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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:
August - September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compiant
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
January 2000
Deviations:
yes
Remarks:
Due to unusual high temperatures during the summer months the room temperature exceeded22 °C (by maximum 24.2°C, corresponding to 8.0%) for altogether 26 days.
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP Certificate: Statement of GLP Compliance from Staatliches Gewerbeaufsichtsamt Hildesheim, Germany, dated 03.01.2017
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 18 - 25°C, dark, in the tightly closed original container
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
Analytical monitoring:
not required
Vehicle:
no
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C (nominal)
18.6 - 24.2°C (measured)
Moisture:
At test initiation the soil was adjusted to approximately 45 % of its maximum water holding capacity. All replicates were weighed once per week to check for water losses by evaporation (recommended maximum 5 %, actual 0.5 – 0.9 %). Demineralized water was added as necessary.
Organic carbon content (% dry weight):
0.544
Nitrogen content (% dry weight):
1.15
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): Plastic boxes (volume 1.0 L, food grade) with perforated tops to enable gas exchange
- Amount of soil: 400 g soil dry weight per replicate
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: Not applicable

SOIL INCUBATION
- Method: Bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: R-439683; H-5449554
- History of site: 2015 - 2019 uncultivated; 2015 - 2019 no fertilisation
- Vegetation cover: no information given
- Treatments with pesticides: No crop protection products applied during the year of sampling and 4 preceding years.
- Accidental contamination: No
- Depth of sampling: 0 - 20 cm
- Soil texture
- % sand: 57.3
- % silt: 35.0
- % clay: 7.6
- Soil taxonomic classification: Silty sand
- Soil classification system: German DIN classification
- pH (in water): 5.9 ± 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 11.5 (Total inorganic nitrogen: 12.1 mg/kg dw)
- Maximum water holding capacity (g/100 g dw): 34.9 ± 1.8
- Cation exchange capacity (meq /100 g): 7.3 ± 1.1
- Pretreatment of soil: The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm, the soil amounts were amended with powdered lucerne-green-grass-meal (0.5% of soil dry weight). Total nitrogen content: 24.6 g/kg, total carbon content: 43.2%, C/N ratio: 17.6, origin: Futtermittel & Naturkost Mühle Gladen, Dorsten, Germany
- Storage (condition, duration): The soil was stored for 60 days (2019-05-31 to 2019-07-29) in the dark at 4-6°C in a climatic room.
- Initial microbial biomass as % of total organic C: 2.88

DETAILS OF PREINCUBATION OF SOIL (if any):
The soil was pre-incubated at room temperature (ca. 20°C) for 23 days (2019-07-29 to 2019-08-21) to adapt the microorganisms to test conditions before the start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Metabolic activity of the microbial biomass, measured as nitrate formation rate was determined after 0, 7, 14 and 28 days.

RANGE-FINDING STUDY
- Test concentrations: 0, 10, 100 and 1000 mg/kg dw
- Results used to determine the conditions for the definitive study:

Test concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 7 d 14 d 28 d
10 -27 -22 -6
100 -61 -52 -1
1000 n.d. n.d. 52

Nominal and measured concentrations:
10, 31.6, 100, 316, 1000 mg/kg soil dry weight (nominal)
Reference substance (positive control):
yes
Remarks:
Cyanoguanidine
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
354 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95%CI 210-614 mg/kg dw
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
566 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CI 393-734 mg/kg dw
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
828 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CI 629-933 mg/kg dw
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Other: Cyanoguanidine a well-known inhibitor of nitrification is tested once per year as toxic reference with 50 mg/kg soil dry weight. Most recent test conducted from 2019-03-13 to 2019-04-10

Test concentration Nitrate-N Formation Rate
[mg/kg soil dry weight] [%] compared to Untreated Control
7 d 14 d 28 d
50 55* 60* 58*
* = difference to control >= 25%

The observed effects caused by the reference item Cyanoguanidine prove the sensitivity of the test system thereby showing its suitability for this kind of study.
Reported statistics and error estimates:
EC-values and confidence intervals: ECx values were calculated by straight line (day 7) and sigmoida dose-response regression (day 14 and 28). Calculations of the confidence intervals for ECx-values were carried out using standard procedures.
Calculations were carried out using software: Excel, Microsoft Corporation, GraphPad Prism, GraphPad Software, Inc.
Test concentration [mg/kg soil dry weight] Day 0 Day 7 Day 14 Day 28
Inhibition compared to control [%]  Mean ± SD [mg NO3-N/kg soil dry weight] CV [%] Inhibition compared to control [%]  Mean ± SD [mg NO3-N/kg soil dry weight] CV [%] Inhibition compared to control [%]  Mean ± SD [mg NO3-N/kg soil dry weight] CV [%] Inhibition compared to control [%] 
Control  Nitrate-N  - 10.9 ± 0.78 7  - 14.7 ± 0.72 5  - 22.6 ± 0.97 4  -
Nitrate-N formation rate   - 0.76 ± 0.11 15  - 0.65 ± 0.05 8  - 0.61 ± 0.04 6  -
  10.0 Nitrate-N 13 10.7 ± 0.17 2 2 14.5 ± 1.55 11 1 21.5 ± 0.70 3 5
Nitrate-N formation rate   -

