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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:

08-06-2020 to 06-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016 ; signature: January 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours). A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS/MS from freshly prepared media after 0 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at room temperature (ca. 20 ± 2 °C) before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item exposures was prepared from stock solution. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland, United Kingdom, PA37 1QA
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: OECD TG 201: (mg/L) - NH4Cl 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with the OECD TG 201 guideline.

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 21.5 ; max 23.5 and mean 22.5 °C

pH:

0 hours: pH 8.1 ± 0.1 (and 8.22 in control); 72 hours: pH 9.21-9.42 (definitive test concentrations) and pH 9.46 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

- Preliminary test: Not applicable. See below
- First definitive test: 0 (control), and 100% of the saturated solution ; the results in the first definitive limit test showed a higher inhibition as expected. Therefore, the study had to be repeated as dose response test (Second definitive test)
- Second definitive test (dose-response test) : 0 (control), 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution
- Equivalent geometric mean measured concentrations (with algae): 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Measured concentrations of the test item were in the samples with algae in the range of 0.0322 to 0.897 µg/L at the start of exposure (0 h) and in the range of: < LOQ (Limit of Quantification) to 0.0545 µg/L (and/or 0 to 6% and 20% of the initial test item concentrations) at the end of exposure (72 h). All effect values given were based on geometric mean measured concentrations after 72 hours for samples.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 5375 cells/mL
- Control end cells density: First definitive (limit test): Mean (of replicates after 72 hours) 354000 cells/ml (or ca. x66 increase in cell density) ; Second definitive (dose-response test): Mean (of replicates after 72 hours) 390680 cells/ml (or ca. x73 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. OECD TG 201 medium. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition). Ultrapure water was used to prepare the dilution water (conductivity max. = 0.1 μS/cm)
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area (or 4440 to 8880 lux)
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the highest preliminary test concentration level of 100% saturated solution.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 In second definitive test justified from the results of the first definitive test (limit test : 0 (control), and 100% of the saturated solution) ; the results in the first definitive limit test showed a higher inhibition as expected. Therefore, the study had to be repeated as dose response test (Second definitive test).
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: No, however first definitive limit test could be considered a range-finder.
- Test concentrations: limit test : 0 (control), and 100% of the saturated solution
- Results used to determine the conditions for the definitive study: Yes. Second definitive test (dose-response test) : 0 (control), 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration

Remarks:

i.e. ErC50 is above the solubility limit

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration

Remarks:

i.e. ErC10 is above the solubility limit

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: or 100 mg/L nominal concentration

Remarks:

i.e. no effects were observed below the solubility limit (set as the NOEC)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: or 100 mg/L nominal concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration

Remarks:

i.e. EyC50 is above the solubility limit

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: > 0.0597 - < 0.221 µg/L ; based on geometric mean measured concentrations ; 72.4 (C.I. > 50 - < 100) mg/L nominal concentration

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.06 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: or 50 mg/L nominal concentration

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.221 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: or 100 mg/L nominal concentration

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: None reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitate or similar observations reported. At the start of the exposure the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 0.0545 μg/L. The analytical values indicate that the sampling points were not completely homogeneous, which can be caused by the low water solubility of the test item, which is in accordance with the results of related studies (Water Solubility and/or Acute Daphnia Toxicity – cited in the full study report). The analysis of additional replicates (not shown) confirmed the inhomogeneous results but also the tendency of declining analytical values at higher diluted test item solutions. Therefore, the values of replicate 1 were used for the evaluation.
- Effect concentrations exceeding solubility of substance in test medium: Yes. See above and below.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.12 mg/L with a 95% confidence interval ranging from 1.08 to 1.15 mg/L with headspace and 0.949 mg/L with a 95% confidence interval ranging from 0.901 to 0.995 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.594 mg/L with a 95% confidence interval ranging from 0.518 to 0.664mg/L with headspace and 0.551 mg/L with a 95% confidence interval ranging from 0.472 to 0.604 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using: As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Table 1. Results of the First Definitive (Limit) Test (0 - 72 hours)

Saturated solution [%]	Nominal test item loading rate [mg/L]	Geometric mean measured test item concentration [µg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	100	6.92	28	70
Control	0	< LOQ	-	-

All biological observations are documented in the full study report and all validity criteria were considered to be met.

The results in the first definitive limit test showed an inhibition, therefore requiring a repetition as a dose response test.

