Safety of a feed additive consisting of folic acid for aquatic species (Chr. Olesen A/S and DSM Nutritional Products Ltd)

TL;DR

This paper evaluates the safety of using folic acid as a feed additive for aquatic species and concludes it is safe when used to meet nutritional needs but cannot set maximum safe levels for all species.

Contribution

The paper provides updated safety conclusions on folic acid for aquatic species based on new literature and reaffirms the need for species-specific supplementation.

Findings

Folic acid is safe for aquatic species when used to meet nutritional requirements.

Maximum safe levels cannot be set for all fish, crustacean, and mollusc species due to narrow margins between requirements and tolerance levels.

Supplementation should not exceed the nutritional needs defined by international scientific organizations.

Abstract

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of a feed additive consisting of folic acid intended for use as a nutritional additive (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effects) for aquatic species. The characterisation, safety and efficacy of the additive have been assessed previously, however the FEEDAP Panel, considering the narrow margin between the requirement and the tolerated levels seen in some aquatic animal species, could not set a maximum safe level for all fish and crustacean species and considered that supplementation should not exceed the requirements of the different aquatic animal species. In addition, the FEEDAP Panel recommended that further research is conducted to allow setting a maximum safe level of folic acid in aquatic animal species. In the present assessment, the applicant submitted new information retrieved with an extensive literature search. After the assessment of the data newly submitted, the FEEDAP Panel reiterates its previous conclusions that the use of folic acid in aquatic animal species to cover their nutritional needs is considered safe. However, the Panel is not in a position to set maximum safe levels for all fish, crustacean and molluscs species. Considering the narrow margin between the requirement and the tolerated levels seen in some aquatic animal species, the FEEDAP Panel considers that supplementation should not exceed the requirements of the different aquatic animal species recognised by International scientific organisations.

Tables

TABLE 1. Description of the substances.

Category of additive	Nutritional additives
Functional group of the additive	Vitamins, pro‐vitamins and chemically well‐defined substances having similar effect
Description	Folic acid
Target animal category	Aquatic species
Applicant	Two companies: Chr. Olesen A/S 2 and DSM Nutritional Products Ltd. 3
Type of request	New opinion ‐ Request for scientific opinion pursuant to Article 29(1)(a) of Regulation (EC) No 178/2002

Full text

INTRODUCTION

1

Background and Terms of Reference as provided by the requestor

1.1

Regulation (EC) No 1831/20031 establishes the rules governing the Community authorisation of additives for use in animal nutrition; in particular, Article 9 defines the terms of the authorisation by the Commission.

The applicant is seeking a Union authorisation of the feed additive described in Table 1.

On 14.11.2023, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), in its opinion on Safety and efficacy of the feed additive folic acid for all animal species, could not conclude on the safety of folic acid for aquatic species.

The European Commission gave the possibility to the applicant to submit supplementary information and data to complete the assessment and to allow a revision of the EFSA's opinion.

The new supplementary information and data have been transmitted by the applicant using the E‐Submission Food Chain Platform (application number FEED‐2024‐29830).4

In view of the above and in accordance with Article 29(1)(a) of Regulation (EC) No 178/2002, the European Commission requests EFSA to deliver a new scientific opinion on the safety of the feed additive folic acid for aquatic species under the conditions of Regulation (EC) No 1831/2003, based on the supplementary information and data submitted by the applicant.

Additional information

1.2

The additive is currently authorised for use in feed and water for drinking for all animal species (3a316).5

EFSA issued an opinion on the safety and efficacy of this product when used in feed for all animal species (EFSA FEEDAP Panel, 2012) and another one on the assessment for the renewal of its authorisation (EFSA FEEDAP Panel, 2023).

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of supplementary information6 to a previous application on the same product.7 The dossier was received on 17/09/2024 and the general information and supporting documentation are available on Open.EFSA at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00576.

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers, other scientific reports and experts knowledge, to deliver the present output.

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety of folic acid is in line with the principles laid down in Regulation (EC) No 429/20088 and the relevant guidance document: Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017).

ASSESSMENT

3

Folic acid is currently authorised with a purity of 96% for its use as nutritional additive (functional group: vitamins, pro‐vitamins and chemically well‐defined substances having similar effects) in feed and water for drinking in all animal species, without a minimum or maximum use level. The applicant reported typical use levels below 20 mg/kg complete feed.

