Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

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:

April 14th 27th to 18th, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 266/2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.
At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates.

Vehicle:

no

Details on test solutions:

Preparation of stock solution: 126 mg test item/l (corresponding to 100 mg a.s./l) were freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Preparation of test item solutions: One limit concentration was tested with a nominal concentration of 126 mg test item/l.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Synonyms Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
- 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 PA37 1QA; Scotland, UK
- Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

34 mg CaCO3/L

Test temperature:

min: 21.0 °C; max: 23 °C; mean value: 22.0 °C

pH:

7.92-8.90 (control)

Conductivity:

136 μS/cm

Nominal and measured concentrations:

126 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Type: closed
- Fill volume: 100 ml
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL; Actual: 5416 cells/mL
- Control end cells density: 1420729 cells/ml (mean, 72hrs)
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- Application: carried out by adding appropriate volumes of the algae inoculum to the test item solutions.
- Preculture: a three days old preculture, prepared in dilution water, was used as inoculum.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERSv
- Source/preparation of dilution water: OECD TG 201 medium. Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 μS/cm). This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.Hardness, conductivity, TOC, acidity and alkalinity are determined
- Total organic carbon: < LOQ (2.00 mg C/l)
- Acidity: 0.6 mmol/l
- Alkalinity: 0.8 mmol/l
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of the limit concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of the limit concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes; sterile glass flasks were used
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity (target): approximately 4440 to 8880 lux, corresponding to 60 to 120 μE*m-2*s-1
- Light homogeneity (target): within ±15 % over incubation area

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the concentration of 126 mg test item/l.
- Microscopic evaluation: 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
- Range finding study: A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility. A stock solution with a concentration of 126 mg test item/l was freshly prepared with dilution water and agitated until the solution was visually clear. Out of this stock solution two concentration levels were prepared (by dilution) with the concentrations 1.26 and 12.6 mg/l. The stability was assessed in an additional stability test with the Limit test concentration under test conditions with algae. All concentration levels were clear throughout the exposure period
- Results used to determine the conditions for the definitive study: The dilution levels are based on the results of a preliminary range finding test (non-GLP). Growth rate inhibition was 0 % in all tested concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. The measured concentration of the test item remained stable within ± 20 % of the nominal measured concentration over the test interval at 88 % of the nominal concentration of the test item. All effect values are given based on the nominal test item concentrations. An aged sample and a freshly prepared calibration standard were measured via LC-Q-TOF high resolution-MS for confirmation of the analyte identity. No degradation products were found. It is noteworthy that on basis of the measured accurate masses, only the molecular formula (C40H40N12O22S6) was predicted in the LC-Q-TOF. The chemical structure of the compounds has not been determined. Besides these two isomers, no significant metabolites have been identified by comparison of freshly prepared and aged samples. In comparison to control samples, no additional peaks were visible. The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits in the definitive study. The test media were clear throughout exposure period (possible turbidity related to algae growth not taken into account).

Results with reference substance (positive control):

ErC50 (72 hrs) = 0.940 mg/l; 95 % CL: 0.732-1.33 mg/l
Valid range: 0.758 ± 0.535

EyC50 (72 hrs) = 0.660 mg/l; 95 % CL: 0.393-0.700 mg/l
Valid range: 0.414 ± 0.335

Reported statistics and error estimates:

The program states no significant difference for the limit concentration compared to the control for growth rate inhibition.

Cell Densities

nominal test item concentration (mg/l)	Replicate no.	Cell density [cells/ml]
		0 hrs	24 hrs	48 hrs	72 hrs
126	1	5416	26124	221039	1369712
	2	5416	26387	213883	1463616
	3	5416	24292	202366	145973
	4	5416	30420	212384	1345421
	5	5416	27485	229140	1673670
	6	5416	24628	196952	1260279
	Mean	5416	26556	212627	1428112
Control	1	5416	31233	240219	1579717
	2	5416	28799	217846	1477928
	3	5416	26086	213474	1331888
	4	5416	30839	228672	1277074
	5	5416	29454	229918	1436111
	6	5416	27351	222120	1421654
	Mean	5416	28960	225375	1420729

