potassium titanium oxide (K2Ti6O13)

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

January 21, 2000

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Potasum hexatitanate, potassium titanium oxide
Batch n. MA-95150
Solid, light-yellow powder

Analytical monitoring:

not required

Remarks:

The analytical monitoring of the test item was not required, according to the test guideline.

Vehicle:

yes

Remarks:

Potassium titanium oxide was not soluble in water or acetone; therefore appropriate amounts of Potassium titanium oxide were weighed and mixed with quartz sand (as dispersant) and compounded afterwards with the soil.

Details on preparation and application of test substrate:

Potassium titanium oxide was not soluble in water or acetone; therefore appropriate amounts of Potassium titanium oxide were weighed and mixed with quartz sand (as dispersant) and compounded afterwards with the soil and additionally 0.5% lucerne meal (related to soil dry weight) by means of a laboratory mixer. Throughout the application the soil (biologically active agricultural soil, Loamy sand) was ventilated and the soil water content for the test item treatments were adjusted to 48% and 49% of WHC with ultrapure water.
To the control, an adequate amount of quartz sand and Lucerne meal was mixed into the soil. The soil water content of the control was adjusted to 49% of WHC.

The test item was applied to the total soil amount for each treatment and soil from each treatment was divided into three replicates after application. The applications were conducted in the following order:
1. Control
2. test rate 1: 62.5 mg Potassium titanium oxide/kg soil dry weight
3. test rate 2: 125 mg Potassium titanium oxide /kg soil dry weight
4. test rate 3: 250 mg Potassium titanium oxide /kg soil dry weight
5. test rate 4: 500 mg Potassium titanium oxide /kg soil dry weight
6. test rate 5: 1000 mg Potassium titanium oxide /kg soil dry weight.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

3 replicates per treatment

Test temperature:

20 +/- 2 °C (with continuous monitoring and recording), in the dark

Moisture:

The water content of one replicate of each treatment group was determined at each sampling date. Water losses were compensated by adding pure water. Throughout the study, the water content ranged from 48% to 49% WHC.

Organic carbon content (% dry weight):

0.89

Nitrogen content (% dry weight):

0.11

Details on test conditions:

SOIL ORIGIN AND FIELD HISTORY

The soil batch used in this study was according to the guidelines and was taken from fallow grassland in Germany.
No organic or mineral fertilizer had been used on the soil for at least four years prior to test initiation.

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SOIL PARAMETERS

The soil particle size, cation exchange capacity, organic carbon content and nitrogen content, water holding capacity, pH is determined at the standard soil F5M at LUFA, Speyer, Germany periodically. The soil specifications are listed below.
The nitrite content, nitrate content and ammonium content of the soil was determined at the test facility.

The soil specifications are listed below:

- Soil texture .......................................................... Loamy sand
- pH .................................................................... 7.4
- Total Org. C (%) based on soil dry weight ............................. 0.89
- Total N (%) based on soil dry weight .................................. 0.11
- NH4+-N (mg/kg dry weight) ............................................. 0.317
- NO2--N (mg/kg dry weight) ............................................. 0.010
- NO3--N (mg/kg dry weight) ............................................. 25.742
- Nmin-N (mg/kg dry weight) ............................................. 26.069
- CEC cation exchange capacity (meq/100 g dry weight) ................... 12.7
- WHC water holding capacity (%) ........................................ 40.8
- Clay (< 0.002 mm, %) .................................................. 11.4
- Silt (0.002-0.063 mm, %) .............................................. 36.0
- Sand (0.063-2.00 mm, %) ............................................... 52.6

The following parameters were determined at the test facility on the soil batch used for test:

- Dry weight (%) ..................................................................... 84.96
- Microbial biomass (mg C/kg dry weight), calculated from respiration activity* ...... 305.89
- Microbial biomass (% of total organic carbon)* ..................................... 3.44
- NO3--N (mg/kg dry weight) .......................................................... 8.225

* based on CO2-production

The soil criteria of the OECD guideline 216 were met.

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SOIL PREPARATION

Soil batch was sampled (sampling depth 0.2 m) and brought to the laboratory. In the laboratory it was air-dried, pre-sieved (mesh 10 mm) and sieved (mesh 2 mm) at room temperature.

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SOIL STORAGE UNTIL USE (PRE-INCUBATION)

The soil was stored at 20°C ± 2°C with appropriate ventilation and periodical moisture adjustment. Total duration of pre-incubation was 16 days. The soil dry weight prior to test start was determined to be 88.0%.

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TEST UNITS

The soil (300 g dry weight) was filled loosely into disposable plastic boxes of approximately 0.5 L, which were covered by perforated lids to allow air exchange to ensure aerobic incubation conditions.