The characterisation, safety and efficacy of the additive have been already assessed previously (EFSA FEEDAP Panel, 2012, 2023). In the context of the last assessment, the FEEDAP Panel concluded that: (i) there was no new evidence to lead it to reconsider the previous conclusions that folic acid is safe for terrestrial species under the authorised conditions of use and (ii) the use of folic acid in aquatic animal species to cover their nutritional needs is considered safe. However, the Panel was not in a position to set a maximum safe level for all fish and crustacean species.

In the current dossier, the applicant submitted additional data to address the gaps identified in the previous assessment regarding the safety of the additive for the target species.

Safety

3.1

The safety of the additive was evaluated in a previous EFSA opinion (EFSA FEEDAP Panel, 2012) and more recently in the context of the renewal of its authorisation (EFSA FEEDAP Panel, 2023). In the latter opinion, the FEEDAP Panel concluded that folic acid remains safe for all terrestrial species, consumers and the environment under the authorised conditions of use and that the use of folic acid in aquatic species to cover their nutritional needs is considered safe. However, the Panel was not in a position to set a maximum safe level for all fish and crustacean species, due to the narrow margin between the requirements and the tolerated levels observed in some species, as reported in published papers provided by the applicant. The FEEDAP Panel further considered that supplementation should not exceed the requirements of the different aquatic species and recommended that further research is conducted to allow setting a maximum safe level of folic acid in aquatic species. Regarding user safety, the additive was neither a skin irritant nor a dermal sensitiser. In the absence of data, no conclusions could be drawn on the eye irritation and on the potential of the additive to be harmful for the respiratory system (EFSA FEEDAP Panel, 2023).

For the current assessment, the applicants conducted an extensive literature search aimed at identifying potential publications to support the safety of the additive for aquatic species.

Safety for the target species

3.1.1

Extensive literature search

3.1.1.1

Several databases were searched (e.g. LIVIVO, OVID, NCBI, Toxinfo, Elsevier, Ingenta, Springer, Wiley). The search was not limited to any specific period. Inclusion and exclusion criteria for the selection of the relevant papers were provided by the applicants. To identify potential additional papers, the reference lists of the relevant publications obtained from the literature search, were further screened. A secondary manual search was also performed. A total of 94 references were selected by the applicants as relevant for the current assessment.

The FEEDAP Panel assessed all the references and considered that only nine provide information relevant to the safety of the additive for the target species. Two of those papers (Asaikkutti et al., 2016; Sesay et al., 2016) were already evaluated by the FEEDAP Panel in the last assessment (EFSA FEEDAP Panel, 2023).

Salmonids

3.1.1.2

Firouz et al. (2013) investigated the effects of the dietary supplementation with 0, 6 and 10 mg folic acid/kg complete feed on growth, haematological and immunological parameters in fingerling rainbow trout (Oncorhynchus mykiss) over a 2‐month period. Both folic acid levels improved body length, body weight (bw), specific growth rate (SGR) and feed conversion ratio (FCR). No differences in survival rate were observed across groups. Haematological analysis showed lower red blood cells (RBC), haemoglobin (Hb) and haematocrit (Hct) in both folic acid treated groups compared to the control, while mean corpuscular volume (MCV) increased. Mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were lower at 6 mg/kg and returned to control levels at 10 mg/kg. White blood cells (WBC) counts were higher in all supplemented groups, with shifts in leukocyte profiles including lower neutrophils and higher lymphocytes, eosinophils and monocytes. Immunological measures such as total immunoglobulin, IgM, lysozyme activity and 50% haemolytic complement activity (CH50) levels were higher in the supplemented groups. Both levels of folic acid supplementation supported comparable responses across most measured parameters, and no additional effects were observed at 10 mg/kg compared to 6 mg/kg for key physiological and immune endpoints. These findings suggest that a dietary folic acid level of approximately 6 mg/kg may be sufficient to meet the physiological needs of juvenile rainbow trout under the conditions tested, with no apparent advantage from 10 mg/kg feed supplementation.

Other finfish

3.1.1.3

The study by Sesay et al. (2016) was already evaluated in the previous assessment (EFSA FEEDAP Panel, 2023). The study concluded that levels up to 2 mg folic acid/kg feed showed an improvement in the zootechnical performance, immune response and antioxidant status in blunt snout bream, while higher levels (5 and 10 mg/kg feed) offered no additional benefit and may reduce functional responses. A follow‐up study with a temperature stress challenge (Sesay et al., 2017) concluded that 1.0, 2.0 and 5.0 mg folic acid/kg feed can enhance acute high temperature resistance ability in blunt snout bream.

