[nitrilotris(methylene)]trisphosphonic acid, sodium salt

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 March 2014 - 15 May 2014

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:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): A respective test item was weighed out for each test item concentration and solved in demineralised water. The spiking solyutions and additional demineralised water were added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 52% of the maximum water holding capacity. Test medium was thoroughly mixed to ensure homogenous distribution

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: laboratory stock population
- Age at test initiation (mean and range, SD): aged 2-12 months, difference of age did not deviate by more than 1 per month
- Weight at test initiation (mean and range, SD): 0.32 to 0.54 g

ACCLIMATION
- Acclimation period: two days
- Acclimation conditions (same as test or not): same as the test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Cocoons and offspring were incubated under the same conditions for further 4 weeks.

Test temperature:

20 ± 2 °C

pH:

6.0 ± 0.5

Moisture:

52% of the maximum water holding capacity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): plastic boxes, inner diameter of 15.0cm corresponding to a bottom surface area of 177cm squared. Lids used were transparent and perforated to enable sufficient gas exchange
- Amount of soil or substrate: approximately 600g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% peat, 20% kaolin, 69% air dried quartz sand, 0.35% calcium carbonate
- Maximum water holding capacity (in % dry weight): 55.8 g / 100g dry weight
- CEC: not reported
- Pretreatment of soil: stored with lid on to prevent the test soil drying out

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light : 8 hour dark
- Light intensity: 400 -800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, biomass and reproduction

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

control - 62.5 – 125 – 250 – 500 – 1000 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

472.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 945 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 945 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 945 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

growth

Remarks:

biomass

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

Significant effects between 1 and 5 mg a.i./kg soil dry weight.

Table 1. Body weight changes of adult earthworms after 28 days' exposure to HEDP-H

Application rates (mg test item / kg soil dry weight)	Mean body weight		Mean body weight change (SD) (%)
	Day 0	Day 28
Control	0.39	0.51	29.2 (5.80)
62.5	0.39	0.50	28.2 (8.63)
125	0.39	0.50	26.9 (4.98)
250	0.39	0.52	32.3 (3.19)
500	0.39	0.50	27.5 (4.72)
1000	0.39	0.49	24.4 (6.43)

Table 2. Adult mortality after 28 days' exposure to HEDP-H.

Application rates (mg test item / kg soil dry weight)	Mean mortality of adult earthworms after 28 days exposure (%)
Control	0
62.5	2.5
125	0
250	0
500	0
1000	2.5

Table 3. Reproduction rate of earthworms after 56 days' exposure to HEDP-H

Application rates (mg test item / kg soil dry weight)	Replicate	Number of juveniles	Mean (SD)	CV (%)	% of control (* significantly reduced compared to control)
Control	1	78	88 (10.8)	12.3	-
	2	94
	3	107
	4	84
	5	76
	6	83
	7	98
	8	82
62.5	1	74	85 (9.43)	11.1	96.6
	2	81
	3	94
	4	92
125	1	110	115 (9.87)	8.58	130
	2	120
	3	126
	4	104
250	1	98	104 (17.3)	16.6	118
	2	125
	3	84
	4	108
500	1	72	82 (8.96)	10.9	93.2
	2	78
	3	93
	4	84
1000	1	52	46 (12.7)	27.5	52.3 *
	2	48
	3	27
	4	55

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, HEDP-H did not induce evident mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure in all tested concentrations. The adult earthworm biomass increased in the control and in all application rates without any statistically significant differences. After eight weeks of exposure, the earthworm reproduction was not statistically significantly reduced at the test item concentrations 62.5, 125, 250 and 500 mg test item/kg soil dry weight. However, at 1000 mg test item/kg soil dry weight the reproduction rate was statistically significantly reduced.
The EC50 - value for reproduction was determined to be >1000 mg test item / kg soil dry weight, which is equivalent to > 945 mg active acid/kg soil dry weight.

