8.3.5 Post exposure period
There was no post exposure period.
8.4 Test conditions
8.4.1 Environmental conditions
The incubation temperature was measured continuously with a thermograph. According to the guideline the permitted range is 20 ± 2 °C. A controlled light/dark cycle of 16 h:8 h was applied. The light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in Lux. According to the guideline the permitted value is about 600 lux.
The test conditions are presented in Table 46.
Table 46: NM-300K – Test with earthworms, incubation conditions.
NM-300K Incubation temperature [°C] 19 – 21 Light intensity [lux] 600 – 750 Soil dry mass [%] 78 - 90 pH [1 mol/L KCl] – test start 5.0 – 5.1 pH [1 mol/L KCl] – test end 6.8 – 7.1
8.4.2 Test concentrations
The following nominal contents were applied in the test containers with Ag:
• 15, 30, 60, 120, 200 mg/kg soil, dry mass (application on soil)
• 15, 30, 60, 120, 200 mg/kg soil, dry mass (application on feed).
8.4.3 Other information on materials and methods
Treatment was performed once at test start.
Frequency of treatment
For silver, eight controls (no addition of dispersant), four controls with dispersant applied in soil, and four controls with dispersant applied in feed were prepared (dispersant amount cor-responded to the dispersant amount used in the highest test concentration).
Control group and treatment
Data evaluation Statistical method
In this report numerical values are frequently rounded to a smaller degree of precision (num-ber of digits) than used in the actual calculation. Minor differences in the results obtained from calculations with rounded values compared to 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
For each concentration the percent mortality, the percent loss/increase in biomass of the adults, and the number of offspring produced in the test was determined.
Mortality, biomass and number of offspring were compared by a suitable test for multiple comparisons with a control after testing variance homogeneity. All statistical tests were per-formed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat® Solutions GmbH).
Air-dried, finely ground cow manure was used as feed.
Feed
All tests were performed in polypropylene containers (Bellaplast GmbH, Alf). Adjusted to 55% of the maximum water-holding capacity, 640 g soil (dm) was filled into containers to a depth of about 5 cm. The containers were covered with transparent plastic lids to prevent worms from escaping and to guarantee access of light. The lids had several small holes to permit gaseous exchange between the medium and the atmosphere.
Test container
Soil and food were spiked. Test soil was added to the test containers and 10 g of air dried, finely ground cow manure per test container was spread onto the soil surface and moistened with water. The next day (start of the test) batches of ten conditioned worms were weighed and placed into each container. Spiking of soil and food, respectively, filling of the test ves-sels and addition of the earthworms could not be performed at the same day due to high number of test variables and test concentrations.
Test procedure
Once a week the worms were fed according to their feed consumption. Feeding behaviour and the quantity of feed applied over the test period was recorded for each test container.
The water content of the soil substrate in the test containers was maintained during the test period by weighing the test containers periodically and replenishing lost water, if necessary.
The adult worms were kept in the substrate over a period of 4 weeks. At the end of this pe-riod, the adults were removed. For each container the total number and mass of living adult worms was recorded.
To allow the offspring to develop, the test containers were kept in the test environment for another period of 4 weeks. After this period the number of offspring per test container hatched from the cocoons was counted by hand.
The test was carried out at 20°C ± 2°C and a controlled light/dark cycle of 16 h:8 h with a light intensity of 400 - 800 lux.
8.5 Results
(Raw data, chapter 21.3)
8.5.1 NM-300K
Zeta potential in dispersion with deionised water (20 g/L; 100 mg/L) was -12 mV.
Zeta potential
Total Ag-concentrations and the Ag+-concentration (ion concentration of Ag) in soil were de-termined. Detailed results for the ion concentration are presented in chapter
Test item concentrations:
21.3.1.
Total Ag-concentration
Two representative concentrations of total Ag in soil were determined. The results are pre-sented in Table 47.
Recovery was 90% (15 mg/kg and 120 mg/kg). Therefore, the use of nominal concentrations for reporting the effects was considered acceptable.
Concentration of Ag+
The results are presented in Table 48. In four concentrations (15 mg/kg, 60 mg/kg,
120 mg/kg, 200 mg/kg) the Ag+ ions were determined via DGT. There was a small increase from the concentration at day 0 to the concentration at day 56. The concentrations of ions were in the range of 0.2 - 2.3 * 10-4% of the nominal concentrations.
Table 47: NM-300K – Test with earthworms: Ag concentrations in spiked soil.
