2-methylpentyl 2-hydroxybenzoate

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:

02-07-2020 to 27-07-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. For the definitive test: equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/L which were based on analysis during the definitive test period.
Singular samples for possible analysis were taken from all test concentrations and the control; compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel containing 10% of the SS prepared at a loading rate of 100 mg/L, but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule: t=0h, t=24h and t=72h. 2 mL volume samples were extracted; the replicates were not pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer (≤ -15°C) until analysis.
- Other: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. For the definitive test: equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/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. Undissolved test item was excluded on the basis of application of the soluble fraction of a pure test item obtained after removal of the undissolved fraction (e.g. by filtration, settlement, centrifugation or other removal steps). In this case siphoning through glass wool.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium 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 21-24°C.

ACCLIMATION
- Acclimation period: 3 days pre-culture.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 7.1 ± 0.3
- 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.

Hardness:

Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 1°C).

pH:

0 hours: pH 7.3 ± 0.1 ; 72 hours: pH 7.4 - 8.1 (definitive test concentrations) and pH 8.1 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Range-finder test: 0 (control), 0.10, 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L. The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report).
Final test: Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. For the definitive test: equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/L which were based on analysis during the definitive test period.
See table 3 for %SS of 100 mg/L loading rate and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 40 mL, containing ca. 40 mL test solution (minimal headspace to prevent test item losses); closed airtight (capped)
- Aeration: Vessel shaken continuously (ca. 175 rpm)
- Initial cells density: 1 x 10^4 cells/mL.
- Control end cells density: Mean (of replicates after 72 hours) 295.418 x10^4 cells/mL (factor x295 increase)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration (1 extra replicate for sampling purposes, after 24 hours exposure).
- No. of vessels per control (replicates): 6 replicates of the control ; 1 or 2 replicates of each test concentration without algae (abiotic control)
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); pH 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported. Media pH was recorded at t=0 and t=72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 to 120 µE/m2/s (in 400 to 700 nm range), typically.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 680 nm with immersion probe (pathlength = 10 mm or 20 mm) or cuvettes (pathlength = 10 mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Range-finder test: 0 (control), 0.10, 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L.
Final test: Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. For the definitive test: equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/L which were based on analysis during the definitive test period.
- Results used to determine the conditions for the definitive study: Yes.
- Other: The preparation procedure for test solutions was based on a non-GLP saturation test and range-finder (cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.93 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.81 - 1.1 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.65 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.58 - 0.73 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.37 - 0.56 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.22 - 0.28 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC based on statistical significance

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells at the concentration closest to the EC50 when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other: (i) At the end of the test, a small response was measured in the sample taken from the control. Considering that the response was detected only at the end of the test, it is considered that this might derive from a contamination during sampling or sample preparation ; (ii) At 24 hours of exposure, the measured concentrations were generally at 53 to 111% relative to the initial concentrations, except for the concentration measured in the sample taken from the 10% SS test group, which was at 222% relative to the initial concentration. A repeated measurement after re-dilution confirmed this result. The reason for this could not be identified. Considering that the concentration measured at the end of the test was comparable to the concentration measured in the sample without algae, and that the TWA concentrations for both groups, i.e. 10% SS with and without algae, were approximately 0.2 mg/L this was accepted and taken into account during the further analysis of the results. That is, following a worst-case approach, it was decided to include the measurement result in the determination of the TWA concentration. Consequently, all replicates were considered for inclusion in the statistical analysis and no data was disregarded
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.015 and 0.041 mg/L TWA growth inhibition was negative between 0-24h. This was low dose stimulation and was limited to < 8.0 % and was taken into account in the data analysis and effect level calculations. For higher doses negative inhibition% was seen at 0.22 and 0.42 mg/L TWA at 48-72h. This was limited to < or = 24.0 % and was taken into account in the data analysis and effect level calculations. This too could be considered low dose stimulation, since at the end of the test, the concentrations had decreased to 12-30% of the initial concentrations within these replicates specifically.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from initial values. See above for further information. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.020, 0.070, 0.21, 0.74 and 3.3 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. At the end of the test, the concentrations had decreased to 12-57% of the initial concentrations. Additionally, the concentration measured in the solution without algae decreased to 31% relative to the initial value at the end of the test, while the concentration in the solution with algae was at 30% of the initial concentration at the end of the test. This indicates that the presence of algae does not appear to influence the actual exposure concentrations. No further observations were made which may account for the differences.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was within the expected range. The reference test was conducted in a separate study (cited in the full study report) under GLP.
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (The Shapiro-Wilk´s Test on Normal Distribution and Levene´s Test on Variance Homogeneity (with Residuals) were performed. For the effect concentrations including NOEC: Step-down Jonckheere- Terpstra test, α=0.05, one-sided, smaller). Additionally, calculation of ECx-values was based on probit analysis using linear max. likelihood (growth rate) and linear weighted (yield) regression with the percentages of growth rate inhibition and
the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item.

