Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th September - 8th October 2021

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material used was HEBMP (1-3Na). The active ingredient of HEBMP-xNa is the polyphosphonic acid form (HEBMP-H).

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: LUFA Standard soil 2.3
- Other:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was weighed out and diluted in demineralized water for each test separately and applied to the surface of the soil. The soil was mixed 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: no vehicle used

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20.0 - 21.5

Moisture:

43.7 - 45.2 % moisture content

Organic carbon content (% dry weight):

0.616

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 as stated below.
- Amount of soil: 400g soil dry weight per vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3


SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, “rechts der Landauer Str.“ Nr. 826/7, Rheinland-Pfalz, Germany. R-439683; H-5449554
- History of site: Cultures: 2017 - 2021: uncultivated. Fertilisation: 2017 - 2021: none
- Vegetation cover: not reported
- Treatments with pesticides or fertilizers: No crop protection products applied during the year of sampling and 4 preceding years. Fertilisation: 2017 - 2021: none
- Accidental contamination: not reported
- Other:
- Depth of sampling: ca. 0 - 20 cm
- Soil texture
- % sand: 57.6
- % silt: 35.1
- % clay: 7.4
- Soil taxonomic classification: Silty sand
- Soil classification system: German DIN classification
- pH (in water): 6.2 +/- 0.3
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 6.55 mg/kg dry weight
- Maximum water holding capacity (in % dry weight): 42%
- Cation exchange capacity (mmol/kg): 6.0 +/- 0.9 meq / 100g
- Pretreatment of soil: The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm.
- Storage (condition, duration): The soil was stored for 2 days (2021-07-21 to 2021-07-23) in the dark at 4 - 6 °C in a climatic room.
- Initial microbial biomass as % of total organic C: 2.12

DETAILS OF PREINCUBATION OF SOIL (if any): The soil was pre-incubated at room temperature (ca. 20 °C) for 18 days (2021-07-23 to 2021-08-10) to adapt the micro-organisms to test conditions before the start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Inorganic nitrate concentration measured at 0, 7, 14 and 28 days. pH and water contents were determined on day 0 and 28


RANGE-FINDING STUDY
- Test concentrations: control, 10, 100 and 1000 mg active acid/kg dry weight
- Results used to determine the conditions for the definitive study:yes. No inhibition observed so proceeded with 1000 mg active acid/kg soil dry weight as highest concentration

Nominal and measured concentrations:

Nominal: Control, 3.16, 9.83, 31.4, 99.4, 314 and 1000 mg active acid/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Cyanoguanidine, tested yearly.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: none reported
- Other: There was a decrease in pH values with increasing test material concentration beginning at 99.4 mg active acid/kg soil dry weight. See 'Any other information on results including tables'

Results with reference substance (positive control):

at 50 mg reference item / kg soil dry weight, 75%, 100% and 93% inhibition of nitrate formation rate was observed after 0-7 days, 0-14 days and 0-29 days compared to the control.

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) and Dunnett’s test was carried out for the determination of statistically significant differences compared to control replicates. A Normality test and an Equal Variance test was conducted first. P-values for both Normality and Equal Variance test are 0.05. As no inhibitions of nitrate-N content were observed, no ECx values could be calculated.

Table 1. Inhibition of Nitrate-N content in soil containing the test material, relative to the control

Test item concentration (mg active acid/kg soil dry weight)	Inhibition (%) compared to control
	Day 0	Day 7	Day 14	Day 28
3.16	-12	-1	0	2
9.83	-6	-4	-3	7
31.4	-10	-14	-17	-16
99.4	-11	-13	-20	-14
314	1	-39	-48	-58
1000	-5	-43	-51	-59

Table 2. Inhibition of Nitrate-N formation rates in soil containing the test material, relative to the control

