Nuclease, deoxyribo-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2021 to 30 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

yes

Remarks:

There was a decline in oxygen < 3 mg/L. This decline was considered to have had no adverse effect on the outcome or integrity of the test given that it did not, in every test group, coincide with significant immobilization.

GLP compliance:

yes

Remarks:

No claim of compliance is made for the analysis of samples. This exception is considered not to affect the integrity or validity of the study.

Analytical monitoring:

yes

Remarks:

Non-GLP

Details on sampling:

- Concentrations: 361.1, 722.2, 1444.4, 2888.9 and 5777.8 mg enzyme concentrate dry matter/L (nominal).
- Sample storage conditions before analysis: Frozen.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to the test item being weighed out, the test item was gently stirred via a magnetic stir for 5 minutes and inverted several times to ensure homogeneity and to avoid damaging the enzyme. A nominal amount of test item (111 g) was dispersed in the test medium and the volume adjusted to 2.0 L to give the 1000 mg aep/L stock solution. This stock solution was then filtered through a 0.2 μm Sartorius Sartopore filter (first initial 500 mL discarded in order to pre-condition the filter). From the filtered stock solution, a series of dilutions were made to give the required test concentrations of 361.1, 722.2, 1444.4, 2888.9 and 5777.8 mg enzyme concentrate dry matter/L.
The stock dispersion and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration of the test item in the unfiltered stock solution and filtered test preparations were verified by chemical analysis from the freshly prepared preparations (including medium renewal), and the 24 hour old medium.
- Controls: Elendt M4 medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The batch contained precipitates, therefore a filtration process was introduced, as stated above.

Test organisms (species):

Daphnia magna

Details on test organisms:

Common name: Daphnia magna
Strain: Straus
Source: Derived from in-house laboratory cultures.
Age at study initiation: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
- Feeding during test: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L CaCO3

Test temperature:

20-21°C

pH:

7.5-9.0

Dissolved oxygen:

0.1-8.9 mg O2/L.
The oxygen concentration in the freshly prepared solutions (at 0 hours and at 24 hours renewal) was 8.7-9.0 mg/L. This dropped to 2.9-6.0 mg/L after 24 hours, while the control was 7.7 mg/L; and to 0.1-2.1 mg/L at the end of the test, while the control was maintained at 8.9 mg/L. This drop was also noticed for the concentrations in the absence of Daphnia.

Nominal and measured concentrations:

Nominal concentration: 361.1, 722.2, 1444.4, 2888.9 and 5777.8 mg enzyme concentrate dry matter/L.
Measured concentration: 231, 404, 852, 1704 and 3495 mg enzyme concentration dry matter/L (calculated from active enzyme protein).

Details on test conditions:

TEST SYSTEM
- Test vessels: 150 mL glass vessels containing approximately 100 mL of test preparation.
- Experimental design: 5 test concentrations plus 1 control each in duplicate. 20 animals per test concentration. An additional replicate per test concentration was prepared, containing only media (no Daphnia), in order to assess the oxygen concentration over the duration of the study.
- Type: Covered to reduce evaporation.
- Aeration: None.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water – Elendt M4 Medium.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
Method of initiation: Daphnia were placed in the test solutions after addition of the test substance.
Loading: 20 mL test solution per organism.
Photoperiod: 16 h light: 8 h dark with 20 minute dawn and dusk transition periods.
Light intensity: 934 to 950 Lux.
Medium renewal: Yes, after 24 hours
Duration of exposure: 48 hours.
Criterion of effect: Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

EFFECT PARAMETERS MEASURED: Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.

RANGE-FINDING STUDY: No

Reference substance (positive control):

no

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12-month period to demonstrate satisfactory conditions of the test.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1 546 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits = 932 – 2571 mg enzyme concentrate dry matter/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

852 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1 704 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No sub-lethal effects of exposure were observed throughout the test
- Mortality of control: None
- Other adverse effects control: No
- Immobilisation of control: 0%
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The precipitation issue was solved by filtering of the batch.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The positive control was conducted between 05 January 2021 and 29 January 2021. The 48-hr EC50 = 0.50 (0.43-0.58) mg/L.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) at 24 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction incorporating Qualitative Trend Analysis by Contrasts. The LOEC and NOEC at 48 hours were calculated using Step-down Cochran-Armitage Test Procedure incorporating Tarone’s test and Qualitative Trend Analysis by Contrasts. All results were calculated using the ToxRat Professional computer software package (ToxRat® Solutions GMBH).