Badran and Ali (2021) conducted a 2‐month feeding trial to evaluate the effects of dietary folic acid on flathead grey mullet (Mugil cephalus). Fish (n = 240; ~ 39 g initial bw) were distributed into four groups and fed diets supplemented with 0, 10, 30 or 50 mg folic acid/kg feed. Fish were fed two times per day with a 5% feeding rate; feed intake was not measured. Weight gain, SGR, plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were significantly higher with 50 mg folic acid/kg feed supplementation. Haemoglobin and Hct were lowest and MCHC was highest at 50 mg folic acid/kg feed, whereas leucocyte counts were highest at 30 and 50 mg/kg. Gonadosomatic index (GSI) was lower at 50 mg/kg than at 10 and 30 mg/kg, but higher than control.

Hang et al. (2022) conducted a 7‐week feeding trial to investigate the effects of 0, 0.5, 1.5, 4.5 and 13.5 mg folic acid/kg complete feed in juvenile largemouth bass (Micropterus salmoides). Fish (~ 14.5 g initial body weight) were distributed into five tanks with three cages per tank. Fish were fed fish meal‐based diets two times per day until apparent satiation at a 2% feeding rate the first week and subsequently modified according to daily feed intake. Weight gain rate, SGR, organ indexes (condition factor – CF, viscerosomatic index – VSI, hepatosomatic index – HSI and relative intestine weight), haematological measurements (Hb, RBC and Hct) and plasma, liver and head kidney clinical chemistry markers followed a dose–response, improving at 0.5 or 1.5 mg folic acid/kg feed, but plateaued or declined at higher levels. Plasma total protein, albumin and liver AST, ALT and malondialdehyde (MDA) increased at the highest dose tested (13.5 mg/kg), compared to the control. The authors suggested that folic acid reduced lipid oxidation but at high concentrations induced oxidative damage.

Shalaby et al. (2019) conducted an 8‐week feeding trial to assess the effects of different dietary folic acid levels (0, 10, 20, 40, 80, 160 mg folic acid/kg complete feed) on growth and physiological parameters in Nile tilapia (Oreochromis niloticus). Fish were fed to apparent satiation twice daily, but feed intake was not recorded. Fish fed 10–20 mg folic acid/kg feed showed significant improvements in SGR, weight gain, haematological parameters (RBCs, Hb, Hct (at 10 mg folic acid/kg feed only)) and indices such as HSI and GSI. At higher supplementation levels (40–160 mg/kg feed), growth performance and blood parameters declined compared to the two lowest supplementation levels, and elevated levels of AST, ALT and plasma lipids indicated potential adverse effects. The study indicated that folic acid at 10–20 mg/kg diet optimally supports growth and physiological health while levels of 40–160 mg/kg led to impairment of liver functions and damage to other organs.

Shi et al. (2015, 2016) conducted an 8‐week feeding trial to examine the effects of folic acid deficiency on gill and intestinal health status in young grass carp (Ctenopharyngodon idella). Fish were fed diets with increasing levels of folic acid at 0.10 (basal diet), 0.47, 1.03, 1.48, 1.88 and 3.12 mg/kg complete feed. Fish were fed to apparent satiation four times daily and uneaten feed was collected to calculate actual feed intake. Feed intake was significantly different across all levels of folic acid supplementation, following a dose–response pattern. A similar dose–response pattern was found in gill oxidative stress markers (ROS, MDA, protein carbonyls), antioxidant enzyme activities (e.g. Cu/ZnSOD, GPx) and regulation of antioxidant‐related genes in gills. Moreover, ALP, Na+/K + ATPase, g‐GT transpeptidase, creatine kinase (CK), acid phosphatase (ACP), lysozyme and complement component (C3) activities in proximal, mid‐ and distal intestine, along with intestinal PC, MDA, ROS, anti‐superoxide anion and antihydroxyl radical activities also followed the same dose–response pattern. Regression analysis estimated folic acid requirements of 1.60–2.08 mg/kg based on weight gain, lysozyme activity and MDA content. Fish fed folic acid levels at 3.12 mg/kg feed had significantly lower feed intake, weight gain, SGR, gill ACP, ROS compared to all the other experimental groups. Gills' MDA content was lower, and lysozyme activity and C3 showed no difference compared to the basal diet.

