Oncorhynchus mykiss

Simakov, Iuriy, Nikiforov-Nikishin, Alexey, Nikiforov-Nikishin, Dmitry & Beketov, Sergey, 2021, Amphibian and fish eye lens used as biomarker of remote and chronic environmental contamination, Ecologica Montenegrina 45, pp. 72-82 : 75

Experimental procedures Oncorhynchus mykiss

The duration of the experiment was 30 days. Chronic contamination was modeled by introducing three substances into the corresponding experimental aquariums: dibutyl sebacate (DBS) plasticizer (“Rosspolimer”, Russia, CAS 109-43-3); polyterpenes (PTP) (lubricant, “Metaprom” company, Russia. GOST 2416-015-56807 2002) and aminopropyltriethoxysilane (AGM-9) (lubricant, “Vita- reactiv” company, Russia, CAS 919-30-2). All of these pollutants enter the fishery reservoirs with wastewater from light industry enterprises. Previously, work was conducted with these substances and established threshold concentrations for them that had a pronounced toxic effect on trout fingerlings. Trout was used in experiments since it lacks sharp seasonal changes in mitotic activity in the epithelium of the lens ( Tsentalovich, 2019). Accordingly, the spectrum of concentrations of pollutants in the experiment was taken upward from the threshold concentrations obtained earlier in the experiment when establishing MACрх (rh – related to fish industry). In the experiment on the effect of pollutants on the lens, the following concentrations were used: DBS and PTP - 0.1; 0.01; 0.001 mg / l, and for less toxic AGM-9 - 1.0; 0.1; 0.01 mg / l.In addition, an aquarium with control fish was set up.

To reveal the complex effect of pollutants on the lens in a chronic experiment, fish were exposed to a solution of all three toxicants, taken at the concentrations indicated above. Three complex solutions were prepared by mixing in equal parts of solutions of one substance: the first solution - DBS - 0.1 mg /l; PTP - 0.1 mg /l; AGM-9 - 1.0 mg/l; the second solution is DBS 0.01 mg /l; PTP - 0.01 mg /l; AGM-9 - 0.1 mg /l; the third solution is DBS- 0.001 mg /l; PTP - 0.001 mg /l; AGM-9 - 0.01 mg /l.

On the 30th day of the experiment, trout fingerlings were slaughtered in compliance with ethical standards, with instant head cutting, in accordance with an international document (Directive 2010/63 / EU of the European Parliament and Council of the European Union, dated September 22, 2010, On the protection of animals used for scientific purposes).

Both eyes were fixed in a mixture of absolute ethanol and algid acetic acid in a 3: 1 ratio ( Lilly, 1969). Dehydrated copper sulfate was placed on the bottom of the vessel to remove the aqueous humor coming from the fixed eyes. The epithelium was removed from the lenses and total planar preparations were prepared. The epithelium of the lens was stained with Mayer’s hemalaun ( Howard, 1952), after which the preparations were embedded in a Canadian balsam. Since we are interested in the effect of pollutants on mitotic activity in the lens, the frequency of mitosis (mitotic index, MI) was determined in the germinal area of the lens epithelium. For this, under a microscope (model Olympus BX43) in three squares of the epithelium, isolated by an eyepiece mesh in the germinal area, the number of mitoses was counted at all stages of division in 1000 cells. MI count is a fairly objective indicator characterizing the proliferative activity in the epithelium of the lens under the influence of chronic adverse factors. The results obtained were subjected to statistical processing according to the Student's test ( Platonov, 2000).