Thus, the No Observed Effect Concentration (NOEC) for HEDP-H summarizing the endpoints mortality and body weight and reproduction of Eisenia fetida after 8 weeks of exposure in artificial soil was determined to be 500 mg test item/kg soil dry weight, which is equivalent to 472.5 mg active acid/kg soil dry weight.

Executive summary:

Effects of HEDP-H on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to the Guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994). The study was conducted under static conditions over 8 weeks with the test item concentrations 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight, which were mixed into artificial soil containing 10 % peat and a control using untreated artificial soil. 80 test organisms were inserted into 8 control replicates and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.32 and 0.54 g at experimental starting.

After 28 days of exposure in soil, no evident earthworm mortalities (< 10%) as well as no evident pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. Moreover, no statistically significant differences in earthworm body weights were observed at all test item concentrations compared to the control. After further four weeks the reproduction rate (average number of juveniles produced) was 88 in the control and ranged between 46 and 115 in the test item treatment rates. Compared to the control, no statistically significant reduction of reproduction was observed at the test item concentrations 62.5, 125, 250 and 500 mg test item/kg soil dry weight. However, at 1000 mg test item/kg soil dry weight the reproduction was statistically significantly reduced.

Overall when expressed in terms of the active acid content, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was determined to be 472.5 mg active acid/kg soil dry weight. The EC₅₀ value for reproduction was determined to be > 945 mg active acid/kg soil dry weight.

 

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 November 2020 - 06 January 2021

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))

Deviations:

yes

Remarks:

Room temperature decreased to 17 degrees C for about 7 hours. Food was provided on day 0 instead of day 1. These deviations are considered to have no impact on the quality of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the control
- Sampling method: samples were taken on day 0 in all test concentrations and the control. Samples of the application solution were used to confirm tes titem concentrations.
- Sample storage conditions before analysis: All original samples were stored at room temperature. Prepared samples were stored at room temperature in an autosampler until analysis.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Test item for each test concentration weighed out and dispersed in demineralised water to give final volume of 250mL. 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.
- Controls: demineralized water

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): Aged 2-12 months, difference of age did not deviate by more than 1 month
- Weight at test initiation (mean and range, SD): 0.40 to 0.54 g

ACCLIMATION
- Acclimation period:Earthworms were acclimatised to the artificial soil to be used for the test two days prior to test start.
- Acclimation conditions (same as test or not): yes
- Health during acclimation (any mortality observed): not reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

17 - 22 °C

pH:

5.93 - 6.19

Moisture:

24.3 - 30.8%

Details on test conditions:

TEST SYSTEM
- Test container (material, size):plastic boxes with inner diameter of 15cm and height of 14cm. Transparent and perforated lids enable sufficient gas exchange, light input and prevent the test substrate from drying.
- Amount of soil or substrate: 600g approximately
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% peat, 20% kaolin, 69% quartz sand, 0.4% CaCO3
- Organic carbon (%): 5.9%
- Maximum water holding capacity (in % dry weight): 48.2g / 100g dwt
- CEC: not reported
- Pretreatment of soil: Two days before test start the artificial soil (18000 g) was pre-moistened with 1933 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
- Storage (condition, duration): Not reported
- Stability and homogeneity of test material in the medium: Not specified

OTHER TEST CONDITIONS
- Photoperiod: 16h light : 8 h dark
- Light intensity: 491 +/- 29 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Body weight of adult earthworms was determined on day 0 and day 28. Mortality and morphological changes of adult worms were recorded on day 28. Number of offspring hatched from the cocoons was counted for each test container


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: Not applicable
- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:

Nominal: control, 95.26, 171.47, 308.64, 555.56, 1000 mg/kg dwt
Measured: control (

Reference substance (positive control):

yes

Remarks:

Carbendazim at 0.5, 1, 2, and 4 mg a.i./kg soil dry weight

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

71.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks on result:

other: 95% C.I. 42.1 - 120

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

337 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks on result:

other: 95% C.I. 221 - 506

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

95.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

990 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

other: biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

556 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

other: biomass

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

Details on results:

- Mortality at end of exposure period: 1.25% in control, 0% in all treatment groups

Results with reference substance (positive control):

Mean reproduction rate for the controls: 67 +/- 12.7, CV = 19%.
NOEC = 0.5 mg a.i./kg dwt. LOEC = 1 mg a.i. / kg dwt.
OECD 222 guidance states significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.

