glutamyl endopeptidase

Ecotoxicological Summary

Hazard for aquatic organisms

Freshwater

Hazard assessment conclusion:

PNEC aqua (freshwater)

PNEC value:

0.17 µg/L

Assessment factor:

1 000

Extrapolation method:

assessment factor

PNEC freshwater (intermittent releases):

1.7 µg/L

Marine water

Hazard assessment conclusion:

PNEC aqua (marine water)

PNEC value:

0.017 µg/L

Assessment factor:

10 000

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

The aquatic toxicity data were obtained from short term toxicity studies in species representing three trophic levels (i.e. algae, crustacean and fish).

Fish: Glutamyl endopeptidase has not been tested in fish, however, a similar enzyme - subtilisin belonging to the same enzyme sub-subclass - has been tested in rainbow trout for 96 hours. The 96-hour LC50 value for subtilisin was nominally 17.7 mg TOS/L, with 95% confidence limits of 13.3 and 23.5 mg TOS/L. Based on the initial measured levels, the 96-hour LC50 was 14.6 mg TOS/L (95% confidence limits, 10.6 and 19.9 mg TOS/L) corresponding to 8.2 mg aep/L. The no-observed-effect concentration (NOEC) was not clearly identified during the definitive test but based on the findings of a range finding test, the NOEC was 10 mg TOS/L (nominal) corresponding to 4.6 mg aep/L. Due to the similarity between the two enzymes, similar results are expected for glutamyl endopeptidase.

Daphnia: Glutamyl endopeptidase was tested in Daphnia magna in a supporting study. The test revealed an 48h EC50 value of 5 mg TOS/L. The NOEC (48h) was determined to be 4.6 mg/L.

Another similar proteolyticenzyme – subtilisin belonging to the same enzyme sub-subclass– was tested using Daphnia magna. The EC value was 0.306 mg TOS/L (95% confidence limits, 0.23 and 0.429 mg TOS/L), and NOEC was 0.066 mg TOS/L (measured concentrations).

Algae: Glutamyl endopeptidase batch PPA 27269 was tested with Pseudokirchneriella subcapitata. It inhibited algal growth at 10 mg TOS/L, whereas no inhibitory effect was detected at lower concentrations. The inhibitory effect at 10 mg TOS/L was below 50% and thus no ErC values could be determined. The NOEC was determined to be 1 mg TOS/L.

Another similar enzyme - subtilisin – was tested with Pseudokirchneriella subcapitata. The 72-hour ErC50 was 1.48 mg TOS/L corresponding to 830 µg aep/L. The NOEC was 0.566 mg TOS/L.

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.

The EC50 for a similar enzyme to glutamyl endopeptidase, subtilisin, was 0.86 mg active enzyme protein/L was used for PNEC derivation and the assessment factors 1000 and 10000 were applied for fresh and marine water, respectively.

The PNEC value for STP is based on actual measurements of enzyme concentration in STP connected to manufacturing site. Up to 65000 µg aep/L 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 the enzyme 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 enzyme is thus not expected.

As no soil ecotoxicity data are available for the test enzyme, the PNEC for soil is based on the PNEC for surface water using the equilibrium partitioning method. PNEC soil was estimated to 568 µg active enzyme protein/kg soil dw.

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

Conclusion on classification

Based on the available data glutamyl endopeptidase is classified as acute aquatic toxic.