ToxRat Professional v 3.2.1 was the software package utilised.

See 'Any other information on results incl. tables' for further information.

Table 1. Mean cell densities (x10^4 cells/mL) during the range-finding test

Time (h)	Test item, % SS prep. At 100 mg/L
	Control	0.10	1.0	10.0	100
0	1.0	1.0	1.0	1.0	1.0
72	259	239	261	206	3.0

 

Table 2. Percentage inhibition of growth rate and inhibition of yield during the range-finding test

	Growth rate (0 – 72h)				Yield (0 – 72h)
Test item, % SS prep. At 100 mg/L	mean	Std. Dev.	n	% inhibition	mean	Std. Dev.	n	% inhibition
Control	1.852	0.0096	3	-	258.163	7.4129	3	-
0.10	1.825	0.0224		1.5	238.150	15.8749		7.8
1.0	1.854	0.0232	3	-0.10	260.013	17.8363	3	-0.72
10.0	1.771	0.0698	3	4.4	204.744	40.2552	3	21
100.0	0.307	0.2689	3	83	2.010	1.7980	3	99

 

Table 3. Measured concentrations versus nominal concentrations: final test

Test item, % SS prep. At 100 mg/L	Measured concentrations (mg/L) at time point			TWA (mg/L)
	t=0h	t=24h	t=72h
0 (Control)	n.d.	n.d.		0
1.0	0.0203	0.0190	0.00800	0.015
3.2	0.0703	0.0513	0.0193	0.041
10	0.210	0.467	0.0626	0.22
10 *	0.212	0.235	0.0668	0.16
32	0.741	0.737	0.0913	0.42
100	3.28	1.74	1.87	2.0

n.d. = not detected

n.a. = not applicable

* = Abiotic control, incubated without algae

 

Table 4. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test item, % SS prep. At 100 mg/L concentration	Test item, TWA concentration (mg/L)	Growth rate (0 – 72h)				Yield (0 – 72h)
		mean	Std. Dev.	n	% inhibition	mean	Std. Dev.	n	% inhibition
0 (Control)	0	1.896	0.0085	6	-	294.418	7.5568	6	-
1.0	0.015	1.889	0.0141	3	0.35	288.692	12.0657	3	1.9
3.2	0.041	1.895	0.0007	3	0.039	293.688	0.5961	3	0.25
10	0.22	1.889	0.0083	3	0.38	288.073	7.2195	3	2.2
32	0.42	1.764	0.0756	3	6.9*	201.250	42.8228	3	32*
100	2.0	0.150	0.1340	3	92*	0.651	0.5934	3	100*

* = effect was statistically significant

 

