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 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:

Study initiation: 22 October 2021. Study completed on 13 February 2023.

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:

See 'principles of method if other than guideline' for deviations

Principles of method if other than guideline:

Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. At test end, the soil moisture deviated by more than 10 % from the initial value in the control and the treatment rates 269 to 907 mg product/kg sdw and 2040 mg product/kg sdw.The room temperature increase to 22.5 °C for about 5 hours. These deviations are considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test item concentrations and the control were diluted with dilution medium prior to analysis. The sample was analyzed within 24 hours. The samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): On day - 1 (experimental starting) the respective test item amount was weighed out for each test item concentration and dissolved in the appropriate amount of demineralized water (544 g) to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the WHCmax. The pH value of each spiking solution as well as of the demineralised water for the control was adjusted to approx. pH 6.5 using K2CO3. The adjusted spiking solutions were added to the respective amount of artificial soil (3600 g SDW). Subsequently, the test medium was thoroughly mixed with a handheld mixer (approx. 30 minutes) to ensure a homogenous distribution. The bulks of spiked artificial soil were kept covered to prevent evaporative water losses and stored at 20 ± 2 °C (in the dark) for equilibration for approx. 24 ± 2 hours. At day 0 the spiked artificial soil was mixed once again (approx. 3 minutes) and about 600 g soil dry weight will be filled into each test vessel.
- Controls: demineralised water

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworms
- Source: breeding stock culture maintained at the test facility.
- Age at test initiation (mean and range, SD): minimum 2 months old, exact age not reported.
- Weight at test initiation (mean and range, SD): 0.44 - 0.45 g.

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions (same as test or not): artificial soil, the same as the test.
- Health during acclimation (any mortality observed): not reported.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18-22.5°C. The room temperature increased to 22.5°C for about five hours. This deviation is considered to have no impact on quality and integrity of the study.

pH:

Soil pH: 5.78 - 6.52.
Test media pH: 5.55-5.87.

Moisture:

21.2 - 25.0%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes with an inner diameter of 15.0cm corresponding to a bottom surface area of 177cm^2 and a 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 soil dry weight.
- 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, 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), 0.32 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5
- Organic carbon (%): not reported
- Maximum water holding capacity (in % dry weight): 53.8 g/100g dry weight
- CEC: not reported
- Pretreatment of soil: Two days before test start the artificial soil (22000 g) was pre-moistened with 2328 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): The bulks of spiked artificial soil were kept covered to prevent evaporative water losses and stored at 20 ± 2 °C (in the dark) for equilibration for approx. 24 ± 2 hours
- Stability and homogeneity of test material in the medium: The stability of a standard stock solution of the test item (1000 mg test item/L in ultra-pure water) was checked after 17 days and was 97% and 99% of the nominal concentrations. The stability of the fortified samples at the 1 x LOQ was checked after 21 days and was 100% of the nominal concentration. The stock solutions and samples were found to be stable over this period.

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

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. Apart from that, the pH-value of the control and each test item concentration were measured once per week using an additional replicate.
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.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5.
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: control, 10, 100, 250, 1000 mg active acid/kg soil dry weight.
- Results used to determine the conditions for the definitive study: no significant effects on earthworm mortality or live weight were observed in any treatment level.

Nominal and measured concentrations:

Nominal: control, 132, 198, 296, 445, 670, 1000 mg active acid/kg soil dry weight.
Measured: see Table 1 in 'Any other information on results inc. tables'

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

132 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

198 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.:

687 mg/kg soil dw

95% CI:

>= 572 - <= 886

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

< 132 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.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

other: mortality and biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: active acid

Basis for effect:

other: mortality and biomass

Details on results:

- Mortality at end of exposure period: Mortality ranged from 0 - 7.5%, see Table 2.
- Total mass of adults at beginning of test: 12.46g across all treatment and control groups.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: mean body weight change ranged from 0.07-0.11g, see Table 2.
- No. of offspring produced: mean number of juveniles produced ranged from 36 - 128, see Table 3.
- No. of unhatched cocoons: none reported.
- Morphological abnormalities: no pathological changes were observed in the control or treatment groups.
- Behavioural abnormalities: no behavioural changes were observed in the control or treatment groups.
- Other biological observations: none reported

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: Significant effects to reproduction rate were observed in treatment levels from 0.5 - 4 mg active substance/kg soil dry weight.
- Other: The OECD 222 guidance states that significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW. In this study, a significant reduction in reproductive rate was observed in the 0.5 mg active substance/kg soil dry weight treatment level, however this is not considered to have impacted the results with the registered substance.

