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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached document in section 13 "Assessment report" for justification and rationale of the analogy approach.
Original letters from the French Competent Authorities requiring the read across to be done with cerium oxide isostearate are attached below.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: No biological relevant effect on the nitrification process in soil
Conclusions:
Iron oxide isostearate is to be considered as Cerium and iron oxide isostearate as having no adverse effects on organic matter turnover, and hence on soil fertility.
Executive summary:

The source and target substances present similar characterization (nanoparticles of very similar parameters), and similar physico-chemical and ecotoxicological properties (high melting point, low vapour pressure, very low water solubility, no expected bioaccumulation potential when considering cerium and iron elements and isostearate parts, no acute toxicity to daphnids up to and including the saturation concentration). This similarity supports the relevance of the read-across.

Thus, the toxicity to soil microorganisms is expected to be the same for the source and target substances.

Iron oxide isostearate is to be considered as cerium and iron oxide isostearate as having no adverse effects on organic matter turnover, and hence on soil fertility.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 21 October 2008 to 31 March 2009
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: lucerne meal. Prior to application all soil aliquots (except of “unamended”) were amended with 0.8 g lucerne meal as a source of organic nitrogen (corresponding to 5.33 g/kg dry soil; in accordance with the Lucerne-soil ratio of 5 g/kg dry soil recommended by the guideline). The lucerne meal contained approximately 2.5% nitrogen.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method:
Application solutions were prepared in flasks using trichloromethane as a solvent. The solvent trichloromethane was used since the test item is not sufficiently soluble in water or in acetone and not homogeneously dispersible in sand.
The application solution for each treatment was added dropwise to 20 g of quartz sand using a Hamilton syringe. The flask containing the application solution was rinsed twice with approximately 200 μL solvent, added also dropwise to the sand to apply the application solution quantitatively. The sand mixtures were homogeneously mixed using a Vortex mixer. Thereafter the solvent was completely evaporated to dryness in a stream of nitrogen. The sand aliquots applied with the concentrations IV (359 mg/kg d.w.) and V (1024 mg/kg d.w.) of the test item were additionally homogenized in a mortar. The treated sand was stored one day at room temperature until application.
The amount of fortified quartz sand added to the soil aliquots (150 g dry soil) was:
- I, II, III: 2.50 g
- IV: 2.55 g
- V: 2.65 g
A higher amount of sand (16.7 g/kg dry soil) as recommended by the testing guideline (10 g/kg dry soil) was used as the two highest test item concentrations were not homogeneously dispersible in a lower amount of sand. As sand is an inert material, no effects on the test results are to be expected by this deviation to the guideline recommendations. 16.7 g/kg dry soil were also added to the control and reference item treatments.
During application, the soil was thoroughly mixed.

VEHICLE:
- Chemical name of vehicle (organic solvent): trichloromethane
- Evaporation of vehicle before use: yes
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20±2 °C
Moisture:
Adjusted to 45% of the water holding capacity by adding purified water.
Details on test conditions:
TEST SYSTEM
- Testing facility: Harlan Laboratories Ltd (Itingen, Switzerland)
- Test container (type, material, size): 1-litre incubation flask. The flasks were stoppered with cotton wool plugs.
- Amount of soil: 150 g dry soil
- No. of replicates per concentration: 1
- No. of replicates per vehicle control: 1