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the Second Definitive (dose response) Test (at 72 hours)

Concentration based on Saturated Solution	Nominal test item loading rate	Geometric mean measured test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Yield Inhibition
[%]	[mg/L]	[µg/L]	No.	[d-1]		[%]	[d-1]		[%]
100.0	100.0	0.221	1		1.38	3		333621	13
			2		1.39	2		345472	10
			3		1.36	5		308502	20
			Mean	(+)*	1.38	4	(+)	329198	15
50.0	50.0	0.0597	1		1.39	19		343175	11
			2		1.39	20		344030	11
			3		1.42	18		376871	2
			Mean	(-)	1.40	19	(-)	354692	8
25.0	25.0	0.0481	1		1.41	5		364995	5
			2		1.40	6		354097	8
			3		1.39	8		342662	11
			Mean	(-)	1.40	6	(-)	353918	8
12.5	12.5	0.0431	1		1.41	2		359180	7
			2		1.40	1		357298	7
			3		1.40	1		352411	9
			Mean	(-)	1.10	1	(-)	356296	8
6.25	6.25	0.0179	1		1.39	2		342833	11
			2		1.42	2		373254	3
			3		1.41	3		360573	6
			Mean	(-)	1.41	2	(-)	358887	7
0% (Control)	0 (Control)	0 (Control)	1		1.42			369662
			2		1.42			370567
			3		1.42			374501
			4		1.44			404385
			5		1.41			359107
			6		1.47			433609
			Mean		1.43			385305

Negative inhibition = increase in growth

n.a. = data not determinable

Statistically significant differences of growth rates and yield compared to control values are marked (+) and non-statistically significant are marked (-)

* = inhibition < 5%, therefore considered not biologically relevant

 

Table 3. Measured Concentration of test item in the Second Definitive (dose response) Test with Algae

Sampling	Fresh Media, 0 hours	Old Media, 72 hours		Geometric mean measured test item concentration
Saturated test item solution [%]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]
100	0.897	0.0545	6	0.221
50	0.134	0.0266	20	0.0597
25	0.231	< LOQ	-	0.0481
12.5	0.186	< LOQ	-	0.0431
6.25	0.0322	< LOQ	-	0.0179
Control	< LOQ	< LOQ
PC	75% / 72% 83% / 80%*	86% / 83% (reanalysed values)

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percent of the initially measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.02 µg/L of the test item).

For Geometric mean measured test item concentration, 1/2 LOQ was used for the calculation.

PC = Procedural recovery, % recovery calculated to: weighed amount of the test item / mean value of the method validation.
* Value for 50% saturated solution, since it had to be separately reanalysed

 

Table 4. Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours) in the Second Definitive (dose response) Test

	Replicate No.	Specific growth rate [d-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.49	1.63	1.13	1.42	0.256	18.1
	2	1.47	1.65	1.13	1.42	0.267	18.9
	3	1.52	1.68	1.06	1.42	0.321	22.6
	4	1.60	1.49	1.24	1.45	0.183	12.7
	5	1.58	1.63	1.01	1.41	0.342	24.3
	6	1.54	1.58	1.28	1.47	0.165	11.3	17.9
				Mean	1.43
				SD ±	0.02
				CV [%]	1.64

SD = Standard deviation

CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. No effects on growth rate were seen below the limit of solubility.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask.The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. In the definitive, dose-response test: five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS/MS at test start and the end of exposure. At the start of the exposure, the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ (0.02 μg/L) to 0.0545 μg/L. Effect concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyC50 (yield) was > > 0.221 µg/L based on geometric mean measured concentrations. The corresponding EyC10 was 0.0998 (C.I. > 0.0597- < 0.221) µg/L. The 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. Under the conditions of this study, no effects on growth rate were seen below the limit of solubility.

Description of key information

ErC50 (algae; growth rate) = > 0.221 µg/L based on geometric mean measured concentrations, or > 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020

ErC10 (algae; growth rate) = > 0.221 µg/L based on geometric mean measured concentrations, or > 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020

NOEC (algae; growth rate) = 0.221 µg/L based on geometric mean measured concentrations, or 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020

 

Conclusion : The substance has apparent lower solubility in algal medium OECD TG 201 (2011), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the exposure matrix and that of pure water. The conclusion of the experimental study was that no effects were observed on algal growth rate at up to 100% saturation, with geometric mean measured concentration of 0.221 µg/L. Consequently, it can be concluded the test item has no effects on growth rate below the water solubility limit, observed experimentally.

Additional information

Key study : OECD TG 202, 2020 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask.The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. In the definitive, dose-response test: five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS/MS at test start and the end of exposure. At the start of the exposure, the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ (0.02 μg/L) to 0.0545 μg/L. Effect concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyC50 (yield) was > > 0.221 µg/L based on geometric mean measured concentrations. The corresponding EyC10 was 0.0998 (C.I. > 0.0597- < 0.221) µg/L. The 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. Under the conditions of this study, no effects on growth rate were seen below the limit of solubility.