TWN Info Service on Biosafety
18 December 2023
Third World Network
www.twn.my
Dear Friends and Colleagues
Norwegian Scientific Committee Finds Many Risks with Proposed Field Trial of New GE Salmon
The Norwegian Scientific Committee for Food and Environment (VKM) has identified many risks, knowledge gaps and uncertainties associated with an application for the experimental release of salmon obtained from new genetic engineering (new GE, also new genomic techniques, NGT) (Item 1). VKM carries out risk assessments for the Norwegian Food Safety Authority and the Norwegian Environment Agency.
CRISPR/Cas was used in the salmon to switch off the genes responsible for the development of the reproductive organs. The intention was to use the sterile salmon for fattening in aquaculture. The proposal is for the new GE fish to be released into enclosures in the sea, which are surrounded by nets, from autumn 2023 to February 2025.
VKM concluded that the field trial is associated with potentially high risk for wild Atlantic salmon populations. First, there was insufficient proof of sterility in all the new GE fish. This is due to a considerable number of genetic differences between the CRISPR salmon in regard to their altered genes and could be due to a lack of precision in the new GE techniques (Item 2).
Second, it was uncertain whether the likelihood of fish escaping from the experimental pens differed from the likelihood of escaping from standard net pens used in fish farming. It was unclear how the new GE salmon would behave in the environment: for example, if they could become competitors of younger fish in wild populations. Furthermore, if they were not completely sterile, they could pass on the artificial genetic defects, and thus weaken wild populations. There was also high uncertainty regarding the potential susceptibility of the new GE salmon to diseases and contribution to the spread of such diseases.
The negative opinion makes it doubtful whether the new GE fish will actually be released in 2024. VKM acknowledges that several of the knowledge gaps identified could be addressed by conducting studies in contained facilities. While this would reduce uncertainties, VKM concludes that it would not necessarily reduce the likelihood of unwanted effects.
With best wishes,
Third World Network
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Item 1
ENVIRONMENTAL RISK ASSESSMENT OF GENETICALLY MODIFIED STERILE VIRGIN® ATLANTIC SALMON FOR USE IN RESEARCH TRIALS IN AQUACULTURE SEA-CAGES
Scientific Opinion of the Norwegian Scientific Committee for Food and Environment (VKM)
SUMMARY
Background
One of the substantial environmental challenges posed by the aquaculture industry is the escape of farmed Atlantic salmon (Salmo salar), which can mate with wild Atlantic salmon and alter the genetic composition of the wild populations. One potential solution to mitigate this issue is the cultivation of sterile salmon in aquaculture. Atlantic salmon can be made sterile by pressure or temperature treatment of newly fertilized eggs to produce triploids, which are functionally sterile due to their unpaired chromosomes. However, these triploids often perform poorly on commercial fish farms and the production of triploid salmon in Norway is put on hold due to welfare issues of the fish.
In this application, the Institute of Marine Research (IMR), Bergen, seeks to rear genetically modified sterile Atlantic salmon (VIRGIN® salmon) in a marine aquaculture environment from the post-smolt stage until harvest. The research trials are to take place in small, open sea cages (net pens) at the IMR Matre Aquaculture Research Station from autumn 2023 until February 2025. The Norwegian Environment Agency has asked VKM to assess the environmental risks associated with this field trial according to the Gene Technology Act and using risk assessment guidance from the European Food Safety Authority, EFSA.
The research trial material and rearing environment
The Atlantic salmon used in the experiment are offspring of parental fish (F0 generation) produced by a precise gene editing method (microinjection of CRISPR-Cas9 at the egg stage) introducing various point mutations leading to a non-functional gene sequence of the dead end (dnd) gene. This dnd gene is essential for germinal cell development since non-functional gene sequences prevent the development of gonads and lead to genetic sterility. To produce fertile parental fish, a stabilized mRNA encoding a functional dnd protein was injected together with the CRISPR-Cas9 constructs. This approach results in genetically sterile parental fish (functional knockout of the dnd gene), while they remain physiologically fertile due to translation of the functional dnd protein, which promotes the development of gonad tissue. These fish are referred to as F0 VIRGIN rescued salmon. In November 2021 two males and four females of the F0 VIRGIN rescued salmon were crossed, and a total of 2200 offspring (F1 generation) was produced. Of these 2200 offspring, 303 genetically sterile fish (F1 VIRGIN salmon) and 485 wildtype fish as control, were selected for the experiment based on their genotype in adipose fin-clips. The 303 experimental fish show genotypes that carry two dnd nonfunctional gene variants (alleles), whereas the control fish carry two functional wildtype alleles. The mosaic nature (not all cells have been mutated) of the parental F0 fish resulting from CRISPR-Cas9 editing enables the presence of both sterile individuals (F1 VIRGIN salmon) and fertile individuals (wildtype farmed salmon) within the same sibling offspring groups.