SOIL INCUBATION
- Method: after application, all soil aliquots were adjusted to 45% of the soil MWC (i.e. 15.5 g water per 100 g dry soil) and incubated in the dark at 20±2 °C.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: fresh field soil, provided by Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), Speyer/ Germany
- History of site: The soil had not been subjected to any pesticide treatment in the sampling year and four former years. Furthermore, no organic fertilizer treatment was applied in the sampling year and two former years. From 2004 to 2005 pumpkins were cultivated, in the following years and in the sampling year the soil remained uncultivated.
- Accidental contamination: no data
- Depth of sampling: about 20 cm
- Soil texture
- % sand: 58.7 - 60.8 %
- % silt: 29.8 - 31.9%
- % clay: 9.4%
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- pH (in water): 7.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 29.9
- Maximum water holding capacity: 34.4 g/100 g
- Cation exchange capacity: 8 mval/100 g soil
- Pretreatment of soil: The soil was sieved through a 2-mm sieve by Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer. After storing the soil for 11 days in the dark at a temperature of 4 °C at Harlan Laboratories, the soil was equilibrated at 20 ± 2 °C and at about 40% of the maximum water-holding capacity (MWC) in the dark before application of the test item for a period of 14 days. Prior to application of the test item, the soil moisture content was adjusted to just below 45% of the MWC.
- Storage (condition, duration): 11 days
- Initial microbial biomass: 1.9 % of organic C

EFFECT PARAMETERS MEASURED: nitrification was determined for all treatments in three samples at 0 (within 3 hours after application) and 28 days after treatment. Potential effects of chemicals on nitrification were assessed by comparing the concentrations of nitrite and nitrate formation in control and treated soils. For this purpose, triplicate aliquots of about 47 g wet soil (corresponding to 40 g dry soil) were taken from the flasks at each sampling day. After extraction using KCl, shaking and centrifugation, the supernatant liquid was decanted through fluted filter paper. Then the whole extraction process was repeated once. The two aqueous extracts were combined and the concentrations of nitrite- and nitrate- nitrogen were determined using a Flow Injection Analyzer (AutoAnalyzer 3, Bran+Luebbe). The system was calibrated using standard solutions. Nitrite was analysed immediately after extraction, as nitrite is not stable. The determination of nitrite is based on a colorimetric method with a limit of quantification of 0.07 mg NO2-/L extract, corresponding to 0.08 mg NO2-/kg dry soil. For Nitrate analysis on day 0, the extraction solutions had to be stored at -20 °C for 6 days prior to analysis (in accordance with the guideline) because of technical reasons. On day 28, the nitrate was analysed on the day of sample preparation. Nitrate is reduced to nitrite in a cadmium reductor. The nitrite is then determined as described above. The limit of quantification is 0.43 mg NO3-/L extract, corresponding to 0.53 mg NO3-/kg dry soil. The initial nitrate concentration of the soil used in the study was measured in unamended and untreated soil samples at the start of the study.

The concentration of nitrite and nitrate in the soil samples was calculated as followed:
M = (M(s) x Vextr) / Ws
where:
M = mg of NO2- or NO3- per kg of dry soil
M(s) = Amount of NO2- NO3- [mg/L] found in the aqueous soil extract
Vextr = Volume of aqueous soil extract [L]
WS = Dry weight of the soil sample taken for analysis [kg]

Additionally a nitrate formation rate for each treatment was calculated as follows, considering the nitrate formation over the 28 days exposure period:
V: Ri(1) = (Mi – Mi-1) / (ti – ti-1)
where:
i = 0 and 1 for interval number corresponding to 0 and 28 days of incubation
Ri = Formation rate of nitrate per kg of dry soil per day on interval i
Mi = mg of nitrate per kg of dry soil on interval i in each replicate
Mi-1 = mg of nitrate per kg of dry soil on interval i-1, mean value of the three replicate values
ti = Incubation time on interval i

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: no data
Nominal and measured concentrations:
Application rate nominal (intended amount): 10, 33, 111, 333 and 1000 mg test item/kg dry soil
Amount applied: 10.3, 40.9, 122.2, 358.5 and 1023.9 mg test item/kg dry soil
Reference substance (positive control):
yes
Remarks:
Nitrapyrin (at 6.7 mg/kg d.w.)
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: No biological relevant effect on the nitrification process in soil
Details on results:
Initially, 29.9 mg nitrate (NO3–) per kg dry soil were detected in untreated and unamended soil.

