[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

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:

From 2022-08-11 to 2022-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

yes

Remarks:

The pH of the control medium had increased by more than 1.5 units the during the test (1.9 units in total). This deviation has no influence on the study, as a pH of 9.83 is within the natural range of growth conditions of the test organism.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

yes

Remarks:

The pH of the control medium had increased by more than 1.5 units the during the test (1.9 units in total). This deviation has no influence on the study, as a pH of 9.83 is within the natural range of growth conditions of the test organism.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Test Concentrations: 0, 0.617, 1.85, 5.56, 16.7 and 50.0% v/v saturated solution (nominal mg/L)
- Sampling method: at 0 and 72 hours for quantitative analysis and pooled.
- Sample storage conditions before analysis: room temperature

Vehicle:

no

Details on test solutions:

A saturated solution with a nominal loading rate of 1.00 mg test item/L was prepared once at room temperature 24 ± 1 hour prior to the start of the exposure.
An appropriate amount of the test item was weighed out and inserted in a glass flask with an appropriate amount of dilution water. The dispersion was stirred for 24 ±1 hour with a magnetic stirrer at a sufficient speed to ensure a vortex depth of about 10 – 35 % of the dispersion height in the preparation vessels.
Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item.
Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter always was kept covered. The Tyndall effect was negative.

Five concentrations were tested in a geometric series with a dilution factor of 3: 0.617 – 1.85 – 5.56 – 16.7 – 50.0% of the saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain : 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 PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

37 mg/L CaCO3/L

Test temperature:

21 – 23 °C

pH:

Control: pH-0hr = 7.93, pH-72h = 9.83
0.375 µg/L*: pH-0hr = 7.88, pH-72h = 9.71
0.634 µg/L*: pH-0hr = 7.81, pH-72h = 9.40
1.97 µg/L*: pH-0hr = 7.79, pH-72h = 8.56
9.85 µg/L*: pH-0hr = 7.78, pH-72h = 8.21
14.1 µg/L*: pH-0hr = 7.75, pH-72h = 7.97

* Geo. mean meas.

Conductivity:

184 µS/cm

Nominal and measured concentrations:

Dilution Meas. Meas. Meas.
Level conc. conc. conc.
[%] [µg/L] [%] [µg/L] [%] [µg/L]
0 h 72 h
50.0 61.6 12 3.25 1 14.1
16.7 18.7 11 5.19 3 9.85
5.56 6.33 11 0.615 1 1.97
1.85 2.01 11 < LOQ 0.634
0.617 0.702 11 < LOQ 0.375
Control < LOQ < LOQ < LOQ

0 Hours measured in Fresh Media ; 72 hours measured in the old media
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percentage of the nominal concentration of the test item
LOQ = limit of quantification (0.40 µg test item/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Type : ealed with cotton wool plugs
- Material, size, headspace, fill volume: 250 ml filled with 100 mL test volume
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): not relevant
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 6864 cells/mL (mean value)
- Control end cells density: 1431416 cells/mL (mean value)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS :
- Source/preparation of dilution water: composition of dilution water according to the OECD 201 guidelines

Conductivity : 184 µS/cm
Total hardness : 37 mg CaCO3/L
Acidity : 0.2 mmol/L
Alkalinity : 0.6mmol/L
Total organic carbon : < LOQ*

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous
- Light intensity and quality : Approximately 4440 to 8880 lux

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 in the preliminary range finding test at the saturated solution

- Other: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: spacing factor of 3
- Range finding study: yes
- Test concentrations: 0.617, 1.85, 5.56, 16.7 and 50.0 % v/v of the saturated solution
- Results used to determine the conditions for the definitive study: used of the results of the range finding study

Reference substance (positive control):

yes

Remarks:

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number BCCG1644, purity 99.9%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2022-05-31 to 2022-06-03.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.61 µg/L

95% CI:

ca. 2.87 - ca. 5.09

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.97 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.634 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.706 µg/L

95% CI:

ca. 0.616 - 0.836

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 0.375 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

<= 0.375 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Unusual cell shape: no unusual cell shape observed
- Colour differences: no colour differences observed

- Any stimulation of growth found in any treatment: no stimulation of growth found mentionned
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: undissolved particles were removed by membrane filtration
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 : 1.22 (1.07 -1.30) mg/L
- EyC50 : 0.599 (0.543-0.654) mg/L

Reported statistics and error estimates:

EC10 , EC20 and EC50- values with confidence intervals of growth analyses rate inhibition and yield inhibition after 72 hours were calculated by sigmoidal dose-response and third order polynomial regression with the GraphPad Prism Software.
The NOEC / LOEC was determined by calculation of statistical analyses significance of growth rate and yield.
The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Dunnett’s multiple -t-test was performed with a significance level of 0.05 for growth rate.
Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for yield.
Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for yield.