Table 1. Nominal and measured concentrations of HEBMP-H

Nominal concentration (mg active acid/L)	Measured concentration (mg active acid/L)	% Recovery
6615	6262	95
4410	4594	104
2941	2898	99
1959	2046	104
1307	1325	101
872	871	100
Control	<LOQ	<LOQ

Table 2. Mortality and change in earthworm biomass after 28 days' exposure

Nominal concentration (mg active acid/kg soil dry weight)	Mean mortality of adult earthworms (%)	Mean body weight change (g) +/- SD^a	Mean body weight change (%) +/- SD^a	Significant?
Control	2.5	0.11 +/- 0.02	24.2 +/- 4.72	No
132	7.5	0.11 +/- 0.03	25.3 +/- 7.61	No
198	0	0.07 +/- 0.05	15.8 +/- 10.7	No
296	0	0.08 +/- 0.05	16.9 +/- 10.3	No
445	5	0.12 +/- 0.02	25.8 +/- 3.91	No
670	2.5	0.09 +/- 0.02	18.0 +/- 4.96	No
1000	5	0.08 +/- 0.01	18.0 +/- 3.40	No

Table 3. Reproduction rates of earthworms after 56 days' exposure

Nominal concentration (mg active acid/kg soil dry weight)	Mean number of juveniles +/- SD^a	CV^b (%)	% of reproduction compared to the control	Significant?
Control	128 +/- 18.7	14.6	-	-
132	113 +/- 12.2	10.8	88.3	No
198	92 +/- 18.2	19.8	71.9	Yes
296	90 +/- 25.8	28.6	70.3	Yes
445	84 +/- 11.9	14.1	65.6	Yes
670	78 +/- 28.9	37.0	60.9	Yes
1000	36 +/- 10.8	30.0	28.1	Yes

a = standard deviation

b = coefficient of variation.

Validity criteria fulfilled:

yes

Remarks:

Adult mortality in the control group was 2.5%, 99-150 juveniles were produced per replicate in the control group and coefficient of variation for reproduction in the control group was 14.6%.

Conclusions:

56-day NOEC, EC10 and EC50 values of 132, <132 and 687 mg active acid/kg soil dry weight have been determined for the effects of the test substance on the reproduction of Eisenia fetida, based on nominal concentrations. Recovery of the test substance in measured concentrations was between 95 and 104%. The NOEC and LOEC values for the effects of the test substance on mortality and biomass are ≥1000 and >1000 mg active acid/kg soil dry weight, respectively.

Description of key information

A 56-day NOEC value of 132 mg active acid/kg soil dry weight has been determined for the effects of HEBMP-H on the reproduction of Eisenia fetida.

Key value for chemical safety assessment

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

132 mg/kg soil dw

Additional information

56-day NOEC and EC₅₀ values of 132 mg active acid/kg soil dry weight and 687 mg active acid/kg soil dry weight, respectively, have been determined for the effects of HEBMP-H on adult reproduction of Eisenia fetida (Noack, 2023). An EC₁₀ value of <132 mg active acid/kg soil dry weight was reported for the effects on reproduction. The statistical analysis found no statistically significant difference for reproduction between the lowest test concentration (132 mg active acid/kg soil dry weight) and the control group. However, the ECx determination produced an EC₁₀ value that was lower than the lowest test concentration, which is why it has been reported as <132 mg active acid/kg soil dry weight (lowest concentration tested). NOEC and LOEC values of ≥1000 mg active acid/kg soil dry weight and >1000 mg active acid/kg soil dry weight, respectively, were determined for the effects on mortality and adult weight. The test was conducted according to OECD TG 222 and in compliance with GLP. The study met the OECD validity criteria and was assigned reliability 1.

 

Analysis of the test substance in the spiking solution on day -1 showed measured concentrations of HEBMP-H were within 80 -120% of nominal values and all values have been reported in terms of nominal values.