Research Document 2018/049

Environmental Risk Assessment of the Glofish® Electric Green® Tetra and the Glofish® Long-Fin Electric Green® Tetra: Transgenic Ornamental Fish, Imported to Canada, For Sale in the Pet Trade

By Leggatt, R. Johnson, N., and McGowan, C.

Abstract

BACKGROUND

On July 5, 2017, GloFish LLC submitted a regulatory package to Environment and Climate Change Canada (ECCC) under the New Substances Notification Regulations (Organisms) [NSNR(O)] of the Canadian Environmental Protection Act, 1999 (CEPA 1999) for GloFish® Electric Green® Tetra (CGT2016), a green fluorescent genetically engineered Tetra (Gymnocorymbus ternetzi), for use as an ornamental fish in home aquaria.

The biotechnology provisions of CEPA take a preventative approach to pollution by requiring all new living organism products of biotechnology, including genetically engineered fish, to be notified and assessed prior to import or manufacture, to ultimately determine whether they are “toxic” or capable of becoming “toxic”. Under CEPA, an organism is considered “toxic” if it can enter the environment in a quantity or concentration or under conditions that (a) have or may have an immediate or long-term harmful effect on the environment or its biological diversity; (b) constitute or may constitute a danger to the environment on which life depends; or (c) constitute or may constitute a danger in Canada to human life or health. Anyone proposing to import or manufacture a living animal product of biotechnology in Canada, including genetically engineered fish, is required to provide ECCC with the information prescribed in NSNR(O) at least 120 days prior to the commencement of import or manufacture of the organism. This information is used to conduct an environmental risk assessment and an assessment of indirect human health (risk to human health from environmental exposure to the living organism), which is then used as the basis to determine if the organism is CEPA-toxic or capable of becoming CEPA-toxic.

Under a memorandum of understanding with ECCC and HC, DFO provides science advice in the form of an environmental risk assessment for fish products of biotechnology under the NSNR(O). This advice is used to inform the CEPA risk assessment conducted by ECCC and HC. Under this arrangement, the Minister of ECCC receives scientific advice from DFO and retains ultimate responsibility for regulatory decision making on the use of notified fish.

It is in this context that DFO conducted an environmental risk assessment of the notified organism (CGT2016) under its proposed use which was peer-reviewed by a panel of experts. Risk is defined as a function of the potential for Canadian environments to be exposed to the notified organism, and the potential for the notified organism to pose hazards to the Canadian environment. Exposure and Hazard assessments are conducted separately and then integrated into an assessment of Risk. Uncertainty in Exposure and Hazard assessments are determined, and uncertainty associated with the final risk assessment discussed.

THE NOTIFIED ORGANISM

CGT2016 is a line of genetically engineered diploid, hemizygous or homozygous, long- or regular-fin, green fluorescent transgenic Tetra of the White morph of Black Tetra (G. ternetzi), containing transgenes driving expression of a green fluorescent protein gene. Expression of the transgene results in green colouration in natural light and fluorescent green colouration in blue or UV light. The protein is expressed in the skin, musculature, fins, eyes, and likely other organs of the organism. All CGT2016 individuals are descendants of a single G0 founding individual with the transgene construct microinjected at the single cell stage. Confirmation of a single transgene copy inserted at a single site with Mendelian segregation was confirmed at the F3 generation. CGT2016 has been marketed in the USA ornamental aquarium trade since 2012 as GloFish® Electric Green® Tetra and GloFish® Long-Fin Electric Green® Tetra. The targeted phenotypic change is the presence of green/fluorescent green colouration as a novel colour morph for the ornamental aquarium trade. Other unanticipated phenotypic changes noted by the company include slightly impaired cold tolerance of CGT2016 when temperature was rapidly decreased (i.e., average LD₅₀ was 8.11C relative to 7.94C for wildtype siblings). As well, CGT2016 had over 40% reduction in expected reproductive success in competition with wild-type siblings, but did not differ in juvenile survival. No other non-target phenotypic changes have been noted in CGT2016.

