5 Pre-tests - spiking of soil
5.1 Materials and methods .1 Nanomaterial .1 Nanomaterial
5.2.2 Soil microflora - nitrification activity
Three experiments differing in application forms (in silica sand, in soil, via suspension) and tested concentrations of nanoparticles were conducted.
Experiments with silver nanoparticles
1st experiment: Comparison of three spiking forms
• Spiking via soil and silica sand as solid carrier; two test concentrations (10 and 100 mg/kg)
• Spiking via dispersion; one test concentration resulting in a concentration of 10 mg/kg in soil; a concentration in soil of 100 mg/kg was not considered suitable; the concen-tration in the test suspension was considered to be too high and large agglomerates were expected.
Inhibition of in biomass increase [%] Inhibiton of reproduction [%]
P25 Ag
Inhibition of biomass increase [%] Inhibition of reproduction [%]
2nd experiment: Comparison of two spiking forms – valdidation of the results for the most promising procedures of the 1st experiment
• Spiking via soil as solid carrier; two test concentrations (10 and 100 mg/kg); based on the results of the first experiment spiking using silica sand was considered less suit-able.
• Spiking via dispersion (10 mg/kg)
3rd experiment: Comparison of two spiking forms – three test concentrations
• Spiking via soil as solid carrier; two test concentrations (10, 50, 100 mg/kg); based on the results of the first experiment spiking using silica sand was considered less suit-able.
• Spiking via dispersion (10, 50, 100 mg/kg)
The microbial nitrification activity was used as indicator for ecotoxicity. A compilation of the results is shown in Figure 3. A comparison of the results obtained for the soil contents of 10 and 100 mg/kg indicates lowest effects for the application via silica sand, followed by the application via soil. At 10 mg/kg the highest effects are obtained with the application via sus-pension. A slightly increased toxicity is obtained at 50 mg/kg for the application via suspen-sion. At 100 mg/kg no difference between an application via soil and suspension is obtained.
Both application forms almost completely inhibit the nitrification activity. From these results it can be concluded that the bioavailability of the nanoparticles is reduced in the presence of silica sand. Therefore, this application form was not considered in the main experiments. Due to the comparable results for spiking via soil and dispersion in the 3rd experiment for the low-est tlow-est concentration (10 mg/kg) which did not correspond to the results of the 2nd experi-ment no information on the most suitable application form was achieved.
The standard deviation of the nitrification activity in the replicates was used as an indicator for the homogeneity of spiking. To determine the nitrification activity four soil samples (20 g each) were analysed. A high standard deviation of the activity values indicated a
non-homogeneous distribution of the test substance. For this assessment only activity values that were in a medium inhibition range compared to the control were suitable as high inhibition results in low activity values. Even small differences in the activity values result in a compa-rable high percent standard deviation. Activity values which are only slightly reduced com-pared to the control are not applicable. In Figure 4 the standard deviation of suitable soil samples is presented. It is obvious that the application of dry powder as well as the applica-tion of suspensions can result in standard deviaapplica-tions which are in the range of the standard deviations of the control. However, for every application form outliers were observed (e.g.:
experiment 1, application via silica sand results in a soil content of 100 mg/kg; experiment 3, application via suspension results in a soil content of 50 mg/kg). Therefore, both application techniques (application of dry powder and suspensions) are theoretically suitable. The rea-son for the outliers, however, is not yet understood.
0
Experiment 1 Experiment 2 Experiment 3
Application via:
Concentration in soil: 10 mg/kg 100 mg/kg 50 mg/kg
Figure 3: Pre-tests: effects of different application forms for Ag nanoparticles on nitrification activity.
Experiment 1 Experiment 2 Experiment 3
--- Soil
0 mg/kg 100 mg/kg 50 mg/kg
Suspension
100 mg Ag/L Sand Suspension
50 mg Ag/L
Figure 4: Pre-tests: standard deviation of nitrification activity in the nitrification tests with Ag resulting in~50% inhibition.
The toxicity of P25 is much lower compared to the toxicity of silver ( Experiments with P25
Figure 3 and 5). There-fore, information concerning suitable application techniques of P25 is limited. Nevertheless, it was observed that P25 application via soil achieved higher bioavailability compared to appli-cation via silica sand (Figure 5). Appliappli-cation via suspension resulted in no inhibition effect at all. It is assumed that the high concentration in the dispersions used for spiking showed a high agglomeration rate. The dispersions were very polydisperse and the particle size could
not be determined. From the results it is concluded that high test concentrations can be achieved by dry application whereas by wet application the maximum test concentration can be limited by the agglomeration behaviour of the nanoparticles in the dispersion.
