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Ecotoxicological information

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Description of key information

Subtilisin has been tested in aquatic species representing three trophic levels i.e. algae, crustacaens and fish. Both long- and short-term toxicity studies were performed. All studies were conducted according to OECD guidelines and in compliance with GLP.


Acute fish:



  • The 96-hour LC50 for Subtilisin, batch PXI48489 (measured) was 15.6 mg enzyme concentrate dry matter/L (equivalent to 8.2 mg active enzyme protein/L) . ≤10% mortality based on measured 10.9 mg enzyme concentrate dry matter/L (equivalent to 5.7 mg active enzyme protein/L). Conducted under semi-static (daily renewal) exposure conditions.


Long-term fish:



  • The EC50 value of Subtilisin, batch PPA25057, measure on mortality was estimated to be 0.74 mg enzyme concentrate dry matter/L (equivalent to 0.21 mg active enzyme protein/L). The EC10 value was 0.06 mg enzyme concentrate dry matter/L (equivalent to 0.017 mg active enzyme protein/L). NOEC on hatching success in fathead minnows is ≥1.49 mg enzyme concentrate dry matter/L. Mortality, body length and weight and time to 95% hatch appear to be the most sensitive parameters in this early life stage test of the fathead minnow, with the NOEC for Subtilisin, batch PPA 25057 being 0.15 mg enzyme concentrate dry matter/L (measured as/equivalent to 0.426 mg active enzyme protein/L). Conducted under continuous flow conditions.

  • The EC10 and LOEC of Subtilisin, batch PBI48037 for larval mortality was 0.047 mg enzyme concentrate dry matter/L (equivalent to 0.024 mg active enzyme protein/L). The NOEC for larval mortality was estimated to be 0.12 mg enzyme concentrate dry matter/L. The lowest observed effect level (LOEC) for larval mortality of the test substance was nominally 0.37 mg enzyme concentrate dry matter/L, which was estimated to be 0.047 mg enzyme concentrate dry matter/L (measured). LC50 ≥0.092 mg enzyme concentrate dry matter/L (highest concentration). Performed under flow-through conditions.


Acute invertebrates:
The geometrical mean of the EC50 values was 2.56 mg enzyme concentrate dry matter/L and the geometrical mean of the four values in active enzyme protein was 0.331 mg aep/L. 



  • The 48-hour EC50 of Subtilisin, batch PXI48489 for the immobilisation of Daphnia magna was 0.327 mg enzyme concentrate dry matter/L (corresponding to 0.172 mg active enzyme protein/L). The 'no-observed effect concentration' NOEC was 0.071 mg enzyme concentrate dry matter/L. Media was renewed after 24 hours.

  • (Reliability: 2) The nominal 48-hour EC50 of the Subtilisin, batch PPA3980 for the immobilisation of Daphnia magna was 0.90 mg enzyme concentrate dry matter/L, equivalent to 0.092 mg/L. NOEC at 48 h was 0.55 mg enzyme concentrate dry matter/L.

  • (Reliability: 2) The nominal 48-hour EC50 of Subtilisin, batch PPA3352 for the immobilisation of Daphnia magna was nominal 2.4 mg enzyme concentrate dry matter/L (equivalent to to 0.062 mg active enzyme protein/L). NOEC at 24 and 48 hours was 1.7 mg enzyme concentrate dry matter/L.

  • (Reliability: 2) The nominal 48-hour EC50 of the Subtilisin, batch PPA4046 for the immobilisation of Daphnia magna was 60.4 mg enzyme concentrate dry matter/L, corresponding to 12.4 mg active enzyme protein/L. NOEC at 24 and 48h: 30.2 mg enzyme concentrate dry matter/L.


Long-term invertebrates:
The long-term effect of Subtilisin on invertebrates was based on the flow-through test, since it was the most reliable study.



  • (Reliability:2) The time integrated mean value of 7% for Subtilisin, batch PBI48037 was used for the calculation of NOEC. The data processing showed that 21-day NOEC was 0.0016 mg enzyme concentrate dry matter/L. The 21-d EC10 was estimated to be between 0.0016 mg/L and 0.0053 mg enzyme concentrate dry matter/L, corresponding to values 0.79 and 2.7 μg active enzyme protein/L. The 21-day EC50 was estimated to be between 0.022 mg enzyme concentrate dry matter/L and 0.11 mg enzyme concentrate dry matter/L. Media renewal every second day.

  • The 21-day EC50 (reproduction) of Savinase, batch PPA25057 could not be calculated due to low levels of inhibition. 21-d NOEC (reproduction) was 1.14 mg enzyme concentrate dry matter/L (equivalent to 0.324 mg active enzyme protein/L). Test was conducted under flow-through conditions.


Algae:



  • (Reliability 2) Based on the initial measured concentrations, after 72 hours of exposure to Subtilisin, batch PXI48489, the ErC50 was 1.58 mg enzyme concentrate dry matter/L, equivalent to 831 µg active enzyme protein (aep)/L. The 72-h NOEC of the test substance (as initially measured) for the growth rate was 0.605 mg enzyme concentrate dry matter/L, equivalent to 0.317 mg active enzyme protein/L.

  • (Reliability 2) Based on the initial measured concentrations, after 72 hours of exposure to Savinase, batch PBI48037, the ErC50 of 9.77 mg enzyme concentrate dry matter/L corresponding to 5.02 mg active enzyme protein/L and the NOEC was 0.042 mg enzyme concentrate dry matter/L.

  • (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Alcalase, batch PPA 4046, the ErC50 of 339 mg enzyme concentrate dry matter/L corresponding to 69.5 mg active enzyme protein/L and the NOEC was 118 mg enzyme concentrate dry matter/L.

  • (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Esperase, batch PPA 3980, the ErC50 of 76 mg enzyme concentrate dry matter/L corresponding to 7.8 mg active enzyme protein/L and the NOEC was 24 mg enzyme concentrate dry matter/L.

  • (supporting study) Based on the nominal concentrations, after 72 hours of exposure to Savinase, batch PPA 3352, the ErC50 of > 1509 mg enzyme concentrate dry matter/L corresponding to > 39.4 mg active enzyme protein/L and the NOEC was > 1509 mg enzyme concentrate dry matter/L.

Additional information

The most sensitive endpoint has been long-term exsposure of Subtilisin to fish.The EC10 value was 0.06 mg enzyme concentrate dry matter/L (equivalent to 0.017 mg active enzyme protein/L).


For short-term EC50 values, the value from the study with reliability 1 was selected 0.327 mg enzyme concentrate dry matter/L (corresponding to 0.172 mg active enzyme protein/L.