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

Toxicity to terrestrial plants

Description of key information

Study by Ibacon (Spatz & Kowalczyk, 2019) preformed according to OECD TG208, Reliability 1. No seedling emergence effects have been observed apart from for Allium cepa, see remarks in table. Seedling growth effects are based on fresh weight results.

The effect concentrations are summarized in the table below.

Species	NOEC mg /kg dw	EC10 mg /kg dw (95%CL)	EC50 mg /kg dw (95%CL)	Remarks
Average		7.63		The average EC10 is used for risk assessment because of the presence of 6 plant species. A.sative is not included because it would increase the EC10 disproportional
Brassica napus	4.12	3.55 (0.864-6.59)	19.8 (12.7-31.1)
Glycine max	37.0	12.3 (4.41-22.2)	246 (169-422)
Solanum lycopersicum	12.3	10.3 (4.3-16.1)	46.5 (35.0-63.0)
Cucumis sativus	12.3	5.20 (1.55-10.9)	141 (98.4-206)
Avena sativa	≥1000	-	-
Allium cepa	4.12	6.78 (5.16-7.84)	12.4 (12.0-12.9)	A significant impact on the emergence was noted at 37.0 and 111 mg/kg dw (70 and 57% resp.)

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

7.63 mg/kg soil dw

Additional information

Three relevant plant studies were identified Ibacon (Spatz, 2019), Wang et al. (2015) and Chen et al. (2010) The OECD guideline GLP study conducted by Spatz & Kowalczyk (Ibacon, 2019) is considered key based because carried out under GLP and Kl 1, while the publications are considered K2. A comparison of the results is presented in the table after the summaries of these 3 studies. 

Key study: Spatz & Kowalczyk (2019; IBACON 145421084), Terrestrial plants: The substance was tested for effects on seedling emergence and seedling growth on six plant species (Avena sativa, Brassica napus, Cucumis sativus, Allium cepa, Solanum lycopersium and Glycine max) according to OECD TG208. The test item was dissolved in acetone and mixed with fine quartz sand before being mixed into LUFA sandy loam soil ( Particle Size: all particles ≤ 0.2 cm; Corg: 0.66 ± 0.07%; pH: 5.9 ± 0.5). Six species, four dicotyledonous and two monocotyledonous species were exposed to nominal concentrations ranging between 0.457, 1.37, 4.12, 12.3, 37.0, 111, 333, 1000 mg test item/kg soil dry weight. 30 seeds were tested per concentration and species. An untreated and a solvent treated control were prepared to compare with the data of the test item treated concentrations. The exposure time was 14 or 21 days after 50% emergence in the controls depending on the growth of the seedlings. The analytical recovery of the test substance in the stock solution was 100% of the nominal value. In the control solutions no test substance was detected. There was a statistically significant impact of the test item on the emergence of A. cepa at 37.0 and 111 mg/kg dry soil (70 and 57%, respectively). The emergence of all other species was not affected. There was no statistically significant mortality for all tested species. Phytotoxic effects observed were necrosis, growth reduction and leaf and stem deformations. All validation criteria were met. The EC50 in terms of fresh weight ranged from 12.4 to 246 mg/kg dry soil for all species except A. sativa. For A. sativa no dose-response curve was calculated due to low effects, hence NOECavena was ≥ 1000 mg/kg dry soil. The EC10 values in terms of fresh weight ranged from 3.55 to 12.3 mg/kg dry soil, on average 7.63 mg/kg dry soil (Avena excluded).

Supporting studies

Chen et al. (2010), Triticum aestivum, Journal of Environmental Sciences, 22(12): 1966-1973

The study by Chen et al. (2010) misses details on experimental design and data. Exposure duration is not specified and whether values were expressed as wet or dry weight. Also, the EC10 values are higher compared to the key study. Therefore, the study is used as supporting information only.

Method: The acute toxic effect of the test substance on seed germination and seedling growth of wheat (Triticum aestivum) were investigated. In this study T. aestivum were exposed to nominal concentrations of 0 (control), 39, 77, 194, 387, 775, 1936, 3873 mg/kg. The soil samples were collected from the surface layer (0–20 cm) of an uncultivated and unpolluted field in Tianjin (Particle Size: all particles ≤ 0.2 cm pH, 6.23; organic matter, 3.86%; CEC, 19.88 cmol/kg; soil texture, 48.90% clay, 36.20% silt, and 14.90% sand). The tested concentrations of pollutants were determined according to 10% – 60% of the inhibitory rates of wheat root and shoot elongation. Results: The results showed that the toxicity to wheat was in the following sequence: seed germination and seedling growth was germination rate > shoot elongation> root elongation. The LC50 and LC10 values for seed germination were 846.6 and 84.1 mg/kg, respectively. The EC50 and EC10 values for root elongation were 2123.0 and 55.7 mg/kg and for shoot elongation 928.5 and 25.0 mg/kg, respectively. 

