Homepage of Institute of educational foundation Fukuyama Akenohoshi

●Method

 

(1) The fertilization and outbreak

 

Using the Hemi centrotus pul cherrimus which I collected in a Fukuyama-shi race island on April 1, 2001, I tested it from April 2 to April 4.
I almost put a fertilized egg obtained from the male and female of one pair in the solution (200?) of 11 following shu* by the same amount and performed aeration. The seawater performed approximately 14 degrees Celsius at 20 degrees Celsius using the thing which filtered seawater of the collection ground of the sea urchin (soshima). In addition, NaNO2, the density of NaCl mean the density of reagent in addition to natural seawater (after seawater dilution).

 

A 0.243% NaNO2+ seawater
A' 0.243% NaCl+ seawater
B 0.081% NaNO2+ seawater
B' 0.081% NaCl+ seawater
C 0.027% NaNO2+ seawater
C' 0.027% NaCl+ seawater
D 0.009% NaNO2+ seawater
D' 0.009% NaCl+ seawater
E 0.003% NaNO2+ seawater
E' 0.003% NaCl+ seawater
F seawater

 

(2) The measurement

 

After having stirred the embryo suspension in the beaker with a pipette uniformly every 2 hours except the night, I made a prepared slide and checked an embryonic outbreak stage and a malformed number about 20 individuals with a microscope.

●Result

 

　100% became malformed (table 1, figure 1), and, in experiment A (0.243% NaNO2), significant difference was detected 90 after the fertilization in comparison with experiment A' (deformity rate 0?20%) which added NaCl of the same density 44 hours later (correct probability assay of P <table 0.05 2 Fisher)

 

　Fertilization membrane was not torn, and the emboly did not happen, and it featured deformity of experiment A that cells increased in fertilization membrane disorderly (figure 2-a). In addition, there was not at all the thing which became the normal gastrula and prism larva (table 3).

 

　100% became malformed (table 1, figure 1), and, in experiment B(0.081%NaNO2), significant difference was detected 90 after the fertilization in comparison with experiment B' (deformity rate 0?10%) which added NaCl of the same density 44 hours later (P <table 0.05 2).

 

　In experiment C(0.027%NaNO2), a malformed rate rose after the fertilization from 50 hours, and it was in 45% (table 1, figure 1), and, in 52 hours, significant difference was detected in 52 hours in comparison with experiment C' which added NaCl of the same density (P <table 0.05 2).

 

　The malformed form of experiment C was similar to experiment B, and it was the type that a primitive gut jumped out of outside (figure 2-b).

 

　In experiment D(0.009%NaNO2), a malformed rate rose after the fertilization from 50 hours (table 1, figure 1), and it was in 25% in 52 hours, and it was not admitted, but, in 52 hours, significant difference was less than 0.1 with less than 0.05 in P=0 .09 than experiment D' using the NaCl (table 2).

 

　Deformity of experiment D was similar to experiment B,C and was the type that a primitive gut jumped out of outside (figure 2-b). The control experiment that added NaCl had little this type of deformity. It may be said that I had effect of the sodium nitrite despite intentionality statistically when I consider this even if there is not it.

 

　In experiment E(0.003%NaNO2), the difference was not accepted in comparison with control experiment after the fertilization until 52 hours by a malformed rate (table 1, figure 1).

 

　Through these experiments, I understood somethings. One has high density of sodium nitrite in seawater, and an embryonic deformity rate of the sea urchin is that it becomes higher from an initial stage of the outbreak (table 1, figure 1, table 3). The second is that a malformed form varies according to the density of sodium nitrite in seawater (figure 2).

 

A similar tendency was confirmed by the experiment that I performed in April, 2000 about these points.

●Consideration

 

　The use standard about the addition to the sausages of the sodium nitrite is set of 70 ppm (0.007%).

 

　The embryo of the sea urchin of 100% became malformed at 0.243%, 0.081% of sodium nitrites levels after the fertilization this time in 52 hours, and, at the 0.027% sodium nitrite levels, the embryo of the sea urchin of 45% became malformed, and, at 0.009% sodium nitrite levels, the embryo of the sea urchin of 25% became malformed. Besides, of the characteristic that the control experiment when added NaCl in these deformity has little morphological; was characteristic. I grew up to the pluteus larva which was approximately normal by the different control experiment that added 0.2% of these experiments KCl which went a few days later in seawater.

 

The time when deformity occurs is delayed so that the density of sodium nitrite is light. If deformity begins to occur about the D group (0.009% sodium nitrite) when it becomes the pluteus larva and continues observing it more as higher than half are still the situation that it is not to a pluteus larva 52 hours later, it is thought about a deformity rate rising.

 

When I continue eating a sausage of 0.007% of sodium nitrite addition that addition from such a result to food is accepted day after day, and an enteral wall continues being exposed to around 0.007% of sodium nitrites, it may not be said that there is not at all the possibility that abnormality gets up at a cell level. However, the influence that the embryo of the sea urchin among the seawater receives by sodium nitrite may be bigger than the influence that the cell of the enteral wall which is surrounded by internal tissue fluid receives from food sodium nitrites.

 

　In any case, I want to suggest this experiment system as a method of the simple and easy screening to check the mutagenesis of food additives. In April, 2001, I experimented even on sorbic acid potassium and the saccharin which were other food additives, but agreement was seen at the point where deformity occurred in density among seawater of the density approximately at the same level as safety standards when I added any case in food. I may reduce a laboratory animal if I test it even if it is the animal experiment using mammals and checks the final safety after pro-this experiment, having tested screening and a spare. For the reason using the sea urchin egg, it is easy to find outbreak abnormality in well known animals in the embryology, and mutagenesis and teratogenicity are easy to appear because a thing, cell division and the differentiation that the control in a timing of the fertilization is easy are prosperous, and, also, this is because in a short time many data are provided.

 

　In addition, I may check a material denying the mutagenesis of food additives in this experiment system. A lot of nitrous acid is included in natural foods such as the Brassica campestris, but it is thought about enough other materials in these food denying nitrite mutagenesis. I may remove hazardousness if I can identify such a material by eating it together with food with nitrous acid.

 

　After performing document retrieval about a past treatise, there was the experiment that used a sea urchin egg for the noxious screening of pharmaceutical products and bioassay of the water pollution, but the article that I used about the hazardousness of the food additives such as sodium nitrites was not found.

 

　In addition, I will be suitable as junior high school, one of the environmental learning in the high school as I can experiment on this experiment system without using an expensive apparatus.

 

　I borrow this place and, about this study, want to thank for having had various advice commencing with documents collection to Mr. Kato Norihisa of the Hiroshima University creature production subject deeply. Thank you.

 | Club introduction ｜ Activity record ｜