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

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-09 to 2020-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: LC-MS/MS
- Sample storage conditions before analysis:All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): Not reported
- Method of mixing into soil (if used): The respective test item amount for each test item concentration was weighed out and, dispersed in demineralised water to give a final volume of (250 mL). A sample of 50 mL was analytically verified via LC-MS/MS. 200 mL spiking solution was added to the artificial soil (2400 g SDW). Additional demineralised water (102 mL) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed with a handheld mixer (approx. 3 minutes) to ensure a homogenous distribution and about 600 g SDW were filled into each test vessel.

- Method of application to soil surface (if used): Not reported
- Controls: Artificial soil moistened with demineralised water without test or reference item was used as control medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/a
- Concentration of vehicle in test medium (stock solution and final test solution):N/a
- Evaporation of vehicle before use:N/a

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: Breeding stock culture maintained at the test facility
- Age at test initiation (mean and range, SD): Not reported but earthworms used were of homogeneous age and body weight
- Weight at test initiation (mean and range, SD): 0.39 to 0.53 g

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): Earthworms were acclimatised to the artificial soil to be used for the test two days prior to test start.
- Health during acclimation (any mortality observed): None reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

20 +/- 2 °C

pH:

5.90 - 6.87

Moisture:

23.7 - 29.8% of dry weight - The soil moisture deviated by more than 10 % from the initial value. This deviation is considered to have no impact on quality and integrity of the study.

Details on test conditions:

TEST SYSTEM
- Test container (material, size):Test units containing about 600 g SDW were round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm. Transparent and perforated lids enable sufficient gas exchange, light input and prevent the test substrate from drying.
- Amount of soil or substrate: 600g per test container
- No. of organisms per container (treatment): 10 worms
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: n/a

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: N/a
- Pesticide use history at the collection site: N/a
- Collection procedures: N/a
- Sampling depth (cm): N/a
- Soil texture (if natural soil)
- % sand: N/a
- % silt: N/a
- % clay: N/a
- Soil taxonomic classification: N/a
- Soil classification system: N/a
- Composition (if artificial substrate):
10 % peat, air-dried and finely ground
20 % kaolin, kaolinite content > 30 %
69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
0.50 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5

- Organic carbon (%): Not reported
- Maximum water holding capacity (in % dry weight): 45.5g / 100 g dw
- CEC: Not reported
- Pretreatment of soil: Two days before test start the artificial soil (18000 g) was pre-moistened with 1810 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil.
- Storage (condition, duration): The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
- Stability and homogeneity of test material in the medium: , the test medium was thoroughly mixed with a handheld mixer (approx. 3 minutes) to ensure a homogenous distribution and about 600 g SDW were filled into each test vessel.

OTHER TEST CONDITIONS
- Photoperiod:16 h light and 8 h dark
- Light intensity:400 - 800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : At experimental starting and experimental completion, the pH-value and moisture content of the test medium were determined in each treatment and the control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate. After the first four weeks, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After a further four weeks, the number of offspring (juveniles) hatched from the cocoons was counted for each test container.


VEHICLE CONTROL PERFORMED: not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:

Nominal: control, 95, 171, 309, 556, 1000 mg active acid/kg dw or 5.01, 9.02, 16.2, 29.2 and 52.6 g/L when expressed as nominal concentration of application solution of the test item
Measured: Control (

Reference substance (positive control):

yes

Remarks:

Carbendazim at 0.5, 1, 2, 4 mg active ingredient/kg dw

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

556 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

543 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks:

95% C.I. 247 - >1000

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:

mortality

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:

behaviour

Remarks:

pathological symptoms and changes in behaviour

Remarks on result:

other: No symptoms or changes of behaviour were found in any of the control or treatment organisms

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:

other: body weight change

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

763 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

95% C.I. 333- >1000

Details on results:

- Mortality at end of exposure period: Only mortality observed in 556 and 1000 mg active acid/kg dw treatments but is not significantly different to control
- Total mass of adults at beginning of test: No statistically significant differences in body weight change between treatments and control
- Changes in body weight of live adults (% of initial weight) at end of exposure period: No statistically significant differences in body weight change between treatments and control
- No. of offspring produced: Statistically fewer offspring produced in 1000 mg active acid/kg dw treatment
- No. of unhatched cocoons: Not reported
- Morphological abnormalities: None reported
- Behavioural abnormalities: Behaviour was observed, however no pathological symptoms or abnormal behaviours were reported.
- Other biological observations:N/a

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: NOEC = 0.5 mg a.i./kg dw, LOEC 1 mg a.i./kg dw

Table 1. Mortality of adult earthworms after 28 days' exposure to HMDTMP(4-7K)

Application rates (mg active acid/ kg soil dry weight)	Mean mortality of adult earthworms after 28 days exposure (%)
Control	0
95	0
171	0
309	0
556	2.5
1000	2.5

Table 2. Body weight change of adult earthworms after 28 days' exposure to HMDTMP(4-7K)

Application rates (mg active acid/ kg soil dry weight)	Mean body weight		Mean body weight change (SD) (%)
	Day 0	Day 28
Control	0.46	0.50	9.61 (6.94)
95	0.46	0.52	14.3 (5.53)
171	0.46	0.53	16.5 (3.84)
309	0.45	0.51	12.2 (5.31)
556	0.45	0.52	14.9 (3.41)
1000	0.46	0.50	10.4 (3.30)

Table 3. Reproduction rate of earthworms after 56 days' exposure to HMDTMP(4-7K)

Application rates (mg active acid / kg soil dry weight)	Replicate	Number of juveniles	Mean (SD)	CV (%)	% of control (* significantly reduced compared to control)
Control	1	106	102 (14.9)	14.6	-
	2	126
	3	88
	4	111
	5	114
	6	99
	7	81
	8	93
95	1	86	107 (16.4)	15.3	105
	2	102
	3	123
	4	116
171	1	96	119 (22.0)	18.5	117
	2	147
	3	108
	4	123
309	1	101	103 (10.4)	10.1	101
	2	92
	3	117
	4	102
556	1	79	95 (15.9)	16.7	93.1
	2	117
	3	9272.6
	4	92
1000	1	42	74 (23.3)	31.5	72.6 *
	2	73
	3	96
	4	85

Validity criteria fulfilled:

yes

Conclusions:

Reliable 56-day NOEC and EC10 values of 556 and 543 mg active acid/kg soil dry weight are available for the effects of HMDTMP(4-7K) on the reproduction of the earthworm, Eisenia fetida.

Description of key information

56-day NOEC and EC₁₀ values of 556 and 543 mg active acid/kg soil dry weight are available for the effects of the read-across substance HMDTMP(4-7K) on the reproduction of the earthworm, Eisenia fetida, reliability 1 (Noack, 2020).

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

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

543 mg/kg soil dw

Additional information

56-day NOEC and EC₁₀ values of 556 and 543 mg active acid/kg soil dry weight have been determined for the effects of HMDTMP(4 -7K) on the reproduction of Eisenia fetida in a reliable, guideline 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.