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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, no quantification of titanium concentration of test soils
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
no
Remarks:
; In deviation to GLP no archiving of the raw data was performed and the quality assurance unit was not involved with respect to the inspection of the test, of the raw data and of the report.
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
SPIKING OF SOIL WITH TIO2 POWDER
- Direct addition of the test substance into test vessel: yes; the TiO2 material was mixed with 1% of the total amount of air-dried test soil. This part was subsequently mixed with a spoon to the rest of the test soil.
- Solvents: no

SPIKING OF SOIL WITH AQUEOUS TIO2 DISPERSION
- Direct addition of the test substance into the test vessel: no
- Sonication of dispersion: yes; The TiO2 material was added to deionized water (max. 212 mg/L), stirred with a magnetic flea (900 rpm; 1 min) and sonicated for three minutes in a bath sonicator. Subsequently, a specific volume of the dispersion was sprayed onto the soil and mixed thoroughly with the test soil.
- Solvents: no
- Appearance of dispersion: no information
- Test medium: deionized water
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2°C
Moisture:
40 - 60%
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): no information
- Amount of soil: 658 g spiked soil (one batch)
- No. of replicates per concentration: 3 subsamples were taken from the batch at each sampling time
- No. of replicates per control: 3 subsamples were taken from the batch at each sampling time
- Light conditions: dark

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Treatments with pesticides or fertilizers: no plant protection products for at least 1 year prior soil sampling, no fertiliser for at least 6 month prior soil sampling
- Depth of sampling: no information
- Soil texture: loamy sand
- % sand: 71%
- % silt: 24%
- % clay: 5%
- Soil taxonomic classification: RefeSol 01-A
- pH (in water): 5.7
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 81.7
- Maximum water holding capacity (in g H20/ kg dry weigth): 264
- Cation exchange capacity (mmol/kg): 37.9
- Pretreatment of soil: soil was sieved to 2 mm
- Storage (condition, duration): outdoors on the grounds of the test facility in high grade stainless steel basins with drainage, and ground contact
- Initial microbial biomass as % of total organic C: 1.0%
- Organic substrate: 5 g/kg soil dw of ground lucerne-grass-green meal
Nominal and measured concentrations:
nominal concentrations (application via powder): 9.3, 21, 45, and 100 mg n-TiO2/kg soil (dw)
nominal concentrations (application via dispersion): 9.3, and 21 mg n-TiO2/kg soil (dw)
Reference substance (positive control):
no
Remarks:
not required according to guideline
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
nitrate formation rate
Remarks:
based on adverse effects
Remarks on result:
other: (application via powder);A significant stimulation at > 9.3 mg/kg dw was observed.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 21 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
nitrate formation rate
Remarks:
based on adverse effects
Remarks on result:
other: (application via dispersion)
Reported statistics and error estimates:
Statistical calculations:
For each concentration, the quantity of nitrate was determined. According to the guidelines for non-agrochemicals, the quantities of nitrate found in the treated samples after 28 days were compared to those found in the controls. Furthermore, the percent deviation values for the test concentrations were calculated. Means were compared by means of the STUDENT-t test and the U-Test (Wilcoxon, Whitney and Mann) for significant differences between control and study groups. EC x ,LOEC, and NOEC calculations were performed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat® Solutions GmbH).

A zeta potential of -18 mV was measured for the the stock dispersion containing 100 mg n-TiO2/L n deionised water.

Validity criteria fulfilled:
yes
Conclusions:
Application of the nano-sized TiO2 material P25 (21 nm, anatase-rutile) to a natural loamy sand via dry powder in a standardized nitrogen transformation test according to OECD 216 (2000), enhanced the nitrate transformation rate of the test soil. However, the effect may be due to an improvement of the test soil. In contrast, application via dispersion did not affect the nitrate transformation rate of the test soil, resulting in a 28-d NOEC of ≥ 21 mg/kg soil dw (nominal).
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, no quantification of titanium concentration of test soils
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
GLP compliance:
no
Remarks:
; In deviation to GLP no archiving of the raw data was performed and the quality assurance unit was not involved with respect to the inspection of the test, of the raw data and of the report.
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
SPIKING OF SOIL WITH TIO2 POWDER
- Direct addition of the test substance into test vessel: yes; the TiO2 material was mixed with 1% of the total amount of air-dried test soil. This part was subsequently mixed with a spoon to the rest of the test oil
- Solvents: no