Reported statistics and error estimates:

Mortality after 28 days was analysed using Chi2 2x2 table test with Bonferroni correction
Growth of adult worms after 28 days: Shapiro-Wilk's test for normality and Levene's test for homogeneity of variance, then Williams multiple sequential t-test and ANOVA
Reproduction after 56 days: Shapiro-Wilk's test for normality and Levene's test for homogeneity of variance, then Multiple sequentially-rejective Welsh t-test after Bonferroni-Holm adjustment and ANOVA

Table 1. Nominal and measured concentrations of the test substance on day 0

Nominal concentration of test solutions as active acid (mg/kg dwt)	Nominal concentration of the application solution of the test item (g/L)	Measured concentration (g/L)	Recovery %
Control	N/a	<LOQ
95.3	3.94	4.10	104
171	7.09	7.31	103
309	12.8	12.9	101
556	23.0	21.7	94
1000	41.4	40.2	97

Table 2. Biological observations of Eisenia fetida adults after 28 and 56 days' exposure to the test substance

Nominal concentration (mg a.i. /kg dwt)	Mean mortality after 28 days’ (%)	Mean body weight per replicate Day 0 (g)	Mean body weight per replicate Day 28 (g)	Mean body weight change (SD) (g)	Mean number of juveniles after 8 weeks (SD)	CV (%)
Control	1.25	0.455	0.524	0.07 (0.04)	146 (5.45)	3.73
95.3	0	0.455	0.505	0.05 (0.03)	128 (21.8)	17.0
171	0	0.455	0.530	0.08 (0.01)	84 (15.7)	18.6
309	0	0.455	0.505	0.05 (0.02)	90 (30.0)	33.3
556	0	0.455	0.490	0.04 (0.02)	60 (7.50)	12.5
1000	0	0.455	0.468	0.01 (0.02)	11 (5.12)	46.6

Validity criteria fulfilled:

yes

Remarks:

In the control group,the following effects were observed: 1.25% mortality, 146 juveniles (average) and 3.73% COV, which all meet the OECD 222 criteria (≤10%, ≥30 and <30%, respectively).

Conclusions:

Reliable, 56-day EC50, EC10 and NOEC values of 337, 71.1 and 95.5 mg active acid/kg soil dry weight have been observed for the effects of ATMP-5Na on the reproduction of the earthworm, Eisenia fetida. 28-day NOEC values of 556 and ≥1000 mg active acid/kg soil dry weight have been determined for the biomass and mortality of E. fetida, respectively.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Annex 4 of the CSR and IUCLID Section 13 for justification of read-across from the HEDP and HMDTMP categories to the ATMP category.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

472.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks on result:

other: Source substance: HEDP-H

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 945 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Remarks on result:

other: Source substance: HEDP-H

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

Remarks on result:

other: Source substance: HMDTMP(4-7K)

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 on result:

other: Source substance: HMDTMP(4-7K)

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

Remarks on result:

other: Source substance: HMDTMP(4-7K)

Description of key information

The following data are available for the registration substance, as part of a weight-of-evidence approach:

ATMP-xNa: 56-day NOEC, EC₁₀ and EC₅₀ values of 95.5, 71.1 and 337 mg active acid/kg soil dry weight, Eisenia fetida.

Data from two structurally analogous substances have been read-across as a weight-of-evidence approach:

HEDP-H: 56-day NOEC and EC₅₀ values of 472.5 and >945 mg active acid/kg soil dry weight, reproduction, Eisenia fetida

HMDTMP(4-7K): 56-day NOEC, EC₁₀ and EC₅₀ values of 556, 543 and >1000 mg active acid/kg soil dry weight , Eisenia fetida.