0 5 10 15 20 25 30
sand soil suspension
100 mg/L
sand soil suspension
940 mg/L
Inhibition of nitrification [%]
Experiment 1 Experiment 2 Application
via:
Concentration in soil: 10 mg/kg 100 mg/kg
no inhibition no inhibition no inhibition
Figure 5: Pre-tests: effect of different application forms of P25 on nitrification activity.
5.2.3 Chemical analyses
The pre-tests presented above were performed with material purchased from Sigma Aldrich and distributed as powder. This material was selectedas the OECD nanomaterial was not yet available. It had been expected that the OECD material would be available in solid form (powder). However, the NM-300K was received as a stabilised dispersion. To get information about the homogenous distribution of the NM-300K material soil samples from the earthworm test (see main test presented below) were used for chemical analyses. Soil spiked with NM-300K using a small amount of soil as carrier was investigated.
Silver analyses NM-300K in the earthworm test
The results for the application via soil are presented in Table 17.
Table 17: Homogeneity of spiking: recovery of NM-300K in soil (earthworm test).
Five replicate samples, each measured twice
Application / sample
With 90% each for the low and high test concentrations, recovery was satisfactory. Powder in the concentration of 120 mg/kg was more homogenously distributed than in concentrations of 15 mg/kg despite the same amount of carrier soil used for both concentrations. Nevertheless, 15% standard deviation was considered to be in an acceptable range.
After elaboration of the results of the main tests, we could also include biological variability in the considerations concerning the acceptance of the homogeneity of spiking. We selected 68 results (mean values and standard deviations) with either TiO2 or Ag nanoparticles from ran-domly-selected tests of this project with different test organisms and test parameters, includ-ing earthworm reproduction, plant growth and microbial nitrogen and carbon transformation.
The same criteria were applied to tests with conventional chemicals performed at the Fraun-hofer-Institute (60 test results, mean values and standard deviations). Each standard devia-tion was expressed as a percentage of the respective mean value. The 90% percentile of the standard deviations for both sets of tests (nanoparticles and conventional chemicals) was
calculated. Ninety percent of the standard deviations in the nanomaterial tests were in the range 2–17% compared to 3–24% in the conventional chemical tests. The variability of the chemical analysis results was comparable to the variability of the ecotoxicological test results with nanoparticles. Furthermore, the variability of the nanomaterial tests based on dry spiking using soil as the carrier was comparable to the variability of the conventional substance tests spiked with aqueous solutions. We therefore concluded that the dry spiking procedure using soil as the carrier achieves adequate spiking homogeneity.
Due to the high background values of titanium in the applied soil only silver analyses of the soil samples are presented. Due to high concentration levels the samples had to be diluted prior to analysis to fit with the working and calibration range of the instrument (ICP-OES).
The silver concentrations measured after digestion in the control soil samples were below the limit of detection (< 0.122 - < 0.819 mg/kg).
Silver analyses (Sigma Aldrich) in soil samples of the nitrification test
Table 18 summarizes the measured silver con-centrations in soil.
Table 18: Homogeneity of spiking: recovery of silver in soil (nitrification test).
Silver: Sigma Aldrich; six replicate samples
Application / sample
The recovery determined for application via dispersion (10 mg/kg) was low. As the experi-ments focused on the characterisation of the homogeneity of spiking, no further sourcing concerning the low recovery was performed. Six replicates sampled at different spots of the soil were analysed. The standard deviation of the recovery was between 1.6 and 8.8 %. To receive information on the homogeneity, the recovery was considered to be 100 % and the standard deviation was calculated as percent of the recovery. Values between 2.0 and 11.5% were calculated (e.g.: 80.6 % was considered to be 100 %; 1.6 of 80.6 amounts to 2.0
%). A variability between 2.0 and 11.5 % is considered to be acceptable for biological analy-ses. The observed outliers in the nitrification test resulting in high standard deviation (Figure 4) cannot be explained by non-homogeneous spiking.
5.2.4 Conclusions
As the toxicity for silver was higher than that for P25, the following conclusions drawn for the most suitable application technique are based on the results obtained for silver:
• Based on the lower effects - indicating lower bioavailability – obtained upon the cation via silica sand compared to the application via soil and suspension, the appli-cation via silica sand is considered less suitable.
• Application via powder allows a high variability of the test concentrations.
• Application via liquid suspension may cause a higher bioavailability of the elements.
• For earthworms, spiking of dung may result in a higher toxicity for earthworms com-pared to the spiking of soil. However, spiking of soil is the method described in the respective guidelines.
To obtain further results the following procedure was applied in the main tests:
• TiO2 nanoparticles: application via suspension and via solid carrier (soil) in soil as well as via suspension (all tests) and directly in the form of powder in dung (tests with earthworms; due to the high amount of nanomaterial added to dung, no carrier was considered to be necessary)
• Ag nanoparticles: application via solid carrier (soil) in soil and directly in dung