Wang et al. (2015), Terrestrial plants, Science of the Total Environment 508: 122-127

The study is used as supporting because although the paper mentions that the study was performed according to OECD TG208, test concentrations are unknown and description of materials, methods and results is limited. It is unknown whether the validity criteria as described in OECD TG208 are met. Based on similar (fresh weight & most critical) results as compared to the key study, this study is considered supporting information.

Method: In this study, terrestrial plants (T. aestivum, Z. mays, A. tuberosum, G. max, S. lycopersicum, C. sativus, B. pekinensis and L. sativa) were exposed to test substance concentrations for 21 days after germination. The main characteristics of the test soil are: 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. Appropriate amounts dissolved in acetone were spiked into air-dried soil to obtain the desired treatment concentrations. The totality was carefully mixed, and the acetone evaporated under darkness in a fume cupboard for 24 h. Moisture content was adjusted to 40–60% of the WHC. Four replicates for each treatment and controls were used, and each replicate contained 400 g of the test soil and 10 seeds. The following conditions were applied: temperature of 22 ± 1°C, humidity of 75±10%, and light/dark of 16/8 h. The emergence and survival were recorded every day, and the growth and biomass of plants were recorded at the end of the test. EC10, EC50 and NOEC values are presented.

Results: Based on the lowest EC10 values, results showed for root length that A. tuberosum was most sensitive (21.67 mg/kg). For shoot length and fresh weight S. lycopersicum was the most sensitive (6.67 and 4.35 mg/kg respectively). 

In the overview below toxicity to plants of the Ibacon and Wang studies are compared. These show fairly similar results: usually the EC10 and 50 are within a factor of 3.

Study owner		Species	(mg/kg dry soil)	EC50	EC10	NOEC
Ibacon	DB (14)	Brassica napus	Oilseed rape	19.8	3.55	4.12
Wang	DB (21)	Brassica Pekinensis (Rapa)	Napa cabbage	95.8	14.3	8.8
Ibacon	DF (14)	Glycine max	Soybean	246	12.3	37.0
Wang	DF (21)	Glycine max	Soybean	152	30.8	13.2
Ibacon	DS (14)	Solanum lycopersicum	Tomato	46.5	10.3	12.3
Wang	DS (21)	Solanum lycopersicum	Tomato	48.8	4.34	5.0
Ibacon	DC (14)	Cucumis sativus	Cucumber	141	5.20	12.3
Wang	DC (21)	Cucumis sativus	Cucumber	143	16.8	8.5
Wang	DA (21)	Lactuca sativa	Lettuce	66.0	12.0	8.8
Ibacon	MP (14)	Avena Sativa	Oat	-	-	>1000
Wang	MP (21)	Zea mays	Corn	291	58.7	19.8
Wang	MP (21)	Triticum aestivum	Wheat	339	33.6	15.6
Chen	MP	Triticum aestivum	Wheat	846.6	55.7	-
Ibacon	ML (21)	Allium cepa	Onion	12.4	6.78	4.12
Wang	ML (21)	Allium tuberosum	Chinese Leek	65.6	13.5	12.8

- DB = Dicotyledonae Brassicaceae (Cruciferae); DF = Dicotyledonae Fabaceae (Leguminosae); DS = Dicotyledonae Solanaceae; DC = Dicotyledonae Cucurbitaceae; DA = Dicotyledonae Asteraceae; MP = Monocotyledonae Poaceae (Gramineae); ML = Monocotyledonae Liliaceae (Amarylladaceae). Number between brackets is total days of exposure

- ECx/NOEC-values in the IFF study are based on fresh weight. In the study by Wang, ECx/NOEC values are based on root length, shoot length and fresh weight. However, as per OECD 208 fresh weight results are presented in the table.

The following studies were also identified, but were either missing critical information, non-guideline, non GLP, not performed in soil or endpoints measured were not relevant for regulatory purpose and are therefore not assessed for the toxicity to terrestrial plants endpoint.