ENVIRONMENTAL RISK ASSESSMENT

The environmental risk assessment is conducted under GloFish LLC’s proposed use scenario: the importation of CGT2016 to four aquarium wholesale locations in Canada, for further distribution to aquarium retail stores across the country for purchase by Canadian consumers for home aquaria.

Exposure

The intended housing for CGT2016 is in indoor, static, physically contained aquaria at wholesalers, retail stores, and in consumer’s homes. Based on historical records of aquarium fish in natural ecosystems in Canada and worldwide, it is highly likely that CGT2016 will be introduced purposefully or accidentally into natural freshwater ecosystems in Canada. Based on the expected number of CGT2016 to be purchased by individual consumers, it is expected that release events will be very low magnitude (e.g., 5 or less), although larger magnitude releases cannot be ruled out. Base on temperature preferences and limitations of wild-type G. ternetzi and recorded water temperature throughout freshwater systems in Canada, CGT2016 have potential to survive in many natural ecosystems in the summer in Canada, and some ecosystems in the fall and spring. However, the lower temperature tolerance of both CGT2016 and wild-type G. ternetzi preclude the fish from surviving over winter in most Canadian freshwater ecosystems. There are some lakes that reach temperatures for a short time in the summer months that are adequate for reproduction of CGT2016. G. ternetzi has a relatively long lifecycle (i.e., minimum time to maturation is 4 months in ideal conditions), and as such the potential exists for only one reproductive cycle prior to termination over the winter. Given the above analysis, the occurrence of CGT2016 in the Canadian environment is expected to be rare, isolated and ephemeral and likely in low numbers. Consequently, the likelihood of exposure of CGT2016 to the Canadian environment is ranked low. The uncertainty associated with this estimation is low, given the quality of temperature tolerance data available for CGT2016 and valid surrogate organisms, and data available on the environmental parameters of the receiving environment in Canada.

Hazard

The potential for CGT2016 to cause a hazard to Canadian environments was examined in the context of environmental toxicity (i.e., potential to be poisonous), through horizontal gene transfer, through interactions with other organisms including hybridization, as a vector of disease, and through impacts to biogeochemical cycling, habitat, and biodiversity. Wild-type G. ternetzi is a small, non-aggressive fish with expected limited activity due to low temperatures in most seasons in Canada, is not known to be susceptible to diseases of concern in Canada, and has no history of invasiveness reported in Canada or worldwide despite its wide use in the aquarium trade spanning greater than 65 years. There are no reports of phenotypic effects of the transgene that may increase hazard potential of CGT2016 above that of wild-type domesticated G. ternetzi, no evidence of toxicity of the fluorescent protein (i.e., not poisonous to organisms or the environment), and no evidence that potential gene transfer will result in harm to Canadian environments. Some evidence suggests CGT2016 may have lower potential to impact other species through trophic interactions relative to wild-type G. ternetzi, as lower cold tolerance may further limit activity in cooler water temperatures. Taken together, CGT2016 is expected to pose negligible to low environmental hazard if released to Canadian aquatic ecosystems. The uncertainty rankings associated with individual hazard assessments ranged from negligible to moderate due to limited data specific to CGT2016, limited direct data on the comparator species, variable data from a surrogate model (red fluorescent protein zebrafish), and the reliance on expert opinion for the assessment of some hazards.

CONCLUSIONS ON RISK

Based on a risk matrix integrating the Low Exposure and Negligible to Low Hazards of CGT2016, CGT2016 poses Low Risk to Canadian environments under the proposed use for the ornamental aquarium trade. While uncertainty associated with some hazard classifications is moderate due to limited or no direct data on the notified organism or comparator species, evidence was not identified that suggests CGT2016 under the proposed use, or other potential uses, could cause harm as a result of exposure to Canadian environments.

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