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

Sediment toxicity

Description of key information

The lowest NOEC for three sediment species is the one from Hyallela azteca, which is 7.1 mg/kg dwt sediment. The organic carbon content in the tests was circa 1.8%. For the key value in risk assesment this value needs to be converted to 5% OC. This results in 19.7 mg/kg sediment (7.1*0.05/0.018 fraction OC)

The key sediment toxicity results are summarised in the following table:

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

19.7 mg/kg sediment dw

Additional information

In the table below an overview of the sediment toxicity studies is presented

Test species	OECD TG	Results^1in mg/kg dwt (95% CL)	Remarks
Insectae Chironomus riparius	218	NOEC >1000 (development rate) LOEC = 500 (emergence ratio) EC50 = 402 (emergence ratio)   (CL: 260 – 829)  NOEC = 200 (emergence ratio, based on measured concentration)	Key study, K1. Egeler & Gilberg 2004a 2.6% OC
Crustaceae, Amphipoda Hyalella azteca	218	NOEC = 34.7 (survival) LC50= 62.5 (CL: 49.8 – 78.4) LOEC = 34.7 (growth) EC50= 53.5 (growth) (CL: 53.0 – 54.0)  NOEC = 14.5 (growth, nominal) NOEC = 7.1 (growth, measured)	Key study, K1. Egeler 2004 1.8% OC
Oligochaeta Lumbriculus variegatus	218	NOEC = 140 (survival)  NOEC = 60.9 (biomass) LOEC = 140 (biomass) LOEC = 60.9 (reproduction) EC50= 74.1 (reproduction) (CL: 42.5 – 165.7)  NOEC = 26.5 (reproduction, nominal) NOEC = 16.2 (reproduction, measured concentrations)	Key study, K1. Egeler & Gilberg 2004b 2.1% OC

¹ nominal concentrations; (95% confidence limits);

Key studies methodology

Toxicity tests were carried out with three species of sediment organisms, according to or in line with the OECD TG 218 (Draft December 2002): Sediment-water chironomid toxicity test using spiked sediment. The sediment was formulated from 5% Sphagnum moss peat, 75% quartz sand (>50% in range 50-200 μm), 20% kaolinite clay and 0.05% calcium carbonate to adjust the pH between 6.5 and 7.1. The organic carbon content was ~2%. At the same time 0.2-0.25% Urtica powder was added as feed. The formulated sediment was conditioned for 7 days prior to application of the test material. The test material was solved in acetone to prepare the stock solutions for each concentration. The proper volumes were mixed first with dry quartz sand allowing the solvent to evaporate. Next the sand was mixed with the formulated sediment to achieve the intended nominal concentration levels. Each glass vessel contained a layer of 1.5 to 3 cm of sediment and the water (Elendt medium M4) volume was 3.5 to 4.5 times the sediment volume. Both a control and a solvent control were included. The test animals were introduced after an equilibration period of 1 week. The tests were carried out at 20 ºC under a 16/8 hours L/D cycle with a light intensity of 400 to 600 lux. The overlying water was slightly aerated during the test. No additional food was given during the test. Test concentrations were measured. Samples of porewater and overlying water were extracted by SPE using Speedisks. Sediment samples were freeze-dried and analysed by GC/MS after solvent extraction. The analytical results are reported by Belfroid & Balk (2005). These tests were carried out under GLP and they are completely documented.

Key study 1, its NOEC is used for risk assessment, Egeler (2004; ECT AF1HA), Hyalella azteca:

