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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:
12 Dec 2016 - 09 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:
AMENDMENT OF SOIL
- Type of organic substrate: Lucerne-grass-green meal; source: Semhof, 86753 Möttingen, Germany

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 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 (freshly collected and sieved (2 mm) field soil (Lufa 2.3) harbouring its natural autochthonous microbial community), sand (or non-treated sand for the control) was mixed thoroughly into portions of 0.6 kg soil dry weight equivalent and soil moisture to 45% WHCmax was achieved by adding deionised water, afterwards the soils were distributed equally to four test vessels

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.3 to 21.1 °C
Moisture:
4.3% dry mass (initial soil moisture)
16% dry mass (soil moisture adjusted for test, equivalent to 45% of WHCmax)
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH, Flörsheim am Main, Germany; Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, D-67346 Speyer, Germany
- Test container (type, material, size): Glass jars with lids (Model Sturz, volume 0.37 L, Weck GmbH, Germany) were used as test vessels.
- Amount of soil: test item spiked sand (or non-treated sand for the control) was mixed thoroughly into portions of 0.6 kg soil dry weight equivalent and was amended with ground lucerne grass green meal (5.0 g/kg soil)
- No. of replicates per concentration: 4
- No. of replicates per control: 4

SOIL INCUBATION
- Method: incubated for 28 days at 19.3 to 21.1 °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
- Treatments with pesticides or fertilizers: uncultivated during the last five years and has not received pesticides within the last 5 years; 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
- Accidental contamination: no
- Other: freshly collected and sieved (2 mm) field soil (Lufa 2.3) harbouring its natural autochthonous microbial community
- Depth of sampling: Depth: ca. 20 cm; Date: 24 Oct 2016; weather conditions: cloudy
- 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: Soil type (USDA)
- pH (in water): 5.9 ± 0.6 (0.01 M CaCl2)
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 53.9
- 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 (only until sievable) on 24 to 27 Oct 2016; final sieving to 2 mm on 27 and 28 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 12 Dec 2016 (date of test start). Soil microbial biomass carbon was determined on 21 Nov 2016
- Initial microbial biomass as % of total organic C: 2.2% of Corg (Cmic)

EFFECT PARAMETERS MEASURED: measurement of nitrate on days 0 and 28

Nominal and measured concentrations:
Control, 260, 364, 510, 714 and 1000 mg/kg soil dw
Reference substance (positive control):
no
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
Details on results:
- Other: See "Any other information on results incl tables"
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.

Conclusion:

Due to the lack of a dose-response relationship, effective concentrations could not be calculated. However, based on the maximum effect of 16.7% observed at the highest tested concentration, the EC50 and EC25 can be considered to be greater than 1000 mg/kg soil dry weight, which was the highest tested test item concentration.

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

53.9

53.5

54.3

0.6

Day 28

124

122

126

1.4

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

40.5

53.9

53.8

54.7

54.0

54.5

55.6

Day 28

n.d.

124

116

117

107

111

103

Day 28 decrease [%]a

n.a.

 n.a.

6.5

5.6

13.8

10.6

16.7

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.

Table 3: N-Transformation Single Data (day 0); Nitrate concentration in soil [mg NO3/kg soil dry weight] as calculated from measured nitrate concentration [mg NO3/L] in CaCl2-extracts from the test soils.

Replicate

Test concentration

[mg NO3/L]

 

[mg NO3/kg soil dw]

Code

[mg/kg soil dw]

(a)

(b)

mean

Ca

0(C)

10.7

10.8

53.8

Cb

0(C)

10.8

10.8

54.0

Cc

0(C)

10.9

10.5

53.5

Cd

0(C)

11.0

10.7

54.3

T1a

260(Ti)

10.9

10.6

53.8

T1b

260(Ti)

10.7

10.7

53.5

T1c

260(Ti)

10.9

10.9

54.5

T1d

260(Ti)

10.7

10.7

53.5

T2a

364 (T2)

11.0

11.2

55.5

T2b

364 (T2)

10.8

10.9

54.3

T2c

364 (T2)

10.5

10.9

53.5

T2d

364 (T2)

10.9

11.3

55.5

T3a

510 (T3)

10.8

10.6

53.5

T3b

510 (T3)

10.7

10.9

54.0

T3c

510 (T3)

10.7

10.7

53.5

T3d

510 (T3)

10.8

11.1

54.8

T4a

714 (T4)

10.8

11.0

54.5

T4b

714 (T4)

10.3

11.0

53.3

T4c

714 (T4)

11.1

11.1

55.5

T4d

714 (T4)

10.7

11.2

54.8

T5a

1000 (T5)

11.0

11.0

55.0

T5b

1000 (T5)

11.2

11.2

56.0

T5c

1000 (T5)

11.7

11.3

57.5

T5d

1000 (T5)

10.8

10.7

53.8

Lufa a

LUFA Sp2.34316(no lucerne)

8.22

8.49

41.8

Lufa b

LUFA Sp2.34316(no lucerne)

7.91

8.18

40.2

Lufa c

LUFA Sp2.34316(no lucerne)

7.96

7.83

39.5

Lufa d

LUFA Sp2.34316 (no lucerne)

8.18

8.10

40.7

Table 4: N-Transformation Single Data (day 28); Nitrate concentration in soil [mg NO3/kg soil dry weight] as calculated from measured nitrate concentration [mg NO3/L] in CaCl2-extracts from the test soils.

Replicate

Test concentration [mg/kg soil dw]

[mg NO3/L]

[mg NO3/kg soil dw]

code

 

(a)

(b)

mean

Ca

0(C)

24.6

24.8

123.5

Cb

0(C)

25.2

25.2

126.0

Cc

0(C)

24.5

24.5

122.5

Cd

0(C)

24.1

24.8

122.3

T1a

260 (T1)

24.2

24.0

120.5

T1b

260 (T1)

23.1

23.0

115.3

T1c

260 (T1)

22.7

23.1

114.5

T1d

260 (T1)

22.4

22.4

112.0

T2a

364 (T2)

23.7

23.0

116.8

T2b

364 (T2)

23.3

22.5

114.5

T2c

364 (T2)

23.8

23.1

117.3

T2d

364 (T2)

23.4

23.7

117.8

T3a

510 (T3)

22.3

22.9

113.0

T3b

510 (T3)

21.7

21.5

108.0

T3c

510 (T3)

19.5

19.8

98.3

T3d

510 (T3)

21.2

21.5

106.8

T4a

714 (T4)

22.0

22.5

111.3

T4b

714 (T4)

21.2

22.1

108.3

T4c

714 (T4)

21.8

21.8

109.0

T4d

714 (T4)

22.9

22.5

113.5

T5a

1000 (T5)

20.2

20.6

102.0

T5b

1000 (T5)

20.8

20.5

103.3

T5c

1000 (T5)

20.9

20.7

104.0

T5d

1000 (T5)

20.6

20.4

102.5

Validity criteria fulfilled:
yes

Description of key information

EC50 (28 d) ≥1000 mg/kg soil (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 16260-09-6) on nitrogen transformation (ECT, 2017) of 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. However, based on the maximum effect of 16.7% observed at the highest tested concentration, the EC50 and EC25 can be considered to be greater than 1000 mg/kg soil dry weight, which was the highest tested test item concentration.