Evaluation after 72 hours - Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

nominal test item concentration (mg/l)	Replicate no.	Growth rate (d^-1)	Inhibition of Growth rate (%)	Yiels (cells/ml)	Inhibition of yield (%)
126	1	1.84	1	1364296	4
	2	1.87	-1	1458200	-3
	3	1.87	0	1450557	-2
	4	1.84	1	1340005	5
	5	1.91	-3	1668254	-18
	6	1.82	2	1254863	11
	Mean	(-) 1.86	0	(-) 1422696	-1
Control	1	1.89		1574301
	2	1.87		1472512
	3	1.84		1326472
	4	1.82		1271658
	5	1.86		1430695
	6	1.86		1416238
	Mean	1.86		1415313

Negative values = growth stimulation

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

	Replicate no	Specific growth rate (d-1) section-by-section			Mean (0-72 hrs)	SD ±	CV (%)	Mean CV (%)
		0-24 hrs	24-48 hrs	48-72 hrs
Control	1	1.75	2.04	1.88	1.89	0.144	7.62	10.5
	2	1.67	2.02	1.92	1.87	0.181	9.65
	3	1.57	2.10	1.83	1.84	0.265	14.4
	4	1.74	2.00	1.72	1.82	0.158	8.70
	5	1.69	2.06	1.83	1.86	0.182	9.80
	6	1.62	2.09	1.86	1.86	0.238	12.8
				Mean	1.86
				SD ±	0.10
				CV (%)	5.51

Environmental Conditions

nominal test item concentration (mg/l)	pH values
	Start 0 hrs	End 72 hrs
126	7.84	8.81
Control	7.92	8.90
Room temperature (°C):     min: 21.0 °C; max: 23 °C; mean value: 22.0 °C
Light density (lux)   n=14 mean value: 5031; range of the measured values: 4517-5339; equalling -10.2 to 7.32 %     CV (%):5.26

Measured Concentrations and Percent of Nominal Concentration of the Test Item conc during the Definitive Test

Sampling date	Fresh media 0 hrs		Old media 72 hrs
nominal test item concentration (mg/l)	Test item concentration
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	% of nominal
126	120	96	111	88
QC	0.978	96	0.941	92
Control	< LOQ		< LOQ

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

% = percent of the nominal test item concentration

LOQ = limit of quantification (1.00 mg/l of the test item)

QC = Analytical quality control (1.00 mg/l of the test item)

Validity criteria fulfilled:

yes

Remarks:

Increase of cell growth in control cultures: exponentially ≥16-fold; Mean CVs for section-by-section specific growth rates(days 0-1,1-2,2-3) in ctrl. cultures ≤35 %; CV of average specific growth rates during whole test period in replicate ctrl. cultures

Conclusions:

ErC50 (72h) > 126 mg/L (nominal)
NOEC (72h, growth rate) = 126 mg/l (nominal)

Executive summary:

The toxicity of the test item 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. The aim of the study was the determination of the effects of growth rate and yield over a period of 72 hours in a limit test. The study was conducted under static conditions with an initial cell density of 5416 cells/ml. A stock solution with a nominal test item concentration of 126 mg/l (corresponding to 100 mg a.s./l) was freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. Six replicates were tested for the limit concentration and six for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits.

The concentration of the test item was analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) in the limit concentration and in the control.The measured concentration of the test item was 96 % at the start of the exposure (0 hours) and 88 % at the end of the exposure (72 hours) of the nominal concentration of the test item during the definitive test.

The test item was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours for growth rate. Effect values were determined as follows (based on nominal test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 126 mg/l. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 126 mg/l.

Description of key information

ErC50 (72h) > 126 mg/L  (nominal)

NOEC (72h, growth rate) = 126 mg/l (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

126 mg/L

EC10 or NOEC for freshwater algae:

126 mg/L

Additional information

The toxicity of the test item 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. The study was conducted for 72 hours under static conditions with an initial cell density of 5416 cells/ml. A stock solution with a nominal test item concentration of 126 mg/l (corresponding to 100 mg a.s./l) was freshly prepared with dilution water. Six replicates were tested for the limit concentration and six for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits. The concentration of the test item was analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) in the limit concentration and in the control.The measured concentration of the test item was 96 % at the start of the exposure (0 hours) and 88 % at the end of the exposure (72 hours) of the nominal concentration of the test item during the definitive test. The test item was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours for growth rate. Effect values were determined as follows (based on nominal test item concentration): The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >126 mg/l. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 126 mg/l.