Five replicate samples, each measured twice
Application / sample
Table 48: NM-300K – Test with earthworms: concentration of Ag ions measured by DGTs in soil.
Control 15 mg/kg 60 mg/kg 120 mg/kg 200 mg/kg
Day 0
Replicate 1 [µg/kg] 1 0.141 * 10-3 21.5 * 10-3 19.6 * 10-3 29.70 * 10-3 33.6 * 10-3 Replicate 2 [µg/kg] 1 0.287 * 10-3 36.1 * 10-3 22.3 * 10-3 technical defect 34.3 * 10-3 Mean value [µg/kg] 1 0.214 * 10-3 28.8 * 10-3 21.0 * 10-3 29.7 * 10-3 34.0 * 10-3 Percentage 2 [%] --- 1.92 * 10-4 0.35 * 10-4 0.25 * 10-4 0.17 * 10-4 Day 28
Replicate 1 [µg/kg] 1 1,16 * 10-3 41.9 * 10-3 46.1 * 10-3 56.7 * 10-3 76.0 * 10-3 Replicate 2 [µg/kg] 1 0.696 * 10-3 25.9 * 10-3 37.6 * 10-3 81.9 * 10-3 64.1 * 10-3 Mean value [µg/kg] 1 0.928 * 10-3 33.9 * 10-3 41.9 * 10-3 69.3 * 10-3 70.1 * 10-3 Percentage 2 [%] --- 2.26 * 10-4 0.70 * 10-4 0.58 * 10-4 0.35 * 10-4 Day 56
Replicate 1 [µg/kg] 1 1.23 * 10-3 35.9 * 10-3 75.5 * 10-3 162 * 10-3 86.2 * 10-3 Replicate 2 [µg/kg] 1 1.16 * 10-3 95.0 * 10-3 51.3 * 10-3 140 * 10-3 91.3 * 10-3 Mean value [µg/kg] 1 1.20 * 10-3 65.5 * 10-3 63.4 * 10-3 151 * 10-3 88.8 * 10-3 Percentage 2 [%] --- 4.37 * 10-4 1.06 * 10-4 1.26 * 10-4 0.44 * 10-4
1 Ag+ was measured in pore water as [µg/L]; for calculation in dry matter [µg/kg] a dry matter content of 88.9% was applied for the calculations at day 0 (mean value of the dry matter content of all test concentrations at day 0); at day 56 a dry matter con-tent of 79.5% was applied (mean value of the dry matter concon-tent of all test concentrations at day 56); for day 28 a dry matter content of 84.2% was used (mean value of the measured dry matter contents at day 0 and 56)
2 Recovery with respect to nominal concentration
Effect concentrations:
Effects:
No mortality was observed. Weight change of the adults varied, with both increases and de-creases, compared to the control. For reproduction, dose-response curves were obtained (Figure 16, Figure 14) and ECx, NOEC and LOEC-values were calculated (Table 49). Two controls were tested: one control without addition of the stabiliser, and a control with dispers-ant (concentration of dispersdispers-ant corresponded to the amount applied in the highest test con-centration of NM 330-K). This concon-centration resulted in about 20% inhibition of reproduction.
Inhibition of the dispersant was independent of the application via feed or via soil. Both con-trol values were considered for the calculation of ECx, NOEC and LOEC. Considering the confidence intervals, both concentration-effect curves overlap. In a second experiment the effect of the dispersant was investigated again. In this experiment no difference between control and dispersant control was observed (number of juveniles: control 347 ± 27; dispers-ant control 329 ± 4). Therefore it is recommended to use ECx, NOEC and LOEC values re-ferring to the control for the assessment of NM-300K.
Table 49: NM-300K – Test with earthworms: summary of effects on number of offspring
1 values in brackets: confidence interval; 2 n.d. = not determined due to mathematical reasons or inappropriate data; 3 results refer to nominal values; 4 results refer to Ag ions measured at day 56; 5ion concentration not measured in test vessels with 30 mg Ag/kg.
Physical/pathological symptoms and changes in behaviour
At the beginning of the test a high tendency of the worms to escape from the soil was ob-served in the tests with Ag-contents ranging from 60 to 200 mg/kg dm. As the vessels were covered with lids, in some of the test vessels the earthworms were observed at the lids and at the walls of the vessels (200 mg/kg: 2 vessels; 120 mg/kg: 2 vessels; 60 mg/kg: 1 vessel).
