Brazilian silverside, Atherinella brasiliensis (Quoy & Gaimard,1825) embryos as a test-species for marine fish ecotoxicological tests

The fish embryo test (FET) is an alternative to the classic freshwater toxicity test used to assess environmental hazards and risks to fish. This test has been standardized and adopted by the Organization for Economic and Cooperation and Development (OECD). As salinity may affect the substances’ toxicity, we describe the development of an alternative euryhaline test species for embryonic ecotoxicological tests: the Brazilian silverside Atherinella brasiliensis (Quoy & Gaimard, 1825). This species is broadly distributed along the coast of South America and is able to inhabit a broad range of environmental and saline conditions. Ours is the first study on the maintenance of a native South American species for natural reproduction and the generation of embryos for tests. The embryos used are transparent and possess fluorescent cells which have only been seen in a few species and which may be used as markers, making it an alternative assessment tool for the lethal and sublethal substances in marine and estuarine environments. We provide a detailed description and analysis of embryonic development under different salinities and temperatures. The embryos and larvae developed in similar ways at different salinities, however as temperatures increased, mortality also increased. We considered the effects of the reference toxicants Zn2+ and SDS using a protocol similar to the FET that was standardized for zebrafish. Brazilian silverside embryos are as sensitive as freshwater, or euryhaline fish, to the surfactant but are more resistant to metals prior to hatching. We were able to show the advantages of the Brazilian silverside as a model for a marine fish embryo test (FETm) with high levels of reproducibility and little contaminated waste.


Toxicological endpoints
Regarding Brazilian silversides morphology during development, toxicological endpoints are suggested below in order to perform MFETs (Table S1). The endpoints are similar to those suggested by Lammer et al. 2009. The table can be used in any technical approach and is subdivided into three main groups: lethal endpoints, sublethal developmental endpoints and teratogenicity endpoints.
Lethal endpoints: morphological characteristics of the embryos that implies embryo death. Coagulation: Embryos die and tend to form a concentrated amorphic mass that is opaque under translucent light in the middle of the chorion (Sup. Fig 2). This coagulation feature is more common in the first days, but this observation of embryo death and degeneration can be observed until the last day the embryo/larval day of the test.
Tail deformation: This includes any kind of tail deformation, torsion or impairment of tail growth will make it stay close to the chorion and could be a result of somite or notochord malformation. In the case of zebrafish, this endpoint was tail detachment, regarding the elongation of the yolk underneath the tail, but in the Brazilian silversides, this elongation of yolk does not exist; thus, we characterize it as tail deformation that can be analyzed until day 12.
Absence of somite formation: This occurs when it is not possible to observe somite development from 27 hpf until 48 hpf. The somites are well defined, and any malformation is easily observed on those stages and later.
Absence of heart beat: This is considered when after 5 seconds of observation, the heart does not contract.
Absence of hatching: This is considered as occurring after 11 days.

Sublethal developmental endpoints
Completion of gastrula: It should be observed until the end of epiboly movements. It is normally observed in 24 hpf at 25°C.
Formation of somites: This is the deformed shape of or undefined separation of the somites from 27 hpf until 48 hpf. The somites are well defined, and any malformation is easily observed during those stages. This endpoint is considered teratogenicity.
Development of the eyes: The retina and lenses are easily observed if the retina has a round-shape and the position of the lenses symmetrically in the middle of the retina.
Heartbeat / blood circulation: The frequency of the heart beat can be considered a great morphological endpoint. Changes in the frequency and presence of clots in the tail, body or brain can be easily analyzed but should not be confused with the spleen that also appears after 9 days as a round red organ. Any changes in the pericardium can also indicate a heart/blood circulation endpoint.
Pigmentation: The pigmentation of xanthophores (the yellow color) but especially the distribution of melanophores can be used as endpoints.
Erratic swimming or no reaction to touch: Swimming may indicate disruption of neurological features and the balance of the larvae.

-Teratogenicity endpoints:
Teratogenic Endpoints: These are malformations of visible structures of the embryos as otoliths (number of otoliths in the otic vesicle), craniofacial (as small or longer mandible), heart (epicardial chamber same as heart edema, thin atrium and ventricle) and tail (curved tail). Table S1. Endpoints considered in the embryo testing with Brazilian silversides.

Exposure time
Lethal endpoints 8 h 24 h