1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Toxicity to soil macroorganisms except arthropods

Description of key information

The sublethal effects of the substance on Eisenia fetida were derived from an ISO 11268, equivalent to an OECD TG 222 study and based on nominal concentrations.

4wk-NOEC (survival) >= 250 mg/kg dw

4wk-NOEC (growth) = 105 mg/kg dw;

8wk-NOEC (reproduction) = 45 mg/kg dw.

Key value for chemical safety assessment

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

45 mg/kg soil dw

Additional information

Three earthworm studies were identified with varying organic matter content. The study by Gossmann is considered key because there is sufficient information to assess validity of the test.

In the studies by Wang et al. (2015) and Chen et al. (2011), the validity could not be assessed, and the organic matter content was lower as indicated in OECD TG222 (2.3 and 4.2 % respectively). The guideline study performed under GLP was therefore preferred. An overview with the results is presented after the summaries of the key and supporting studies.

Key study: Gossmann (1997, Ibacon 2032022) Eisenia fetida:

The chronic earthworm test was carried out according to ISO 11268 (similar to OECD TG 222) and GLP. Adult worms (Eisenia fetida) were exposed to nominal concentrations in soil of 8, 19, 45, 105 and 250 mg/kg. The test materials at appropriate concentrations were dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil  containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.1-0.5 mm) and 0.5% calcium carbonate to adjust to pH 6.0±0.5. After preparation of the test concentrations and an equilibrium period of one week, the test organisms were added to the soil. The test medium was not refreshed during the test period. Weights of the adult worms ranged between 340 and 540 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Results: Mortality of the adults was not affected after 4 weeks in concentrations up to 250 mg/kg. Growth was significantly inhibited (15%) in the highest concentration of 250 mg/kg after 4 weeks. Reproduction was not significantly affected up to concentrations of 45 mg/kg (7% inhibition, NOEC, 8 weeks). At the level of the LOEC (105 mg/kg), the reproduction was 57% of the control, whereas in the highest concentration reproduction was inhibited completely. NOECs for survival, growth and reproduction were: > 250, 105 (4 weeks) and 45 mg/kg dw at 8 weeks, respectively based on nominal concentrations.

Supporting studies 

Wang et al. (2015), Eisenia fetida, Science of the Total Environment 508: 122–127d

An acute and long-term test was performed. The main characteristics of the test soil for both tests were: organic matter content of 23.19±0.47 g/kg (2.3%), pH of 8.05±0.05, cation exchange capacity of 17.10 cmol/kg, 3.91% clay, 32.04% silt and 64.05% sand. In addition, a control and a solvent control (acetone) were tested in parallel. At the end of the test, the survival was recorded. 

The acute toxicity test was performed according to the standard guideline OECD TG207 (OECD, 1984). Earthworms were exposed to nominal concentrations of 60.0, 90.0, 135.0, 202.5, 303.8 and 455.6 mg/kg for 14 days.  Four replicates per treatment were used, and each replicate contained 500 g of the test soil and 10 well-developed adults (300–400 mg). The test materials at appropriate concentrations were dissolved in acetone, and spiked into air dried natural soil. 

The reproduction test was performed according to the standard guideline OECD TG222 (OECD, 2004) with the same test soil as described above.  The final treatment concentrations were 6.0, 9.0, 13.5, 20.3, 30.4 and 45.6 mg/kg. Test duration was 56 d and food (5 g oatmeal) was added weekly during the first 28 d. The adults were removed from the soil and were counted and weighted at day 28 of the test. A further 5 g of food was then administered to each test container. No further feeding took place during the remaining 28 days of the test. At the end of the second 28-d period, the number of juveniles was counted using a warm water bath method. The cocoon numbers were checked using a 0.5 mm mesh size sieves method. 

Results: No significant differences occurred between control and solvent control in all measured parameters. In the acute test: the 14-d-LC50 for E. fetida survival was 188.36 mg/kg. In the reproduction study the EC50 for cocoon production and number of juveniles were 20.95 and 16.69 mg/kg respectively. The NOEC values for cocoon production and number of juveniles of E. fetida were 9.0 and 6.0 mg/kg soil, respectively. This value was normalized according to the method for organic substances, though from the paper it is not clear what that means. Possibly it is standardised to 3.4% organic matter as prescribed in the pre-REACH TGD guidance. 

Chen et al. (2011), Eisenia fetida Ecotoxicology 20:1949–1958:

The acute soil toxicity test was performed according to the standard guideline OECD TG 207 (OECD, 1984) with slight modifications. Earthworms were exposed to nominal concentrations of 100, 140 196, 274, 384, 538, 753, 1054, and 1476 mg/kg dw soil for 14 days. Three replicates of 10 worms were used for each container containing 500 g soil and placed in an incubation chamber (20 ± 1 °C and a light/dark cycle of 12/12 h). The test materials at appropriate concentrations were dissolved in acetone, and spiked into air dried natural soil. The main characteristics of the test soil were an organic matter content of 4.2%, pH of 7.2, cation exchange capacity of 20.35 cmol/kg, 47.4% Clay, 38.0% silt and 14.6% sand. In addition, a control and a solvent control (acetone) were tested in parallel. Dry cow manure (2 g) was spiked at the same concentration of the test substance as the tested soil and then supplied to each treatment. Any remaining food was removed, and new food added once a week. At the end of the test, the survival was recorded after 7 and 14 days.