An et al. (2009), Triticum aestivum L. Chemosphere 76:1428-1423: Study on seed germination, shoot elongation and root elongation, chlorophyll content, soluble protein concentration, peroxidase and superoxide dismutase activity. Seed germination experiments were performed on filter papers placed in Petri dishes and moistened with 5.0 mL solution of test material. Controls were obtained by moistening the filter papers with 5 mL deionized water. Twenty wheat seeds were placed in each dish, covered by the lid. All treatments were replicated three times to minimize experimental errors. Wheat seeds germinated in darkness at 25 ± 2°C for 7 days. Concentrations tested were 0, 50, 100, 150, 200 and 250 mg/L. When the length of the growing roots cultured in the control reached 20.0 mm, this exposure experiment was finished. Other germinated seeds underwent further growth using solution hydroponics in a growth chamber operated with 12 h light: 12 h dark cycles at a constant temperature of 25 ± 2°C. Concentrations were equal to 0.2, 0.4, 0.7, 1.5, and 3.0 mg/L. The test solutions were renewed every day. Results: No significant difference in the germination frequency of wheat seeds were found. EC50 of the test substance was 143.4 and 422.3 mg/L based on shoot and root elongation, respectively.

Belz et al. (2018), Lactuca sativa, Science of the Total Environment 631–632: Study on hormesis. Dose-response germination experiments were conducted with L. sativa and shoot and root elongation were endpoints. Assays were conducted in 6-well cell culture plates. Each well was prepared with one layer of filter paper and prepared by adding increasing amounts of the ethanolic stock solutions. The ethanol was allowed to evaporate before 1.5 ml demineralized water and 60 lettuce seeds were added to each well/replicate. There were 11–13 treatments per toxin and a control with demineralized water only. The number of replicates (one replicate equals one well) per treatment was 24 arranged in blocks of six replicates (one 6-well-plate) that were randomly placed in a climate chamber after plates were sealed with parafilm (12/12 h day/night cycle starting at 8 am with 24/18 °C and a 12 h light period of 50–70 μmolm−2 s−1 photosynthetic active radiation). 5-day exposure with dose range 6.7e-7 - 1.3 mM. Results: ED50 were 3.208 mM (829 mg/L) and 4.300mM (1111 mg/L) for root and shoot elongation, respectively.

Patama et al. (2019), Gypsophila elegans Bieb, Ecotoxicology 28:732–743: Study on hormesis with similar set up as Belz et al. (2018) above. An untreated control and total 12 concentrations ranging from 0.0000067 to 2.00 mM were applied. Results: Results for shoot growth: Average (slow+fast) ED50 is 0.316 mM (81.7 mg/L). Slow-growing EC50 is 0.076 mM (19.6 mg/L). Fast-growing EC50 is 0.876 mM (226 mg/L). Results for root growth: Average (slow+fast) ED50 for shoot growth is 0.216 mM (55.8 nmg/L). Slow-growing EC50 is 0.267 mM (69.0 mg/L). Fast-growing EC50 is 0.200 mM (51.7 mg/L). The test substance exposure did not cause selective effects on root elongation of the slow- and fast-growing seedlings.

Sinkkonen et al. (2011), Gypsophila elegans & Portulaca oleracea L. ssp. sativa, Dose-Response 9:130–143: Seeds per dish were germinated for one day in tightly closed 53 mm diameter polyethylene Petri dishes on a filter paper in 1 ml aqueous solution. After harmonization, treatments started as randomized replicates by adding 2 ml of deionized water that had been enriched with micronutrients and treatment. Six replicates of 50 Gypsophila elegans seeds had 3-day exposure with 0, 1.25 or 1.75 mg/L. Six replicates of 47 Portulaca oleracea seeds had 4-day exposure with 0, 0.01, 0.10 or 1.00 mg/L. Root length was measured. Results: There were no significant between-treatment differences in mean root length of G. elegans. The substance affected mean root length of P. oleracea at 0.01 and 1.00 mg/L but not at 0.10 mg/L, as compared to control.

Chen & Cai (2015), Triticum aestivum, Bull Environ Contam Toxicol 95:272–277: Seedlings of wheat (Triticum aestivum) were exposed in soil to the test material for 21 days, to evaluate its effect upon chlorophyll, lipid peroxidation and the antioxidant system. Soil and set up are similar as described in Chen et al. (2010) as described above. Treatments were 1, 10 and 100 mg/kg. Results: The investigated enzymes in T. aestivum may be considered as effective biomarkers of oxidative stress caused by test substance pollution in soil. No regulatory relevant endpoints were investigated, and EC50/EC10/NOECs have not been concluded.