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REPLICATES

- No. of replicates per concentration: 3
- No. of replicates per control (sodium chloride): 3
- No. of replicates per vehicle control (quartz sand): 3

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EFFECT PARAMETERS MEASURED

Nitrate Determination: For the determination of nitrogen content, soil samples were taken within 6 hours after application and at day 28. The nitrogen content was determined in each sample of treated and control soils.
For extraction, 24 g to 25 g soil were suspended in 100 mL 0.1 M KCl-solution and agitated for one hour. The suspension was centrifuged (Multifuge 3s+, 4350 rpm) and the extracts were stored deep frozen (days 0, 28) and analysed afterwards.
Amounts of 14.8 mg and 72.2 mg sodium nitrite and potassium nitrate, respectively, were diluted in 200 mL (sodium nitrite) and 100 mL (potassium nitrate) 0.1 M KCl to prepare the standard stock solutions for nitrite-N and nitrate-N + nitrite-N determination. Appropriate aliquots of the stock solutions were automatically diluted by the dilution unity with 0.1 M KCl to prepare 6 standard solutions at a range of 0.5 mg/L to 3.0 mg/L for nitrite-N and 7 standard solutions at a range of 1.0 mg/L to 12.0 mg/L for nitrate-N + nitrite-N determination. Before photometric determination, frozen soil extracts were thawed. For determination, undiluted extracts were used. To get the nitrate-N concentrations, the nitrite-N values were subtracted from the nitrate-N + nitrite-N values. The nitrogen-N content was determined using a AA3 Continuous Flow Analyzer and equipment.

Amounts of NO3--N were calculated based on concentrations determined in soil extracts and the amount of extracted soil. Results are reported as mg NO3--N/kg soil dry weight. The following LOQ was determined for extracted soil: nitrate-N: 0.152 mg/kg soil dry matter.

The nitrate-N formation rate was calculated using the difference between soil nitrate-N contents from day 28 and day 0 according to the following equation:
NO3--N per day = [NO3--N (d 28) - NO3--N (d 0)] /28 days
The amount of nitrate is presented as mg NO3--N/kg soil dry weight.

Nominal and measured concentrations:

Nominal concentration used were the following:
- test rate 1: 62.5 mg Potassium titanium oxide/kg soil dry weight
- test rate 2: 125 mg Potassium titanium oxide/kg soil dry weight
- test rate 3: 250 mg Potassium titanium oxide/kg soil dry weight
- test rate 4: 500 mg Potassium titanium oxide/kg soil dry weight
- test rate 5: 1000 mg Potassium titanium oxide/kg soil dry weight.

Reference substance (positive control):

yes

Remarks:

Effects of sodium chloride (used as reference, positive item) were determined at a rate of 16 g/kg dry soil in a separate GLP study perfomed at the same facility within one year before start of the experimental phase of this study.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Results with reference substance (positive control):

The reference item sodium chloride was tested in a GLP study performed at the same facility. Sodium chloride was tested at 16 g/kg soil dry weight.
At day 28 after applications, the deviations drom control samples were the following:
- soil nitrate content -42.60%
- soil nitrate formation rate -111.81%

Validity criteria

The validity criteria were met: the coefficient of variation % of replicate control samples for nitrogen trasformation nitrate-N content were 2.83 on day 0 and 2.62% on day 28. Detailed data are presented in the following Table.

