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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:
02 Aug 2017 to 04 Sep 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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The highest test item concentration solution was prepared by weighing in an appropriate amount of test item and dissolving it in an appropriate amount of acetone. The lower test item concentration solutions were prepared by diluting solution. Necessary amounts of the test item application solutions were applied drop-wise to appropriate amounts of quartz sand (1 % of soil dry weight per treatment group). For the solvent control group an equal amount of pure acetone was applied and untreated quartz sand was prepared for the control. After evaporation of the solvent the prepared quartz sand was mixed into the pre-moistened soil using an electrical mixer.
Simultaneously 57.6 g of deionised water were applied to each soil batch of pre-moistened soil to achieve the desired final soil moisture content of 43 % of the WHCmax.

Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.3 to 21.0 °C
Moisture:
water content: 13.0 % to 14.2 %, corresponding to 38.9 % to 42.5 % of the maximum water holding capacity.
Organic carbon content (% dry weight):
0.69
Nitrogen content (% dry weight):
0.062
Details on test conditions:
TEST SYSTEM
- Test container : 1 Liter glass bottles were used as test units. During the exposure period the bottles were loosely closed with screw caps to allow gaseous exchange whilst limiting evaporation.
- Amount of soil: Per replicate approx. 500 g of finally moistened test soil were applied per test unit )
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3

SOIL INCUBATION
- Method: 12000 g of the stored soil was pre-moistened by mixing 618 g of deionized water into the soil. Afterwards, the pre-moistened soil was incubated under similar conditions as for the exposure period for 5 days until application of the test item.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Germany, Rhineland-Palatinate, community: 76877 Offenbach, plot: “rechts der Landauer Str.”, No. 826/7, coordinates: UTM Zone 32: E 439713, N 5449850
- History of site: the sampling site was not cultivated since 2013. For at least four years prior to soil sampling, no plant protection products had been applied to the sampling site. To increase the soil pH, calcium oxide and magnesium oxide had been applied to the site in 2014. The soil was finally sieved using a mesh size of 2 mm.
- Vegetation cover: uncultivated area from 2013 to 2017.
- Treatments with pesticides or fertilizers: No organic fertilizer had been applied to the site for at least six months prior to soil sampling.
- Depth of sampling: ~ 20 cm

SOIL PARAMETERS
- Soil batch: F2.3 2217
- pH (in 0.01 M CaCl2 solution): 7.1
- Total Organic Carbon (TOC) [%] based on soil dry weight: 0.69
- Microbial biomass [mg C/100g soil dry weight] calculated from respiration activity: 18.1
- Microbial biomass [% of the TOC]: 2.62
- NO3--N (mg/kg soil dry weight): 14.8
- Total nitrogen [% N]: 0.062
- Density [g/L]: 1398
- Cation exchange capacity [Cmol + /kg]: 6.1
- WHCmax [%]: 33.4
Particle size distribution
According to USDA classification:
- Clay [%] (< 0.002 mm): 8.0
- Silt [%] (0.002 mm – 0.050 mm): 32.9
- Sand [%] (0.050 mm – 2.00 mm): 59.1
- Soil texture sandy loam
According to DIN classification:
- Clay [%] (< 0.002 mm) 8.0
- Silt [%] (0.002 mm – 0.063 mm): 35.1
- Sand [%] (0.063 mm – 2.0 mm): 56.9
- Soil texture: medium loamy sand

EFFECT PARAMETERS MEASURED
Soil samples of 10 g (wet weight) per replicate were taken 0 and 28 days after application. The samples were stored deep frozen until determination of the nitrate-N concentrations. Nitrate-N concentrations were determined by measuring aqueous soil extracts by means of Segmented Flow Analysis (SFA) using a photospectrometric procedure. Each soil sample that had been taken was mixed with 40 mL of a 0.0125 M CaCl2 solution. The samples were vigorously shaken for approx. one hour. Afterwards the samples were carefully centrifuged and the supernatant was used for nitrate-N determination. Calibration was performed with eight freshly prepared standard dilutions (1.0, 2.0, 5.0, 0.316, 20.0, 50.0, 100, 200 mg NO3-/L corresponding to 0.226, 0.452, 1.13, 2.26, 4.52, 11.3, 22.6 and 45.2 mg NO3--N/L) derived from a nitrate standard solution (10 g NO3-/L).




Nominal and measured concentrations:
Test item concentrations: 0 (solvent control and control), 0.316, 1.00, 3.16, 10.0 and 31.6 mg test item/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Details on results:
No statistically significant difference for the nitrate-N contents on day 28 could be detected in any of the test concentrations and the control compared to the solvent control. Hence, the 28-day LOEC could not be determined. Hence, the 28-day NOEC was considered to be ≥31.6 mg test item/kg soil dry weight, the highest concentration tested. The EC10 and EC50 could not be calculated since an appropriate dose-response relationship was absent at the tested concentration range after 28 days of exposure. The EC50 can be regarded as > 31.6 mg test item/kg sdw, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested.
Results with reference substance (positive control):
In a separate GLP-study, the toxic reference item (sodium chloride) was applied to demonstrate the sensitivity of the test system. A reduction of microbial nitrogen turnover activity of 134 % and 107 % for the exposure periods 0 to 28 and 14 to 28 days after application, respectively, and for the short-term respiration a reduction of 92.8 % compared to the control was determined in a field soil tested at a concentration of 20.0 g sodium chloride/kg soil dry weight. Hence, acceptable sensitivity of the test method is assured.
Reported statistics and error estimates:
Calculation of treatment means and deviation from solvent control in %. Level of significanceα= 0.01 for the pre-tests,α= 0.05 for the hypothesis tests. Test on normality and homogeneity of data using Shapiro-Wilk and Levene’s test followed by Dunnett’s t-test (two-sided) based on nitrate-N contents determined at day 28 of exposure. Calculation of the EC10,50data using 3-parameter normal cumulative distribution function; determination of the EC10,50values was not possible since an appropriate dose-response relationship was absent at the tested concentration range.

