Registration Dossier

Ecotoxicological information

Toxicity to soil microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05/11/2015 - 12/02/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD guideline and in compliance with GLP, without deviations that influence the results.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
0 and 28 days after treatment
Details on preparation and application of test substrate:
The soil batch used was according to the guidelines and was taken from fallow grassland: Darmstadt-Dieburg, 64380 Rossdorf, Germany
No pesticides or organic or mineral fertilizer had been used on the soil for at least four years prior to test initiation. The field has been rented by ibacon since 2004.
The soil particle size, cation exchange capacity, organic carbon content and nitrogen content of the soil is determined at LUFA, Speyer, Germany periodically.
The maximum water holding capacity, pH, nitrite content, nitrate content and ammonium content of the soil is determined periodically in the soil batch of the reference test. The soil has the following specifications:
Dry weight 87.38%
pH 6.5
Total Org. C (%) based on soil dry weight 0.95
Total N (%) based on soil dry weight 0.107
Microbial biomass (mg C/kg dry weight) calculated from respiration activity 308.38
Microbial biomass (% of total organic C) 3.25
NH4+-N (mg/kg dry weight) 0.355
NO2--N (mg/kg dry weight) 0.487
NO3--N (mg/kg dry weight) 4.940
Nmin-N (mg/kg dry weight) 5.782
CEC (mmol Ba/kg dry weight) 42
WHC (%) 40.3
Clay (%) 8.6
Silt (%) 30.2
Sand (%) 61.2

The soil batch used for test had the following parameters:
Dry weight (%) 88.4
Microbial biomass (mg C/kg dry weight) calculated from respiration activity 203.25
Microbial biomass (% ot total organic carbon) 2.01
NO3--N (mg/kg dry weight) 6.503

Soil batch was sampled (according to DIN 10381-6 sampling depth 0.05 to 0.2 m) and brought to the laboratory. In the laboratory it was air dried and sieved (mesh 2 mm) at RT.

The soil was stored at 20°C ± 2°C with appropriate ventilation and periodical moisture adjustment. Pre-incubation time was 9 days.

A total amount of soil of 1354.8 g soil wet weight was mixed with 6 g Lucerne meal and 70 mL water was added to adjust the soil water content between 47% and 48% WHC. Afterwards, about 60 g of prepared soil (corresponding to 50 mg soil dry weight) was filled in each test flask (volume 500
mL) and test item added if appropriate (0.2 μL, 0.5 μL, 1.6 μL, 5.2 μL and 16.4 μL for test rates 1 to 5). The bottles were immediately tightly closed, shaken and incubated in the dark (day 28 bottles) or directly used for extraction of nitrate-N (day 0 bottles). There were separate bottles for each test rate (control, test rate 1-5), sampling date (day 0 and 28) and replicate (n=3). For each treatment and sampling date, one additional bottle was prepared for determination of dry weight and pH.
The applications were conducted in the following order:
Control: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test Rate 1: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test Rate 2: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test Rate 3: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test Rate 4: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test Rate 5: 8 bottles (4 for day 0, 4 for day 28 sampling)
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20°C ± 2°C
Moisture:
The water content of one replicate of each treatment group was determined at each sampling date. Throughout the study, the water content ranged from 47% to 48% WHC.
Details on test conditions:
The pH was determined at test start and on day 28 in one replicate of each treatment group. Over the course of the study, the pH value was 6.7.
Nominal and measured concentrations:
Test Rate 1: 3.16 mg Iso E Super/kg soil dry weight
Test Rate 2: 10 mg Iso E Super/kg soil dry weight
Test Rate 3: 31.6 mg Iso E Super/kg soil dry weight
Test Rate 4: 100 mg Iso E Super/kg soil dry weight
Test Rate 5: 316 mg Iso E Super/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
sodium chloride
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrate content
Details on results:
See also the table in 'Overall remarks'.
Nitrogen transformation - Nitrate content
No adverse effects (deviations to control below 25%) of the test item on nitrate content in soil were observed at day 28 up to a concentration of 100 mg Iso E Super/kg soil dry weight (test rates 1-4). For a concentration of 316 mg Iso E Super/kg soil dry weight (test rate 5), the deviation was
outside the trigger range. At day 28 differences to the control were 11.70%, 2.45%, 10.53%, 9.78% (all not significant) for test rate 1 to 4 and 26.79% (statistically significant) for 316 mg Iso E Super /kg soil dry weight treatment.