The research trials will be carried out in sea cages (open net pens) at the Smørdalen fish-farming facility (locality no. 12154) at Matre Aquaculture Research Station. All 788 individuals are PIT-tagged, vaccinated and length-measured and weighed prior to release into the sea cages at approximately 400 g average weight. Double net pens are used with four 5 x 5 m net pens (5 m deep), situated within a single 12 x 12 m net pen (14 m deep) and covered by double netting. Each of the 5 x 5 m nets will receive 197 (788/4) individuals that are monitored by PIT antennas. The fish will be sampled after 5 months, 10 months and at slaughter when the fish weigh approximately 5 kg. Sampling includes measurement of length and weight and recording of welfare indicators, such as malformations, injuries, and other signs of health issues. The experimental fish will be inspected daily, and if there are any dead fish, they will be collected. Environmental conditions like conductivity, temperature, and depth profiling will be recorded. If there is an escape, the applicant will follow standard procedures for capturing escaped farmed salmon. The study facility has access control, and the sea cages are marked with GMO labelling. However, the PIT tags used are small and internal, so there is no external marking to identify the fish as GMO. Upon detection of disease, the experiment will be terminated. If all fish survive until slaughter, a total of approximately 1515 kg of experimental fish will be incinerated (303 individuals at 5 kg).
Methodology and data
The VKM project group for this risk assessment has expertise in molecular biology, biochemistry and toxicology, Atlantic salmon genetics and ecology, fish diseases, and interactions between fish farming and wild salmonids, including escaped farmed salmon. VKM also appointed an approval group with expertise in CRISPR-Cas9, aquaculture genetics, fish genomics, aquatic ecosystems, parasitology, and virology.
VKM has assessed the application according to national and international (EFSA) guidelines for environmental risk assessment of genetically modified organisms. VKM has put emphasis on identifying knowledge gaps and uncertainties in the risk assessment of the research trial.
Molecular characterization
VKM finds that the data provided in the application are not sufficient to confirm that the genotypes of all 303 homozygous (mut/mut) F1 VIRGIN salmon and all 485 homozygous (WT/WT) fish are correct for all individuals.
Furthermore, VKM finds that it is not sufficiently documented that all genotypes identified among the 303 F1 VIRGIN salmon indeed result in germ cell free and/or sterile individuals. Some of the double allelic mutations identified among the 303 F1 VIRGIN salmon are assumed by the applicant to result in germ cell free sterile fish, but this assumption is not supported by confirmative experimental data in the application.
Overall, based on the information provided in the application, VKM concludes that there is a possibility that an unknown number of potentially fertile individuals of F1 VIRGIN salmon with allelic mutation(s) in the dnd gene are erroneously registered as double allelic mutants (n = 303). Likewise, an unknown number of the homozygous wildtype controls (n = 485) may in fact be fertile heterozygous fish, carrying a mutated dnd allele.
Environmental risk assessment
No experiments have been carried out in contained facilities that could inform the environmental risk assessment (ERA) for this application:
* Crosses between F0 VIRGIN rescued or F1 VIRGIN salmon and wild salmon have to our knowledge not been carried out for studies of behaviour, reproduction, and offspring survival. Such crosses could be carried out in contained facilities and informed the assessment of possible risk to wild salmon in cases where potentially fertile fish escape.
* The feeding behaviour of F1 VIRGIN salmon has to our knowledge not been studied in contained facilities. This could have informed the risk assessment with respect to feeding behaviour in sea water and fresh water, especially with respect to potential predation on juvenile salmonids.
* Neither a targeted research design nor laboratory methods have so far been applied to investigate potential differences in immunocompetence and susceptibility to infectious agents between F1 VIRGIN salmon and the wildtype controls.
Uncertainties and data gaps
Correspondence between the dnd knockout genotype in adipose fin-clips and the germ cell-free phenotype in F0 VIRGIN rescued salmon has been shown in a limited number of individuals and discrepancy has been reported due to mosaicism.
Information on a confirmative second genotyping of all individuals included in the study, both wildtype (485) and fish with double knockout alleles (303), is missing. Undeliberate errors may have been introduced in the analysis of 2200 samples due to sample quality, sample handling, selection and marking of the individuals to be included in the study. Confirming the data is essential for the determination of the genetical background included in the study.
