Subtilisin

Ecotoxicological Summary

Hazard for aquatic organisms

Freshwater

Hazard assessment conclusion:

PNEC aqua (freshwater)

PNEC value:

1.7 µg/L

Assessment factor:

10

Extrapolation method:

assessment factor

PNEC freshwater (intermittent releases):

0.9 µg/L

Marine water

Hazard assessment conclusion:

PNEC aqua (marine water)

PNEC value:

0.17 µg/L

Assessment factor:

100

Extrapolation method:

assessment factor

STP

Hazard assessment conclusion:

PNEC STP

PNEC value:

65 000 µg/L

Assessment factor:

1

Extrapolation method:

assessment factor

Sediment (freshwater)

Hazard assessment conclusion:

no exposure of sediment expected

Sediment (marine water)

Hazard assessment conclusion:

no exposure of sediment expected

Hazard for air

Air

Hazard assessment conclusion:

no hazard identified

Hazard for terrestrial organisms

Soil

Hazard assessment conclusion:

PNEC soil

PNEC value:

568 µg/kg soil dw

Assessment factor:

1 000

Extrapolation method:

assessment factor

Hazard for predators

Secondary poisoning

Hazard assessment conclusion:

no potential for bioaccumulation

Additional information

Aquatic toxicity studies with subtilisin revealed that the species most sensitive to the test substance was the invertebrate Daphnia magna for acute effects and fish for chronic effects. Results from the acute toxicity to Daphnia magna are thus used for estimation of PNEC (intermittent release) and from the long-term study in fish to derive PNEC (water).

The PNEC value for STP is based on actual measurements of subtilisin levels in STP connected to manufacturing site. Up to 65000 µg active enzyme protein (aep) were detected in STP connected to manufacturing site and since there was no negative impact observed, this concentration is the estimated PNEC value for STP.

PNEC values for sediment exposure have not been derived because subtilisin is readily biodegradable, highly water soluble and has a very low potential for adsorption to sediments. Exposure of the sediment to toxicologically significant concentrations of the substance is thus not expected.

The PNEC value for soil has been derived from the acute toxicity to earthworm study (PNEC soil).

Subtilisin is not expected to cause any significant secondary poisoning as it is ready biodegradable and has no bioaccumulation potential. Furthermore, as subtilisin is a protein it is expected to be degraded in the gastrointestinal tract. Thus PNEC oral is thus not relevant.

Inhibition control carried out with non-proteolytic enzymes in the test of ready biodegradability showed no inhibition of the activated sludge inoculum at an enzyme concentration above the expected levels in inlet to sewage treatment plants (STPs). Monitoring of enzymes in the inlet to municipal STPs (in Denmark) resulted in concentrations of less than 2 µg active enzyme protein (aep)/L which are below the initial concentration used in tests for ready biodegradability, where no inhibitory effects were observed. It is concluded that a study on activated sludge respiration inhibition does not need to be conducted. Therefore, the test enzyme is not considered to be toxic to microorganisms.

Conclusion on classification

Based on the aquatic toxicity data subtilisin is classified as acute toxic to aquatic organism category 1 and chronic toxicity to aquatic organisms category 2.

Classification is based on active enzyme protein content because the toxicity of subtilisin is caused by its catalytic activity.

Acute aquatic classification is based on the results from aquatic acute toxicity studies to Daphnia magna, which was observed to be the most sensitive species towards acute effects.

The EC50 values for acute toxicity to Daphnia magna ranged between 90 and 12400 µg active enzyme protein (aep)/L with a geometric mean of 586 µg aep/L. Based on these results subtilisin is classified as acute toxic to aquatic organism category 1. The effect of long-term aquatic exposure to subtilisin has been assessed in the early life stage test (ELS), daphnia reproduction assay and in algae. Fish were found to be the most sensitive species. In total two ELS have been performed in flow through systems in order to keep the exposure concentrations constant. Although both tests were performed in flow-through systems, exposure concentration could only be kept constant in one of the two studies. Subtilisin is unstable in aquatic media and it is thus crucial when performing a longterm aquatic toxicity study with subtilisin to optimize and adjust the test design in order to avoid degradation/inactivation during exposure. In addition, samples must be handled very carefully in order to avoid degradation/inactivation of enzyme protein during storage and transport. The selected key study was designed taking the special properties of subtilisin in consideration in order to keep exposure concentrations constant. Not only was the flow velocity optimized, but also preparation of the test media. In addition, exposure concentration analysis were performed immediately after sample collection in order to avoid any loose of activity during storage. This resulted in constant exposure concentrations making it possible to calculate actual effect levels. The results from this study are thus used for classification of subtilisin. Based on the derived EC10 value of 0.017 mg AEP/L from the selected key study in fish subtilisin is classified as aquatic chronic 2.