now IFF) to produce a thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in milk processing for cheese production. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.98 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,320 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,300. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and five matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but is considered low except for individuals sensitised to mustard proteins, but this risk will not exceed that of mustard consumption. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme chymosin from the genetically modified Aspergillus niger strain DSM 29546.

The food enzyme chymosin (EC 3.4.23.4) is produced with the genetically modified Aspergillus niger strain DSM 29546 by Chr. Hansen. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in milk processing for cheese production and for the production of fermented milk products. Dietary exposure was estimated to be up to 0.52 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 410 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 790. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure, although unlikely, cannot be excluded, particularly for individuals sensitised to cedar pollen allergens. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme α-amylase from the genetically modified Bacillus licheniformis strain NZYM-BC.

The food enzyme α-amylase (4-α-d-glucan glucanhydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain NZYM-BC by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The production strain was shown to qualify for the qualified presumption of safety (QPS) status. The food enzyme was free from viable cells of the production organism and its DNA. It is intended to be used in six food manufacturing processes, namely starch processing for the production of glucose syrups and other starch hydrolysates, distilled alcohol production, brewing processes, cereal-based processes, refined and unrefined sugar production and fruit and vegetable processing for juice production. Since the residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups, dietary exposure was not calculated for these two food manufacturing processes. For the remaining four processes, the dietary exposure to the food enzyme-TOS was estimated to be up to 0.05 mg TOS/kg body weight per day in European populations. Genotoxicity tests did not raise safety concern. The similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood was considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme dextranase from the Collariella gracilis strain AE-DX.

The food enzyme dextranase (6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) is produced with the non-genetically modified Collariella gracilis strain AE-DX by Amano Enzyme Inc. The food enzyme is considered free from viable cells of the production organism. The food enzyme is intended to be used in refined sugar production from sugar beet or sugar cane. Since residual amounts of total organic solids (TOS) are removed by crystallisation during the production of refined white sucrose, dietary exposure was not considered necessary for refined sugars. However, beet molasses and cane syrups, by-products from sugar production, could enter the food chain. Based on the maximum use levels recommended, dietary exposure was estimated to be up to 0.39 mg TOS/kg body weight (bw) per day via the consumption of unrefined sugars. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a lowest observed adverse effect level (LOAEL) of 940.5 mg TOS/kg bw per day, the lowest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of more than 800. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme glucose oxidase from the genetically modified Aspergillus niger strain DP-Aze23.

The food enzyme glucose oxidase (ß-D-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4) is produced with the genetically modified Aspergillus niger strain DP-Aze23 by Danisco US, Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in baking processes, cereal-based processes and egg processing. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.05 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 19.55 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure above 380. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme catalase from porcine liver.

The food enzyme catalase (EC 1.11.1.6) is obtained from porcine liver by Laboratorios Arroyo S.A. It is intended to be used in a broad range of food processes. The Panel noted that the manufacturing process involved the use of a solvent not permitted in the production of food ingredients which include food enzymes. In addition, the evidence provided showed that the manufacturing process could not be guaranteed to inactivate viruses originating from the starting material, including the human zoonotic pathogen Hepatitis E virus. Consequently, the Panel concluded that the use of catalase extracted from porcine liver may present a health risk.

Safety evaluation of the food enzyme trypsin from porcine pancreas.

The food enzyme trypsin (EC 3.4.21.4) is extracted from porcine pancreas by Ningbo Linzyme Biosciences Co., Ltd. It is intended to be used for the hydrolysis of whey proteins for use in infant formulae and follow-on formulae. Based on maximum use levels and the maximum permitted protein content in infant formula, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be 16.8 mg TOS/kg body weight (bw) per day for infants. In the toxicological evaluation, clinical studies with pancreatic enzymes were considered. Hypersensitivity to the pharmaceuticals was identified as the major side effect. However, allergic reactions to porcine pancreatic enzymes in hydrolysed foods have not been reported. The Panel considered that a risk of allergic sensitisation to this food enzyme after consumption of products prepared by hydrolysis of milk proteins could not be excluded in infants, but it considered the likelihood to be low. Based on the origin of the food enzyme from an edible tissue of pigs, the data provided by the applicant, the information from the evaluation of clinical studies based on pancreatic enzymes and the estimated dietary exposure, the Panel concluded that the trypsin from porcine pancreas does not give rise to safety concerns under the intended conditions of use.

Scientific Guidance for the submission of dossiers on Food Enzymes.

Following a request from the European Commission, EFSA developed an updated scientific guidance to assist applicants in the preparation of applications for food enzymes. This guidance describes the scientific data to be included in applications for the authorisation of food enzymes, as well as for the extension of use for existing authorisations, in accordance with Regulation (EC) No 1331/2008 and its implementing rules. Information to be provided in applications relates to source, production and characteristics of the food enzyme, toxicological data, allergenicity and dietary exposure estimation. Source, production and characteristics of the food enzyme are first considered only for enzymes of microbial origin and subsequently for those enzymes derived from plants and for enzymes from animal sources. Finally, the data requested for toxicology, allergenicity and dietary exposure applies to all food enzymes independent of the source. On the basis of the submitted data, EFSA will assess the safety of food enzymes and conclude whether or not they present a risk to human health under the proposed conditions of use.

Safety evaluation of the food enzyme catalase from the genetically modified Aspergillus niger strain DP-Azw58.

The food enzyme catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) is produced with the genetically modified Aspergillus niger strain DP-Azw58 by Danisco US, Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in egg processing. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 1 µg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,288 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 1.3 × 106. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Development of a method for assessing the accumulation and metabolization of antidepressant drugs in zebrafish (Danio rerio) eleutheroembryos.

Antidepressant drugs are widely used for the treatment of common mental or other psychiatric disorders such as depression, which affect about 121 million people worldwide. This widespread use has contributed to the input of these pharmaceuticals and their metabolites into the environment. The aim of this work was to develop an analytical method to quantify the most widely used antidepressant drugs, selective serotonin reuptake inhibitors (SSRI), and their main metabolites in the environment. For this, a new and reliable miniaturized extraction method based on dispersive SPE cleanup procedure for extraction of SSRI followed by derivatization with n-heptafluorobutyrylimidazole, and detection by GC-MS was developed. The methodology, including a first-order one-compartment model, was then applied to a bioconcentration study in zebrafish (Danio rerio) eleutheroembryos. The results showed low bioaccumulation of these compounds; however, a biotransformation evidence of the parent compounds into their metabolites was observed after 6 h of exposure. These results indicate the need to integrate metabolic transformation rates to fully model and understand the bioaccumulation patterns of SSRI and their metabolites. Graphical abstract.