Nuclease, deoxyribo-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2021 to 14 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

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: 90.3, 180.6, 361.1, 722.2 and 1444.4 mg enzyme concentrate dry matter/L (nominal).
- Sampling method: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a hemocytometer and light microscope.
- 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 magnetic stir for 5 minutes and inverted several times to ensure homogeneity and to avoid damaging the enzyme.
A nominal amount of test item (56 g) was dispersed in culture medium and the volume adjusted to 2.0 L to give a 500 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 was made to give further stock solutions of 90.3, 180.6, 361.1, 722.2 and 1444.4 mg enzyme concentrate dry matter/L. An aliquot (500 mL) of each of the 90.3, 180.6, 361.1, 722.2 and 1444.4 mg enzyme concentrate dry matter/L stock solutions was separately inoculated with algal suspension (5.5 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock dispersions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration of the test item in the unfiltered stock solution and filtered test preparations (without algae and with centrifuged algae) were verified by chemical analysis at 0 and 72 hours.
- Controls: ASTM medium

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Derived from Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L CaCO3.

Test temperature:

24 ±1 ºC

pH:

7.5-8.9

Salinity:

30 ppt

Nominal and measured concentrations:

Nominal concentrations: 90.3, 180.6, 361.1, 722.2 and 1444.4 mg enzyme concentrate dry matter/L.
Measured concentrations: 5.3, 47, 123, 253 and 448 mg enzyme concentrate dry matter/L (calculated from active enzyme protein).

Details on test conditions:

TEST SYSTEM
- Test vessels: 250 mL glass conical flasks containing 100 ml test solution.
- Type: The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm).
- Aeration: None. Gaseous exchange and suspension of algal cells maintained by orbital shaker.
- Initial cells density: approximately 1*10^3 cells/mL
- Control end cells density: 1.1*10^6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, ASTM

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous (intensity approximately 7000 lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Samples were taken at 23, 45 and 72 hours and the cell densities determined using a hemocytometer and light microscope.

Medium renewal: None.
Duration of exposure: 72 hours.
Measurement of growth: Samples were taken from the control cultures at 23, 45 and 72 hours and the cell densities determined using a hemocytometer and light microscope. Samples of the control and each test concentration to which no algal cells had been added were taken for analysis at 0 hours. A further aliquot of the control and each test concentration was incubated alongside the test from which samples were taken for analysis at 72 hours.

Reference substance (positive control):

yes

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:

72 h

Dose descriptor:

EC50

Effect conc.:

59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits = 26 - 108 mg enzyme concentrate dry matter/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: Effect conc.: <5.3 mg enzyme concentrate dry matter/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: enzyme concentrate dry matter

Basis for effect:

growth rate

Remarks on result:

other: Effect conc.: ≤5.3 mg enzyme concentrate dry matter/L

Details on results:

Nominal cell density of control @ 0 h: 5.00 x 10^3 cells per mL
Mean cell density of control @ 72 h: 1.10 x 10^6 cells per mL

- Exponential growth in the control: yes
- Observation of abnormalities: Extreme clumping of cells was observed in the 5.3, 47 and 123 mg enzyme concentrate dry matter/L test cultures. Enlarged cells were observed to be present in the 253 and 448 mg enzyme concentrate dry matter/L test cultures.
- Adherence to test vessels: The 123 mg aep/L test cultures were observed to have adherence to the flask walls above the media surface and the 253 and 448 mg aep/L test cultures were observed to have adherence to the flask walls above the media surface.
- Aggregation of algal cells: All test and control cultures were inspected at 72 hours.

Results with reference substance (positive control):

The positive control was conducted between 21 December 2020 and 24 December 2020 and gave the following results:

ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s test on Variance Homogeneity and Shapiro-Wilks’ test on Normal Distribution.

Verification of Test Concentrations:

Analysis of the test preparations to which algal cells had been added showed measured concentrations, following centrifugation, to range from 27% to 32% of nominal values at 0 hours. Analysis of the corresponding preparations at 72 hours showed measured concentrations to range from less than the LOD to 40% of nominal. Details of the analysis are included in Annex 4 for information only. No claim of GLP compliance is made for the analysis of samples.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:
EC50 = 59 (26 – 108) mg enzyme concentrate dry matter/L, equivalent to 8.1 mg active enzyme protein (aep)/L (analytically measured as aep).
NOEC = <5.3 mg enzyme concentrate dry matter/L.
LOEC = ≤5.3 mg enzyme concentrate dry matter/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

Raphidocelis subcapitata was exposed to aqueous dispersions of Deoxyribonuclease, batch PPW51643 at nominal concentrations of 90.3, 180.6, 361.1, 722.2 and 1444.4 mg enzyme concentrate dry matter/L, respectively (three replicate flasks per concentration) over 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a hemocytometer and light microscope.

Analysis of the test solutions at 0 and 72 hours was performed. Samples were prepared in the presence and absence of algae to investigate whether any loss of test item occurred due to either adsorption to algae or through the process of centrifugation.
Analysis of the test preparations to which algal cells had been added showed measured concentrations, following centrifugation, to range from 27% to 32% of nominal values at 0 hours. Analysis of the corresponding preparations at 72 hours showed measured concentrations to range from less than the LOD, to 40% of nominal. Details of the analysis are included in Annex 4 for information only. No claim of GLP compliance is made for the analysis of samples.
Analysis of the test preparations at 0 hours, to which no algal cells had been added showed measured concentrations of the active enzyme protein to range from less than the limit of detection (LOD) of the analytical method employed (0.316 mg aep/L), to 32% of nominal values. Analysis of the corresponding preparations at 72 hours showed measured concentrations to range from 30% to 33% of nominal values. Despite the low recovery, these results indicated that the test item was stable under the conditions of the test.
It was considered appropriate to calculate results based on the measured concentrations obtained from the test preparations which contained algal cells and had been centrifuged prior to analysis in order to give an estimate of the dissolved and hence bioavailable fraction concentrations to which the algae had been exposed.
Given that the measured concentrations obtained at 0 hours were not within ±20% of the nominal concentrations, and also the slight variability between the measured concentrations obtained at 0 and 72 hours, 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 0.73, 6.5, 17, 35 and 62 mg aep/L; equivalent to 5.3, 47, 123, 253 and 448 mg enzyme concentrate dry matter/L, respectively.
Exposure of Raphidocelis subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable		Concentration (mg active enzyme protein/L)	Equivalent Concentration (mg enzyme concentrate dry matter/L)†
Growth Rate	EC50	8.1	59
	95% Confidence Limits	3.6 - 15	26 - 108
	No Observed Effect Concentration (NOEC)	<0.73	<5.3
	Lowest Observed Effect Concentration (LOEC)	≤0.73	≤5.3
Yield	EC50	Not determined*	Not determined*
	95% Confidence Limits	Not determined	Not determined
	No Observed Effect Concentration (NOEC)	<0.73	<5.3
	Lowest Observed Effect Concentration (LOEC)	≤0.73	≤5.3

† Results calculated based on a conversion factor of 7.222 rather than by separate statistical analysis.
* It was not possible to determine an EyC50 value as at the lowest test concentration employed, 85% inhibition of yield occurred.

Description of key information

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:
EC50 = 59 (26 – 108) mg enzyme concentrate dry matter/L, equivalent to 8.1 mg active enzyme protein (aep)/L (analytically measured as aep).
NOEC = <5.3 mg enzyme concentrate dry matter/L.
LOEC = ≤5.3 mg enzyme concentrate dry matter/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

59 mg/L