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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Dec 2016 - 11 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: the test item was ground with fine quartz sand and mixed with the sand into the test soil; this spiked sand (or non-treated sand for the control) was mixed into portions of 0.6 kg soil dry weight equivalent and deionised water was added to the soil to adjusted soil moisture to 45% WHCmax; soils were distributed equally to four test vessels which constituted the replicates of the respective treatment.

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.3 to 20.8 °C
Moisture:
45% WHCmax (adjusted)
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH
- Test container (type, material, size): Glass jars with lids (Model Sturz, volume 0.58 L, Weck GmbH, Germany)
- Amount of soil: 0.6 kg soil dry weight equivalent
- No. of replicates per concentration: 4
- No. of replicates per control: 4

SOIL INCUBATION
- Method: bulk / series of individual subsamples
All test vessels were incubated for 28 days at 19.3 to 20.8 °C in the dark. Test vessels were weighed once per week and weight loss was compensated with deionised water to keep the soil moisture within a range of ± 5 % of the water content at test start.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Germany, Rhineland-Palatinate, Offenbach, field name „Rechts der Landauer Str.“ field number 826/7
- Supplier: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer,Obere Langgasse 40, D-67346 Speyer, Germany
- History of site: was uncultivated during the last five years
- Treatments with pesticides or fertilizers: has not received pesticides within the last 5 years; in 2014 the sampling location was fertilised three times (Jun, Sep and Dec) with 3500 kg/ha CaO, each, and once (Dec) with 1463 kg/ha MgO
- Depth of sampling: ca. 20 cm
Soil texture:
- % sand: 59.6% ± 1.4%
- % silt: 33.6% ± 0.5%
- % clay: 6.8% ± 1.6%
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- pH (in water): 5.9 ± 0.6 (0.01 M CaCl2)
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 40.5 ± 1.0 mg kg-1 soil dry weight (mean nitrate concentration of the bulk soil two days prior to lucerne meal amendment), after adding lucerne meal, mean initial nitrate concentration was 53.3 mg kg-1 soil dry weight (control) and 51.7 to 56.3 mg/kg soil dry weight (test item treatments T1 to T5).
- Maximum water holding capacity [g/100 g]: 35.6 ± 1.4
- Cation exchange capacity (mmol/kg): 7.6 ± 0.8 meq/100 g
- Pretreatment of soil: Air drying on 24 Oct 2016; final sieving to 2 mm on 27 Oct 2016
- Storage (condition, duration): soil arrived at the test facility on 08 Nov 2016 and was stored at 16.8 to 21.3 °C in the dark until 13 Dec 2016 (date of test start)
- Initial microbial biomass as % of total organic C: 2.2%
- Microbial Biomass Carbon (Cmic): 146 μg Cmic g-1 soil

DETAILS OF PREINCUBATION OF SOIL: no preincubation

EFFECT PARAMETERS MEASURED: Nitrate of the soil was measured after test item application on day 0, and 28 days after test item application

RANGE-FINDING STUDY: no range-finding test was performed
Nominal and measured concentrations:
Control, 260, 364, 510, 714 and 1000 mg/kg soil dry weight
Reference substance (positive control):
no
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
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
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
Due to the lacking of adverse effects of the test item at tested concentrations of up to and including 1000 mg/kg soil dry weight, effective concentrations could not be calculated. Consequently, the EC50, EC25, and EC10 can be considered to be higher than 1000 mg/kg soil dry weight.
Reported statistics and error estimates:
Probit Analysis was used to determine the effective concentrations EC10, EC25 and EC50. All statistical tests were performed with the ToxRat® pro statistical software Version 2.10.

Validity criteria:

The study was valid. Coefficient of variance (CV) between replicate control samples was 1.8 (day 0) and 2.5 (day 28). Hence, variability was always below the critical value of 15 % as required by the OECD test guideline No. 216

Biological Results:

Mean nitrate concentration of the bulk soil as determined two days prior to lucerne meal amendment (day 0) was 40.5 ± 1.0 mg kg-1 soil dw. Adding lucerne meal to the bulk soil on day 0 increased the mean initial nitrate concentration in the test soil to 53.3 mg kg-1 soil dw (control) and 51.7 to 56.3 mg/kg soil dry weight (test item treatments T1 to T5). At the end of the test (day 28) mean nitrate concentration of the control soil had increased to 118.1 mg/kg soil dw (day 28). Nitrate concentration in the test item treated soils ranged from 120.0 mg/kg soil dw to 128.4 mg/kg soil dw. No adverse effects of the test item on nitrate formation were observed. The coefficient of variation within the four replicates of the control was 1.8% on day 0 and 2.5% on day 28. Hence, the validity criterion defined by the OECD Guideline No. 216 was fulfilled as the variability within the control was clearly below 15%.

Table 1: Mean, minimum and maximum nitrate concentration of the untreated soil (control), and coefficient of variance (CV), on day 0 and day 28 after test item application (n = 4).

Time

NO3 [mg/kg soil dry weight]

CV [%]

 

Mean

Min.

Max.

 

Day 0

53.3

52.5

54.5

1.8

Day 28

118

115

122

2.5

 

Table 2: Mean nitrate concentration [mg NO3/kg soil dry weight] in soil of the different treatments at starting (day 0) and at the end of the test (day 28). Test item concentration [mg/kg soil dry weight] and treatment code (in brackets).

Measurement time point

0.0 (BS)

0.0 (C)

260 (T1)

364 (T2)

510 (T3)

714 (T4)

1000 (T5)

Day 0

40.6

53.3

56.3

54.0

53.6

53.6

51.7

Day 28

n.d.

118

121

120

128

127

124

Day 28 decrease [%]a

n.a.

n.a.

-2.2

-1.6

-8.7

-7.9

-4.6

BS = bulk soil without lucerne meal; C = control soil; T1 to T5 = test item treated soil; n.d. = not determined; n.a. = not applicable; a = decrease as percentage related to the control (negative values indicate increase).

Validity criteria fulfilled:
yes

Description of key information

EC50 (28 d) ≥1000 mg/kg soil dw (OECD 216)

Due to the lacking of adverse effects of the test item at tested concentrations of up to and including 1000 mg/kg soil dry weight, effective concentrations could not be calculated.

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the potential effects of the test item (CAS 10094-45-8) on the nitrogen transformation (ECT, 2017) by terrestrial microorganisms. The study was performed according to OECD guideline 216 under GLP conditions. Test item was ground with fine quartz sand using a pestle and mortar prior to being mixed together with the sand into the test soil (Lufa standard soil type 2.3). The soil test was amended with ground lucerne grass green meal (5.0 g/kg soil). Test item was tested at five concentrations ranging from 260 mg/kg soil dry weight (T1) to 1000 mg/kg soil dry weight (T5) against an untreated control (C) with four replicates, each. Nitrate of the soil was measured after test item application on day 0 and 28 days after test item application. Due to the lack of a dose-response relationship, effective concentrations could not be calculated. Consequently, the EC50, EC25, and EC10 can be considered to be higher than 1000 mg/kg soil dry weight.