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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Potasum hexatitanate, potassium titanium oxide
Batch n. MA-95150
Solid, light-yellow powder
Analytical monitoring:
not required
Remarks:
The analytical monitoring of the test item was not required, according to the test guideline.
Vehicle:
yes
Remarks:
fine quartz sand
Details on preparation and application of test substrate:
Dosages of the Test Item:
62.5, 125, 250, 500 and 1000 mg test item/kg soil

Application:
Potassium titanium oxide was weighed separately for each concentration using an analytical balance and 20 g fine quartz sand was added.
After mixing with a spoon to reach a homogeneous distribution of the test item within the sand the mixture was added to artificial soil equivalent to 2080 g dry weight, resulting in the nominal concentrations of the test item of 62.5, 125, 250, 500 and 1000 mg test item/kg soil.
There were no significant deviations to the nominal target concentration (< 5%). The control was treated with the same amount of 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.

Application Order:
1. control, 2. test item (from low to high concentrations)
Test organisms (species):
Eisenia andrei
Animal group:
annelids
Details on test organisms:
Taxonomic Group:
Earthworms (Annelida: Oligochaeta)

Species:
Eisenia andrei

Life Stage:
Adults

Age:
Approximately 6 months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks

Body Weight:
301 mg to 599 mg

Origin:
Bred under standardised conditions in the testing facility 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, under test conditions.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
Within the range of 18°C to 22°C
pH:
pH was determined at the start and end of the test for each treatment group, according to ISO 10390 (CaCl2).
pH range at experimental start: 6.0 to 6.3 and thus did not deviate from the values given by the guidelines (6.0 ± 0.5)
pH range at experimental end: 6.4 to 6.7
Moisture:
Water content was determined at the start and end of the experiment for each test concentration, according to ISO 11465.
At experimental start: 29.4% to 30.0% (54.4% to 55.5% of the maximum water holding capacity, i.e. within the recommended range 40 60 % of the total water holding capacity).
At experimental end: 29.5% to 30.5% (54.6% to 56.6% of the maximum water holding capacity)
Details on test conditions:
No. of Replicates:
8 per control, 4 per test item treated group

No. of Individuals:
10 per replicate, i.e. 80 individuals per control, 40 individuals per test item treated group

Test Units:
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. Each container was filled with 641.0 g of the prepared soil (500 g dry weight plus water). The height of the soil layer in the containers was approximately 5 cm.

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)
• 69.5% 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)
• 0.5% 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.

Maximum Water Holding Capacity of the Artificial Soil:
54% of the dry weight

Test Environment:
Controlled environment room, in a ventilated area

Temperature:
Within the range of 18°C to 22°C

Light Intensity:
Within the range of 400 to 800 lux

Light Regime:
16 h light : 8 h dark

Introduction of Individuals:
All worms were rinsed with tap water, dried with dry paper towels, weighed individually and randomly assigned to batches of 10 worms. The different batches were sorted into four classes on the basis of the total weight and one batch of each weight class was assigned to each treatment group (two batches for the control) to ensure weights were homogeneous. The earthworms were placed on the surface of the artificial soil after application.

Test Duration:
8 weeks

Exposure Time of Adult Worms:
After 4 weeks, the artificial soil was transferred to a tray and adult worms were counted, removed and weighed per replicate after being rinsed under tap water and dried on paper towels. The remaining soil (without the adult worms) was then returned to the respective test containers.

Exposure Time of Offspring:
Additional 28 days

Removal of Juveniles:
Juveniles were removed by placing the test units in a water bath at 50 - 60 °C and counting all emerging worms. In addition the soil of each container was emptied out onto a tray and checked visually for any remaining juvenile worms.

EFFECT PARAMETERS MEASURED

Mortality:
Number of dead adult earthworms at day 28 after application (including any missing ones)

Behavioural 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
Nominal and measured concentrations:
Nominal concentrations
62.5, 125, 250, 500 and 1000 mg test item/kg soil
Reference substance (positive control):
yes
Remarks:
Carbendazim
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
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):
In the most recent test with the reference item Carbendazim (performed at the testing facility), there were statistically significant effects on reproduction at a concentration of 0.695 mg a.i./kg soil and above, 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 0.92 mg a.i./kg soil.

Results and Discussion

1. Validity Criteria of the Study

Control Mortality:

Control mortality was 1.4% and so this validity criterion was met.

Reproduction of Control:

The number of juvenile worms per replicate was 131 to 225 and so this validity criterion was met.

Coefficient of Variation of Reproduction in Control:       

Was 19.8% and so this validity criterion was met.