Nitrogen transformation - nitrate formation rate:
The soil nitrate formation rates per day did not exceed the trigger range of ± 25% set by OECD guideline 216 calculated from the 0 - 28 day interval up to a concentration of 100 mg Iso E Super /kg soil dry weight (test rates 1 to 4). For a concentration of 316 mg Iso E Super /kg soil dry weight (test rate 5) the deviation was above the trigger range (statistically significant). Differences to the control were 16.60% 3.67%, 14.97%, 13.74% for test rate 1 to 4 and 36.60% for 316 mg Iso E Super /kg soil dry weight treatment.
Results with reference substance (positive control):
The reference item sodium chloride was tested in a GLP study. Sodium chloride was tested at 16 g/kg soil dw.

Results: Deviation from control
day 28 day 99
Soil nitrate content -83.29% -92.81%
Soil nitrate formation rate1 -100.43% -100.58%
1 incremental calculation (intervals days 14 - 28, days 86 - 98)

The variation of replicate control samples was less than 15%. The reference item had a retarding effect of more than ± 25% compared to the control at days 28 and 98 after application. The results of the study proved sensitivity of the test system and provided assurance that the laboratory test conditions are adequate.
Reported statistics and error estimates:
Calculation of mean values per treatment, standard deviation and coefficient of variation. Normality and homogeneity of variances were
tested using the Shapiro-Wilk`s Test (α = 0.01) and Levene´s test (α = 0.01), respectively and pair-wise comparisons of treated and control
values according to Williams Multiple Sequential t-test Procedure were conducted after trend analysis by Contrasts.
Validity criteria fulfilled:
yes
Remarks:
The variation between the replicate control samples didnot exceed the validity criterion of 15% throughout the study.
Conclusions:
The test item Iso E Super had no impact on nitrogen transformation (nitrate content and nitrate formation rate) of soil microorganisms when
applied up to 100 mg test item/kg soil dry weight treatment. An application of 316 mg Iso E Super caused a significant increase above the
trigger value for nitrate content and nitrate rate. The NOEC determination revealed that the NOEC appears to be higher than or
equal 100 mg test item/kg soil dry weight treatment for nitrate content and nitrate rate.
Executive summary:

Iso E Super was tested for its effect on the activity of soil microflora (nitrogen transformation) according to OECD TG 216 Nitrogen Transformation Test. Nitrogen-transformation in soil enriched with lucerne meal was determined. Test item treated soil was compared with a non-treated soil. NO3 -nitrogen formed in the nitrification process was determined by continuous flow analysis. The following test rates of Iso E Super were used: 3.16, 10, 31.6, 100 and 316 mg test item/kg soil dw. Sodium chloride was used as reference item. Moisture content was 47% to 48% of WHCmax and temperature was 20°C ± 2°C in the dark. No adverse (deviations to control below 25%) of the test item on nitrate content in soil were observed at day 28 up to a concentration of 100 mg test item/kg soil dw. The soil nitrate formation rates per day did not exceed the trigger range of ± 25% from the 0 - 28 day interval up to a concentration of 100 mg test item/kg soil dw. In conclusion, Iso E Super had no impact on nitrogen transformation (nitrate content and nitrate formation rate) of soil microorganisms when applied up to 100 mg test item/kg soil dw treatment. An application of 316 mg test item/kg soil dw caused a significant increase above the trigger value for nitrate content and nitrate rate. The NOEC appears to be 100 mg test item/kg soil dw.

Description of key information

See below.

Key value for chemical safety assessment

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

Additional information

Iso E Super was tested for its effect on the activity of soil microflora (nitrogen transformation) according to OECD TG 216 Nitrogen Transformation Test. Nitrogen-transformation in soil enriched with lucerne meal was determined. Test item treated soil was compared with a non-treated soil. NO3 -nitrogen formed in the nitrification process was determined by continuous flow analysis. The following test rates of Iso E Super were used: 3.16, 10, 31.6, 100 and 316 mg test item/kg soil dw. Sodium chloride was used as reference item. Moisture content was 47% to 48% of WHCmax and temperature was 20°C ± 2°C in the dark. No adverse (deviations to control below 25%) of the test item on nitrate content in soil were observed at day 28 up to a concentration of 100 mg test item/kg soil dw. The soil nitrate formation rates per day did not exceed the trigger range of ± 25% from the 0 - 28 day interval up to a concentration of 100 mg test item/kg soil dw. In conclusion, Iso E Super had no impact on nitrogen transformation (nitrate content and nitrate formation rate) of soil microorganisms when applied up to 100 mg test item/kg soil dw treatment. An application of 316 mg test item/kg soil dw caused a significant increase above the trigger value for nitrate content and nitrate rate. The NOEC appears to be 100 mg test item/kg soil dw.