After two days, the earthworms had moved into the soil again. After 28 days, neither physical / pathological symptoms nor changes in behaviour were observed. All specimens gave the impression of healthy condition.
Weight change of the adults
The results of weight change are presented in Figure 13, Figure 14 and Table 50. For raw data of the biomass see chapter 21.3.2. Due to feeding, the biomass of the worms increased in all test approaches during the incubation period. Compared to the control, the increases due to treatments were varied and ranged from a small change to large change in weight.
Application in feed showed a concentration-effect relationship which did not occur for appli-cation in soil. The weight increase for appliappli-cation in soil exceeded the weight increase for the controls. Differences in weight between the three controls (control; control with dispersant on feed; control with dispersant on soil) during the incubation period were small. Therefore, the higher increase of the weight compared to the controls is assumed to be caused by the silver addition. The mode-of-action as well as the missing concentration-effect-relationship when soil was spiked cannot be explained so far.
Spiked feed, control: without further additions
Spiked feed, control: dispers-ant control
Figure 13: NM-300K – Test with earthworms: spiked feed, weight change - concentration-effect curve.
Spiked soil, control: without further additions
Spiked soil, control: dispersant control
Figure 14: NM-300K – Test with earthworms: spiked soil, weight change - concentration-effect curve.
Table 50: NM-300K – Test with earthworms: mean weight at test start and weight change at test end.
Concentrations given as nominal values
Control Control
Application on feed [mg/kg] Application on soil [mg/kg]
15 30 60 120 200 15 30 60 120 200
No mortality was observed.
Reproduction:
The results for reproduction are presented as mean values (Table 52). For single values of the replicates see chapter 21.3.2. Independent of whether the soil or feed was spiked, the dispersant had a small impact on reproduction, i.e. there were fewer juveniles in the control with dispersant (e.g. for soil spiking: 268) than in the control (e.g. for soil spiking: 341), repre-senting a statistically significant 22% inhibition of reproduction. The small inhibitory effect was not reproducible. Therefore, it is concluded that the effect of the dispersant observed in this test reflects biological variability. Both control values were considered with respect to the effect of Ag-NPs and inhibition was calculated in comparison to both the control and the con-trol with dispersant.
We found a concentration-effect relationship and observed strong inhibition of earthworm reproduction (Figure 15, Figure 16, Table 49). For the experiment with spiked soil, in com-parison to the control, the inhibition of reproduction ranged from 26% at the lowest concen-tration to 72% at the highest concenconcen-tration, yielding an EC50 of 80 mg/kg compared to the control and an EC50 of 146.0 mg/kg for the dispersant control. Soil spiking and feed spiking resulted in comparable effects and EC50 values.
The size of the worms in the different treatments differed considerably. In the control, the worms were much smaller than in the samples treated with 120 and 200 mg/kg. The
juve-niles in the control worms showed the typical expected sizes. Ten representative worms were selected and are depicted in Figure 17. These worms were also weighed. As some of the selected worms escaped before weighing, the result is presented as mean weight per remaining worms (Table 51).
The effect was observed in all replicate test vessels. However, the effect was not reproduci-ble when the test was repeated and different dung charges and grinding degrees were tested. Therefore, the reason for the increase in size is still unknown.
Spiked feed, control: without further additions
Spiked feed, control: dispers-ant control
Figure 15: NM-300K – Test with earthworms: spiked feed, reproduction - concentration-effect curve.
Spiked soil, control: without further additions
Spiked soil, control: dispers-ant control
Figure 16: NM-300K – Test with earthworms: spiked soil, reproduction - concentration-effect curve.
Control 120 mg/kg soil applied via feed
200 mg/kg soil applied via feed
Control 120 mg/kg soil
applied via soil
200 mg/kg soil applied via soil Figure 17: NM-300K - Representative sizes of earthworms after 56 days.
(Every photograph shows 10 worms; Ø of petri dishes: 100 mm; small black dots in petri dishes: gut content)
Table 51: NM-300K – Test with earthworms: mean weight of the offspring presented in Figure 17.
Content Calculated weight per worm [mg]
Control 11
120 mg / kg (soil) 69.3
200 mg / kg (soil) 53.7
120 mg / kg (feed) 108.6
200 mg / kg (feed) 72.0
Table 52: NM-300K – Test with earthworms: number of juveniles at test end.
Mean values and coefficient of variance (CV)
Application on feed [mg/kg] Application on soil [mg/kg]
15 30 60 120 200 15 30 60 120 200