Nitrogen Transformation Test: Results of Nitrate-N Determination

	measured value [mg NO3-N/L]	volume of KCl-solution [mL]	amount of extracted moist soil [g]	soil water content [mL]	% soil dry weight	mg NO3-N/ kg soil dw	mean             mg NO3-N/ kg soil dw	SD	% deviation from control	% CV
day 0
Control	2.937	100	24.17	4.04	83.3	15.180	15.432	0.436	---	2.83
	2.926	100	24.07	4.02	83.3	15.180
	3.073	100	24.08	4.02	83.3	15.935
test rate 1	3.069	100	24.56	4.05	83.5	15.569	15.624	0.318	1.24	2.04
	2.995	100	24.32	4.01	83.5	15.338
	3.113	100	24.29	4.01	83.5	15.966
test rate 2	2.933	100	24.56	4.10	83.3	14.923	14.921	0.032	-3.31	0.21
	2.915	100	24.47	4.09	83.3	14.888
	2.879	100	24.05	4.02	83.3	14.951
test rate 3	2.932	100	24.32	4.06	83.3	15.059	15.376	0.427	-0.36	2.78
	3.091	100	24.35	4.07	83.3	15.862
	2.982	100	24.50	4.09	83.3	15.208
test rate 4	2.939	100	24.80	4.09	83.5	14.772	15.064	0.438	-2.38	2.91
	3.113	100	24.94	4.12	83.5	15.568
	2.918	100	24.48	4.04	83.5	14.853
test rate 5	2.868	100	24.19	3.99	83.5	14.765	14.672	0.167	-4.92	1.14
	2.928	100	24.71	4.08	83.5	14.772
	2.869	100	24.69	4.07	83.5	14.480
day 28
Control	7.010	100	24.36	4.09	83.2	35.998	35.145	0.921	---	2.62
	6.619	100	24.23	4.07	83.2	34.169
	6.790	100	24.07	4.04	83.2	35.269
test rate 1	6.601	100	24.25	3.98	83.6	33.861	33.251	0.533	-5.39	1.60
	6.439	100	24.26	3.98	83.6	33.014
	6.449	100	24.40	4.00	83.6	32.877
test rate 2	6.267	100	24.55	4.08	83.4	31.865	32.029	0.741	-8.87	2.31
	6.042	100	24.01	3.99	83.4	31.384
	6.507	100	24.74	4.11	83.4	32.838
test rate 3	6.564	100	24.32	4.06	83.3	33.714	33.239	0.436	-5.42	1.31
	6.379	100	24.25	4.05	83.3	32.858
	6.395	100	24.09	4.02	83.3	33.144
test rate 4	6.866	100	24.35	4.02	83.5	35.130	36.398	1.481	3.57	4.07
	6.987	100	24.14	3.98	83.5	36.037
	7.432	100	24.35	4.02	83.5	38.026
test rate 5	7.280	100	24.42	4.00	83.6	37.077	36.891	0.703	4.97	1.91
	6.997	100	24.09	3.95	83.6	36.114
	7.387	100	24.52	4.02	83.6	37.483
LOQ: 0.152 mg/kg soil dry weight SD: standard deviation CV: coefficient of variation (calculated as SD / mean value * 100) dw = dry weight --- = not applicable

Results - Nitrogen Transformation Test

Nitrate Content: At day 28, concentrations of the test item up to 1000 mg/kg soil dry weight caused no relevant increasing nitrate-N contents in dependency of the test concentrations. All deviations between test item concentrations up to and including 1000 mg/kg soil dry weight and control were within a range of 25% deviation to control. At day 28, the differences to the control were -5.39%, -8.87%, -5.42%, 3.57% and 4.97% for test rate 1 (62.5 mg test item/kg soil dry weight) up to test rate 5 (1000 mg test item/kg soil dry weight). The differences of test rates 4 and 5 were not statistically significant compared to the control at day 28 (Student-test procedure, one-sided smaller, α = 0.05).

Detailed results (as mean values) are reported in the following Table. Individual values are rported under the paragraph "Validity criteria".

Nitrogen Transformation Test: Effects of the test item on Nitrate-N Content and Nitrate-N Rate (Mean Values)

	day 0			day 28			interval day 0-28
rate	NO3-N mg/kg dry weight	Dev. %^1	sig.^2	NO3-N mg/kg dry weight	Dev. %^1	sig.^2	NO3-N mg/d/kg dry weight	Dev. %^1	sig.^2
control	15.432	--	--	35.145	--	--	0.704	--	--
test rate 1	15.624	1.24	n.s.	33.251	-5.39	*	0.629	-10.65	*
test rate 2	14.921	-3.31	*	32.029	-8.87	*	0.611	-13.21	*
test rate 3	15.376	-0.36	n.s.	33.239	-5.42	*	0.638	-9.37	*
test rate 4	15.064	-2.38	n.s.	36.398	3.57	n.s.	0.762	8.24	n.s.
test rate 5	14.672	-4.92	*	36.891	4.97	n.s.	0.794	12.78	n.s.
^1: Dev., deviation from control ^2: sig.: significance to control according to Studentt-test, one-sided smallerα= 0.05 (* = significant; n. s.: not significant)   test rate 1: 62.5 mg Potassium titanium oxide/kg soil dry weight test rate 2: 125 mg Potassium titanium oxide/kg soil dry weight test rate 3: 250 mg Potassium titanium oxide/kg soil dry weight test rate 4: 500 mg Potassium titanium oxide/kg soil dry weight test rate 5: 1000 mg Potassium titanium oxide/kg soil dry weight

Nitrogen Transformation - Nitrate Formation Rate: The test item caused no relevant increasing cumulative soil nitrate formation rates in dependency of the test concentration (up to 1000 mg/kg soil dry weight). All deviations between test item concentrations up to and including 1000 mg/kg soil dry weight and control were within a range of 25% deviation to control. Differences to the control were -10.65%, -13.21%, -9.37%, 8.24% and 12.78% for test rate 1 (62.5 mg test item/kg soil dry weight) up to test rate 5 (1000 mg test item/kg soil dry weight). The differences of test rates 4 and 5 were not statistically significant compared to the control at day 0-28 interval (Student-test procedure, one-sided smaller, α = 0.05).

