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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:
2014-07-08 to 2014-08-05
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Principles of method if other than guideline:
The soil was stored at 6 ± 2 °C instead of 4 ± 2 °C due to organisational reasons.

The temperature was under 18° C for 39 hours and over 22° C for 16 hours in total, due to technical reasons.
These deviations are considered to have no impact on the quality and integrity of the study.

GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No analytical monitoring
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: The soil amounts were amended with powdered lucerne-green-grass-meal (0.5% of soil dry weight). The C/N ratio was between 12/1 and 16/1. A ratio of 5 g Lucerne per kg of soil (dry weight was used.
Content of total inorganic nitrogen: 2.8% (dry weight)
Content of total organic carbon: 42.4% (dry weight)
C/N-ratio: 15.31
Particle size: 0.0288 - 1.008 mm
Origin: Alfalis, la maîtrise des luzernes, DESIALIS,
Société par actions simplifiée BP 124,
F-51007 Chalons en Champagne, France



APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The respective test item amounts were weighed out for each test item concentration and blended thoroughly with quartz sand (1 % of the entire soil amount per concentration). Afterwards the test item - quartz sand mixture was mixed carefully into the soil with a mixer to ensure a homogeneous distribution of the test item in the soil. Subsequently the soil was distributed to the replicates.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
nominal: 20 +/- 2 °C, actual: 16.4* - 22.1* °C
* The temperature was < 18° C for 39 hours and > 22 for 16 hours in total.
Moisture:
Dry weight (DW) before application: 87.0 g/100 g soil
Maximum water holding capacity: 36.2 ± 3.2 g/100 g DW
Details on test conditions:
TEST SYSTEM
- Testing facility: DR.U.NOACK-LABORATORIEN
- Test container (type, material, size): Plastic boxes (volume 1.0 L, food grade) with perforated tops.
- Amount of soil: 400 g soil DW
- No. of replicates per concentration: 3
- No. of replicates per control: 3


SOIL INCUBATION
- Method: bulk approach


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Im Bildgarten Nr. 585, Rheinland-Pfalz, Germany
- History of site:
Cultures:
2009 - 2013: uncultivated

Fertilisation:
No organic fertilisation.
2009 - 2013: none

- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: No crop protection products applied during sampling year and 4 former years.

- Accidental contamination: No
- Depth of sampling: 20 cm
- Soil texture
Sand:
2.0 - 0.63 mm % dry weight 2.1 ± 0.6
0.63 - 0.2 mm % dry weight 30.1 ± 0.1
0.2 - 0.063 mm % dry weight 25.4 ± 2.0
Silt:
0.063 - 0.02 mm % dry weight 20.2 ± 2.1
0.02 - 0.006 mm % dry weight 11.4 ± 1.0
0.006 - 0.002 mm % dry weight 5.1 ± 0.1
Clay:
< 0.002 mm % dry weight 5.9 ± 2.5


- Soil taxonomic classification: silty sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 6.0 +/- 0.9
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 13.4
- Maximum water holding capacity (in % dry weigth): 36.2 +/- 3.2
- Cation exchange capacity: 6.9 +/- 1.0 meq/100 g
- Pretreatment of soil:
LUFA Speyer:
The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm (carried out by LUFA Speyer).
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 maximal water holding capacity with demineralised water.
Drying out of the soil was prevented by moistening with demineralised water as necessary.
The soil was checked for a detectable microbial biomass (result in terms of percentage of total organic carbon) and the amount of total inorganic nitrogen.
The soil amounts were amended with powdered lucerne-green-grass-meal (0.5% of soil dry weight). The C/N ratio was between 12/1 and 16/1. A ratio of 5 g Lucerne per kg of soil (dry weight was used.

The soil was incubated in the above mentioned boxes. The top of the boxes was perforated to enable gas exchange.

- Storage (condition, duration): The soil was stored for 65 days (2014-04-10 to 2014-06-13) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 4.07

DETAILS OF PREINCUBATION OF SOIL (if any): Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 25 days (2014-06-13 to 2014-07-08) before experimental starting to guarantee a temperature adaptation of the micro-organisms

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Measurements of inorganic nitrate were carried out after 0, 7, 14 and 28 days. The pH values and water contents were determined on day 0 and 28. The room temperature was measured and recorded continuously once per hour.

Determination of nitrate concentrations:
Nitrate was extracted from soil with a mineral salt solution. For the elimination of coloured organic matter in the extraction solution a cleaning step with SPE cartridges was carried out. Thereafter photometric determination took place.
Wave length: 588 nm for Nitrate
Chemicals: Potassium chloride, p.a.
4-Ethylresorcin, p.a.
2-Propanol, p.a.
Sulphuric acid, 86 %
Methanol (for conditioning of the cartridges)
Solutions: Extraction solution: Potassium chloride, 1 M and 2 M, respectively
Reagent: 2 g/L 4-Ethylresorcin in 2-Propanol
Standard: Potassium nitrate > 99 %
Preparation of standard solutions:
A stock solution of 100 mg nitrate/L was prepared in demineralised water. 6 concentrations were prepared by dilution with 1 M potassium chloride and used for calibration.
Calibration: Calibration was performed prior to study initiation.
Working steps:
15 g soil of each replicate was weighed into shaking flasks. 60 mL extraction solution was added. Shaking was carried out for 1 h with 150 - 200 rpm. Subsequently, filtration was carried out. The first 20 mL of filtrate were rejected.
Sample cleaning:
10 mL of the filtrated extract was cleaned via C18-SPE-cartridges to remove dissolved and coloured organic matter which would have influence on photometric determination. Conditioning of the cartridges was done with 2 x 2.5 mL methanol and thereafter with 2 x 2.5 mL demineralised 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 demineralised water. A cuvette was filled with 1.8 mL sulphuric acid (86 %). 0.5 mL sample were added. The cuvette was closed and shaken gently. After 15 min 0.3 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 14 and 28 samples were diluted 1:4 before measurements.
Method validation:
The nitrate method was evaluated on linearity and limit of quantification (LOQ). In the range of calibration standards the second lowest standard was chosen as limit of quantification.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY: None




