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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:
2019-03-04 to 2019-05-28
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:
July 2006
Deviations:
yes
Remarks:
The room temperature was temporarily > 22 °C (23 °C) due to technical reasons.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
The test item was weighed out and diluted with demineralized water for each test concentration separately and applied to the surface of the soil. The soil was mixed to ensure a homogeneous distribution of the test item in the soil. Afterwards, the soil was distributed to the replicates.

Frequency of application
The application was carried out once at experimental starting.

Application Conditions of the Soil
Dry Weight (DW) before application [g/100 g soil] 87.63
Maximum water holding capacity (MWHC)* [g/100 g soil DW] 34.9 ± 1.8*
Soil moisture content in terms of percent MWHC before application [%] 40.4
Water amount to adjust to 45 % MWHC [g/100 g soil DW] 1.61
Water amount / test group [mL] 19.32
*) data provided by LUFA Speyer
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
Temperature [°C]
nominal measured
20 ± 2 18.6 - 23.0
Moisture:
Maximum water hoding capacity: 34.9 g/100 g DW

Test concentration Moisture content as [%] of Maximum Water Holding Capacity
[mg/kg soil dry weight] day 0 day 28
Control 43.7 44.8
10.0 44.3 43.2
31.6 43.7 44.7
100 43.7 44.7
316 44.0 44.2
1000 43.1 43.0
Organic carbon content (% dry weight):
0.601
Nitrogen content (% dry weight):
6.87
Details on test conditions:
TEST SYSTEM
- Testing facility: Noack Laboratorien GmbH
- Test container (type, material, size):
Plastic boxes (volume 1.0 L, food grade) with perforated tops to enable gas exchange. Incubation was performed in bulk (and sub-samples were taken.
- Amount of soil: 400 g soil DW per replicate
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude):
Offenbach, “rechts der Landauer Str.“ Nr. 826/7, Rheinland-Pfalz, Germany
Gauß-Krüger-Coordinates R-439683; H-5449554
- History of site:
Cultures: 2014 - 2018: uncultivated
Fertilisation: 2018: none
2017: none
2016: none
2015: none
2014: 3500 kg/ha CaO (2014-06-05 and 2014-09-24); 3500 kg/ha CaO and
1463 kg/ha MgO (2014-12-15)
Pesticides No crop protection products applied during the year of sampling and 4
preceding years.
Soil Parameters
Parameter LUFA-soil 2.3
Batch-No. F2.31219
sampling depth* 0 - 20 cm
pH value* 5.9 ± 0.5
Dry weight (DW) before application [g/100 g soil] 87.63
Maximum water holding capacity* [g/100 g DW] 34.9 ± 1.8
Particle size distribution acc. to DIN*
Sand:
2.0 - 0.63 mm [% dry weight] 2.7 ± 0.5
0.63 - 0.2 mm [% dry weight] 29.4 ± 1.2
0.2 - 0.063 mm [% dry weight] 25.2 ± 1.4
Silt:
0.063 - 0.02 mm [% dry weight] 18.7 ± 1.3
0.02 - 0.006 mm [% dry weight] 11.0 ± 0.7
0.006 - 0.002 mm [% dry weight] 5.3 ± 0.8
Clay:
< 0.002 mm [% dry weight] 7.6 ± 0.4
Organic carbon content [%]1) 0.601
Microbial biomass [%] of total organic carbon 1.44
Nitrate-N [mg NO3-N/kg DW] 6.24
Ammonium-N [mg NH4-N/kg DW] 0.625
Total inorganic Nitrogen [mg/kg DW] 6.87
Cation exchange capacity [meq / 100 g]* 7.3 ± 1.1
Soil texture* silty sand (uS)#
*) data provided by LUFA Speyer
#) acc. to German DIN classification
1) determined by AGROLAB Agrar und Umwelt GmbH, based on dry weight (non GLP)

Soil handling
LUFA Speyer:
The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm.
The maximum water holding capacity and the pH value were determined.
Test facility:
The soil moisture content was determined.
The soil was adjusted to about 42 % of its maximum water holding capacity with demineralized water. Drying out of the soil was prevented by moistening with demineralized water as necessary. The amount of total inorganic nitrogen was determined at test start.
The soil was checked for a detectable microbial biomass (result in terms of percentage of total organic carbon).
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, Bahnhofstraße 42, 46286 Dorsten, Germany

Soil storage
The soil was stored for 19 days (2019-03-21 to 2019-04-09) in the dark at 4 - 6 °C in a climatic room. Subsequently, the soil was pre-incubated at room temperature (ca. 20 °C) for 21 days (2019-04-03 to 2019-04-30) to adapt the micro-organisms to test conditions before the start of the study.

