ISO 21115:2019 pdf free download – Water quality一Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1).
Using DMSO as a solvent, the chemical stock solutions need to be 200-times higher concentrated than the final exposure concentrations of the chemical to gain a final DMSO concentration in the exposure media of 0,5 % volume fraction. The highest exposure concentration of 3,4-DCA in the wells is 100 mg/i. Therefore, prepare a stock of 20 g/l in DMSO by weighing the appropriate amount of 3,4-DCA in a 4 ml amber glass vial and addition of the corresponding DMSO volume. Mix this first stock solution extensively by using a shaker for at least 15 mm. This stock solution is now the starting point for a serial dilution in DMSO as depicted in Figure 4, B, 5. 3,4-DCA should be tested in five concentrations spaced by a constant factor of two. These concentrations were established to obtain a full concentration response curve in L-15/ex, leading to 0 % and 100 % cell viability compared to the control for the highest and lowest concentration, respectively. The 3,4-DCA dilution series shall be prepared freshly for each experimental day.
Special attention should be drawn to keep the DMSO free of contaminations. It is recommended to use a new bottle and take aliquots, which are then used to prepare the chemical dilution series.
8.3.2.3 Preparation of the whole-water sample/ex
In a first step, the osmolality of the raw water sample is measured in triplicates using a standard osmometer (see Figure 4, B, 1). If the raw osmolality is below 90 mmol/kg, a total of S ml of 6.3.8 is added to 95 ml of water sample (see Figure 4, B, 2). If the raw osmolality is above 90 mmol/kg, a total of 4 ml of 6.3.9 is added to 96 ml of the water sample (see Figure 4. B, 2). By adding the solutions (6.3.8 and
6.3.9) as described above, the water sample is being diluted by 4 % or 5 %. In some cases, precipitates may form upon addition of salts (see 51=).
After adding the respective amount of salts to the water sample, the sample is now referred to as “wholewater sample/ex” and the whole-water sample/ex is filtered through a 022 urn membrane filter in a glass-filter apparatus (see Figure 4. B, 3) before the final osmolality is measured in triplicates. The final osmolality of the whole-water sample/ex should be within the acceptable range between 290 mmol/kg and 360 mmol/kg.
8.3.2.4 Preparation of the dilution series of the whole-water sample/ex
Steps 8.3.2.4 and 8.3.2.5, as well as 8.3.2.7 and 8.3.2.8, are performed under sterile conditions. Thus, turn the biosafety cabinet on and let the air flow for approximately 10 mm to 15 mm. Clean the cabinet and the material needed for preparation of dosing mixtures and exposure according to the sterile procedures of the facility and put them under the cabinet.
For the preparation of the dilution series of the whole-water sample/ex, place seven sterilized 15 ml glass vials under the biosafety cabinet. Add to each glass vial the respective amount of L-1S/ex and whole-water sample/ex to obtain 10 ml each of the following dilutions: 100 %, 80 %, 60 %, 40 %, 20 %, 10 % and 0 % (see Figure 4. B, 4), where 100 % contains whole-water sample/ex oniy and 0 % contains L-15/ex only. Mix them by gently pipetting up and down.
8.3.2.5 Whole-water sample/ex exposure: step-by-step performance
Twenty-four hours after plating (see 8.2.3), cells should have formed a confluent monolayer within each well (see Figure 2). This is observed by microscopy. If confluence is not yet reached after 24 h of incubation, the plate can be incubated another 24 h before whole-water sample/ex exposure.
Even distribution of the cells across the whole well should be observed. RTgill-W1 cells grow in monolayer. Once a confluent monolayer is formed, they can still increase in cell number, though very slowly, which leads to tighter packing of cells. The cells do not form multi-layers. As well, if in L-15/ex, the cells no longer grow.