SPIKING OF SOIL WITH AQUEOUS TIO2 DISPERSION
- Direct addition of the test substance into the test vessel: no
- Sonication of dispersion: yes; The TiO2 material was added to deionized water (92 and 202 mg/L), stirred with a magnetic flea (900 rpm; 1 min) and sonicated for three minutes in a bath sonicator. Subsequently, 202 ml and 208 ml of the dispersion were sprayed onto 2 kg test soil (dw) and mixed thoroughly with the test soil, resulting in TiO2 soil concentrations of 9.3 and 21 mg/kg soil dw.
- Solvents: no
- Appearance of dispersion: no information
- Test medium: deionized water
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2°C
Moisture:
40-60 % (of max. WHC)
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size):day 0: Sapromat® Voith Inc.; day 28: Sensomat, Aqualytik
- Amount of soil: 733 g (one batch)
- No. of replicates per concentration: 3 subsamples were taken from the batch at each sampling time
- No. of replicates per control: 3 subsamples were taken from the batch at each sampling time

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Treatments with pesticides or fertilizers: no plant protection products for at least 1 year prior soil sampling, no fertiliser for at least 6 month prior soil sampling
- Vegetation cover:
- Soil texture: loamy sand
- % sand: 71%
- % silt: 24%
- % clay: 5%
- Soil taxonomic classification: RefeSol 01-A
- pH (in water): 5.7
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 81.7
- Maximum water holding capacity (in g H20/ kg dry weigth): 264
- Cation exchange capacity (mmol/kg): 37.9
- Pretreatment of soil: soil was sieved to 2 mm
- Storage (condition, duration): outdoors on the grounds of the test facility in high grade stainless steel basins with drainage, and ground contact
- Initial microbial biomass as % of total organic C: 1.0%
- Microbial biomass [mg C/kg dry mass soil], calculated from respiration activity: 92
- Organic substrate: 5 g/kg soil dw of ground lucerne-grass-green meal
Nominal and measured concentrations:
nominal concentrations (application via powder): 9.3, 21, 45, and 100 mg n-TiO2/kg soil (dw)
nominal concentrations (application via dispersion): 9.3, and 21 mg n-TiO2/kg soil (dw)
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 21 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
respiration rate
Remarks on result:
other: (application via dispersion)
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
respiration rate
Remarks on result:
other: (application via powder)
Reported statistics and error estimates:
Statistical calculations:
For each concentration the quantity of consumed oxygen was determined. Performed according to the guidelines for non-agrochemicals, the glucose-induced respiration rates found in the treated samples after 28 days were compared to the respiration rates found in the controls. Furthermore, the percent inhibition value for the test concentrations was calculated. The percent deviation of the respiration rates were calculated in comparison to the control. ECx, LOEC and NOEC calculations were performed with the computer software ToxRat Professional version 2.10.4.1 (ToxRat® Solutions GmbH).

The zeta potential in the stock dispersion containing 100 mg n-TiO2/L was determined as a negative value of -18 mV in deionised water.