Nominal and measured concentrations:
nominal test concentrations: 1000 - 316 - 100 - 31.6 - 10 - 3.16 mg/kg soil dry weight (factor √10)
Reference substance (positive control):
yes
Remarks:
Cyanoguanidin
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Mean Nitrate-N Content in the Reference Item Test
Reference Item Concentration
[mg/kg soil dry weight] Mean ± SD of Nitrate-N Content [mg NO3-N/kg soil dry weight]
0 d 7 d 14 d 28 d
Control 16.1 ± 0.93 24.5 ± 0.46 33.6 ± 1.67 43.8 ± 1.66
50 16.0 ± 0.21 18.7 ± 1.67 18.3 ± 0.27 19.9 ± 0.65
100 17.8 ± 0.46 18.1 ± 0.21 18.5 ± 1.08 19.0 ± 0.96

Inhibition of Nitrate-N Formation Rates of the Reference Item Test
[%] compared to Control
Day 7 14 28
50 68* 87* 86*
100 97* 96* 96*

*) difference to control ≥ 25 %
Reported statistics and error estimates:
No ECx values were calculated, since a lacking concentration-effect relationship on day 7 and 14 and no effects > 25 % occurred at day 28.



Inhibition of Inorganic-N Contents

Nitrate-N Content

Test concentration

Inhibition [%] compared to control

[mg/kg soil dry weight]

0 d

7 d

14 d

28 d

      3.16

5

1

-8

10

    10

8

4

-9

0

    31.6

15

2

0

11

  100

5

1

-5

11

  316

13

-1

-10

2

1000

1

-2

-15

3

positive values = inhibition             negative values = increase               

Inhibition of Inorganic-N Formation Rates

Nitrate-N Formation Rate

Test concentration

Inhibition [%] compared to control

[mg/kg soil dry weight]

7 d

14 d

28 d

      3.16

-15

-42*

15

    10

-7

-50*

-6

    31.6

-46*

-38*

8

  100

-15

-32*

16

  316

-50*

-67*

-7

1000

-16

-56*

-6

*) Difference to control ≥ 25%                            positive values = inhibition                                negative values = increase

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

 

Day 28

 

[mg/kg soil dry weight]

LOEC

> 1000

NOEC

   1000

EC25

> 1000

95 % confidence interval

EC50

> 1000

95 % confidence interval

Validity criteria fulfilled:
yes
Conclusions:
After 7 days of exposure Celitement stimulated the microbial nitrate transformation to 46 and 50 % compared to the control when applied at the test item concentrations 31.6 and 316 mg/kg soil dry weight. After 14 days of exposure the microbial nitrate transformation rate was stimulated > 25 % in all test item concentrations. After 28 days of exposure no significant effects (< 25 %) on the nitrate transformation were determined for all test item concentrations. These findings thereby exactly reflect the results obtained from the range-finding study with significant but reversible stimulations on day 14.

No EC-values of the nitrate-N formation rate were determined, since effects > 25 % occurred after 28 days of exposure. The NOEC for nitrate transformation in soil on day 28 was determined to be 1000 mg/kg soil dry weight.

Executive summary:

The effects of Celitement (Batch no. Not specified) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, D-31157 Sarstedt, Germany from 2014-07-08 to 2014-08-05. The test item was applied via quartz sand with the test item concentrations 1000 - 316 - 100 - 31.6 - 10 - 3.16 mg/kg soil dry weight. Untreated soil was tested as control under the same test conditions as the test item replicates. Standard soil, moistened to nominally 45 % of its maximum water holding capacity, was used. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were applied.

 

The effects of the test item on the metabolic activity of the nitrogen-N formation rate (nitrate) were measured on the day of treatment (day 0) and after 7, 14 and 28 days.

 

After 7 days of exposure Celitement stimulated the microbial nitrate transformation to 46 and  50 % compared to the control when applied at the test item concentrations 31.6 and 316 mg/kg soil dry weight. After 14 days of exposure the microbial nitrate transformation rate was stimulated > 25 % in all test item concentrations. After 28 days of exposure no significant effects (< 25 %) on the nitrate transformation were determined for all test item concentrations.

 

No EC-values of the nitrate-N formation rate were determined, since no effects > 25 % occurred after 28 days of exposure. The NOEC for nitrate transformation in soil on day 28 was determined to be 1000 mg/kg soil dry weight.

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

 

Day 28

 

[mg/kg soil dry weight]

LOEC

> 1000

NOEC

   1000

EC25

> 1000

95 % confidence interval

EC50

> 1000

95 % confidence interval

Description of key information

No EC-values of the nitrate-N formation rate were determined, since effects > 25 % occurred after 28 days of exposure. The NOEC for nitrate transformation in soil on day 28 was determined to be 1000 mg/kg soil dry weight.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
1 000 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information

After 7 days of exposure Celitement stimulated the microbial nitrate transformation to 46 and 50 % compared to the control when applied at the test item concentrations 31.6 and 316 mg/kg soil dry weight. After 14 days of exposure the microbial nitrate transformation rate was stimulated > 25 % in all test item concentrations. After 28 days of exposure no significant effects (< 25 %) on the nitrate transformation were determined for all test item concentrations. These findings thereby exactly reflect the results obtained from the range-finding study with significant but reversible stimulations on day 14.