In the study conducted with ATMP-xNa, effects are attributed to the sodium cation, not the ATMP anion, therefore it is not appropriate to use this value for chemical safety assessment. Instead, the lowest value from the read-across data from two structurally analogous substances (HEDP-H and HMDTMP(4 -7K)) is used, which is the 56-day NOEC value of 472.5 mg active acid/kg soil dry weight.

Please refer to IUCLID Section 13 for further information on the effects of sodium in soil macroorganism studies conducted with phosphonate substances (PFA, 2021).

Key value for chemical safety assessment

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

472.5 mg/kg soil dw

Additional information

In a 56-day exposure of ATMP-xNa, reproduction of Eisenia fetida was significantly reduced and NOEC, EC₁₀ and EC₅₀ values of 95.5, 71.1 and 337 mg active acid/kg soil dry weight were determined, respectively. 28-day NOEC values of 556 and ≥1000 mg active acid/kg soil dry weight have been determined for the biomass and mortality, respectively and an EC₁₀ value of 990 mg active acid/kg soil dry weight based on biomass (Noack, 2021). However, due to high concentrations of sodium in the study, the effects observed are attributed to the sodium cations, instead of the ATMP anions. This is further explained in a separate report, attached in IUCLID Section 13 and Annex 6 of the CSR (PFA, 2021).

Therefore, two studies have been read-across from structurally analogous substances, HEDP-H and HMDTMP(4 -7K) as weight-of-evidence studies. In these studies, the sodium salts of the phosphonates were not used, and the NOEC/EC₁₀ values were higher. It was determined that these studies more accurately represent the toxicity of the phosphonate anions, than the ATMP-xNa study.

56-day NOEC and EC₅₀ values of 472.5 and >945 mg active acid/kg soil dry weight were determined for the effects of HEDP-H on the reproduction of Eisenia fetida (Noack, 2014). In this study, no effects were observed to mortality and growth, therefore giving NOEC values of ≥ 945 mg active acid/kg soil dry weight for both endpoints.

56-day NOEC, EC₁₀ and EC₅₀ values of 555, 543 and >1000 mg active acid/kg soil dry weight were determined for the effects of HMDTMP(4-7K) on the reproduction of Eisenia fetida (Noack, 2020). No effects were observed to earthworm mortality or growth, giving NOEC values of ≥ 1000 mg active acid/kg soil dry weight.

For further information on the read-across from HEDP and HMDTMP to ATMP, please refer to IUCLID Section 13 and Annex 4 of the CSR.

The hypothesis that the sodium cations are responsible for the toxicity observed to Eisenia fetida in the ATMP-xNa study is further supported by a 14 -day exposure of ATMP-H to E. fetida (Henkel, 1984). In this study, 14-day LC₅₀ and NOEC values of >960 and 960 mg/kg soil dry weight were reported respectively. However, as the documentation was insufficient for reliability assessment, the study was assigned reliability 4.

The acid, sodium, potassium and ammonium salts in the ATMP category are freely soluble in water. The ATMP anion can be considered fully dissociated from its sodium, potassium or ammonium cations when in dilute solution. Under any given conditions, the degree of ionisation of the ATMP 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 ATMP-H, ATMP.4Na, ATMP.7K or another salt of ATMP.

 

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

1. ATMP is present as ATMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether ATMP (3-5K) salt, ATMP (3-5Na) salt, ATMP-H (acid form), or another salt was used for dosing.
2. Disassociated potassium, sodium or ammonium cations. The amount of potassium or sodium present depends on which salt was dosed.
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 ATMP than potassium, sodium and ammonium. This could result in ATMP-dication (e.g. ATMP-Ca, ATMP-Mg) and ATMP-trication (e.g. ATMP-Fe) complexes being present in solution.