Crustaceans

3.1.1.4

The study by Asaikkutti et al. (2016) was already evaluated in the previous assessment (EFSA FEEDAP Panel, 2023). Growth performance, feed intake, digestive enzyme activity, muscle protein content and immune markers in freshwater prawn (Macrobrachium rosenbergii) improved with folic acid supplementation, peaking at 2 mg/kg feed. However, higher concentrations (4–8 mg folic acid/kg feed DM) led to a decline in these parameters. Notably, survival dropped significantly at 8 mg/kg feed. Antioxidant (SOD, catalase – CAT) and liver enzyme activities (AST, ALT) remained near control levels up to 2 mg/kg but increased markedly at higher concentrations, suggesting oxidative and hepatic stress.

Xu et al. (2024) conducted a 60‐day feeding trial to evaluate the effects of dietary folic acid on growth, digestion and antioxidant activity in red swamp crayfish (Procambarus clarkii). Six diets at 0, 0.56, 1.08, 2.11, 4.05, 8.13 mg folic acid/kg feed were tested. Crayfish were fed twice daily and feeding rate was at 5% of body weight throughout the experiment. Feed intake was similar across treatments, but numerically lower at 8 mg/kg feed (calculated values). Growth performance (final bw, SGR, FCR) was significantly improved at 2 mg/kg, which was identified as the optimal folic acid level. However, at higher levels (4 and 8 mg/kg feed), several parameters declined or plateaued. Specifically, lipase and amylase activity in the intestine decreased at 8 and 4 mg/kg feed, and intestinal SOD activity declined at 4 mg/kg feed, indicating potential redox imbalance at excessive doses.

Molluscs

3.1.1.5

Miao et al. (2013) conducted a 16‐week feeding trial to estimate the dietary folic acid requirement of juvenile abalone (Haliotis discus hannai). Six diets containing graded levels of folic acid at 0.06, 1.44, 2.86, 5.88, 14.27 and 35.71 mg/kg feed were tested. Weight gain increased significantly with folic acid levels up to 2.86 mg/kg, with no further increases at higher levels. Based on broken‐line regression analysis, the dietary folic acid requirement was estimated at 2.62 mg/kg feed using weight gain ratio (WGR) as the response criterion. Additionally, folic acid concentration in viscera increased with dietary levels up to 5.88 mg/kg feed, with the requirement estimated at 5.29 mg/kg feed based on this parameter. No significant effects were observed on survival or soft body composition. These results indicate that the folic acid requirement of juvenile abalone lies between 2.6 and 5.3 mg/kg feed, depending on the physiological endpoint used. Under the experimental conditions, no negative effects of high dietary folic acid supplementation were identified in juvenile abalone. The results suggest that H. discus hannai tolerates a wide range of folic acid intake without observable detriment to growth, survival or tissue composition.

Discussion on the data available

3.1.1.6

In the previous assessment (EFSA FEEDAP Panel, 2023), the FEEDAP Panel noted that the margin of safety of folic acid in feed for fish is small based on the study by Sesay et al. (2016). In the current literature screening, five additional studies conducted in finfish were found showing potential adverse effects of excess dietary folic acid. In O. niloticus, Shalaby et al. (2019) found that dietary folic acid levels of 40–160 mg/kg feed led to reduced growth and altered liver function markers (AST, ALT), while in M. salmoides, Hang et al. (2022) observed that performance and biochemical markers plateaued or declined above 1.5 mg/kg feed, with 13.5 mg/kg feed inducing oxidative stress. In M. cephalus, Badran and Ali (2021) reported increased liver enzymes and reduced red blood cell counts at 30–50 mg/kg feed, though feed intake was not measured. In contrast, Shi et al. (2015, 2016) conducted a study on C. idella where feed intake was recorded and followed a clear dose–response pattern, with intake and physiological parameters declining at the highest supplementation level (3.12 mg/kg feed). Although differences in feed intake can partly explain some of the observed effects, a consistency across several studies with a plausible pathway to harm in terms of oxidative stress, suggest that folic acid levels above species‐specific requirements may compromise physiological function in finfish.

