Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

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:

Oct 2015 - Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

version from 2004

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Version / remarks:

version from 2012

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS no: 68919-76-6

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

A stock solution was prepared by dissolving 8400.3 mg of 400112 in 20 mL acetone. A sequential 1:2 dilution series was prepared, starting with the 420.015 mg/mL stock solution by adding 10 mL of acetone to 10 mL of the stock solution or dilution. Then 5 mL of the corresponding dilutions were added to 20 g fine quartz sand. The treated sand was left for two hours in a fume hood until the solvent had evaporated and was mixed with a spoon. Then artificial soil equivalent to 2100 g dry weight was added to test item blended sand.
There were no significant deviations to the nominal target concentration (< 5%).
The control was treated with the same amount of acetone and sand as the test item groups. While mixing the artificial soil in a laboratory mixer for approximately 5 min the soil of each treatment group was moistened with deionised water. Each group was treated in one batch (two in the control) which was then split into the replicates.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Species: Eisenia fetida (Savigny 1826)
Life Stage: adults
Age: approximately 11 months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks
Body weight: 300 - 600 mg
Origin: bred under standardised conditions in ibacon laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.
Acclimatisation: 1 day, in artificial soil

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18 - 22 °C

pH:

pH was determined at the start and end of the test for each treatment group, according to ISO 10390 (CaCl2).
start of experiment: 6.0 - 6.1
after 8 weeks: 6.4 - 6.5

Moisture:

Water content was determined at the start and end of the experiment for each test concentration, according to ISO 11465.
At experimental start: 27.7% to 28.4% (51.4% to 52.6% of the maximum water holding capacity, i.e. within the recommended range 40-60% of the total water holding capacity).
At experimental end: 27.3% to 30.6% (50.6% to 56.7% of the maximum water holding capacity)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (18.3 cm x 13.6 cm x 6 cm, tapered towards the bottom, with a soil surface of approximately 16.5 cm x 11.5 cm = 189.75 cm²) with perforated transparent lids to enable exchange of air, to minimise evaporation from the artificial soil, and to prevent the worms from escaping.
- Amount of soil or substrate: Each container was filled with 627.0 g of the prepared soil (500 g dry weight plus 127.0 g deionised water). The height of the soil layer in the containers was approximately 4 - 5 cm.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per vehicle control: 8

PROPERTIES OF SUBSTRATE
Artificial soil: according to OECD 222:
• 10% Sphagnum-peat, air-dried and finely ground (2 mm with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
• 20% Kaolin clay (Kaolinite content >30%) (Erbslöh, 65558 Lohrheim, Germany)
• 69.6% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
• 0.4% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.


OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 400 - 800 lux
- Feeding: Finely ground cattle manure was used as food. 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 - 3 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult worms.

EFFECT PARAMETERS MEASURED:
Mortality: Number of dead adult earthworms at day 28 after application (including any missing ones)
Behavioural and morphological abnormalities: Number of affected adult earthworms (e.g. lack of movement, rigidity) at day 28 after application
Food given: Cumulative amount of food added to each test container during the test period
Mean body weight: Body weights were determined at start (day 0) and 28 days after application
Reproduction: Number of juveniles 56 days after application

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 62.5, 125, 250, 500 and 1000 mg test item/kg soil

Nominal and measured concentrations:

62.5, 125, 250, 500 and 1000 mg test item/kg soil

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality

Key result

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth

Remarks:

body weight

Duration:

4 wk

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth

Remarks:

body weight

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

reproduction

Details on results:

1. Validity criteria of the study
Control mortality: Control mortality was 0% and so this validity criterion was met.
Reproduction of control: The number of juvenile worms per replicate was 199 to 281 and so this validity criterion was met.
Coefficient of variation of reproduction in control: 10.2% and so this validity criterion was met.

2. Mortality of the adult worms
No mortality was observed in any treatment group. The NOEC for mortality was estimated to be higher than or equal 1000 mg test item/kg soil.

3. Body weights of the adult worms
The body weight changes in the test item treated groups were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil (Williams t-test, α = 0.05, two-sided). The NOEC for body weight changes was determined to be higher than or equal 1000 mg test item/kg soil, the LOEC was estimated to be greater than 1000 mg test item/kg soil.

4. Reproductive assessment
The reproduction rates were not significantly different compared to the control up to the concentration of 1000 mg/kg soil (Dunnetts t-test, α = 0.05, one-sided smaller), expect the lowest concentration of 62.5 mg test item/kg soil. However, the effect was not dose related and the reduction was less than 20% and is therefore not considered to be biologically relevant. The NOEC for reproduction was determined to be higher than or equal 1000 mg test item/kg soil, the LOEC was estimated to be greater than 1000 mg test item/kg soil.

5. Feeding activity and behavioural abnormalities
Feeding activity: In all treatment groups, food was consumed. The results show that the turnover of biomass of those earthworms exposed to the different rates of the test item was comparable to the control.
Behavioural abnormalities: No behavioural abnormalities were observed and all worms burrowed into the soil within 15 minutes after introduction.