A toxicity test was carried out with the Amphipoda Hyalella azteca, according to the OECD TG 218 (Draft December 2002). Hyalella azteca was tested in five concentrations ranging from 6 to 200 mg/kg sediment with step size factor 2.4. The test animals were 7 to 14 days old, and between 355 and 500 mm. Four replicates each with 10 animals were used per test concentration and in the solvent control, whereas six replicates were used in the control. The test concentrations were measured on day 0, 9, 19 and 28 in the control, 35 and 200 mg/kg dw. At the start of the test the test concentration was on average 54% of the intended nominal test concentration. Results: The actual concentrations at day 0 were on average 49% of the initial concentrations and remained at this level during the test.. At termination of the test, the average concentration was 96% of the concentration at the start. Survival was not affected up to 35 mg/kg dw, whereas the mortality in 83 mg/kg dw was 88% and 100% in 200 mg/kg dw. Thus the LC50 was 62.5 mg/kg dw. EC50biomass was 53.5 mg/kg dw. Growth was inhibited at 35 mg/kg dw (LOEC): The length of the amphipods was 9% below the pooled control at 35 mg/kg dw, whereas the inhibition for the (total) biomass per replicate was 15%. The At the NOECgrowth was 14.5 mg/kg dw, the inhibition was negligible 3% for length and none for biomass. The NOECgrowth measured was 7.1 mg/kg dw in this study.

Key study 2, Egeler and Gilberg (2004; ECT AF1ME), Chironomus riparius, Kl. 1

A toxicity test was carried out with Chironomus riparius, in line with OECD TG 218 (Draft December 2002): Sediment-water chironomid toxicity test using spiked sediment. The test was carried out with five concentrations ranging from 62.5 to 1000 mg/kg dw with step size factor 2. Twenty animals, first instar larvae, were used in each of the four replicates per test concentration, in the control and in the solvent control. The test concentrations were measured on day 0, 14, 20 and 28 in the control, 125 and 1000 mg/kg dw. Results: After the equilibrium period the test concentration was on average 80% of the intended nominal test concentration. At termination of the test, the average concentration was 82% of the concentration at the start. The development rate was not affected up to 1000 mg/kg dw for both males and females. The emergence ratio was the more sensitive endpoint. A clear dose response relation was found for the emergence of the midges. The data for males and females were pooled for the statistical analysis. The NOECemerg. was 250 mg/kg dw (-4%), the EC15 was 259 mg/kg dw (with 95% confidence limits 164 – 435) and the EC50 was 402 (CL: 260 – 829) mg/kg dw. The actual concentrations remained largely within 80% of the initial concentrations at day 0. The NOEC in this study was 200 mg/kg dwt based on measured concentration.

Key study 3, Egeler and Gilberg (2004; ECT AF1LA), Lumbriculus variegatus, Kl. 1

A toxicity tests was carried out with the aquatic oligochaete worm Lumbriculus variegatus, in line with the OECD TG 218 (Draft December 2002): Sediment-water chironomid toxicity test using spiked sediment. Lumbriculus variegatus was tested in concentrations ranging from 5 to 140 mg/kg dw with step size factor 2.3. The test animals were 'synchronised' before the start of the test to avoid high variation in the test results. Four replicates each with 10 regenerated animals were used per test concentration and in the solvent control, whereas six replicates were used in the control. The test concentrations were measured on day 0, 9, 20 and 28 in the control, 26.5 and 140 mg/kg dw. Result: At the start of the test the test concentration was on average 60% of the intended nominal test concentration. At termination of the test, the average concentration was 85% of the concentration at the start. Survival was not affected up to the highest test concentration. The total number of worms (including adult and regenerated worms) at the end of the test was evaluated as a parameter of reproduction. The NOEC biomass is 61, the EC15 was 62.4 and the EC50 was 97.7 mg/kg dw. The NOECrepro is 26.5 mg/kg dw (-8%) based on nominal and 16.2 mg/kg dw based on measured concentration. The EC15repro was 24.1 mg/kg dw. The EC50repro was 74.1 mg/kg dw (CL: 42.5 – 165.7). 

Less valid studies

Some less valid studies are shortly summarised for completeness. These studies lack important information and do not follow OECD guidelines. These are considered Kl. 3 studies. These are: Tamura et al. (2013), Artola-Garicano et al. (2003), Ramskov et al. (2009), and Pedersen et al. (2009).

 

The study is not performed under GLP, it is unclear whether results are dry weight or wet weight results and test concentrations over time are not reported.

