*2.1. Toxicity*

As a cellular model to study spermotoxic and embryotoxic properties, we used the spermatozoa and developing embryos of the sea urchin *S. intermedius*.

The spermotoxicities of Ech, CRGs, and the complexes Ech/CRGs were investigated by the Sea Urchins Sperm Cell Toxicity Test (SUSCT)-test [23,24]. These activities were determined by the degree of inhibition of the spermatozoa's ability to fertilize the sea urchin eggs. Previously, all types of CRGs (κ, λ, ι/κ) were investigated for spermotoxicity in seawater, at a concentration 50 to 200 μg mL−1. The results showed that all of the studied CRGs had no toxic effect on the spermatozoa fertilization ability, at these concentrations. The current study with Ech in the concentration range from 1 to 10 μg mL−<sup>1</sup> revealed that this substance exhibited spermotoxicity. The spermotoxicity of Ech was expressed in the 50% inhibition of the spermatozoa's ability to fertilize the egg-cells (IC50 values were of 3 μg mL−1), at a sperm:egg ratio of 300:1. When the Ech was added to a solution of CRGs, with a concentration of the 100 μg mL<sup>−</sup>1, the spermotoxicity of the Ech decreased significantly. The higher the concentration of the Ech, the greater was the protective effect of the CRG (Figure 1a). The protection of various CRGs types, against the spermotoxicity of the Ech (C = 3 μg mL−1) was studied by the SUSCT-test, at the spermatozoa to eggs ratios of 300:1 and 150:1 (Figure 1b). From the data presented in Figure 1b, it can be seen that λ-CRG, with a higher degree of sulfation, showed a greater protective activity. This dependence of the protective effects of the CRGs on their structures, was particularly noticeable at the spermatozoa to eggs ratio of 150:1, when the sensitivity of the method was the highest.

To determine the embryotoxic effects of the Ech and its complex, with the CRG (100 μg mL−1), fertilized eggs from the sea urchins were used. In a concentration range from 2–36 μg mL−1, the Ech did not affect the division and development of early embryos of the sea urchin *Strongylocentrotus intermedius*, as well as its complex with the CRGs.

**Figure 1.** (**a**) The influence of Echinochrome (Ech) and its complex with the κ-carrageenans (CRGs) (100 μg mL−1), on the sea urchin spermatozoa fertilizing ability (spermatozoa to eggs ratio 300:1). (**b**) The spermatozoa fertilizing ability of various types of CRGs (100 μg mL<sup>−</sup>1), in the presence of Ech (3 mg mL<sup>−</sup>1). \* *p* < 0.05.