Mean nitrate-N contents determined for the control soil and soil treated with the test item

Treatment group

 

Soil NO3--N contents

(Day 0)

Soil NO3--N contents (Day 28)

 

Test item
concentration
[mg/kg sdw]

[mg/kg sdw]

Deviation [%]

[mg/kg sdw]

Deviation [%]

Solvent control

0

14.8

-

33.9

--

Control

0

14.8

 

0.000

34.5

1.77

test item

0.316

14.7

-0.676

31.9

-5.9

 

1

14.7

-0.676

32.8

-3.24

 

3.16

14.9

+0.676

34

0.295

 

10

15.0

+1.35

34.5

1.77

 

31.6

15.2

+2.70

33.7

-0.59

Endpoints

[mg test item/kg sdw]

EC10

n.d. 

EC50

> 31.6 

LOEC

n.d. 

NOEC

> 31.6 

 

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on nitrogen transformation in a medium loamy sand soil was determined in a 28 day dose response test. For the determined nitrate-N contents no dose-response relationship could be observed after 28 days within the tested concentration range that would have been applicable for calculation of the EC10 and EC50. The 28-day EC50 can be regarded to be > 31.6 mg test item/kg soil dry weight, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested. The LOEC could not be determined; however, the 28-day NOEC can be considered to be equal to or greater than 31.6 mg test item/kg soil dry weight, the highest concentration tested.
Executive summary:

Determination of the effects of the test item on the microbial soil nitrogen transformation activity was performed with an OECD guideline No 216 study under GLP compliance. EC10, 50 (10 % and 50 % inhibition on soil microflora nitrogen transformation after exposure over 28 days), where possible. No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC), where possible. The following test concentrations were used 0 (solvent control and control), 0.316, 1.00, 3.16, 10.0 and 31.6 mg test item/kg soil dry weight. Standard soil according to OECD guideline 216; soil pH and dry weight were determined for each sampling day for all treatments; temperature was measured continuously; soil pH: 6.9 to 7.1; water content: 13.0 % to 14.2 % (corresponding to 38.9 % to 42.5 % of the maximum water holding capcacity); temperature: 19.3° C to 21.0° C.

The EC10 and EC50 could not be calculated since an appropriate dose-response relationship was absent at the tested concentration range after 28 days of exposure. Hence, the EC50 can be regarded as > 31.6 mg test item/kg sdw, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested. The 28-day EC50 can be regarded as > 31.6 mg test item/kg soil dry weight, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested. No statistically significant difference between the nitrate-N contents of the day 28 soil samples of any test item treatment group or the control compared to the solvent control could be detected. Hence, the LOEC could not be determined, but the NOEC is considered to be > 31.6 mg test item/kg soil dry weight, the highest concentration tested. In a separate study the toxic reference item (sodium chloride) was applied to demonstrate the sensitivity of the test system. A reduction of microbial nitrogen turnover activity of 134 % and 107 % for the exposure periods 0 to 28 and 14 to 28 days after application, respectively, and for the short-term respiration a reduction of 92.8 % compared to the control was determined in a field soil tested at a concentration of 20.0 g sodium chloride/kg soil dry weight. Hence, acceptable sensitivity of the test method is assured. The effect of the test item on nitrogen transformation in a medium loamy sand soil was determined in a 28 day dose response test. For the determined nitrate-N contents no dose-response relationship could be observed after 28 days within the tested concentration range that would have been applicable for calculation of the EC10 and EC50. The 28-day EC50 can be regarded to be > 31.6 mg test item/kg soil dry weight, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested. The LOEC could not be determined; however, the 28-day NOEC can be considered to be equal to or greater than 31.6 mg test item/kg soil dry weight, the highest concentration tested.

Description of key information

The test item was assessed in OECD guideline 216 study under GLP compliance. The NOEC after 28 days of exposure was determined to be 31.6 mg test item/kg soil dry weight, the highest concentration tested.

Key value for chemical safety assessment

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

Additional information

The effect of the test item on nitrogen transformation in a medium loamy sand soil was determined in a 28 day dose response test. For the determined nitrate-N contents no dose-response relationship could be observed after 28 days within the tested concentration range that would have been applicable for calculation of the EC10 and EC50. The 28-day EC50 can be regarded to be > 31.6 mg test item/kg soil dry weight, since the soil nitrate-N content deviated -0.590 % compared to the solvent control at the highest concentration tested. The LOEC could not be determined; however, the 28-day NOEC can be considered to be equal to or greater than 31.6 mg test item/kg soil dry weight, the highest concentration tested.