Table 5. Mean cell densities (x10^4 cells/mL) during the final test

	Replicate	Test item, TWA concentration (mg/L)
		0 (Control)	0.015	0.041	0.22	0.42	2.0
t=0h	1	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
	n	6	3	3	3	3	3
	Mean	1.0	1.0	1.0	1.0	1.0	1.0
	Std. Dev.	0.0	0.0	0.0	0.0	0.0	0.0
	CV	0.0	0.0	0.0	0.0	0.0	0.0
t=24h	1	10.161	10.905	11.698	9.944	5.807	1.856
	2	8.161	11.338	13.337	10.851	7.020	2.222
	3	13.187	11.524	14.178	9.920	5.405	1.892
	4	11.986
	5	12.010
	6	9.998
	n	6	3	3	3	3	3
	Mean	10.917	11.256	13.071	10.238	6.077	1.990
	Std. Dev.	1.817	0.318	1.261	0.531	0.841	0.202
	CV	16.641	2.822	9.649	5.184	13.834	10.137
t=48h	1	88.737	87.992	84.995	73.580	41.873	18.550
	2	72.937	85.842	84.629	77.447	49.307	6.479
	3	95.114	81.194	92.226	77.099	36.907	9.164
	4	92.742
	5	84.233
	6	79.477
	n	6	3	3	3	3	3
	Mean	85.540	85.009	87.283	76.042	42.696	11.398
	Std. Dev.	8.379	3.475	4.284	2.139	6.241	6.338
	CV	9.796	4.087	4.909	2.813	14.617	55.607
t=72h	1	290.861	296.061	295.358	282.832	219.352	1.790
	2	285.876	297.238	294.217	287.408	233.879	1.000
	3	303.316	275.776	294.488	296.980	153.519	2.162
	4	294.902
	5	292.122
	6	305.428
	n	6	3	3	3	3	3
	Mean	295.418	289.692	294.688	289.073	202.250	1.651
	Std. Dev.	7.557	12.066	0.596	7.220	42.823	0.593
	CV	2.558	4.165	0.202	2.497	21.173	35.949

 

Table 6. Section by section growth rate (per day) during the final test

	Replicate	Test item, TWA concentration (mg/L)
		0 (Control)	0.015	0.041	0.22	0.42	2.0
t=0h - t=24h	1	2.319	2.389	2.459	2.297	1.759	0.618
	2	2.099	2.428	2.591	2.384	1.949	0.798
	3	2.579	2.444	2.652	2.295	1.687	0.638
	4	2.484
	5	2.486
	6	2.302
	Mean	2.378	2.421	2.567	2.325	1.798	0.685
	Std. Dev.	0.173	0.028	0.098	0.051	0.135	0.099
	n	6	3	3	3	3	3
	CV	7.287	1.172	3.827	2.198	7.511	14.432
t=24h - t=48h	1	2.167	2.088	1.983	2.001	1.976	2.302
	2	2.190	2.024	1.848	1.965	1.949	1.070
	3	1.976	1.952	1.873	2.051	1.921	1.578
	4	2.046
	5	1.948
	6	2.073
	Mean	2.067	2.022	1.901	2.006	1.949	1.650
	Std. Dev.	0.098	0.068	0.072	0.043	0.027	0.619
	n	6	3	3	3	3	3
	CV	4.750	3.356	3.793	2.132	1.399	37.523
t=48h - t=72h	1	1.187	1.213	1.246	1.346	1.656	-2.338
	2	1.366	1.242	1.246	1.311	1.557	-1.869
	3	1.160	1.223	1.161	1.349	1.425	-1.444
	4	1.157
	5	1.244
	6	1.346
	Mean	1.243	1.226	1.218	1.335	1.546	-1.884
	Std. Dev.	0.093	0.015	0.049	0.021	0.116	0.447
	n	6	3	3	3	3	3
	CV	7.482	1.193	4.022	1.568	7.482	23.740

Notes on statistical analysis :

(i) Inhibition of growth rates increased with increasing concentration of test item from 0.42 mg/L upwards resulting in 92% inhibition at 2.0 mg/L. Statistically significant inhibition of growth rates was found at the two highest test concentrations.

(ii) Inhibition of yield increased with increasing concentration of test item from 0.42 mg/L upwards resulting in 100% inhibition at 2.0 mg/L. Statistically significant inhibition of growth rates was found at the two highest test concentrations.

(iii) For the 72h-ECx determination, it was decided to not include the two lowest test concentrations in the regression analysis. Including these values would result in ErC10 and ErC20 values that are not in agreement with the effects observed in the present study. This is due to mathematical reasons, i.e. during the non-linear regression approach the observed effects at the two lowest concentrations cause the resulting regression model to not well with the obtained experimental data. The followed approach was deemed justified and sufficiently conservative as the concentration removed from analysis clearly did not affect algal growth, i.e. were below the statistically confirmed NOEC of 0.22 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 for growth rate reduction was 0.93 (C.I. 0.81 – 1.1) mg/L based on time weighted average concentrations. The corresponding ErC10 was 0.47 (C.I. 0.37 – 0.56) mg/L. The NOEC was 0.22 mg/L.

