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Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-01-29 to 2020-03-02
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: control, 10, 31.6, 100, 316, 1000 mg active acid/kg soil dry weight
- Sampling method: solid phase extraction, then photometric determination
- Sample storage conditions before analysis: Not reported
Details on preparation and application of test substrate:
- Type of organic substrate: A field fresh, silty sand soil (LUFA standard soil 2.3) was used in the study. Nominal soil parameters were:
Sand content 50 - 75 %
pH value 5.5 - 7.5
Organic carbon content 0.5 - 1.5 %
Microbial biomass (expressed as carbon content) at least 1 % of the total soil organic carbon.No crop protection products were used for a minimum of one year before sampling. No organic fertiliser was applied for at least six months prior use.

- Method: The test item was weighed out and diluted in demineralized water for each test concentration separately and applied to the surface of the soil. The soil was mixed thoroughly with a mixer (approximately 3 minutes) to ensure a homogeneous distribution of the test item in the soil. Afterwards, the soil was distributed to the replicates.

Test organisms (inoculum):
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
At experimental starting the pre-moistened 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 0.2 - 1.5 %). Compensation with demineralized water was done. Replicates were weighed for this procedure.
Organic carbon content (% dry weight):
Nitrogen content (% dry weight):
Details on test conditions:
- Testing facility: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- 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: 450 g soil dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: n/a

- Method: bulk / series of individual subsamples

- Geographical reference of sampling site (latitude, longitude):
- History of site:
- Vegetation cover:
- Treatments with pesticides or fertilizers:
- Accidental contamination:
- Other:
- Depth of sampling: 0-20 cm
- Soil texture: silty sand (uS)
- % sand: approx. 58%
- % silt: approx. 35%
- % clay: approx. 7.3%
- Soil taxonomic classification: LUFA soil
- Soil classification system: LUFA soil 2.3, Batch number F2.31820
- pH (in water): 6.1 +/- 0.4
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weight):35.2 ± 1.8g/100g DW
- Cation exchange capacity (mmol/kg): 6.8 ± 1.4 meq / 100 g
- Pretreatment of soil: 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: 43.9 %
C/N ratio: 17.8

- Storage (condition, duration): The soil was stored for 2 days (2020-05-05 to 2020-05-07) 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
(2020-05-07 to 2020-05-28) to adapt the micro-organisms to test conditions before the start of the study.

- Initial microbial biomass as % of total organic C: 2.26%


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Nitrate-N content increase compared toc ontrol: every 7 days. Nitrate-N formation rate at 7d, 14d, and 28d


- Test concentrations: 0, 100, 1000 mg active acid/kg dry weight
- Results used to determine the conditions for the definitive study: yes
Nominal and measured concentrations:
Nominal: control, 10, 31.6, 100, 316, 1000 mg active acid/kg soil dry weight
Reference substance (positive control):
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
other: active acid
Basis for effect:
nitrate formation rate
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
other: active acid
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):
- Results with reference substance valid? Yes
- Relevant effect levels: at 25 mg/kg soil dry weight, 28d of 32% difference to control in Nitrate-N, 39% in nitrate-n formation rate
- Other:
Reported statistics and error estimates:
ECx values were calculated by sigmoidal dose-response regression using GraphPadPrism. Four parameter were used for dose-response fitting. Calculation of the confidence intervals for EC values was carried out using standard procedures provided by GraphPadPrism.
Validity criteria fulfilled:
Variatin between control replicates was <15% at days 0, 7, 14 and 28 for the determination of inorganic nitrogen (nitrate-N content) (7%, 6%, 5% and 4%, respectively)
The 28d EC10 and EC50 values for the effects of HMDTMP(4-7K) on the nitrogen formation rate of soil microorganisms is >1000 and >1000 mg active acid/kg dry weight, respectively, based on nominal concentrations. The study report is currently in draft format, but the values are not expected to change in the final version.

Description of key information

28-day EC10 >1000 mg active acid/kg dry weight, reliability 1, soil microorganisms (read-across from HMDTMP(4-7K), Noack, 2020).

This study is currently in draft form, however the values are not expected to change in the final study report.

Key value for chemical safety assessment

Additional information

A 28-day EC10 value of >1000 mg active acid/kg soil dry weight has been determined for the effects of HMDTMP-(4-7K) on the nitrogen formation rate of soil microorganisms, based on nominal concentrations. A 28-day EC50 value of >1000 mg active acid/kg soil dry weight based on nitrogen formation has also been reported in the same study (Noack, 2020).

The acid, sodium and potassium salts in the HMDTMP category are freely soluble in water. The HMDTMP anion can be considered fully dissociated from its sodium or potassium cations when in dilute solution. Under any given conditions, the degree of ionisation of the HMDTMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HMDTMP-H, HMDTMP.4Na, HMDTMP.7K or another salt of HMDTMP.

Therefore, when a salt of HMDTMP is introduced into test media or the environment, the following is present (separately):

1. HMDTMP is present as HMDTMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HMDTMP (4-7K) salt, HMDTMP (4-7Na) salt, HMDTMP-H (acid form), or another salt was used for dosing. At pH 5.5 - 6, the HMDTMP anions would be present on average as the HMDTMP trivalent anion according to the pH curves.  At neutral pH (7), the HMDTMP anions would be present on average as the HMDTMP pentavalent anion according to the pH curves. At pH 8, the HMDTMP anions would be present on average as the HMDTMP hexavalent anion according to the pH curves.

2. Disassociated potassium or sodium cations. The amount of potassium or sodium present depends on which salt was added.

3. It should also be noted that divalent and trivalent cations would preferentially replace the sodium or potassium ions. These would include calcium (Ca2+), magnesium (Mg2+) and iron (Fe3+). These cations are more strongly bound by HMDTMP than potassium and sodium. This could result in HMDTMP-dication (e.g. HMDTMP-Ca, HMDTMP-Mg) and HMDTMP-trication (e.g. HMDTMP-Fe) complexes being present in solution.