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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:

2021-01-11 - 2021-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP, and designed according to OECD GD 23.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

March 2006 (corrected July 2011)

Qualifier:

according to guideline

Guideline:

other: OECD Series on testing and assessment, No.23 “Guidance document on aqueous phase aquatic toxicity testing of difficult test chemicals”,

Version / remarks:

February 2019

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Remarks:

2021-03-17

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.5, 2.4, 3.9, 6.2, and 10.0 mg.L-1
- Sampling method: Destructive sampling - additional replicates of each treatment with algae were prepared for sampling. Samples taken at the start and the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The amount of test item required was weighed and added to the mixing vessel which was filled to the brim to avoid a headspace. The solution was mixed for 112h in the dark and then left undisturbed for 1 hour before use. The first 100 mL drained were discarded. Samples from the stock solution were collected for analytical determination of the concentration. All treatments were checked for the Tyndall effect and none was observed.
- Test concentration separation factor: 1.61
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 -75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes
- Age of inoculum (at test initiation):
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (= test
water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Same as test (Medium as outlined in OECD TG 201)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.8 - 24.5 °C

pH:

Start: 8.28 - 8.53
End: 8.80 - 9.86

Nominal and measured concentrations:

Nominal: 0, 1.5, 2.4, 3.9, 6.2, and 10.0 mg.L-1
Measured (geometric mean): 0, 1.34, 2.14, 3.33, 5.83, 8.74 mg.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass closed flasks with ground glass stopper
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: minimum headspace, filled to the rim
- Aeration: No
- Initial cells density: 5x10^3 cells.mL-1
- Control end cells density: 82.1 x 10^4 cells.mL-1
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201)
- Detailed composition if non-standard medium was used: Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 were added to the sterilized water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 5062 lux (range 4845-5491 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.61
- Range finding study
Test concentrations: 0.32, 1.00, 3.20, 10.00 and 13.00 mg test item.L-1
Results used to determine the conditions for the definitive study: Inhibition of growth rate: 1.1% at 0.32 mg.L-1, 4.1% at 1.00 mg.L-1, 10.6% at 3.20 mg.L-1, 92.0% at 10.0 mg.L-1 and 114.7% at 13.00 mg.L-1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.166 mg/L

95% CI:

7.517 - 9.155

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Since the deviation of the exposure concentrations of the test item was greater than 20% of the initial concentrations, the evaluation of the effects was based on the geometric means of the analytically confirmed concentrations.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.29 mg/L

95% CI:

3.161 - 5.012

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Since the deviation of the exposure concentrations of the test item was greater than 20% of the initial concentrations, the evaluation of the effects was based on the geometric means of the analytically confirmed concentrations.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.934 mg/L

95% CI:

3.516 - 4.41

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Remarks on result:

other: Since the deviation of the exposure concentrations of the test item was greater than 20% of the initial concentrations, the evaluation of the effects was based on the geometric means of the analytically confirmed concentrations.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.645 mg/L

95% CI:

1.236 - 1.988

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Remarks on result:

other: Since the deviation of the exposure concentrations of the test item was greater than 20% of the initial concentrations, the evaluation of the effects was based on the geometric means of the analytically confirmed concentrations.

Details on results:

- Exponential growth in the control (for algal test): yes - 164.1 fold increase within 72h
- Observation of abnormalities (for algal test): no
- Effect concentrations exceeding solubility of substance in test medium: no

The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 18.3% in 72 hours.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.5%.

Results with reference substance (positive control):

72h ErC50 0.759 mg.L-1
Within the range proposed in the ISO 8692 method (0.65 - 1.73 mg.L-1). No range is provided in the OECD method.

Table 1. Mean specific growth rate in P. subcapitata during the final test

	Replicate	Concentration (geometric mean; mg.L-1)
		Control (0)	1.34	2.14	3.33	5.83	8.74
0-24h	1	1.974	2.262	1.723	1.163	0.875	1.163
	2	1.856	1.974	1.974	1.723	1.163	0.470
	3	1.856	1.569	1.856	1.723	0.470	0.875
	4	2.175	-	-	-	-	-
	5	1.723	-	-	-	-	-
	6	1.974	-	-	-	-	-
	Mean	1.926	1.935	1.851	1.536	0.836	0.836
	Standard deviation	0.1533	0.3482	0.1257	0.3231	0.3482	0.3482
	% Inhibition	-	-0.4	3.9	20.3	56.6	56.6
0-48h	1	1.892	1.757	1.901	1.666	1.242	0.582
	2	1.814	1.892	1.814	1.707	1.334	0.693
	3	1.892	1.901	1.757	1.707	1.305	0.861
	4	1.936	-	-	-	-	-
	5	1.781	-	-	-	-	-
	6	1.883	-	-	-	-	-
	Mean	1.866	1.850	1.824	1.694	1.294	0.712
	Standard deviation	0.0575	0.0806	0.0725	0.0237	0.0466	0.1409
	% Inhibition	-	0.9	2.3	9.2	30.7	61.8
0-72h	1	1.745	1.720	1.671	1.492	1.447	0.870
	2	1.757	1.678	1.641	1.498	1.290	0.523
	3	1.685	1.645	1.619	1.507	1.342	0.754
	4	1.673	-	-	-	-	-
	5	1.675	-	-	-	-	-
	6	1.653	-	-	-	-	-
	Mean	1.698	1.681	1.644	1.499	1.360	0.716
	Standard deviation	0.0426	0.0377	0.0529	0.0075	0.0799	0.1767
	% Inhibition	-	1	3.2	11.7	19.9	57.9