Results with reference substance (positive control):

Reference item: Carbendazim 600 g/L SC
The reference item test (dose response) is performed at least once a year at the test facility as a means of ensuring that the laboratory test conditions are adequate and to verify that the response of the test organisms does not change significantly over time. The GLP conducted experiment was performed from June 2015 to August 2015.

Tested concentrations: 1.39 / 2 / 2.88 / 4.16 / 6 mg test item/kg soil dry weight (= 0.695 / 1 / 1.44 / 2.08 / 3 mg active ingredient/kg soil dry weight)
No mortality occured. There were statistically significant effects on reproduction at a concentration of 2.08 mg a.i./kg soil and higher, which is in line with the guideline OECD 222 (effects should be observed between 1 and 5 mg a.i./kg soil). The EC50 for reproduction was calculated as 1.91 mg a.i./kg soil.
NOEC (day 28, mortality): ≥ 3.00 mg a.i./kg soil dw
NOEC (day 28, weight): 1.44 mg a.i./kg soil dw
NOEC (day 56, reproduction): 1.44 mg a.i./kg soil dw
EC10 (reproduction): 1.58 mg a.i./kg soil dw
EC20 (reproduction): 1.70 mg a.i./kg soil dw
EC50 (reproduction): 1.91 mg a.i./kg soil dw

Validity criteria fulfilled:

yes

Conclusions:

In an earthworm reproduction and growth study with 400112 the NOEC for mortality, growth, reproduction and feeding activity of the earthworm Eisenia fetida was determined to be ≥1000 mg test item/kg soil, i.e. the highest concentration tested. The LOEC for all parameters was estimated to be > 1000 mg test item/kg soil.

Executive summary:

The effects of 400112 on the mortality, body weight, feeding activity and reproduction of the adult earthworm Eisenia fetida in artificial soil was investigated. The GLP-compliant study was based on OCED Guideline 222 and ISO-Guideline 11268-2. Different concentrations of the test item were incorporated into the soil; 6 treatment groups (5 test item concentrations, 1 control); 4 replicates for the test item treatments and 8 replicates for the control with 10 worms each. Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application). The endpoints mortality, weight change, feeding activity, and reproduction rate were determined.

The following conccentrations were tested: control, 62.5, 125, 250, 500 and 1000 mg 400112/kg soil dry weight.

All study validity criteria were met. No mortality was observed in any treatment group. The body weight changes of the earthworms after 4 weeks exposure to 400112 were not statistically significantly different compared to the control at ≥1000 mg test item/kg soil (Williams t-test, α = 0.05, two-sided). The reproduction rates were not significantly different compared to the control at ≥ 1000 mg/kg soil (Dunnetts t-test, α = 0.05, one-sided smaller), expect the lowest concentration of 62.5 mg test item/kg soil. However, the effect was not dose related and the reduction was less than 20% and is therefore not considered to be biologically relevant. No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all the treated groups was comparable to the control.

Therefore, it can be concluded that in an earthworm reproduction and growth study with 400112 the NOEC for mortality, growth, reproduction and feeding activity of the earthworm Eisenia fetida was determined to be ≥1000 mg test item/kg soil, i.e. the highest concentration tested. The LOEC for all parameters was estimated to be >1000 mg test item/kg soil.

Description of key information

The effects of 400112 on the mortality, body weight, feeding activity and reproduction of the adult earthworm Eisenia fetida in artificial soil was investigated. The GLP-compliant study was based on OCED Guideline 222 and ISO-Guideline 11268-2. Different concentrations of the test item were incorporated into the soil; 6 treatment groups (5 test item concentrations, 1 control); 4 replicates for the test item treatments and 8 replicates for the control with 10 worms each. Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application). The endpoints mortality, weight change, feeding activity, and reproduction rate were determined.

The following concentrations were tested: control, 62.5, 125, 250, 500 and 1000 mg 400112/kg soil dry weight.

All study validity criteria were met. No mortality was observed in any treatment group. The body weight changes of the earthworms after 4 weeks exposure to 400112 were not statistically significantly different compared to the control at ≥ 1000 mg test item/kg soil (Williams t-test, α = 0.05, two-sided). The reproduction rates were not significantly different compared to the control at ≥ 1000 mg/kg soil (Dunnetts t-test, α = 0.05, one-sided smaller), expect the lowest concentration of 62.5 mg test item/kg soil. However, the effect was not dose related and the reduction was less than 20% and is therefore not considered to be biologically relevant. No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all the treated groups was comparable to the control.

Therefore, it can be concluded that in an earthworm reproduction and growth study with 400112 the NOEC for mortality, growth, reproduction and feeding activity of the earthworm Eisenia fetida was determined to be ≥ 1000 mg test item/kg soil, i.e. the highest concentration tested. The LOEC for all parameters was estimated to be > 1000 mg test item/kg soil.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw