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

Long-term toxicity to aquatic invertebrates

Description of key information

Surface water: The 21d-NOEC for Daphnia magna (reproduction) according to OECD TG 202, part II (update June 1993) was 0.111 mg/l.
Marine water: The lowest long-term EC10 was obtained for the marine copepod Acartia tonsa in a larval development study according to the OECD Draft Guideline for Testing of Chemicals: Guideline for life cycle test with Acartia tonsa (2004). It was 0.044 mg/l. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

111 µg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

44 µg/L

Additional information

Surface water: With Daphnia magna, a semi-static 21-d toxicity test was carried out according to OECD Test Guideline 202, part II, proposed updated version of June 1993 (Wüthrich 1996). The test medium was refreshed three times per week.  Nominal concentrations ranged from 0.062 to 1.0 mg/l (step size 2). Concentrations were measured at the start and end of the first and last exposure period. Mobility of the parent generation was not affected at concentrations up to and including 0.205 mg/l, whereas 100% were immobile in the next higher concentration – 0.419 mg/l. At 0.111 mg/l the mean reproduction was inhibited by 21% as compared to the solvent control. According to the Dunnett test (P0.05), this was statistically not significantly different from the control. At the next higher concentration, 0.205 mg/l (LOEC) the mean reproduction was inhibited by 26%. At 0.419 and 0.842 mg/l, reproduction of the surviving adults was inhibited completely. The test was carried out as a GLP study. The validity criteria were met.

Marine water: The chronic toxicity to marine copepod Acartia tonsa was tested according to OECD Draft Guideline for Testing of Chemicals (2004) describing a life cycle test, with specific adaptations to prevent volatilisation and accumulation of organic debris (Bjornstadt, 2007, being a follow up study of the one described below). For this test HHCB was mixed with 0.1% of radio-labelled [3-14C]-HHCB with a radiochemical purity of 94.8%. Ethanol was used as a solvent. Test concentrations were 37.5, 75, 150, 300 and 600 µg/l, with four replicates per concentration and 6 replicates in the (solvent) control. For the test 110-ml glass flask were used, closed with screw caps with Teflon seals and filled almost completely with natural seawater, salinity 30.2%o, and test substance leaving a headspace of 5 ml. The test was carried out at 20 ± 1.0°C in a climate room with a daily light/dark period of 16:8 hours. One day before addition of the eggs, the flasks were saturated with the appropriate test concentration. The test was initiated with approximately 80 eggs in each flask and approx. 7,000 cells/ml of the algae Rhodomonas salina.The algal concentration was readjusted daily to 7,000 cells/ml and the flasks were slowly rotated to keep the algae from settling. This was confirmed to be the optimal design to prevent the formation of organic debris and the loss of test substance by sorption and volatilisation. Samples of the test solution for LSC were taken daily. After 5.5 days the number of nauplii, copepodites and non-hatched eggs was counted and the lengths of the larvae was measured. The actual test concentrations were >80% of nominal during the whole study, implying that the nominal concentrations can be used for the calculations. The larval mortality in the control was below below the quality criterion of 30%: 27.1 % and 29.1% in the control and solvent control, respectively. The larval development ratio (LDR) was 73% in the control and 72% in the solvent control. The NOEC(LDR) was 37.5μg/l with an inhibition of 7.5% as compared to the solvent control. The inhibition was 27% in 75μg/l (LOEC). At 300μg/l, the length of the nauplii was significantly smaller than in the controls. Moreover no copepodites were observed. Up to 150μg/l, there was no impact on growth. The EC10(LDR) was 43.8μg/l (95% confidence interval 30.1 – 55.3μg/l), and the EC50 was 115μg/l (131-153μg/l). The test was carried out under GLP and is meeting the quality criteria.

In 2003 Wollenberger et al. published a study on the larval development rate for Acartia tonsa resulting in a 5d-EC10 of 0.037 mg/l. The test showed a number of methodological deficiencies (e.g. nominal test concentrations whereas it is known from a similar set-up that concentrations decreased to only 10 -30% after 2 days; and the renewal scheme of the medium by 50% combined with daily feeding probably left residual organic matter in the medium to which the test substance could readily sorb, leading to oral exposure in addition to the intended exposure through the water phase. Based on these deficiencies a new study was done as presented above (Bjornstadt, 2007).