 

Table 2. Concentrations of the test item in test water in the final test

Nominal concentration (mg.L-1)	Measured concentration (mg.L-1)		Relative loss to initial value (%)	Geometric mean analytically confirmed concentrations
	Start (0h)	End (72h)		mg.L-1	%Nominal
Control	<LOD	<LOD	N/A	N/A	N/A
1.5	1.55	1.15	26.00	1.34	89
2.4	2.56	1.79	30.00	2.14	89
3.9	3.82	2.90	24.00	3.33	85
6.2	6.66	5.11	23.00	5.83	94
10.0	9.48	8.06	15.00	8.74	87

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon the geometric mean of the analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 8.166 mg.L-1 and 3.934 mg.L-1, respectively. The 72-hour EC10 for growth rate was 4.290 mg.L-1 and for yield was 1.645 mg.L-1. The 72-hour NOEC for growth rate was 2.140 mg.L-1 and for yield was 1.340 mg.L-1.

Executive summary:

The toxicity of the test item to Green algae (Pseudokirchneriella subcapitata) was investigated in a study conducted according to the OECD TG 201 (2006, corrected 2011) and in compliance with GLP. The design further included the recommendations of the OECD GD 23 (testing difficult substances).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 1.5, 2.4, 3.9, 6.2, and 10.0 mg.L-1. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

Specific analyses revealed slight losses of test item, mainly at the lowest concentrations. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than 20% of the initial concentrations between the start and the end of the test, the results were based on the geometric means of the analytically confirmed concentrations.

Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 8.166 mg.L-1 and 3.934 mg.L-1, respectively. The 72-hour EC10 for growth rate was 4.290 mg.L-1 and for yield was 1.645 mg.L-1. The 72-hour NOEC for growth rate was 2.140 mg.L-1 and for yield was 1.340 mg.L-1.

Parameter	Growth rate in mg.L-1	Yield in mg.L-1
	(and 95% conf. limits)	(and 95% conf. limits)
72-hour EC10	4.290 (3.161 – 5.012)	1.645 (1.236 – 1.988)
72-hour EC20	5.351 (4.398 – 5.965)	2.219 (1.802 – 2.567)
72-hour EC50	8.166 (7.517 – 9.155)	3.934 (3.516 – 4.410)
72-hour LOEC	3.33	2.14
72-hour NOEC	2.14	1.34

Description of key information

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon the geometric mean of the analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 8.166 mg.L-1 and 3.934 mg.L-1, respectively. The 72-hour EC10 for growth rate was 4.290 mg.L-1 and for yield was 1.645 mg.L-1. The 72-hour NOEC for growth rate was 2.140 mg.L-1 and for yield was 1.340 mg.L-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.166 mg/L

EC10 or NOEC for freshwater algae:

4.29 mg/L

Additional information

The toxicity of the test item to Green algae (Pseudokirchneriella subcapitata) was investigated in a study conducted according to the OECD TG 201 (2006, corrected 2011) and in compliance with GLP. The design further included the recommendations of the OECD GD 23 (testing difficult substances).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solution of the test item over 72 hours at the required nominal test concentrations 1.5, 2.4, 3.9, 6.2, and 10.0 mg.L-1. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

Specific analyses revealed slight losses of test item, mainly at the lowest concentrations. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than 20% of the initial concentrations between the start and the end of the test, the results were based on the geometric means of the analytically confirmed concentrations.

Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters growth rate and yield were determined to be 8.166 mg.L-1 and 3.934 mg.L-1, respectively. The 72-hour EC10 for growth rate was 4.290 mg.L-1 and for yield was 1.645 mg.L-1. The 72-hour NOEC for growth rate was 2.140 mg.L-1 and for yield was 1.340 mg.L-1.