1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene

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

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))

Version / remarks:

July 29, 2016

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

Pre-treatment of the Test Item
The test item was dissolved in a volume of acetone sufficient to prepare a stock solution. This stock solution was used to produce the various dosage solutions of the test item. An appropriate volume of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil. After evaporation of the solvent by placing the application vessels under a fume hood, the quartz sand was coated by the test item.
The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in for 3 minutes.
Finally, the contaminated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw was used.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) have been kept at ECT Oekotoxikologie GmbH since February 1994. They were originally delivered by Co. Landenberger (D-72355 Schömberg). The adult earthworms were fed with finely ground cow manure.
Only adult earthworms (with clitellum) with a fresh weight (FW) between 300 and 600 mg were used. The worms were at least two months, but not more than one year old, and the age of individuals did not differ by more than four weeks. Animals which fulfilled these requirements, were taken from a synchronised culture. The worms selected for the test were acclimatised in artificial soil under test conditions at least 24 hours before starting the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20 ± 2 ºC

pH:

5.7 - 6.4

Moisture:

51.5 - 59.0 % of WHCmax using deionised water

Details on test conditions:

- Concentrations: 25, 45, 81, 145.8, 262.4, 472.4 mg a.i./kg artificial soil dry weight (dw)
- Exposure: mixed with artificial soil
- Controls: solvent treated, water treated
- Toxic reference item: Control, 150, 300 and 600 mg boric acid/kg (separate GLP study, ECT Study No.: IRR1803)
- Test design: Six treatments plus water control and solvent control, 4 replicates per treatment, 8 for the solvent control and 4 for the water control
- Test system: 10 Eisenia fetida per test vessel
- Feeding: After the start of the experiment, food was first provided one day after application of the test item and introduction of the adult earthworms. Thereafter, the adult earthworms were fed weekly during the first 4 weeks of the test. An amount of 15 ± 0.5 g of moistened dung was spread on the soil surface of each test container. If food remained uneaten, the ration was reduced on demand. After removing of the adults on day 28 further 15 ± 0.5 g of the moistened dung was mixed with the artificial soil of each test vessel.
- Test vessels: Bellaplast containers (Company Kastelplast, D-55120 Mainz) consisting of inert (non-toxic) plastic (Polystyrol), base area of 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm, covered with a transparent and perforate lid
- composition of the artificial soil was based on OECD Guideline No. 222
- water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated
- Temperature: 20 ± 2 ºC
- Light/dark cycle: 16 h light : 8 h dark, light intensity of 400 – 800 lx
- adult earthworms were removed from the substrate after 28 days
- Test parameters: Mortality and biomass after 28 days; reproduction after 56 days
- Endpoints: NOEC/LOECMortality, LC50, NOEC/LOECBiomass, NOEC/LOECReproduction, EC10 and EC50 values for reproduction

Nominal and measured concentrations:

Nominal: 25, 45, 81, 145.8, 262.4, 472.4 mg active ingredient/kg soil dw
Reference: 150, 300, 600 mg/ kg

Reference substance (positive control):

yes

Remarks:

boric acid

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

81 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

81 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

37.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

Observations: After 28 days cocoons were observed in all test vessels except in the highest concentration, i.e. 472.4 mg a.i./kg soil (dw), where in one replicate one cocoon was found and the other ones none.

Results with reference substance (positive control):

In a separate GLP study, the EC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 329.8 mg boric acid/kg soil (dw). The estimated EC50 value in this study was slightly lower than aspired by the guideline. However, since the calculated EC50 was lower by factor of 1.2 than the expected range of 400-600 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Reported statistics and error estimates:

Statistical evaluation:
- Mortality: Data were checked for homogeneity of proportions by Tarone’s test followed by Rao-Scott-Cochran-Armitage test (one-sided, α = 0.05). The LC50 was computed by Weibull analysis using linear max. likelihood regression
- Biomass change: Data were checked for normality by Shapiro-Wilk’s test procedure and for homogeneity by Levene’s test. Afterwards, treatment means were compared Williams t-test (α = 0.05, one-sided) and tested for statistically significant differences compared to the solvent control.
- Reproduction: Data were checked for normality by Shapiro-Wilk’s test procedure and for homogeneity by Levene’s test. Treatment means were compared by ANOVA followed by William’s test (α = 0.05, one-sided) and tested for statistically significant differences compared to the solvent control. The ECx values were computed by Weibull analysis using linear max. likelihood regression (Sachs 1982).
The statistical software package ToxRat Professional 3.2.1 was used for these calculations.

Validity criteria fulfilled:

yes

Remarks:

Mortality of adult animals in solvent control: 0.0%(required: ≤ 10%), mean number of juveniles / replicate in solvent control (+- standard deviation): 292.1 ± 45.9 (required: ≥ 30), CV for no. of juveniles in solvent control: 15.7% (required: ≤ 30%)

Conclusions:

The effects of the test item Perkalink 900 on the mortality, biomass and number of juveniles of Eisenia fetida were tested under laboratory conditions.
Adult mortality and the number of juveniles are the most prominent endpoints of Perkalink 900.
The overall lowest effect levels were:
NOEC: 81 mg a.i./kg soil (dw)
EC10 = 37.9 mg a.i./kg soil (dw)

Executive summary:

A study was conducted to assess the toxicity of Perkalink 900 to earthworms according to OECD Guideline No. 222 "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" which equals ISO 11268-2 Part 2 (2012) "Soil Quality – Effects of Pollutants on Earthworms – Part 2: Determination of Effects on Reproduction to Eisenia fetida/Eisenia andrei".

The earthworms were exposed to 6 concentrations (25, 45, 81, 145.8, 262.4, 472.4 mg a.i./kg artificial soil dry weight (dw)), solvent control and water control. Quarts sand was mixed with Perkalink 900 solved in acetone, dried and mixed with the artificial soil to achieve the different contentrations. Per treatment 4 replicates were executed, 8 replicates for the solvent control and 4 for the water control. 10 Eisenia fetida were exposed per test vessel. The composition of the artificial soil was based on OECD Guideline No. 222. The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated. The worm were kept at 20 ± 2 ºC with a Light/dark cycle of 16 h light : 8 h dark (light intensity of 400 – 800 lx). Adult earthworms were removed from the substrate after 28 days. Test parameters were mortality and biomass after 28 days and reproduction after 56 days

Adult mortality (28 d) and the number of juveniles (56 d) are the most prominent endpoints of Perkalink 900.

The overall lowest effect levels were:

NOEC: 81 mg a.i./kg soil (dw) (mortality and reproduction)

EC10 = 37.9 mg a.i./kg soil (dw) (reproduction).

Description of key information

A study was conducted to assess the toxicity of Perkalink 900 to earthworms according to OECD Guideline No. 222 "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)".

The overall lowest effect levels were:

NOEC: 81 mg a.i./kg soil (dw) (mortality and reproduction)

EC10 = 37.9 mg a.i./kg soil (dw) (reproduction).

Key value for chemical safety assessment

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

37.9 mg/kg soil dw