Validity criteria fulfilled:
yes
Conclusions:
In a soil microbial carbon transformation test according to OECD 217, there were no significant differences between the treatments with nano-sized TiO2 material (P25; 21 nm, anatase-rutile) and the control after an incubation period of 28 d. Thus, the NOEC amounts to ≥100 and ≥ 21 mg n-TiO2/kg soil dw for powder and dispersion spiked soil (nominal, highest test concentration).
Endpoint:
toxicity to soil microorganisms
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2008-02-19 to 2008-04-28
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study conducted according to the ISO 14238 protocol. TiO2 concentrations were not measured.
Qualifier:
according to
Guideline:
ISO 14238
Deviations:
not specified
GLP compliance:
no
Remarks:
Compliance statement to ISO 14238 guideline attached (see below)
Analytical monitoring:
not specified
Vehicle:
not specified
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Slags were added to the soils on 17th of March 2008 by weighing the appropriate amount of air dry soil and slag (0; 0.03; 0.1; 0.3; 1; 3 and 10 wt%) and mixing in a clean concrete mixer to a final total amount of 4292 g dry matter. The soil/slag mixture was homogenized for 5 minutes, and stored in plastic bags till use. All treated soils were moistened at the same time (26th of March 2008) to 18.5% moisture content by adding deionised water and mixing manually. A 144 g subsample was set apart for the soil nitrification test.

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
Immediately after wetting the soils up to 18.5% moisture content, samples were preincubated for 5 days at 20°C to allow activiation of the soil microbial community.
The soils were aerobically incubated in dark at 20°C (test)
Moisture:
18.5% moisture (preincubation)
The moisture content after adding the ammonia solution was 23.5% (constant moisture content for 28 days)
Details on test conditions:
SOIL INCUBATION
- Method: the soil was mixed thoroughly using a spatula, and divided in three 50 g subsamples (wet weight)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical location: Heverlee, Belgium
- Soil texture
- % sand: 10
- % silt: 76
- % clay: 14
- Soil taxonomic classification: Haplic Luvisol with a silt-loam texture
- Soil classification system: WRB soil classification
- pH (in water): 7.00 (Preliminary experiments showed that the addition of slag2 to soil acidified the soil by 0.6-0.7 pH units, and that this pH decrease can be neutralized by adding 10 mmol OH--equivalents per kg dry soil. Therefore, a ‘slag+CaO’ treatment was included for the soil amended with 1 and 10% of slag 2 that received an extra 5 mmol CaO/kg (corresponding to 1.2 g CaO powder) on top of the pure slag to compensate for the pH drop)
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): no data
- Water holding capacity (ml/100g dry soil): 43.4
- Bulk density (g/cm3): 1.38
- Water content at pF2 (100 cm suction) (ml/100g dry soil): 28.9
- Effective Cation exchange capacity (mmol/kg): 98
- Ammonium oxalate extractable Al, Fe and Mn oxides:
Feox (mg/kg): 2021
Alox (mg/kg): 536
Mnox (mg/kg): 254
- Pretreatment of soil: soil was immediately air dried at 25°C for 8 days and sieved (4mm)
- Storage (condition, duration): the air-dried soil was stored in plastic bags at room temperature
- Initial microbial biomass as % of total organic C: no data
- Aqua regia extractable metals:
Cd (mg/kg): 0.3
Cu (mg/kg): 18.5
Co (mg/kg): 6.6
Ni (mg/kg): 13.1
Pb (mg/kg): 26.4
Zn (mg/kg): 52.2
Sb (mg/kg): 0.6

