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

Abstract

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.

Keywords: Atherinella brasiliensis; Brazilian silverside embryos; Developmental toxicity tests; Marine fish embryo test.

Figure 1. Ontogeny of Brazilian silverside from the 1-cell to 18-somite stage.

(A, B, F–M, O, and P) lateral view and (C, D, E, and N) dorsal view. (A) 1-cell stage, considered 0 h from fertilization until first cleavage; (B) 2-cell stage, 0,67 hpf; (C) 4-cell stage, 1.33 h; (D) 8-cell stage, 2 h; (E) 16-cell stage, 2.67 h; (F) 32-cell stage, 3.3 h; (G) 64-cell stage, 4 h; (H) 128-cell stage; 4.67 h; (I) oblong stage; 9 h; (J) sphere stage, 15.5 h; (K) 25% epiboly, 17.5 h the embryo is on the right side (arrow); (L) 40% epiboly, 19 h embryo is on the right side (arrow); (M) 90% epiboly, 23.2 h; (N) optic vesicles forming at 90% epiboly stage (arrow); (O) 14-somite stage embryo, at 34 h showing Kupffer’s vesicle (arrowhead); (P) 18 somite stage, 38.3 h, the otic vesicle is apparent (arrow) (Scale—0.5 mm)

Figure 2. Fluorescent cells, dorsal view.

(A) Bright field image showing the pigmented cells: dark brown (arrow) and black (arrowhead), (B) the same embryo image under a GFP filter, (C) dsRed filter and (D) merged images showing that only the dark brown pigmented cells are fluorescent in GFP and dsRED filters. Scale—0.5 mm.

Figure 3. Ontogeny of Brazilian silverside from 20-somite stage until hatching.

(A and C–I) lateral view, (B and I) dorsal view. (A) 40 h 20-somite stage; (B) dorsal view of 20-somite embryo with first pigmented cells appearing; (C) 48 h; (D) 72 hpf; (E and I) 96 h in I the arrow shows the tail tip at the level of the otic vesicle; (F, J and K) 120 h or 5 dpf, in J the spleen is visible only in a dorsal view (arrow), and in K, the tail tip (arrow) passes the otic vesicle (dashed ellipse); (G and L) 144 h or 6 dpf; (H and M) 168 h or 7 dpf; (M) dorsal view of 7 dpf larvae showing the spleen as a red spot (arrow); (N) composition picture of a 8-9 dpf hatching larvae. Scale—0.5 mm. The pictures A, C, D, E, F, G, H and N use the same scale bar.

Figure 4. Relative mean of cumulative hatching (A) and mortality (B) rates from all salinities (15-35ppT) tested, in percentage, and ± Standard Errors, at 25 °C and 28 °C.

An asterisk (*) indicates significant statistical difference between the temperatures (ANOVA p < 0.0001)

Figure 5. Brazilian silversides embryos exposed to different SDS concentrations.

(A) Hatching rate, in percentage, in mg·L⁻¹. (B) Mortality rate (±Standard Error). (A and B) The graphs correspond to cumulative data. An asterisk (*) indicates significant difference between the values on the corresponding day (ANOVA p < 0.0001).

Figure 6. Brazilian silversides embryos exposed to different Zn concentrations.

(A) Hatching rate of live embryos, in percentage, in mg·L⁻¹. (B) Mortality rate of embryos, in percentage (± Standard Error), in mg·L⁻¹. (A and B) the graphs correspond to cumulative data. An asterisk (*) indicates significant difference between the values on the corresponding day (ANOVA p < 0.001).