Limited information is available about the less common mutations included in the study, since the presented data confirming sterility only covers the most common mutations. About 7% of the dnd mutations represented in the 303 F1 VIRGIN salmon (n=20) are less well-characterized genotypes, where lack of gonad development has not been confirmed by dissection, i.e., morphological, or histological examination.
The likelihood of genetic confounding increases when only two males by four females are used to produce the 303 F1 VIRGIN salmon and their 485 wildtype sibling controls. In addition, information about family representation among the fish in the different experimental groups is missing. This may introduce biased estimates of effects and hence reduce the validity of the results.
The experimental design does not follow EFSA (2013) guidelines on choice of comparators in situations where escape from captivity is possible.
The lack of external marks on the experimental fish (PIT-tagged F1 VIRGIN salmon and wildtype siblings) adds uncertainty as to where experimental fish might spread if escaping from the facility at Matre.
No plan on how to study the response of experimental fish to infectious agents, that are likely to be introduced to the research material within the planned 1.5 years in net pens, has been presented.
Assessment of hazards
VKM assesses that it is uncertain whether the likelihood of fish escaping from the experimental net pens differs from the likelihood of escaping from standard net pens used in fish farming. On the one hand, small net pens anchored within a larger net pen may better contain fish during most operations, and on the other, this system may perform less well during periods with extreme weather conditions.
Hazards identified for this research trial include the potential fertility of some of the presumedly sterile fish generated for the experiment. The provided information regarding methodology and molecular characterization does not fully support the assumption that all genotypes of the 303 experimental fish with double allelic mutations lead to sterility, particularly, those with less common mutation types.
If heterozygote fish (with one fertility allele and one sterility allele in the dnd gene) escape, they could introduce the sterility allele into wild salmon populations by vertical gene transfer to the offspring generation and to the following generations. The spread of the sterility allele would go unnoticed as it would be recessive in heterozygote individuals. The recessive homozygote offspring (two sterility alleles) of heterozygote pairs would compete for food and space in rivers as juveniles and thereby reduce population productivity and reduce the viability in vulnerable populations. VKM assesses this as a potentially high risk.
Another hazard considered by VKM is that escaped sterile individuals from the experiment could enter rivers at a larger size than immature conventional farmed salmon, and act as predators on juveniles of wild Atlantic salmon and brown trout (Salmo trutta). It is known that conventional farmed salmon may enter rivers as immature as well as sexually mature individuals, with immature escapees typically smaller than mature escapees. Sterile large-sized individuals could therefore constitute an ecological novelty in rivers if they acted predators. VKM considers this risk to be low. The risk is associated with a high uncertainty, as the necessary experiments have not been carried out.
The status of wild Atlantic salmon populations is poor in the region (Vestland county), where Matre is situated, according to the Quality Standard for wild Atlantic salmon populations. The pressure from aquaculture on wild Atlantic salmon and sea-run brown trout in the same region (production zone PO4) is high. This suggests that escaped salmon from the research trials can decrease the viability of recipient wild populations in the region.
The typically wide dispersal of escaped farmed salmon and the absence of external tags distinguishing them from ordinary farmed escapes, imply that salmon populations can be (unknowingly) affected even when located far away from Matre.
The hazards identified in relation to pathogens, infections and diseases are that F1 VIRGIN salmon get infected and spread infectious agents when held in confined aquaculture (open net pens) at Matre or after escape from confined aquaculture at Matre. In line with the comparatove approach of the ERA, VKM assessed whether F1 VIRGIN salmon have a more negative outcome for the environment than the comparators would have had.
Conclusions
A thorough molecular characterization of the fish intended for release into sea cages is needed to fully assess the potential risks to the environment in case of accidental escape. This characterization should include confirmational genotype analysis of all individuals included in the experiment, and confirmation that each unique mutated dnd allele present among the experimental fish leads to loss-of-function.
VKM assesses that the research trial on releasing VIRGIN salmon into sea cages from autumn 2023 to February 2025 is associated with potentially high risk for wild Atlantic salmon populations. This is mainly based on two concerning elements. First and foremost, it is based on the information provided in the application, VKM concludes that there is insufficient documentation on proof of sterility in all the 303 F1 VIRGIN individuals and a possibility that an unknown number of potentially fertile individuals of F1 VIRGIN salmon with allelic mutation(s) in the dnd gene are erroneously registered as double allelic mutants (n = 303). Likewise, an unknown number of the homozygous wildtype controls (n = 485) may in fact be fertile heterozygous fish, carrying a mutated dnd allele.
