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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, 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
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.
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: 2012 - 2016 uncultivated; 2012,2013,2015, 2016 no fertilisation (3500 kg/ha CaO - 05.06.2014 and 24.09.2014; 3500 kg/ha CaO, 1463 kg/ha MgO - 5.12.2014)

- Vegetation cover:
- Treatments with pesticides: No crop protection products applied during the year of sampling and 4 preceding years.
- Accidental contamination: No
- Depth of sampling: ca. 0.2m
- Soil texture
- % sand: 57.5
- % silt: 35.6
- % clay: 6.9
- Soil taxonomic classification: Silty sand
- Soil classification system: German DIN classification
- pH (in water): 5.9 ± 0.6
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 14.2 (Total inorganic nitrogen: 14.7 mg/kg dw)
- Maximum water holding capacity (g/100 g dw): 35.6 ± 1.4
- Cation exchange capacity (meq /100 g): 7.6 ± 0.8
- Pretreatment of soil: None
- Storage (condition, duration): The soil was stored for 58 days (2016-09-05 to 2016-11-02) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 3.53

DETAILS OF PREINCUBATION OF SOIL (if any):
Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 23 days (2016-11-02 to 2016-11-25) to adapt the micro-organisms 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 -16 7 0
100 -6 23 24
1000 13 84 81

Nominal and measured concentrations:
12.3, 37.0, 111, 333 and 1000 mg/kg soil dry weight
Reference substance (positive control):
yes
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
111 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
192 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95%CI 158-238 mg/kg dw
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
293 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CI 246-352 mg/kg dw
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CI 420-586 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 and 100 mg/kg soil dry weight in LUFA soil of the same origin as that used for studies with test items.

Test concentration Nitrate-N Formation Rate
[mg/kg soil dry weight] [%] compared to Untreated Control
7 d 14 d 28 d
50 68 76 82
100 75 80 92

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:
One Way Analysis of Variances (ANOVA) and DUNNETT’S test, were carried out for the determination of statistically significant differences of the nitrate formation rate at day 7, 14 and 28 compared to control replicates. When running a One Way Analysis of Variances a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The α-value for ANOVA and DUNNETT’S test (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. Calculations were carried out using software Excel (MICROSOFT CORPORATION), SigmaPlot (SPSS INC.), and 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  - 26.6 ± 0.265 1  - 29.6 ± 0.961 3  - 40.7 ± 0.709 2  -
Nitrate-N formation rate   - 1.27 ± 0.036 3  - 0.852 ± 0.068 8  - 0.823 ± 0.025 3  -
  12.3 Nitrate-N 3 25.7 ± 0.651 3 3 31.4 ± 0.862 3 -6 41.3 ± 1.47 4 -1
Nitrate-N formation rate   - 1.21 ± 0.095 8 5 1.02 ± 0.061 6 -20 0.861 ± 0.052 6 -5
37.0 Nitrate-N 4 26.9 ± 0.833 3 -1 31.3 ± 1.04 3 -6 42.3 ± 2.70 6 -4
Nitrate-N formation rate   - 1.42 ± 0.115 8 -12 1.02 ± 0.076 7 -20 0.902 ± 0.096 11 -10
111 Nitrate-N 3 26.1 ± 0.611 2 2 29.0 ± 0.551 2 2 40.2 ± 1.78 4 1
Nitrate-N formation rate   - 1.27 ± 0.087 7 0 0.840 ± 0.040 5 1 0.821 ± 0.064 8 0
333 Nitrate-N 1 26.6 ± 0.115 0 0 24.8 ± 0.458 2 16 33.5 ± 0.721 2 18
Nitrate-N formation rate   - 1.30 ± 0.012 1 -2 0.522 ± 0.033 6 39* 0.572 ± 0.026 5 30*
1000 Nitrate-N 0 25.7 ± 0.902 4 3 17.5 ± 0.896 5 41 23.5 ± 0.416 2 42
Nitrate-N formation rate   - 1.15 ± 0.131 11 9 n.d. - n.d. 0.206 ± 0.015 7 75*
*) Statistically significant inhibition compared to control (ANOVA, p ≤ 0.05)
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:
The no-observed effect concentration (NOEC) was 1000 mg/kg soil dry weight on day 7 and 111 mg/kg soil dry weight on day 14 and 28. The lowest-observed effect concentration (LOEC) was determined to be 333 mg/kg soil dry weight after 14 and 28 days of exposure.
Executive summary:

The effects of 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) from 2016-11-25 to 2016-12-23. 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 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters.

The test item was applied via quartz sand at concentrations 12.3, 37.0, 111, 333 and 1000 mg/kg soil dry weight. 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 the test item 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.

No significant inhibition of the microbial nitrate transformation was observed after 7 days of exposure.

After 14 days of exposure to 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters, a significant inhibition of the microbial nitrate transformation was observed for the test item concentration 333 mg/kg soil dry weight. For the test item concentration 1000 mg/kg soil dry weight, no nitrate formation rate was determined, as no nitrate was measured.

After 28 days of exposure, a significant inhibition of the microbial nitrate transformation was observed for the test item concentrations 333 and 1000 mg/kg soil dry weight. These inhibitions followed a dose-response relationship.

After 28 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was 293 mg/kg soil dry weight. The EC50-value was 500 mg/kg soil dry weight.

The corresponding no-observed effect concentration (NOEC) was 111 mg/kg soil dry weight on day 14 and 28. The LOEC was determined to be 333 mg/kg soil dry weight after 14 and 28 days of exposure.

All validity criteria were met.

Description of key information

The effects of 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216. The no-observed effect concentration (NOEC) was 1000 mg/kg soil dry weight on day 7 and 111 mg/kg soil dry weight on day 14 and 28. The lowest-observed effect concentration (LOEC) was determined to be 333 mg/kg soil dry weight after 14 and 28 days of exposure. The EC50 for microbial nitrate transformation inhibition was determined to be > 1000 mg/kg dw.

Key value for chemical safety assessment

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

Additional information