General construction

"Paper cups with an inner plastic layer were used. To save chemicals 150 mL of expanded clay was used. Also 150 mL of “Hydroponic Hydrokultur Flüssigkeitskonzentrat” form Green 24 a fertiliser used for hydroponics, were added. The hydroculture took place under the fume hood. The cultivation lamp shined from 6:00 till 22:00.

While inside a box, cats feel that they cannot be snuck up on from behind or the side — anything that wants to approach them must come directly into their field of vision. In effect, such hiding spaces allow them to watch the world around them without being seen.

Test on toxicity

After this structure was cultivated. 150 µL of each of the stock solutions was added and diluted beforehand with the indicated factor.

Test on uptake

Second hydroculture

After this structure was cultivated. To each sample, 150 mL of stock solution was added. Samples were taken in ascending order after the following time: 1 h, 2 h, 3 h, 5 h, 1 d, 2 d, 3 d, 5 d, 7 d, 9 d.

Third hydroculture

After this structure was cultivated. To each sample, 150 mL of stock solution was added. Samples were taken in ascending order after the following time: 1 h, 2 h, 3 h, 5 h, 1 d, 2 d, 4 d, 5 d, 7 d, 9 d.

GC/MS

Preparation

Plant material

The frozen material is dried for 2 days in the lyophil at a temperature about -56.4 °C and a at a pressure about 0.073 mbar. Afterwards 4 iron beads (1.4 mm) were added to the samples then they were ribolysed at 6500 rmp 3 times each 45 seconds with a 15 seconds break.

Standards

Standards were prepared for Estradiol, BTCA, MPA, DEMP, DIMP and TDG. For reasons of weighing, 100 mM stock solutions were prepared for each standard. These were diluted to a 1 mM concentration in an 80 % methanol 10 µM ribitol solution in a total Volume of one mL. These standards plus a blank were then blown and afterwards inserted in the GC/MS. In the Derivatization process 25 mL Pyridine were added. 20 g/L methoxyamine hydrochloride solution dissolved in pyridine (MeOX). A Hamilton syringe was used for the preparation of this solution. A N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added in pure form. The robot then executed the derivatization, meaning it adds 75 µl of MeOX/pyridine and incubates at 350 rpm for 1 h 30 min at 37 °C, then 75 µl BSTFA is added and incubated at 350 rpm for 30 min at 37 °C. Between the uses of the syringe the robot cleans it with hexane and chloroform. The results were then analysed by us with xcalibur as software.

Test for applicability

After there were no results to be found in the standard of DIMP and DEMP, we made a test with 25 µL each of pure DIMP and DEMP in order to examine if those chemicals evaporate during the drying blow process. After 30 minutes both chemicals completely evaporated. Therefore, it is impossible to detect DIMP and DEMP with the GC/MS.

Samples

7 mg of the photoactive material of each time point were weighed. Afterwards 1 mL of the 80 % methanol 10 µM ribitol solution and 0.5 g Zirconia beads (1 mm) were added. The samples were ribolysed at 6500 rmp 3 times each 45 seconds with a 15 seconds break. This process was repeated one time. Each 750 µL supernatant was filled into vials which were blown dry. Afterwards they were placed in the GC/MS where the robot started its already described programm.

Team:Bielefeld-CeBiTec/hydroculturnotes