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 study also followed the OECD Guidance Document number 23 on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals (2019). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The preparation procedure for test solutions was based on a non-GLP saturation test and range finder (cited in the full study report). Following the preliminary range finding test in the 0.1 to 100 %SS (% saturated solution) preparation of the 100 mg/L loading rate, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. The preparation procedure for test solutions was based on a non-GLP saturation test andrange-finder(cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel containing 10% of the SS prepared at a loading rate of 100 mg/L, but without algae. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via UPLC-TUV at 0 hours (test start), at 24 hours and at 72 hours (test end) of the exposure. During the exposure period the measured concentrations decreased in the range finder and definitive test from initial values. See above for further information. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.020, 0.070, 0.21, 0.74 and 3.3 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. At the end of the test, the concentrations had decreased to 12-57% of the initial concentrations. Additionally, the concentration measured in the solution without algae decreased to 31% relative to the initial value at the end of the test, while the concentration in the solution with algae was at 30% of the initial concentration at the end of the test. The equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/L which were based on analysis during the definitive test period. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 0.93 (C.I. 0.81 – 1.1) mg/L based on time weighted average concentrations. The corresponding ErC10 was 0.47 (C.I. 0.37 – 0.56) mg/L. The NOEC was 0.22 mg/L.

Description of key information

72h-EC50 (aquatic algae; growth rate) = 0.93 (C.I. 0.81 – 1.1) mg/L based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2020

72h-EC10 (aquatic algae; growth rate) = 0.47 (C.I. 0.37 – 0.56) mg/L based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2020

72h-NOEC (aquatic algae; growth rate) = 0.22 mg/L based on TWA concentrations, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2020

Key value for chemical safety assessment

EC50 for freshwater algae:

0.93 mg/L

EC10 or NOEC for freshwater algae:

0.47 mg/L

Additional information

Key study : OECD TG 201, 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 study also followed the OECD Guidance Document number 23 on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals (2019). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The preparation procedure for test solutions was based on a non-GLP saturation test and range finder (cited in the full study report). Following the preliminary range finding test in the 0.1 to 100 %SS (% saturated solution) preparation of the 100 mg/L loading rate, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item concentrations in a geometric series with a separation factor of 3.2, were prepared as follows: 0 (control), 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. The preparation procedure for test solutions was based on a non-GLP saturation test andrange-finder(cited in the full study report). Preparation of test solutions started with a loading rate of 100 mg/L applying a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning through glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colourless at the end of the preparation procedure. During the preparation, glassware was closed airtight with minimal headspace to prevent vaporization of test item during preparation. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.Thereafter, the replicates were closed airtight with minimal headspace to prevent potential vaporization of test item. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel containing 10% of the SS prepared at a loading rate of 100 mg/L, but without algae. Test vessels were completely filled and closed to prevent any volatilization of the test item. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via UPLC-TUV at 0 hours (test start), at 24 hours and at 72 hours (test end) of the exposure. During the exposure period the measured concentrations decreased in the range finder and definitive test from initial values. See above for further information. Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 0.020, 0.070, 0.21, 0.74 and 3.3 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. At the end of the test, the concentrations had decreased to 12-57% of the initial concentrations. Additionally, the concentration measured in the solution without algae decreased to 31% relative to the initial value at the end of the test, while the concentration in the solution with algae was at 30% of the initial concentration at the end of the test. The equivalent time weighted average concentrations were: 0 (control), 0.015, 0.041, 0.22, 0.42 and 2.0 mg/L which were based on analysis during the definitive test period. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item 72h-ErC50 for growth rate reduction was 0.93 (C.I. 0.81 – 1.1) mg/L based on time weighted average concentrations. The corresponding ErC10 was 0.47 (C.I. 0.37 – 0.56) mg/L. The NOEC was 0.22 mg/L.