Exposure period

The exposure period was 14 days starting after germination of 50% of the seeds in the con-trol vessels.

• Phaseolus aureus: 2^nd November 2009 - 17 November 2009

• Avena sativa: 5 January 2010 - 21 January 2010

• Sinapis alba: 5 January 2010 - 21 January 2010

11.3.6 Post exposure period

There was no post exposure period.

11.4 Test conditions

11.4.1 Environmental conditions

The test was carried out in a plant growth chamber at 20 ± 2 °C, 70 ± 25% humidity, and an illumination period of 16 h per day with a light intensity of > 7000 lux (light colour 25, univer-sal white).

The incubation temperature was measured continuously with a thermograph. With 19 – 22 °C measured throughout the test, the permitted range of 20 ± 2°C was maintained.

The light intensity was measured in Lux using an illuminance meter (MINOLTA) with photo-metric sensor. With 9000 – 10,000 lux measured throughout the test, the permitted value of at least 7000 lux was maintained.

At 60 – 80% humidity, the permitted range of 70% ± 25% was maintained.

11.4.2 Concentration levels

The nominal concentrations in the test containers with TiO₂ nanoparticles were 10, 20, 30, 44, 67, 100 mg P25/kg soil, dry mass (application via powder) and 10 and 20 mg/kg (applica-tion via dispersion).

11.4.3 Other information on materials and methods

The treatment was applied once at test start.

Frequency of treatment

The control treatment group consisted of soil and plants. Four replicates were conducted per control.

Control group and treatment

Spiking of soil with TiO₂ powder Test item – Preparation protocol

For the first application the TiO₂ powder was mixed directly into the soil using air-dried test soil (1% of the total amount) as a carrier for the TiO2 powder. Suitable amounts of TiO2 pow-der to achieve the desired final soil content were mixed homogenously with the dry soil. Care was taken to avoid a modification of the TiO2 crystalline structure. Uncontaminated test soil (between 20 - 30% of WHC_max) was spread on a plate, the carrier material with the TiO₂ powder distributed on the test soil, and all was then mixed carefully. For the test with con-taminated soil, the soil was adjusted to a water-holding capacity of 60% of the maximum wa-ter-holding capacity (WHCmax).

Spiking of soil with aqueous TiO₂ dispersion

The second application trial utilised a TiO₂ dispersion (TiO₂ nanoparticles in deion. water) that had been prepared with a magnetic flea (900 rpm; 1 min) and ultrasonication (3 min) in a bath sonicator. The test soil was dried to about 10% of WHC_max and spread on a plate. Im-mediately after preparation of the TiO₂ dispersion was sprayed onto the soil by means of a syringe coupled with a cannula, and then mixed thoroughly. Finally, the test soil was adjusted to a water-holding capacity of 60% of WHC_max. A maximum concentration of about 200 mg/L application dispersion of TiO₂ nanoparticles was considered adequate for the tests. Higher concentrations would have sedimented rapidly preventing a homogenous distribution of the nanomaterial in the soil. Furthermore, it was assumed that higher concentrations in the appli-cation dispersion would result in larger agglomerates. Based on the present water content of the soil, 165 mg/L application dispersion of TiO₂ nanoparticles was used in the test with Phaseolus aureus, and of 177 mg/L in the test with Avena sativa and Sinapis alba. The sus-pensions were continuously stirred to achieve homogeneity during spiking.

Due to these limitations, only the soil contents of the treatments 10 and 20 mg/kg were tested.

Test concentrations used in the test with Avena sativa and Sinapis alba were: dispersion with 89 and 178 mg/L deionised water; application of 225 ml test dispersion to 2.0 kg test soil (dm), corresponding to 10 and 20 mg/kg soil (dm).

Test concentrations in the test with Phaseolus aureus were: dispersion with 83 and 165 mg/L deionised water; application of 185 ml test dispersion to 1.5 kg test soil (dm), corresponding to 10 and 20 mg/kg soil (dm).

Floragard Grünpflanzendünger was used as fertiliser., manufactured by Floragard Vertriebs GmbH für Gartenbau, P.O. Box 9006, 26138 Oldenburg, Germany. The concentration ap-plied in the test was 1 mL fertiliser per litre water.

Fertiliser

Nutrient content of Floragard Grünpflanzendünger:

Ammonium nitrogen 23 mg/L , iron (Chelate) 0.50 mg/L; nitrate nitrogen 23 mg/L; copper (chelate) 0.30 mg/L; phosphate 30 mg/L; manganese (chelate) 0.30 mg/L; potassium oxide 60 mg/L; molybdenum 0.01 mg/L; zinc (chelate) 0.05 mg/L; and boron 0.10 mg/L

Round containers made of nonporous plastic with a diameter of 85 - 95 mm were used. A glass fibre wick originating from a water reservoir and passing through the bottom of the con-tainer was used to ensure consistent soil moisture. The concon-tainers were filled up with ap-proximately 280 g moist soil.

Test containers

For each plant test species a control and several concentrations were tested. Five seeds were planted in each replicate immediately after incorporation of the test item. For each test and each species seeds of the same size class were used. Twenty-four hours after test start, the glass fibre wicks passing through the bottom of the container were connected with a res-ervoir of fertiliser to ensure consistent soil moisture.

Test procedure

The test was carried out in a plant growth chamber at 20 ± 2°C, 70 ± 25% humidity and an illumination period of 16 h per day with a light intensity of > 7000 lux (light colour 25, univer-sal white).

Watering/fertilisation: Continuous bottom watering of the test container via glass fibre wicks was performed. Fertiliser was used for watering.

The 14-day-growth-phase started when 50% of the seedlings in the control group had emerged. This day was determined as "growth day 1". On this day, the number of emerged seedlings of all containers was recorded. Observations concerning emergence and visual phytotoxicity and mortality were performed throughout the exposure period of 14 days. At

"growth day 14", all seedlings were counted and the aboveground biomass was measured.

The wet mass of the plants was measured immediately after harvesting and the length of the roots was determined. The roots were carefully rinsed with tap water. The length of the main root biomass and the shortest and longest root of individual roots were measured.

Data evaluation Statistical method

Numerical values in this report are frequently rounded to a smaller degree of precision (num-ber of digits) than were used in the actual calculation. Minor differences in results obtained

from calculations with rounded values compared to the results obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and of no practical concern.

Statistical calculations

The percentage inhibition of seedling emergence was calculated for each plant species as an absolute value and in comparison to the control. Survival of emerged seedlings was calcu-lated as an absolute value. The percentage inhibition of fresh weight was calcucalcu-lated in com-parison to the control. Germination and biomass were compared by a suitable test for multi-ple comparisons with a control after testing variance homogeneity. All statistical tests were performed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat^® Solu-tions GmbH).

11.5 Results

(Raw data, chapter 21.4)

11.5.1 Zeta potential

The zeta potential in deionised water is documented in Table 72. A negative zeta potential of -18 mV (in deion. water) was measured.

Table 72: P25 – Test with plants: zeta potential of the stock dispersion for application via dispersion.

Sample Zeta potential [mV]

100 mg/L -18 mV

11.5.2 Effects

문서에서 Investigation of widely (페이지 147-150)