Validity Criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	209-fold (specific growth rate 1.78 day^-1)
Mean coefficients of variation for section-by-section specific growth rates (days  0-1, 1-2 and 2-3) in the control cultures	≤ 35%	24.5%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	0.86%

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 - 72 hours)

	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	1.32	1.96	2.10	1.79	0.419	23.4	24.4
	2	1.39	1.97	2.01	1.79	0.348	19.5
	3	1.25	2.02	2.09	1.79	0.465	26.0
	4	1.27	1.96	2.08	1.77	0.437	24.7
	5	1.23	1.99	2.16	1.79	0.498	27.8
	6	1.25	1.98	2.03	1.75	0.435	24.8
				Mean	1.78
				SD ±	0.02
				CV [%]	0.86

SD = Standard deviation  CV = Coefficient of variation

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
14.1	1	6864	<LOQcell density	<LOQcell density	<LOQcell density
	2	6864	<LOQcell density	<LOQcell density	<LOQcell density
	3	6864	2741	<LOQcell density	<LOQcell density
	Mean	6864	n.a.	n.a.	n.a.
9.85	1	6864	3773	3218	<LOQcell density
	2	6864	5915	8766	20108
	3	6864	4183	4709	4518
	Mean	6864	4624	5564	n.a.
1.97	1	6864	7543	39431	302306
	2	6864	7954	45259	392108
	3	6864	9463	49345	460275
	Mean	6864	8320	44678	384896
0.634	1	6864	11870	81404	677012
	2	6864	12126	88529	724511
	3	6864	13177	90778	799942
	Mean	6864	12391	86904	733822
0.375	1	6864	18309	105919	1038336
	2	6864	18329	137486	1187130
	3	6864	17038	132871	1082102
	Mean	6864	17892	125425	1102523
Control	1	6864	25566	181827	1482738
	2	6864	27417	195771	1458989
	3	6864	23999	179965	1458449
	4	6864	24404	173793	1384412
	5	6864	23385	170861	1482289
	6	6864	24008	173299	1321620
	Mean	6864	24797	179253	1431416

LOQ_{cell density}                = Limit of quantification of the cell density (2280 cells/mL).           n.a.         = not applicable

 

Validity criteria fulfilled:

yes

Conclusions:

Effect values were determined as follows (based on geometric mean measured test item concentrations):
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 3.60 (2.87 – 5.08) µg/L and 0.706 (0.616 – 0.836) µg/L, respectively.
The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.634 µg/L and < 0.375 µg/L, respectively.

Executive summary:

The toxicity of the Substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2022-08-15 to 2022-08-22, with the definitive exposure phase from 2022-08-16 to 2022-08-19 at the test facility. The aim of the study was the determination of 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 6864 cells/mL. A saturated solution with a nominal concentration of 1.00 mg test item/L was prepared with dilution water (see Table 2). The dispersion was stirred for 24 ±1 hours with a magnetic stirrer at a sufficient speed to ensure a vortex depth of about 10 – 35 % of the dispersion height in the preparation vessels. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The Tyndall effect was negative. Five concentrations were tested in a geometrical series with a dilution factor of 3: 0.617 - 1.85 - 5.56 - 16.7 - 50.0 % of the saturated solution, corresponding to the geometric mean measured test item concentration of 0.375 – 0.634 – 1.97 – 9.85 – 14.1 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The concentrations of the test item and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS. Details of the analytical method are presented in section 14.

At the start of the exposure the measured concentrations were in the range of 0.702 µg/L to 61.6 µg/L test item concentration. At the end of the exposure after 72 hours, the measured test item concentrations were in the range of < LOQ to 5.19 µg/L. The geometric mean measured test item concentrations were calculated to be 0.617 – 1.85 – 5.56 – 16.7 – 50.0% of the saturated solution, corresponding to the geometric mean measured test item concentration of 0.375 – 0.634 – 1.97 – 9.85 – 14.1 µg/L. Therefore, all effect values are given are based on geometric mean measured test item concentrations.

NOEC, LOEC and EC_x-values with 95% Confidence Intervals of the test item (0-72 hours) based on the geometric mean measured test item concentrations [µg/L]

	Inhibition of Growth Rate
NOEC	0.634
LOEC	1.97
ErC10	0.732 (< 0.375 – 1.27)
ErC20	1.60 (1.19 – 2.12)
ErC50	3.61 (2.87 – 5.09)
	Inhibition of Yield
NOEC	< 0.375
LOEC	≤ 0.375
EyC10	< 0.375
EyC20	< 0.375
EyC50	0.706 (0.616 – 0.836)

Description of key information

EC50(72h) for freshwater algae (Pseudokirchneriella subcapitata): 3.61 µg/L (OECD TG 201; static)

Key value for chemical safety assessment

EC50 for freshwater algae:

3.61 µg/L

Additional information

The EC50 (72 hours) was 3.61 µg/L (geometric mean of the measured concentrations)

The NOEC (72 hours) was 0.634 µg/L