0.84 ± 0.03

3

-10

0.69 ± 0.11

16

-6

0.59 ± 0.03

4

3

31.6

Nitrate-N

24

11.5 ± 0.27

2

-6

14.3 ± 0.32

2

3

19.3 ± 0.35

2

15

Nitrate-N formation rate 

 -

1.04 ± 0.04

3

-36

0.72 ± 0.02

3

-10

0.54 ± 0.01

2

11

100

Nitrate-N

15

12.9 ± 0.59

5

-18

19.9 ± 0.75

4

-35

22.0 ± 1.59

7

3

Nitrate-N formation rate 

 -

1.17 ± 0.09

7

-53

1.09 ± 0.06

5

-68

0.62 ± 0.06

9

-1

316

Nitrate-N

19

3.24 ± 0.26

8

70

14.3 ± 0.32

2

3

20.1 ± 0.57

3

11

Nitrate-N formation rate 

 -

-

-

n.d.

0.70 ± 0.02

3

-7

0.56 ± 0.02

4

9

1000

Nitrate-N

12

< LOQ

-

100

n.d.

-

100

11.1 ± 0.85

8

51

Nitrate-N formation rate 

 -

- -

n.d.

n.d.

-

n.d.

0.22 ± 0.03

14

64

n.d. not determinable, due to negative Nitrate-N Formations rates

Validity criteria fulfilled:
yes
Remarks:
Variation between control replicates was < 15 % at days 0, 7, 14 and 28 for the determination of inorganic nitrogen (nitrate-N content).
Conclusions:
EC10-, EC25- and EC50-values were determined for day 7, 14 and 28.
ECx values of Nitrate-N Formation Rate:
Day 7 Day 14 Day 28
EC10 161 mg/kg DW 387mg/kg DW 354 mg/kg DW
95% conficence 150-172 353-425 210-614
interval
EC25 180 mg/kg DW 456 mg/kg DW 566 mg/kg DW
95% confidence 168-193 416-498 393-734
interval
EC50 217 mg/kg DW 590 mg/kg DW 828 mg/kg DW
95% confidence 203-234 543-637 629-933
interval
Executive summary:

The effects of 2-butyloctanoic acid on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at the test facility from 2019-08-21 to 2019-09-19, with the definitive exposure phase from 2019-08-21 to 2019-09-18. The test guideline presents alternative testing procedures that apply to agrochemical and non-agrochemical substances. The approach for non-agrochemical substances, which entails a dose-response test design and focuses primarily on inhibitory effects was adopted in this study performed with 2-butyloctanoic acid.

The test item was applied via quartz sand at concentrations of 1000 - 316 - 100 - 31.6 - 10 mg/kg soil dry weight (factor10). Untreated field soil, adjusted to a moisture content corresponding to nominally 45 % of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne-greengrass-meal), was tested as the control under the same test conditions as the test item replicates. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were used as test vessels.

In the presence of a viable community of soil microflora, organic nitrogen undergoes conversion to mineralised nitrogen and a sequence of microbially mediated transformations that result in the formation of a series of inorganic nitrogen species: ammonification (product = NH4+-N), followed by Stage 1 nitrification (product = NO2--N) and finally Stage 2 nitrification (product = NO3--N). The effects of the test item on nitrogen transformation by soil microflora were determined by comparing rates of formation of the terminal transformation product, nitrate-nitrogen (NO3--N), in soil treated with 2 -butyloctanoic acid against the rate observed in the untreated control. Measurements of nitrate-nitrogen concentrations in soil were made in all treatments and the control on the day of treatment application (day 0) and after 7, 14 and 28 days.

EC10-, EC25- and EC50-values were determined for day 7, 14 and 28.

ECx values of Nitrate-N Formation Rate:

                                  Day 7                     Day 14                     Day 28       

EC10                   161 mg/kg DW       387mg/kg DW       354 mg/kg DW

95% conficence       150-172              353-425                     210-614

interval

EC25                   180 mg/kg DW       456 mg/kg DW       566 mg/kg DW

95% confidence       168-193              416-498                     393-734

interval

EC50                   217 mg/kg DW       590 mg/kg DW       828 mg/kg DW

95% confidence       203-234              543-637                     629-933

interval

Description of key information

The effects of 2 -butyloctanoic acid on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216.

The derived EC10 value was as follows: 354 mg/kg soil dry weight on day 28 and the EC50 for microbial nitrate transformation was determined to be 828 mg/kg soil dry weight on day 28.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
354 mg/kg soil dw

Additional information

The test item was applied via quartz sand at concentrations of 1000 - 316 - 100 - 31.6 - 10 mg/kg soil dry weight (factor10). Untreated field soil, adjusted to a moisture content corresponding to nominally 45 % of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne-greengrass-meal), was tested as the control under the same test conditions as the test item replicates.Measurements of nitrate-nitrogen concentrations in soil were made in all treatments and the control on the day of treatment application (day 0) and after 7, 14 and 28 days.