Regarding crustaceans, in the previous assessment (EFSA FEEDAP Panel, 2023), the Panel noted that ‘higher levels of folic acid (4–8 mg/kg feed DM) seem not to be well tolerated by the prawns’ in its assessment of the study by Asaikkutti et al. (2016). However, the feed intake was different among the supplemented groups with the group at 8 mg/kg feed having significantly lower feed intake. Differences in feed intake between treatment groups represent a critical factor in interpreting the effects of folic acid supplementation, as they can act both as a confounding variable and as a physiological response to the nutrient. Reduced intake of macronutrients resulting from folic acid supplementation may be a key driver of the physiological effects observed in experimental studies, rather than a direct consequence of folic acid itself. Both inadequate and excessive folic acid levels can impair feed intake due to e.g. effects on appetite, potentially reducing feed intake and subsequent performance. Therefore, the observed differences in physiological parameters may reflect both the direct action of folic acid and secondary effects mediated by altered nutrient intake. To accurately interpret treatment effects, feed intake should be carefully monitored, and ideally, accounted for in statistical analyses or controlled through pair‐feeding where appropriate. The study by Asaikkutti et al. (2016) showed differences in the feed intake between groups which could have impacted the results in the highest dose tested. However, in the study by Xu et al. (2024) feed intake was comparable among treatments but with similar outcome in performance and biomarkers as in the study by Asaikkutti et al. (2016) at higher intake of folic acid, strengthening the assumption that folic acid above 4–8 mg/kg feed seem not to be well tolerated by prawns and crayfish. Although some species exhibit higher or lower folic acid needs and beneficial effects on growth, enzyme activity and immune function are often observed at these levels, several studies report physiological impairments and oxidative stress at doses above 4–8 mg/kg feed, indicating a narrow safety margin.

Regarding molluscs, the limited information available shows high tolerance to folic acid.

Overall, the available literature provides a broad background of folic acid requirements and tolerance in various aquatic animal species. For most crustaceans and finfish, the estimated folic acid requirement, which could be derived from the literature, lies between 1 and 3 mg/kg feed, consistent with NRC (2011) recommendations.9

The limited number of studies meeting the FEEDAP Panel safety assessment criteria, particularly with regard to study duration and endpoints, restricts the ability to derive definitive maximum safe levels of folic acid. However, the weight of evidence suggests that supplementation levels beyond species‐specific requirements may compromise health and performance. Therefore, until more robust tolerance data becomes available, folic acid levels should be tailored to meet the species‐specific requirements, rather than providing a surplus amount.

Conclusions on safety for the target species

3.1.1.7

Based on the data available, the FEEDAP Panel reiterates its previous conclusions that the use of folic acid in aquatic animal species to cover their nutritional needs is considered safe. However, the Panel is not in a position to set maximum safe levels for all fish, crustacean and molluscs species. Considering the narrow margin between the requirement and the tolerated levels seen in some aquatic animal species, the FEEDAP Panel considers that supplementation should not exceed the requirements of the different aquatic animal species recognised by International scientific organisations.

CONCLUSIONS

4

The FEEDAP Panel concludes that the use of folic acid in aquatic species to cover their nutritional needs is considered safe. However, the Panel is not in a position to set maximum safe levels for all fish, crustacean and molluscs species. Considering the narrow margin between the requirement and the tolerated levels seen in some species, the FEEDAP Panel considers that supplementation should not exceed the requirements of the different aquatic species.

REMARK

5

The FEEDAP Panel acknowledges that indications on the requirements for folic acid are available for some species only; in particular, the National Research Council (NRC, 2011) suggests requirements of 1–2 mg/kg feed (DM basis) for marine and freshwater finfish (including salmonids) and of 2–5 mg/kg feed (DM basis) for shrimps.10 However, the requirements could be influenced by several factors (e.g. fish species, developmental stage) as observed in more recent studies available in the literature. The FEEDAP Panel is not, therefore, in the position to identify requirements levels for folic acid for all aquatic species.

ABBREVIATIONSACPacid phosphataseALTalanine aminotransferaseASTaspartate aminotransferaseBWbody weightCATcatalaseCFcondition factorCH5050% haemolytic complement activityCKcreatine kinaseFCRFeed Conversion RatioFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedGSIgonadosomatic indexHbhaemoglobinHcthaematocritHSIhepatosomatic indexMDAmalondialdehydeMCVmean corpuscular volumeMCHmean corpuscular haemoglobinMCHCmean corpuscular haemoglobin concentrationPlasma TGplasma trygliceridesSODsuperoxide dismutaseSGRspecific growth rateVSIviscerosomatic indexWGweight gainWGRweight gain ratio

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00576

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PANEL MEMBERS

Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Ilen Röhe, and Katerina Theodoridou.