Test item concentration (mg active acid/kg soil dry weight)	Inhibition (%) compared to control
	Day 0 - 7	Day 0 - 14	Day 0 -28
3.16	4	4	5
9.83	-3	-2	10
31.4	-16	-18	-17
99.4	-15	-22	-15
314	-55	-59	-70
1000	-59	-62	-69

Table 3. Moisture and pH values of the soil after 0 and 28 days

Test concentration	Soil dry weights (%)		Moisture content as (%) of MWHC*		pH values
(mg active acid/kg soil dry weight)	day 0	day 28	day 0	day 28	day 0	day 28
Control	86.4	86.5	44.2	43.7	6.45	6.36
3.16	86.2	86.3	45.0	44.6	6.46	6.34
9.83	86.1	86.2	45.1	44.9	6.39	6.34
31.4	86.1	86.3	45.2	44.3	6.36	6.28
99.4	86.1	86.3	45.1	44.5	6.40	6.22
314	86.1	86.5	45.1	43.9	6.32	6.02
1000	86.3	86.4	44.4	44.2	6.09	5.76

* MWHC - maximum water holding capacity

Results of the additional highest concentration (1000 mg active acid/kg dry weight) without the addition of the Lucerne-grass-green meal:

Table 4. Inhibition of Nitrate-N content

Nitrate-N Content
Test item concentration (mg active acid/kg soil dry weight)	Inhibition (%) compared to control
	d 0	d 7	d 14	d 28
Additional control 1000	-183	-23	-2	3

Table 5. Inhibition of Nitrate-N formation rate

Nitrate-N Formation Rate
Test item concentration (mg active acid/kg soil dry weight)	Inhibition (%) compared to control
	d 0-7	d 0-14	d 0-28
Additional control 1000	42	40	41

Table 6. Moisture and pH values of the soil after 0 and 28 days

Test concentration	Soil dry weights (%)		Moisture content as (%) of MWHC		pH values
(mg active acid/kg soil dry weight)	day 0	day 28	day 0	day 28	day 0	day 28
Control	86.4	86.5	44.2	43.7	6.45	6.36
Additional control 1000	86.7	86.7	43.1	42.9	5.70	5.64

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:

28-day NOEC, EC10 and EC50 values of ≥1000, >1000 and >1000 mg active acid/kg soil dry weight have been determined for the effects of the test substance on the inhibition of nitrate formation of soil microbes, based on nominal concentrations.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to Annex 3 of the CSR or IUCLID Section 13 for justification of read-across within the HEBMP category.

Reason / purpose for cross-reference:

read-across source

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

nitrate formation rate

Description of key information

28-day NOEC, EC₁₀ and EC₅₀ values of ≥1000, >1000 and >1000 mg active acid/kg soil dry weight have been determined for the effects of HEBMP-xNa on the nitrate formation rate of soil microbes.

Additional information

28-day NOEC, EC₁₀ and EC₅₀ values of ≥1000, >1000 and >1000 mg active acid/kg soil dry weight respectively have been determined for the effects of HEBMP-xNa on the nitrate formation rate of soil microbes, expressed in terms of nominal concentrations (Noack, 2021). The study was conducted according to OECD TG 216, in compliance with GLP and met the validity criteria, therefore it was assigned a reliability value of 1.

The acid and sodium salts in the HEBMP category are freely soluble in water. The HEBMP anion can be considered fully dissociated from its sodium cation when in dilute solution. Under any given conditions, the degree of ionisation of the HEBMP 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 HEBMP-H, HEBMP-xNa, or another salt of HEBMP.

 

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

1. HEBMP is present as HEBMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HEBMP-xNa salt, HEBMP-H (acid form), or another salt was used for dosing.
2. Disassociated sodium cations. The amount of sodium present depends on which salt was added.
3. It should also be noted that divalent and trivalent cations would preferentially replace the potassium ions. These would include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺).

The terrestrial environment is exposed to HEBMP predominantly via application of WWTP sludge, and as such this phenomenon also directly affects soil in the receiving environment.