Tamura et al. (2013), Chironomus yoshimatsui, Environmental Technology, 34(12): 1567–1575, Kl 3

Chironomus yoshimatsui were exposed for 20 days to 0.785, 1.57, 3.14, and 6.28 mg/kg according to OECD TG 218. The test substance was added in quartz sand (70% of the total weight) with acetone, and the acetone completely evaporated. Peat moss (5%) and kaolin (25%) were added to prepare an artificial sediment. M4 medium was added, shaken, and 10 first-instar larvae added to each vessel. Three replicates were prepared at five concentrations, with a dilution factor of 2. The 20d-NOEC was determined to be 3.1 mg/kg. The main reason for the Kl. 3 assignment is that it is not clear whether the results are based on wet weight or on dry weight.

Artola-Garicano et al. (2003), C. riparius & L. variegatus, Environmental Toxicology and Chemistry, 22(5): 1086–1092, Kl 3

Fourth instar midge larvae Chironomus riparius and the worm Lumbriculus variegatus were used for acute toxicity testing. All organism exposures were carried out at 19 +/- 2ºC, pH of 6.0 to 6.5, and oxygen levels of 50% up to 97% of saturation. A photoperiod of 12 h was applied in all cases. 10 midge larvae were individually exposed in 40-ml vials containing 10 ml of test solution (24-h renewal). 10 worms were exposed in triplicate in 1.5-L bottles filled with 1 L of test solution. Chironomus riparius were exposed for 96 hours under semi-static conditions. Results: The LC50 for Chironomous was 0.288 mg/L. Lumbriculus variegatus was exposed for 5 days under static conditions and the EC50 for mobility was 0.394 mg/L. Exposure occurred via water only and only acute toxicity was determined in this non-GLP study.

Ramskov et al. (2009), Capitella sp, Environmental Toxicology and Chemistry, 28(12): 2695–2705, Kl3: Study on Capitella sp. I (Marine species) exposed to 0, 1.5, 26, 123, 168 mg/kg dw sediment. Juveniles were exposed until sexual maturation (max 7 weeks) and adults were exposed from maturation till death (max age was 119 days). The substance showed no detectable effects on adult survival, age at first reproduction, length of the reproductive period, number of broods, individual worm body volumes, or body size–specific egestion rates. The substance significantly affected juvenile survival (≥123 mg/kg), maturation time (168 mg/kg), total number of eggs produced (≥26 mg/kg), and brood size (≥123 mg/kg) and marginally increased time between breeding attempts (≥26 mg/kg). A declining trend was observed for population growth rate with increasing test substance concentrations, but differences between the control and exposed groups were not significant.

It proved difficult to determine the reliability of this study and no limit values were determined. Furthermore, the dose selection is irregular as there is a factor 17, 4.7, and 1.4 between the subsequent dose levels. Consequently, the difference between the NOAEL and LOAEL is 17- fold.

Pedersen et al. (2009), Potamopyrgus antipodarum, Ecotoxicology and Environmental Safety 72:1190–1199, Kl 2/3

Snails were exposed to 0, 0.1, 1, 10, 30 and 100 mg/kg dw sediment (nominal). Juveniles were exposed for approx. 22 weeks and adults for 12 or 18 weeks. Snails were exposed individually with 10 replicates per treatment. The snails were exposed in 5ml multi-well dishes containing 0.5 ml wet sediment (0.3 g dw sediment) and 2.5 ml oxygenated artificial freshwater. The experiments were conducted in the dark at 17ºC and 2 ml of the overlying water was renewed every second day to avoid oxygen deficiency. Adult survival and growth were not affected. Juvenile growth (NOEC 10 mg/kg dw) and survival (NOEC 10 mg/kg dw), reproduction (NOEC 1 mg/kg dw), time to first reproduction (NOEC 30 mg/kg dw) and adult feeding rate (NOEC 10 mg/kg dw) declined with increasing test substance concentration. Discussion by the registrant: Confirmation of concentrations is not available. Non GLP and no guideline was followed, therefore the study was considered less reliable than the key guideline studies above.