Sodium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 Na:1)

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-01-29 to 2020-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

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

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- 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.


APPLICATION OF TEST SUBSTANCE TO SOIL
- 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.

VEHICLE: N/a

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C

Moisture:

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.

Details on test conditions:

TEST SYSTEM
- 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

SOIL INCUBATION
- Method: bulk / series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- 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%

DETAILS OF PREINCUBATION OF SOIL (if any):N/a

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

VEHICLE CONTROL PERFORMED: n/a

RANGE-FINDING STUDY
- 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):

yes

Remarks:

Cyanoguanidine

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

54.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% C.I. <10 - 128

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

802 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% C.I. 558 - >1000

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: No
- Other: N/a

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 ED-values was carried out using standard procedures provided by GraphPadPrism.

Increase of Nitrate-N formation rate in soil containing HMDTMP(4 -7K) aqueous solution, relative to the control.

Nitrate-N Formation Rate
Test item concentration	Increase [%] compared to control
[mg a.i./kg DW]	7 d	14 d	28 d
10.0	33	13	4
31.6	71	31	14
100	61	31	7
316	65	30	36
1000	76	78	52

Validity criteria fulfilled:

yes

Conclusions:

The 28d EC10 value for the effects of HMDTMP(4-7K) on the nitrogen formation rate of soil microorganisms is 54.9 mg active acid/kg dry weight (95% C.I of <10 - 128)

Description of key information

28d EC₁₀ 54.9 mg active acid/kg dry weight, reliability 1, soil microorganisms (read-across from HMDTMP(4-7K), Noack, 2020).

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

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

54.9 mg/kg soil dw

Additional information

A 28-d EC₁₀ value of 54.9 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. An EC₅₀ value of 802 mg active acid/kg soil dry weight based on nitrogen formation has also been reported in the same study (Noack, 2020).