Determination of nitrate concentrations
Nitrate was extracted from soil samples with a mineral salt solution. For the elimination of coloured organic matter in the extraction solution a cleaning step with SPE (solid phase extraction) cartridges was carried out. Thereafter, photometric determination took place.
Wave length 588 nm for nitrate
Solutions Extraction solution: Potassium chloride, 1 M and 2 M, respectively
Reagent solution: 2 g/L 4-Ethylresorcinol in 2-Propanol
Standard Potassium nitrate > 99%
Preparation of standard solutions
A stock solution of 100 mg nitrate/L was prepared in demineralized water. 6 concentrations were prepared by dilution with 1 M potassium chloride and used for calibration.
Calibration Calibration was performed at experimental starting.
Working steps
15 g soil of each replicate were weighed into shaking flasks. 60 mL extraction solution was added. Shaking was carried out for 1 h with 150 - 200 rpm. Filtration was carried out thereafter. The first 20 mL of filtrate was rejected.
Sample clean-up
5 - 10 mL of each filtered extract was cleaned via C18-SPE-cartridges to remove dissolved and coloured organic matter which could have influences on photometric determination. Conditioning of the cartridges was done with 2 x 2.5 mL methanol and, thereafter, with 2 x 2.5 mL demineralized water. Dryness of the cartridges was avoided. After conditioning, the sample was applied. The first 2.5 mL of each cleaned extract were rejected. The following volumes were stored in reagent tubes.
Nitrate determination
1 mL of the cleaned extract was diluted with 1 mL demineralized water. A cuvette was filled with 3.6 mL sulphuric acid (86 %). 1.0 mL sample were added. The cuvette was closed and shaken gently. After 15 min 0.6 mL of the reagent solution were added. After 45 min photometric determination at 588 nm was carried out. Extraction solution was used as ground signal. On day 0, 7 and 14 all samples were diluted 1:2. On day 28 all samples were diluted 1:4.
Method validation
The linearity and limit of quantification (LOQ) of the nitrate method were evaluated. In the range of calibration standards the second lowest standard was chosen as the limit of quantification.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY (non-GLP)
The preliminary study was performed under the same laboratory conditions as specified for the definitive study with 2 replicates per concentration and 2 replicates per control. Test duration was 28 days.
Inhibition of Nitrate-N Content
Test item concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 0 d 14 d 21 d 28 d
10 -4 -10 6 1
100 -2 -5 -1 -2
1000 -1 21 -7 -28

Inhibition of Nitrate-N Formation Rates
Test item concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 14 d 21 d 28 d
10 -23 19 4
100 -13 1 -2
1000 66 -16 -47
DW = soil dry weight negative value = promotion of nitrate-N formation rate / nitrate-N content
Nominal and measured concentrations:
1000 - 316 - 100 - 31.6 - 10 mg /kg soil dry weight (factor √10)
Reference substance (positive control):
yes
Remarks:
Cyanoguanidine
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
112 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other:
Remarks:
95 % confidence interval: 33.4 - 263 mg/kg soil dw
Key result
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
258 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other:
Remarks:
95 % confidence interval: 117 - 575 mg/kg soil dw
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
684 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other:
Remarks:
95 % confidence interval: 346 - > 1000 mg/kg soil dw
Details on results:
Deviation of Nitrate-N Content in Soil Containing Sodium methyl oleoyl taurate, Relative to the Control
Nitrate-N Content
Test item concentration Deviation [%] compared to control
[mg/kg soil dry weight] 0 d 7 d 14 d 28 d
10.0 -5 -17 3 4
31.6 0 -8 -4 1
100 12 -20 -11 -2
316 9 3 -21 -19
1000 -3 87 1 -45

