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Short-term toxicity to fish

Reliable results obtained in a guideline studies are available. The acute toxicity of alpha-methylstyrene to Brachydanio rerio was investigated in a study conducted according to EU Method C.1 (Acute Toxicity for Fish; 2008) and OECD Guideline 203 (Fish, Acute Toxicity Test; 1992). 7 Fish were exposed under static conditions to nominal concentrations of 0 (control), 10, 20, 40, and 80 mg/L nominal (WAF) corresponding to initial measured concentrations of 0 (control), 2.613, 4.552, 10.164, and 30.527 mg/L (WAF). The test substance concentrations were determined via GC/FID. After 96 h, the test substance concentrations had decreased to concentrations < 1 mg/L. Therefore the calculation as geometric mean of t (0 h) vs. t (96 h) is not appropriate. The 96 h LC50, NOEC and LOEC were determined to be 2.97, 2.13, and 3.19 mg/L based on measured concentrations (geom. mean). The validity criteria given by the guidelines were fulfilled (control: no dead animals were observed; DO>=60% of air saturation value).

The acute toxicity of alpha-methylstyrene to Oryzias latipes was investigated in a study conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). 10 Fish were exposed to alpha-methylstyrene at nominal concentrations of 0 (control), solvent control, 3.0, 6.0, 12.0 24.0, and 48.0 mg/L for 96 h under semi-static conditions. The test substance concentrations were determined by HPLC in freshly prepared solutions and in 24 h old test solutions. The geometric mean measured concentrations at concentrations of 3-12 mg/L nominal were in the range between 78 and 85% of the initial nominal concentrations. Due to the death of fish within 24 h at concentration levels >=24 mg/L, the calculation of the mean geometric concentrations was not performed. A 96 h LC50=7.28 mg/L (geometric mean measured) was determined.

The prolonged toxicity of alpha-methylstyrene to Oryzias latipes was investigated in a study conducted according to OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study). 10 Fish were exposed to alpha-methylstyrene at nominal concentrations of 0 (control), solvent control, 0.5, 1.3, 3.1, and 8 mg/L for 14 d under flow-through conditions. The test substance concentrations were monitored via HPLC/UV. A 14 d LC50>6.78 mg/L was determined based on mean measured concentrations. The 14 d NOEC (growth: length and weight of fish) was determined to be 1.04 mg/L. Based on these results it can be concluded that the long-term toxicity of fish is comparable to the short-term toxicity.

Long-term toxicity to fish

No data from guideline studies on chronic fish toxicity are available. Based on the results of the prolonged toxicity test with fish (see above) it can be concluded that the long-term toxicity of fish is comparable to the short-term toxicity.

According to Annex IX of Regulation (EC) No 1907/2006 chronic fish toxicity tests should be proposed if the chemical safety assessment indicates the need to investigate further the effects on fish. However, overall information is sufficient for risk assessment. Therefore, testing of on chronic fish toxicity is not regarded necessary. Furthermore, water is not the target compartment of the substance, due to its physico-chemical properties.

Short-term toxicity to aquatic invertebrates

Reliable results obtained in guideline studies are available. The acute toxicity of alpha-methylstyrene was investigated in a study conducted according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) and EU Method C.2 (Acute Toxicity for Daphnia) under static conditions. Based on the results of a screening-test, the concentrations in the main test were fixed. 20 Daphnia per concentration step were exposed to alpha-methylstyrene at nominal concentrations of 0 (control), 1, 2, 4, 8, and 16 mg/L (aqueous eluates, 'WAF') for 48 h in 100 mL carboys sealed with glass stoppers (no gas phase was present in the test vessels). The 48 h NOEC, LOEC, and EC50 values were determined to be 0.64, 1.21, and 1.645 mg/L effective. The measured concentrations deviated more than 20% from the nominal values. Therefore the reported effect concentrations refer to mean measured concentrations (geometric mean). The test substance proved to be stable during the test duration (recovery: 88.2 -97.1% of initial concentration). The validity criteria of the guideline were fulfilled.

The acute toxicity of alpha-methylstyrene to Daphnia magna was investigated in a study conducted according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). 20 Daphnia magna were exposed to alpha-methylstyrene at nominal concentrations of 0 (control), solvent control, 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L under semi-static conditions for 48 h. The test substance concentrations were determined by HPLC in freshly prepared solutions and in 24 h old test solutions. The geometric mean measured concentrations of freshly prepared test solutions and 24 h old solutions at concentrations of 1-10 mg/L nominal were in the range between 78 and 87% of the initial nominal concentration. A 48 h EC50=2.62 mg/L (geometric mean measured) was determined.

Long-term toxicity to aquatic invertebrates

Reliable results obtained in a guideline study are available. The long-term toxicity of alpha-methylstyrene to Daphnia magna was investigated in a study conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test; cited as OECD Guideline 202 (1984)). 40 Daphnids were exposed to alpha-methylstyrene at nominal concentrations of 0 (control), solvent control, 0.08, 0.2, 0.5, 1.2, and 3.0 mg/L for 21 d under semi-static conditions. The test substance concentrations were monitored via HPLC/UV. The parental mortality (21 d LC50) was determined to be 1.56 mg/L (95% c.i.: 1.02 -2.40 mg/L) based on measured concentrations (TWA during 4 d). The calculation of inhibition of reproduction yielded a 21 d EC50=1.11 mg/L (95% c.i.: 1.03 -1.21 mg/L) and a 21 d NOEC=0.401 mg/L. At the concentrations tested (<=1.2 mg/L nominal) no effect on time of first brood was observed. The 21 d NOEC of 0.401 mg/L will be used for the derivation of PNECaqua (freshwater).

Toxicity to aquatic algae and cyanobacteria

In total, there are 4 study results available to cover this endpoint. However only the most recent one (P&D REACH Consortium, 2010) is considered appropriate for risk assessment purposes. The other studies have been evaluated to be non-reliable based on the described test conditions/results or based on the fact that the validity criteria set forward in the OECD Guideline 201 (K3) were not met. Details on the non-suitability of these studies is provided in the respective RSS.

The toxicity of alpha-methylstyrene to Desmodesmus subspicatus CHODAT was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test; 2006) and EU Method C.3 (Algal Inhibition Test; 2008) under static conditions. In the main test, the algal cultures were exposed to nominal concentrations of 0 (control), 10, 20, 40, 80, and 100 mg/L corresponding to mean measured concentrations of 0 (control), 2.26, 8.30, 10.72, 26.56, and 32.39 mg/L (geometric mean of t=0 h) for 72 h. The test substance concentrations were determined by GC/FID. The recoveries of the test substance concentrations after 72 h were < 80% of those determined at time 0, and the measured concentrations deviated more than 20% from the nominal values. Based on these findings, the reported effect concentrations refer to mean measured concentrations (geometric mean) The 72 h NOEC, LOEC and EC50 based on growth rate were determined to be 2.26, 8.3, and 11.441 mg/L based on mean measured concentrations (geometric mean). The 72 h NOEC, LOEC and EC50 based on yield were determined to be <2.26, <=2.26, and 4.347 mg/L based on mean measured concentrations (geometric mean). The validity criteria of the guideline were fulfilled.

Toxicity to microorganisms

Reliable results obtained in a guideline study are available. The respiration inhibition of alpha-methylstyrene was investigated in a study conducted according to EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test) and OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test). The 3 h EC10, EC20, EC50, and EC100 were determined to be 661.5, 998.7, >2000, and >2000, respectively.