NITRITE:
On Day 0, the NO2– concentrations were 0.36 and 0.26 mg NO2–/kg dry soil for the control and reference item treatment, respectively. In the test item treatments the nitrite concentrations ranged between 0.31 and 0.40 mg NO2–/kg dry soil. This resulted in deviations of -14 to 11% from the control.
After 28 days, the NO2– concentrations were below the level of quantification in all treatments.
Thus, neither nitrapyrin nor the test item showed an inhibiting effect on the turnover of nitrite. Therefore, the over all influence of the test item on the turn-over of nitrite was negligible.

NITRATE:
The mean initial nitrate concentrations (day 0) ranged between 30 and 41 mg NO3-/kg dry soil in all treatments and control. The calculated deviations to control were -11 to -27%, resulting from natural variability between soil aliquots.
After 28 days, nitrate concentrations in soil increased to 78 mg NO3-/kg dry soil in the control. In the reference item treatment, concentrations dropped to 2.8 mg NO3-/kg dry soil resulting in a deviation of the nitrate concentration of -96% compared to the control. Thus, the reference item nitrapyrin showed a strong significant inhibiting effect on nitrification, thereby proving the sensitivity of the test system and validity of the experimental design.
Nitrate concentrations in the test item treatments ranged between 65 and 72 mg NO3-/kg dry soil corresponding to a 7 to 17% inhibition in nitrification. But the decrease did not follow a concentration-response relationship.
The nitrate formation rate per day determined for the incubation period of 28 days was 1.3 mg NO3-/kg dry soil for the control. Nitrate formation rates for the test item treatments ranged between 1.1 mg NO3-/kg dry soil/day and 1.4 mg NO3-/kg dry soil/day corresponding to deviations of 9% to -16.9% from the control.
The two highest test item concentrations showed a tendency to a dose-response-relationship, resulting in a reduction in nitrate formation per day of 8 and 17% compared to the control, but the inhibition on nitrate formation up to a test item concentration of 1000 mg test item/kg dry soil was well below 25%.
Results with reference substance (positive control):
Nitrapyrin does not show an inhibiting effect on the turnover of nitrite.
Regarding nitrates, concentrations dropped to 2.8 mg NO3-/kg dry soil resulting in a deviation of the nitrate concentration of -96% compared to the control. Thus, the reference item nitrapyrin showed a strong significant inhibiting effect on nitrification, thereby proving the sensitivity of the test system and validity of the experimental design.

Table 1: Influence of the Test and Reference Item on Nitrite turn-over after Amendment of Lucerne Meal

Treatment [mg/kg dry soil]

Incubation time [days]

Nitrite [mg NO2-/kg dry soil]

% deviation from control

Mean

SD

%SD

Control

0

28

0.363

<0.082

0.010

<0.001

2.8

n.a.

-

-

Test item - 10

0

28

0.322

<0.082

0.006

<0.001

1.9

n.a.

-11.1

n.a.

Test item - 41

0

28

0.340

<0.082

0.010

<0.001

3.0

n.a.

-5.6

n.a.

Test item - 122

0

28

0.311

<0.082

0.005

<0.001

1.5

n.a.

-13.9

n.a.

Test item - 359

0

28

0.371

<0.082

0.016

<0.001

4.2

n.a.

2.8

n.a.

Test item - 1024

0

28

0.400

<0.082

0.002

<0.001

0.6

n.a.

11.1

n.a.

Nitrapyrin -

6.7

0

28

0.261

<0.082

0.006

<0.001

2.4

n.a.

-27.8

n.a.