The reproduction test was performed according to the standard guideline OECD TG222 (OECD, 2004) with the same test soil, feed and feeding frequency.  Based on the acute toxicity test, the final treatment concentrations were 3, 10, 30, 50 and 100 mg/kg dw soil, including a solvent control. Four replicates of 10 worms were used for each container. After 28 days cocoon production was counted by wet sieving of the tested soil.

Results: Acute toxicity test. The calculated 7 and 14 day (LC50) were 489.0 and 392.4 mg/kg dw soil, respectively. Reproduction test: During the 28-day chronic the NOEC values for growth rate is 50 mg/kg bw. The NOEC for reproduction rate (cocoon/worm) was 30 mg/kg dw. soil.

Overview of earthworm toxicity results

			Growth (mg/kg dw)		Reproduction (mg/kg dw)
			NOEC	LOEC	NOEC	LOEC
Ibacon (1997)	Eisenia fetida	4 weeks	105	250
Ibacon (1997)	Eisenia fetida	8 weeks			45	105
Chen et al. (2011)	Eisenia fetida	4 weeks	50	100	30	50
Wang et al. (2015)	Eisenia fetida	4 weeks	Not provided
Wang et al. (2015)	Eisenia fetida	8 weeks			6.0

 

Less valid studies

Four other earthworm studies were identified but are not according to OECD guidelines, non-GLP, lacking important information or the endpoint and/or units are not considered relevant for regulatory assessment under REACH. 

Chen et al. (2011a), Eisenia fetida, Chemosphere 83: 1147-1154: 

Study on acute toxicity, biochemical and transcriptional changes of representative antioxidant enzymatic (SOD, CAT) and stress-response gene (Hsp70). The acute toxicity was determined in a study according to OECD TG207 (1984).

Test substance dissolved in acetone solvent (1 mL) were added into the filter paper and the test tube was rotated for uniform distribution of the toxicant. Acetone solvent was dried under a stream of compressed air and then deionized water (1 mL) was added to each tube. Each tube has a depurated earthworm in it and the tubes were capped firmly with a small ventilation hole to prevent volatilization. The concentrations were prepared as 2.0, 3.2, 5.1, 8.2, 13.1, 21.0, 33.6, 53.7, and 85.9 μg/cm2, which are equivalent to 125–5376 μg/mL. Twenty replicates for each concentration treatment and each test tube containing one depurated worm were performed. Acetone and deionized water test were performed in all tests as control. The worms were visually examined every 6 h and the dead ones were removed. After incubation periods of 24 h, 48 h, 72 h, mortality and abnormality were recorded respectively. All tests were carried out in the climate chamber with 20 ± 1C, 75% humidity and 12/12 h day/dark cycling period. Results: The 48h and 72h-LC50 values were determined to be 11.87 and 6.14 μg/cm2 respectively. These units and other endpoints are considered not relevant for regulatory assessment.

Liu et al. (2011), Eisenia fetida, Chemosphere 83: 1080-1086: Study of responses of antioxidant systems and lipid peroxidation as biomarkers for oxidative stress after exposure. The only regulatory relevant finding is the acute toxicity. The experiments were conducted in 1 L glass containers with 500 g dry soil. The test substance was applied to soil for final treatment concentrations of 0, 10, 50 and 100 mg/kg. The soil samples were vented for 72 h to remove all the solvent, wetted to 50% of water holding capacity, and then stabilized for 24 h prior to adding earthworms. Each treatment has three replicates, consisting of ten earthworms per container. Two earthworms were collected from each replicate container on the 3rd, 7th, 14th and 28th day following application for enzyme assays. Results: No mortality was observed throughout the experimental period (28d NOEC >= 100 mg/kg). Other endpoints are considered not relevant for regulatory assessment.

Liu et al. (2010) Eisenia fetida, Environ Toxicol 27(8): 472-2479: Study on effects on activities of antioxidant enzymes, including superoxide dismutase, peroxidase, catalase, and malondialdehyde after exposure. Normal adults with well-developed clitella, which had individual wet weight of 350–450 mg, were selected for the toxicity test which was carried out according to OECD TG207 (1984), with modifications. The test unit was a flat-bottomed glass vial, covered by perforated plastic film. Each vial was lined with an 85x75 mm filter paper. The test substance was dissolved in acetone and used at final concentrations ranging from 0.1 to 200 mg/1. One milliliter of 0.1, 1, 10, 100, and 200 mg/1 solution (corresponding to 0.00157, 0.0157, 0.157, 1.57, and 3.14 μg/cm2) was pipetted into each glass vial and evaporated to dryness in the open air. The control vials were treated with 1 mL of appropriate organic solvent, which was evaporated later. The dry filter paper was moistened with 1 mL of distilled water. Each treatment had five replicates, consisting of one individual per vial. The experiments were carried out for 1, 2, and 3 day(s) at a constant temperature (20 C+/- 1C) and in the dark. There was no mortality of E. fetida in all tests (72h-NOEC>= 3.14 μg/cm2). Other endpoints are considered not relevant for regulatory assessment.

Mori et al. (2006) Caenorhabditis elegans, Journal of Health Sciences, 52(3): 276-282: Study on acute and chronic toxicity using plates with growth medium. Endpoints measured were lethality, growth, maturation and reproduction. The LC50 was found to be 194.6 mg/L. In growth and maturation tests, the LOEC was 9.8 mg/L, while in the reproduction test the LOEC was 19.5 mg/L. The study was not performed in soil and can therefore not be used for regulatory assessment.