2. Mortality of the Adult Worms

A slight mortality of 2.5% was observed at the concentrations of 62.5 mg test item/kg soil and 500 mg test item/kg soil, which was not statistically significantly different compared to the control where 1.4% of the worms died (Fisher’s Exact Test, one-sided greater, alpha = 0.05). The NOEC for mortality was determined to be >=1000 mg test item/kg soil.

3. Body Weights of the Adult Worms

The body weight changes of the adult worms after 4 weeks exposure to Potassium titanium oxide are shown in Table 4. 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 (Dunnett´s t-test, alpha = 0.05, two-sided). The NOEC for body weight changes was determined to be >= 1000 mg test item/kg soil.

4. Reproductive Assessment

The numbers of juvenile worms found 8 weeks after application are shown in Table 5. The reproduction rates were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil (Dunnett´s t-test, alpha = 0.05, one-sided smaller). The NOEC for reproduction was determined to be >= 1000 mg test item/kg soil.

The EC10, EC20 and EC50 values could not be determined statistically but were estimated to >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.

Validity criteria fulfilled:
yes
Conclusions:
In an earthworm reproduction and growth study with Potassium titanium oxide the No Observed Effect Concentration (NOEC) for mortality, growth and reproduction of the earthworm Eisenia andrei was determined to be >=1000 mg test item/kg soil, i.e. the highest concentration tested. The EC10, EC20 and EC50 values were estimated to >1000 mg test item/kg soil.
Executive summary:

The purpose of this study was to investigate the effects of Potassium titanium oxide on the mortality, body weight, feeding activity and reproduction of adult Eisenia andrei.

This study was designed to comply with the following methods:

- OECD, Guideline for the testing of chemicals No. 222, Earthworm, Reproduction Test (Eisenia fetida/Eisenia andrei; adopted July 29, 2016)

- ISO-Guideline 11268-2, Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei, International Organization for Standardization, 2012.

Material and Methods

Test Item:

Potassium titanium oxide; batch no.: MA-95150

Test Species:

Earthworm (Eisenia andrei), adult worms (with clitellum and weight range 301 to 599 mg), approximately 6 months old, from an in-house culture.

Test Design:

56-day test in treated artificial soil according to OECD 222; different concentrations of the test item were incorporated into the soil; 6 treatment groups (5 test item concentrations, control); 4 replicates for the test item treatments, 8 replicates for the control, 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).

Endpoints:

Mortality, weight change and reproduction rate were determined.

Reference Item:

Carbendazim (600 g/L nominal). The effects of the reference item were investigated in a separate GLP study.

Test Concentrations:

Control, 62.5, 125, 250, 500 and 1000 mg Potassium titanium oxide/kg soil

Test Conditions:

Artificial soil according to OECD 222; pH 6.0 to 6.3 at experimental start, 6.4 to 6.7 at experimental end; water content at experimental start 29.4% to 30.0% (54.4% to 55.5% of the maximum water holding capacity) and 29.5% to 30.5% (54.6% to 56.6% of the maximum water holding capacity) at experimental end; temperature: within the range of 18°C to 22°C; photoperiod: 16 h light : 8 h dark, light intensity: within the range of 400 lux to 800 lux.

Statistics:

Standard procedures, Fisher´s Exact Test (mortality), Dunnett´s t-test (weight changes and reproduction).

Results and Discussion

All study validity criteria were met.

A slight mortality of 2.5% was found at the concentrations of 62.5 and 500 mg test item/kg soil, respectively, which was not statistically significantly different compared to the control, where 1.4% of the worms died (Fisher´s Exact Test, alpha = 0.05).

Body weight of the earthworms exposed to Potassium titanium oxide were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test iem/kg soil (Dunnett´s t-test, alpha  = 0.05 two-sided).  

No statistically significant effects on reproduction were observed up to and including concentration of 1000 mg test item/kg (Dunnett´s t-test, one-sided smaller, alpha =0.05). No behavioural abnormalities were observed in any of the treatment groups and the feeding activity in all the treated groups was comparable to the control.

Conclusion

In an earthworm reproduction and growth study with Potassium titanium oxide the No Observed Effect Concentration (NOEC) for mortality, growth and reproduction of the earthworm Eisenia andrei was determined to be >= 1000 mg test item/kg soil, i.e. the highest concentration tested. The EC10, EC20 and EC50 values were estimated to >1000 mg test item/kg soil.

Description of key information

No effects were observed in the study. The NOEC is estimated to be >= 1000 mg/kg soil.

Key value for chemical safety assessment

Additional information