SD: standard deviation; n.a. = not applicable

Table 2: Influence of the Test and Reference Item on Nitrate Formation after Amendment of Lucerne Meal

Treatment [mg/kg dry soil]

Incubation time [days]

Nitrate [mg NO3-/kg dry soil]

% deviation from control

Mean

SD

%SD

Control

0

28

40.69

77.74

1.7

1.2

4.2

1.6

-

-

Test item

10

0

28

29.53

69.84

1.8

0.5

6.2

0.7

-27.4

-10.2

Test item

41

0

28

32.65

72.29

0.9

0.4

2.9

0.6

-19.8

-7.0

Test item

122

0

28

31.39

69.96

1.2

2.0

4.0

2.9

-22.9

-10.0

Test item

359

0

28

36.11

70.24

4.6

0.8

12.7

1.2

-11.3

-9.6

Test item

1024

0

28

34.05

64.84

0.4

0.7

1.2

1.0

-16.3

-16.6

Nitrapyrin

6.7

0

28

34.07

2.82

0.5

0.0

1.4

1.0

-16.3

-96.4

SD: standard deviation

Table 3: Influence of the Test and Reference Item on Nitrate Formation Rates after Amendment of Lucerne Meal

Treatment [mg/kg dry soil]

Incubation time [days]

Nitrate rate [mg NO3-/kg dry soil/day]

% deviation from control

Mean

SD

%SD

Control

28

1.32

0.04

3.3

-

Test item - 10

28

1.44

0.02

1.3

8.8

Test item - 41

28

1.42

0.01

1.0

7.0

Test item - 122

28

1.38

0.07

5.2

4.1

Test item - 359

28

1.22

0.03

2.5

-7.9

Test item - 1024

28

1.10

0.02

2.2

-16.9

Nitrapyrin - 6.7

28

-1.12

0.00

0.1

-184.3

SD: standard deviation

Validity criteria fulfilled:
yes
Remarks:
variation of the nitrite and nitrate concentrations as well as of the nitrate rates between replicate control samples < 15%.
Conclusions:
Regarding the trigger value of 25% given by the testing guideline, no biologically relevant influence of the test item cerium and iron oxide isostearate on microbial nitrification in soil was observed and the EC50 is determined to be >1000 mg test item/kg dry soil.
Executive summary:

The influence of the test item, cerium and iron oxide isostearate, on soil microorganisms was determined by measuring nitrification (mineralization) of lucerne meal in soil. The study was carried out according to a OECD Guideline 216 and in compliance with GLP.

A fresh agricultural soil, a sandy loam, moistened to 45% of its maximum water-holding capacity was incubated in the dark at 20 ± 2 °C following treatment with the test item at 10, 41, 122, 359, and 1024 mg cerium and iron oxide isostearate/kg dry soil. Control soil was not treated with the test item but was incubated under identical conditions as treated soil. In order to show the validity of the test system and method used, soil aliquots were treated with the reference item nitrapyrin (6.7 mg nitrapyrin/kg dry soil).

 

No biological relevant effect of cerium and iron oxide isostearate on the nitrification process in soil, as measured by the transformation of organic matter (lucerne meal) to nitrite and finally nitrate was documented. At concentrations of up to 1000 mg test item/kg dry soil deviations from the control concerning nitrite and nitrate concentrations as well as nitrate rates were well below the trigger value of 25% given by the test guideline after an incubation period of 28 days. So it can be concluded that no adverse effects of the test item on organic matter turnover, and hence on soil fertility, are to be expected.

The EC50 of cerium and iron oxide isostearate is determined to be >1000 mg test item/kg dry soil.

The variation between replicate control samples was less than 15%. Thus, the validity criterion for the test was fulfilled. The reference item nitrapyrin had a clear influence on the microflora, thereby showing the sensitivity of the test system and validity of the experimental design.

Description of key information

By analogy with cerium and iron oxide isostearate (active matter of DPX10), iron oxide isostearate (active matter of DPX13) should not show any adverse effect on nitrogen-mineralizing soil micro-organisms.

Key value for chemical safety assessment

Additional information

One GLP-compliant study performed according to OECD guideline 216 is available on the analogue, cerium and iron oxide isostearate (active matter of DPX10). It is quoted as reliability 1 according to Klimisch criteria and flagged as a key study.