DETAILS OF PREINCUBATION OF SOIL (if any): Immediately after wetting the soils up to 18.5% moisture content, samples were preincubated for 5 days at 20°C to allow activiation of the soil microbial community.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The NH4+ concentration of the soil was measured immediately after ammonia addition, and after 28 days incubation, whereas the NO3- concentration was measured immediately after ammonia addition and after 3, 7 and 28 days incubation.
Nominal and measured concentrations:
Nominal concentrations (weight %): 0; 0.03; 0.1; 0.3; 1; 3; 10
Measured (mg/L): <0.01
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Exxaro SO4
Basis for effect:
other: Potential Nitrification Rate
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
> 10 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Exxaro SO4
Basis for effect:
other: Potential Nitrification Rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Exxaro SO4
Basis for effect:
other: Substrate Induced Nitrification
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
> 10 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Exxaro SO4
Basis for effect:
other: Substrate Induced Nitrification
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
RBM SO4
Basis for effect:
other: Potential Nitrification Rate
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
3 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
RBM SO4
Basis for effect:
other: Potential Nitrification Rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
RBM SO4
Basis for effect:
other: Substrate Induced Nitrification
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
3 other: %
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
RBM SO4
Basis for effect:
other: Substrate Induced Nitrification
Reported statistics and error estimates:
The highest No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were determined by ANOVA with a Duncan test.
Because the activity of the natural microbial population is tested in the nitification assay and the largest slag doses resulted in a significant dilution of the natural soil, the PNR was corrected, taking into account the amount of natural soil present in the samples tested.
Validity criteria fulfilled:
yes
Conclusions:
The effect of two upgraded imenite slags (UGI, Exxaro SO4 and RBM SO4) on the nitrification activity in a natural soil was measured. For Exxaro SO4, no effect was observed at all tested concentrations, resulting in an unbounded NOEC of >=10%. A bounded NOEC of 1% and LOEC of 3% were retrieved for the UGI RBM SO4.
Upgraded ilmenites (UGI) consist primarily (> 80%) of a titanate phase i.e., Ti (as TiO2) with Fe, Al, Si, Mg and other metals. Transformation/dissolution tests were performed with UGI products by analysing total dissolved concentrations of the trace and minor elements Al, Co, Cr, Cu, Fe, Mn, Mo, Ni, Nb, Ti, V and Zn over periods up to 28 days at loadings of 100, 10 and 1 mg/L. The T/D data revealed that only Fe, Mn and V among the 12 metals dissolved to any significant extent (maximum increase in Fe, Mn and V concentrations 26.2, 8.9 and 3.5 µg/l, respectively). Within the limits of experimental error, the other metals remained refractory to release, none dissolving from any of the slags at any of the loadings. Because of the structural similarity and the low solubility of both UGI slags and TiO2, read-across for ecotoxicological hazard data from UGI to TiO2 is considered relevant.

Description of key information

 Chronic data are available for the toxicity of two upgraded ilmenite slags (>80% TiO2) to the nitrification activity of soil microorganisms in a natural soil, and the lowest NOEC amounts to 10,000 mg/kg dw. Regarding the toxicity of TiO2-NP to soil microbes, in soil microbial nitrogen and carbon transformation tests according to OECD 216 and OECD 217, there were not any differences between treatments with 100 mg n-TiO2/kg (highest test concentration) and the control, respectively (Hund-Rinke and Klawonn, 2013). In sum, toxicity data from standard toxicity tests seems to indicate that micro-and nanosized TiO2 has a low potential for toxicity to soil microbes. 

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Chronic data are available for the toxicity of two upgraded ilmenite slags (>80% TiO2) to the nitrification activity of soil microorganisms in a natural soil, and the lowest NOEC amounts to 10,000 mg/kg dw.

Upgraded ilmenites (UGI) consist primarily (> 80%) of titanate, i.e., TiO2 with Fe, Al, Si, Mg and other metals, and are poorly soluble and not biodegradable. The transformation/dissolution of 3 UGI products at loadings of up to 100 mg/L over 28 d did not result in any significant release of titanium. Bulk and nano-TiO2 forms are also poorly soluble in environmental media (Brouwers, 2009; Klawonn, 2017 a-f). Based on similarity in composition and the same poor solubility of UGI and TiO2, read-across of ecotoxicological data from UGI to TiO2 is justified.

Nanosized TiO2:

Regarding the toxicity of TiO2-NP to soil microbes, in nitrogen and carbon transformation tests according to OECD 216 and OECD 217, there were not any differences between treatments with 100 mg n-TiO2/kg (highest test concentration) and the control, respectively (Hund-Rinke and Klawonn, 2013). 

Data on soil microbial toxicity are also available from a supporting study, in which a non-standard test was performed. After 84 days of cultivating wheat (Triticum ssp.) in a sand-soil mixture spiked with TiO2-NP up to 1000 mg/kg, roots were colonised by AMF, abnormalities in mycorrhizal colonisation could not be observed, and N2O emissions and the amount of extracted DNA were similar to the control (Klingenfuss, 2014).