Secondly, VKM assesses that it is uncertain whether the likelihood of fish escaping from the experimental pens differs from the likelihood of escaping from standard net pens used in fish farming.
Escaped experimental salmon, should they be fertile carriers of mutated dnd alleles, may spawn with wild Atlantic salmon. If such salmon are carrying a sterility allele, they may introduce sterility alleles to wild Atlantic salmon populations by vertical gene transfer. The sterility alleles will be recessive and therefore hidden to purging in wild populations, except when in the homozygote state in the second and later generations. If interbreeding occurs, it may reduce the productivity of wild populations and reduce the viability of already vulnerable populations.
VKM assesses that spread of sterility alleles to future generations of wild salmon populations would be a massive impact on wild Atlantic salmon. The overall likelihood of such spread, including salmon escaping from the experiment, is assessed as very unlikely. However, sterility alleles can be introduced to wild populations by very few individuals. VKM therefore concludes that the experiment poses a potentially high risk to wild salmon populations.
VKM assesses that both the magnitude of impact and the likelihood of impact with regards to spread of infectious agents are affected by the number of F1 VIRGIN salmon and wildtype siblings in the experiment. Based on a comparative approach, the environmental risk with regards to spread of infectious agents from release of 303 F1 VIRGIN salmon is assessed to be low, both if F1 VIRGIN salmon are contained at Matre, and if F1 VIRGIN salmon escape. VKM assesses the level of uncertainty to be high in both assessments since available information on the topic is limited, and mostly expert judgements have been used.
Several of the knowledge gaps identified by VKM can be filled by conducting studies in contained facilities. This would reduce uncertainties related to the risk assessment but would not necessarily reduce the likelihood of unwanted effects.
Key words: Atlantic salmon, aquaculture, GMO, genetically modified, dead end, dnd, allelic mutation, environmental impact.
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Item 2
NEW GE SALMON: LET’S TALK ABOUT THE RISKS!
Testbiotech
https://www.testbiotech.org/en/news/new-ge-salmon-let-s-talk-about-risks
21 November 2023
Will salmon with artificially-induced genetic defects be released in Norway?
An application for the experimental release of salmon obtained from new genetic engineering (New GE, also new genomic techniques, NGT) was submitted in Norway in April 2023. This is the very first application in Europe. Now, a risk assessment of the genetically engineered salmon undertaken by Norwegian scientists came to a negative conclusion. CRISPR/Cas was used in the salmon to switch off the genes responsible for the development of the reproductive organs. The intention was to use the sterile salmon for fattening in aquaculture. A patent application has already been filed (WO2021198424). The fish would be released into enclosures in the sea which are surrounded by nets.
The genetically engineered fish would supposedly be advantageous to fish farming. One particular aim was to reduce the risk of the salmon spreading in the environment because of its sterility. In addition, the New GE salmon could possibly be fattened for a longer period of time than conventionally-produced fish, thus allowing them to reach a higher final weight. Conventionally-bred fish are only reared until they reach sexual maturity.
However, the Norwegian Scientific Committee for Food and Environment (VKM) came to a negative conclusion, as it found there were too many uncertainties in the evaluation of environmental risks. The VKM said that it had not been demonstrated that all the genetically engineered fish were actually sterile. This is due to a lack of precision in New GE techniques, which resulted in a considerable number of genetic differences between the CRISPR salmon in regard to their altered genes. This could lead to confusion when selecting animals for fattening, as only some of the fish derived from the CRISPR salmon had the desired characteristics.
According to VKM, it is also unclear how the CRISPR salmon would behave in the environment: for example, they could become competitors of younger fish in natural populations living in rivers around the fish farms. If they were not completely sterile, they could, in addition, pass on the artificial genetic defects, and thus weaken the natural populations. According to VKM, there is also a risk that the CRISPR salmon would be more susceptible to diseases and contribute to the spread of dangerous pathogens in the affected regions.
The negative VKM opinion makes it doubtful whether the fish will actually be released in 2024. The applicants, however, also make reference to a second report by another expert commission, which considers the planned releases to be less risky.
The genetically engineered salmon under discussion are also interesting against the backdrop of the planned deregulation of new genetic engineering in the EU: if the EU Commission succeeds with its plans to deregulate New GE plants, similar initiatives for animals could soon follow. This means that in the EU – unlike in current Norwegian legislation – mandatory risk assessment may also no longer be required for New GE animals in future.
Contact:
Christoph Then, info@testbiotech.org, Tel + 49 151 54638040