Deviation of Nitrate-N Formation Rates in Soil Containing Sodium methyl oleoyl taurate, Relative to the Control
Nitrate-N Formation Rate
Test item concentration Deviation [%] compared to control
[mg/kg soil dry weight] 7 d 14 d 28 d
10.0 -33 9 8
31.6 -19 -6 1
100 -64 -26 -8
316 -3 -41 -30
1000 n.d. 4 -61
negative values = increase
n.d.= not determinable, due to negative Nitrate-N Formation rates
Results with reference substance (positive control):
Changes of Inorganic-N Contents
Reference Item Concentration
(Cyanoguanidine) Nitrate-N
[%] compared to Untreated Control
[mg/kg soil dry weight] 0 d 7 d 14 d 28 d
50 -12 24 26* 33*
negative values = represent increase positive values = represent inhibition
*) difference to control > 25 %


Changes of Inorganic-N Formation Rates
Reference Item Concentration
(Cyanoguanidine) Nitrate-N Formation Rate
[%] compared to Untreated Control
[mg/kg soil dry weight] 7 d 14 d 28 d
50 55* 60* 58*
negative values = represent increase positive values = represent inhibition
*) difference to control > 25 %

Nitrate-N Contents - Day 0

Test item concentration

[mg/kg soil dry weight]       Nitrate-N

      Repl.       MV +/- SD       CV

      [mg NO3-N/kg soil dry weight]       [%]

Control       6.58       7.09 ± 0.97       14

      6.48              

      8.21              

10.0       6.87       7.46 ± 0.52       7

      7.81              

      7.71              

31.6       7.42       7.06 ± 0.35       5

      7.02              

      6.73              

100       5.88       6.26 ± 0.53       9

      6.87              

      6.03              

316       6.13       6.48 ± 0.48       7

      7.02              

      6.28              

1000       8.06       7.30 ± 1.27       17

      5.83              

      8.01              

Repl.:       Replicate       MV:       Mean value

SD:       Standard deviation       CV:       Coefficient of variation

Nitrate-N Contents and Nitrate-N Formation Rates - Day 7

Test item concentration

[mg/kg soil dry weight]       Nitrate-N       Nitrate-N Formation Rate

      Repl.       MV +/- SD       CV       Repl.       MV +/- SD       CV

      [mg NO3-N/kg soil dry weight]       [%]       [mg NO3-N  (kg soil dry weight  d)-1]       [%]

Control       12.2       12.1 ± 0.90       7       0.73       0.72 ± 0.13       18

      11.2                     0.59              

      13.0                     0.84              

10.0       14.0       14.2 ± 0.47       3       0.93       0.96 ± 0.07       7

      14.7                     1.03              

      13.8                     0.91              

31.6       13.1       13.1 ± 0.25       2       0.86       0.86 ± 0.04       4

      12.8                     0.82              

      13.3                     0.89              

100       15.5       14.5 ± 0.85       6       1.32       1.18 ± 0.12       10

      14.2                     1.13              

      13.9                     1.09              

316       11.9       11.7 ± 0.25       2       0.77       0.74 ± 0.04       5

      11.4                     0.70              

      11.7                     0.75              

1000       1.54       1.54 ± 0.0       -       n.d.       –       -

      < LOQ                     n.d.              

      < LOQ                     n.d.              

Repl.:       Replicate       MV:       Mean value

SD:       Standard deviation       CV:       Coefficient of variation

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

Nitrate-N Contents and Nitrate-N Formation Rates - Day 14

Test item concentration

[mg/kg soil dry weight]       Nitrate-N       Nitrate-N Formation Rate

      Repl.       MV +/- SD       CV       Repl.       MV +/- SD       CV

      [mg NO3-N/kg soil dry weight]       [%]       [mg NO3-N  (kg soil dry weight  d)-1]       [%]

Control       19.0       17.5 ± 1.31       7       0.85       0.75 ± 0.09       12