Detailed results are reported in the following Table.

Nitrogen Transformation Test: Calculation of Nitrate-N Formation Rate

	mg NO3-N/kg soil dw per day^1	mean mg NO3-N/kg soil dw per day^1	SD	% deviation from control	% CV
days 0 - 28
Control	0.744	0.704	0.035	---	4.97
	0.678
	0.691
test rate 1	0.653	0.629	0.025	-10.65	3.97
	0.631
	0.604
test rate 2	0.605	0.611	0.026	-13.21	4.26
	0.589
	0.639
test rate 3	0.666	0.638	0.030	-9.37	4.70
	0.607
	0.641
test rate 4	0.727	0.762	0.057	8.24	7.48
	0.731
	0.828
test rate 5	0.797	0.794	0.030	12.78	3.78
	0.762
	0.822
SD: standard deviation dw = dry weight CV: coefficient of variation (calculated as SD / mean value * 100) --- = not applicable ^1: calculated from the difference of the mean values between the sampling date and day 0

Validity criteria fulfilled:

yes

Conclusions:

The test item had no relevant inhibiting impact on nitrogen transformation (nitrate-N content, nitrate-N formation rate) of soil microorganisms when applied at test item concentrations up to and including 1000 mg/kg soil dry weight treatment. Therefore, no ECx for inhibiting effects of Potassium titanium oxide could be calculated. The statistical analysis revealed that the NOEC for an inhibition of the nitrogen transformation in soil is higher than or equal to 1000 mg test item/kg soil dry weight.

Executive summary:

The purpose of the study was to assess the effects of the test item on the activity (nitrogen transformation) of soil microorganisms in the laboratory.

Materials and Methods

The study was based on the OECD Guideline for the Testing of Chemicals, Soil Microorganisms: Nitrogen Transformation Test, Guideline 216 (January 21, 2000).

The test item was potassium titanium oxide (potassium hexatitanate, CAS n. 12056 -51 -8); quartz sand and sodium chloride were used as negative and positive control, respectively. Since potassium titanium oxide was not soluble in water or acetone, appropriate amounts of potassium titanium oxide were weighed and mixed with quartz sand (as dispersant) and compounded afterwards with the soil. The following concentrations of the test item were used:

- Test Rate 1: 62.5 mg Potassium titanium oxide /kg soil dry weight

- Test Rate 2: 125 mg Potassium titanium oxide /kg soil dry weight

- Test Rate 3: 250 mg Potassium titanium oxide /kg soil dry weight

- Test Rate 4: 500 mg Potassium titanium oxide /kg soil dry weight

- Test Rate 5: 1000 mg Potassium titanium oxide /kg soil dry weight.

Three replicates per treatment were used. The determination of nitrogen-transformation in soil enriched with lucerne meal was performed 6 hours and 28 days after treatment. NO3-nitrogen formed in the nitrification process was determined by continuous flow analysis.

Findings

Nitrogen Transformation - Nitrate Content: At day 28, concentrations of the test item up to 1000 mg/kg soil dry weight caused no relevant increasing nitrate-N contents in dependency of the test concentrations. All deviations between test item concentrations up to and including 1000 mg/kg soil dry weight and control were within a range of 25% deviation to control. At day 28, the differences to the control were -5.39%, -8.87%, -5.42%, 3.57% and 4.97% for test rate 1 (62.5 mg test item/kg soil dry weight) up to test rate 5 (1000 mg test item/kg soil dry weight).

Nitrogen Transformation - Nitrate Formation Rate: The test item caused no relevant increasing cumulative soil nitrate formation rates in dependency of the test concentration (up to 1000 mg/kg soil dry weight). All deviations between test item concentrations up to and including 1000 mg/kg soil dry weight and control were within a range of 25% deviation to control. Differences to the control were -10.65%, -13.21%, -9.37%, 8.24% and 12.78% for test rate 1 (62.5 mg test item/kg soil dry weight) up to test rate 5 (1000 mg test item/kg soil dry weight).

Conclusion

The test item had no relevant inhibiting impact on nitrogen transformation (nitrate-N content, nitrate-N formation rate) of soil microorganisms when applied at test item concentrations up to and including 1000 mg/kg soil dry weight treatment. Therefore, no ECx for inhibiting effects of Potassium titanium oxide could be calculated. The statistical analysis revealed that the NOEC for an inhibition of the nitrogen transformation in soil is higher than or equal to 1000 mg test item/kg soil dry weight.

Description of key information

No effects observed up to 1000 mg/kg soil dw (the maximum concentration tested).