Validity
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control. However, it was noted that not all the test concentrations achieved this criterion and it was shown to be a test item effect. As immobilization was observed in the 236 and 484 mg aep/L test groups only and the depletion in dissolved oxygen concentration was observed in all test concentrations, it was considered that the drop in dissolved oxygen concentration below 3 mg/L had not affected the results of this study, and therefore the study was deemed to be valid.

Verification of Test Concentrations: 
Analysis of the freshly prepared test solutions showed measured concentrations of the active enzyme protein to range from 58% to 74% of nominal values at 0 hours, and from 49% to 60% of nominal values at 24 hours (freshly prepared). Analysis of the corresponding 24-hour aged preparations showed measured concentrations to range from 61% to 69% of nominal values at 24 hours, and from 52% to 61% of nominal values at 48 hours. The low recovery can be attributed to the presence of precipitation and filtration process.
No claim of GLP compliance is made for the analysis of samples.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on results incl. tables'.

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:
EC50 = 1546 (932 – 2571) mg enzyme concentrate dry matter/L, equivalent to 214 mg active enzyme protein (aep)/L (analytically measured as aep).
NOEC = 852 mg enzyme concentrate dry matter/L.
LOEC = 1704 mg enzyme concentrate dry matter/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

20 daphnids (4 replicates of 5 animals) were exposed to aqueous dispersions of Deoxyribonuclease, batch PPW51643 at nominal concentrations of 361.1, 722.2, 1444.4, 2888.9 and 5777.8 mg enzyme concentrate dry matter/L, respectively, for 48 hours at a temperature of 20 °C to 21 °C under semi-static test conditions with medium renewal after 24 hours. The number of immobilized Daphnia was recorded after 24 and 48 hours.

Analysis of the freshly prepared test solutions showed measured concentrations of the active enzyme protein to range from 58% to 74% of nominal values at 0 hours, and from 49% to 60% of nominal values at 24 hours (freshly prepared). Analysis of the corresponding 24-hour aged preparations showed measured concentrations to range from 61% to 69% of nominal values at 24 hours, and from 52% to 61% of nominal values at 48 hours. Details of the analysis are included in Annex 4 for information only. The low recovery can be attributed to the presence of precipitation and filtration process. No claim of GLP compliance is made for the analysis of samples.
Given that the measured concentrations obtained from the freshly prepared test solutions were not within ±20% of the nominal concentrations, and also the slight variability between the measured concentrations from each media renewal it was considered justifiable to calculate the results based on the geometric mean measured test concentrations.
The geometric mean measured test concentrations were determined to be 32, 56, 118, 236 and 484 mg aep/L; equivalent to 231, 404, 852, 1704 and 3495 mg enzyme concentration dry matter/L.

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Time Point (Hours)		Concentration (mg active enzyme protein/L)	Equivalent Concentration (mg enzyme concentrate dry matter/L)*
48	EC50	214	1546
	95% Confidence Limits	129 – 356	932 – 2571
	NOEC	118	852
	LOEC	236	1704

*Results calculated based on a conversion factor of 7.222 rather than by separate statistical analysis.
NOEC = No Observed Effect Concentration
LOEC  = Lowest Observed Effect Concentration

Description of key information

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:
EC50 = 1546 (932 – 2571) mg enzyme concentrate dry matter/L, equivalent to 214 mg active enzyme protein (aep)/L (analytically measured as aep).
NOEC = 852 mg enzyme concentrate dry matter/L.
LOEC = 1704 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

mg enzyme concentrate dry matter/L

Effect concentration:

1 546 mg/L