      17.1                     0.72              

      16.5                     0.67              

10.0       17.8       16.9 ± 0.78       5       0.74       0.68 ± 0.06       8

      16.3                     0.63              

      16.7                     0.66              

31.6       17.9       18.2 ± 1.42       8       0.77       0.79 ± 0.10       13

      16.9                     0.70              

      19.7                     0.90              

100       20.3       19.5 ± 0.74       4       1.00       0.94 ± 0.05       5

      19.2                     0.92              

      18.9                     0.90              

316       20.8       21.1 ± 0.31       1       1.02       1.05 ± 0.03       2

      21.2                     1.05              

      21.4                     1.07              

1000       17.3       17.3 ± 0.25       1       0.71       0.72 ± 0.02       3

      17.1                     0.70              

      17.6                     0.74              

Repl.:       Replicate       MV:       Mean value

SD:       Standard deviation       CV:       Coefficient of variation

Nitrate-N Contents and Nitrate-N Formation Rates - Day 28

Test item concentration

[mg/kg soil dry weight]       Nitrate-N       Nitrate-N Formation Rate

      Repl.       MV +/- SD       CV       Repl.       MV +/- SD

CV

      [mg NO3-N/kg soil dry weight]       [%]       [mg NO3-N  (kg soil dry weight  d)-1]       [%]

Control       24.6       25.0 ± 0.75       3       0.63       0.64 ± 0.03       4

      24.6                     0.63              

      25.9                     0.67              

10.0       26.2       24.0 ± 1.97       8       0.67       0.59 ± 0.07       12

      23.2                     0.56              

      22.5                     0.54              

31.6       24.8       24.8 ± 0.40       2       0.63       0.63 ± 0.02       2

      24.4                     0.62              

      25.2                     0.65              

100       28.6       25.6 ± 2.73       11       0.80       0.69 ± 0.10       14

      24.8                     0.66              

      23.3                     0.61              

316       29.4       29.8 ±1.93       6       0.82       0.83 ± 0.07       8

      28.1                     0.77              

      31.9                     0.91              

1000       36.9       36.2 ± 4.35       12       1.06       1.03 ± 0.16       15

      31.5                     0.86              

      40.1                     1.17              

Repl.:       Replicate       MV:       Mean value

SD:       Standard deviation       CV:       Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
After 28 days of exposure the EC10-, EC25- and EC50-values for stimulation of the nitrate-N formation rate were 112, 258 and 684 mg/kg soil dry weight, respectively.
On the other sampling dates distinct stimulations as well as inhibitions were measured concurrently. These findings inhibit an EC-determination meaningful for these sampling dates.
Executive summary:

The effects of Sodium methyl oleoyl taurate (batch no. DEB4008582) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at the test facility from 2019-03-04 to 2019-05-28. 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 Sodium methyl oleoyl taurate.

The test item was applied via demineralized water at concentrations of 1000 - 316 - 100 - 31.6 - 10.0 mg/kg soil dry weight (factor √10). 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 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 Sodium methyl oleoyl taurate 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.

After 28 days of exposure the EC10-, EC25- and EC50-values for stimulation of the nitrate-N formation rate were 112, 258 and 684 mg/kg soil dry weight, respectively. On the other sampling dates distinct stimulations as well as inhibitions were measured concurrently. These findings inhibit an EC-determination meaningful for these sampling dates.

Nitrate-N Formation Rate: NOEC-, LOEC- and ECx- Values

      Day 28

      [mg/kg soil dry weight]

EC10       112

95 % confidence interval       33.4 - 263

EC25       258

95 % confidence interval       117 - 575

EC50       684

95 % confidence interval       346 - > 1000

Description of key information

The effects of Sodium methyl oleoyl taurate on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at the test facility from 2019-03-04 to 2019-05-28. 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 Sodium methyl oleoyl taurate.

The test item was applied via demineralized water at concentrations of 1000 - 316 - 100 - 31.6 - 10.0 mg/kg soil dry weight (factor √10). 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 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 Sodium methyl oleoyl taurate 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.

After 28 days of exposure the EC10-, EC25- and EC50-values for stimulation of the nitrate-N formation rate were 112, 258 and 684 mg/kg soil dry weight, respectively. On the other sampling dates distinct stimulations as well as inhibitions were measured concurrently. These findings inhibit an EC-determination meaningful for these sampling dates.

Nitrate-N Formation Rate: NOEC-, LOEC- and ECx- Values

      Day 28

      [mg/kg soil dry weight]

EC10       112

95 % confidence interval       33.4 - 263

EC25       258

95 % confidence interval       117 - 575

EC50       684

95 % confidence interval       346 - > 1000

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
684 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
112 mg/kg soil dw

Additional information