Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl-, ethanedioate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

28 July 2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 50, 18.5, 6.9, 2.6, 1.0, 0.4 and 0.1 mg/l
- Sampling method: The analytical determination of the test item concentrations was performed at the beginning
of the test, after 24, 48 hours and at the end of the test in all test concentrations. The samples for analysis were taken at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory. The samples for analysis at 24, 48, 72 hours were delivered to analytical laboratory immediately after the end of appropriate testing period. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of solutions of the test item for preliminary and definite test:

The three stock solutions of the test item with different pH (4.0, 6.0 and 8.1) value were prepared in the test medium for the three preliminary tests. The one stock solution of the test item with pH 8.1 was prepared in the test medium for the full test.

The test item is ionised chemical and coloured dye that shows different forms at different pH, namely EC 255-288-2 (acidic pH) and its carbinol base EC 208-109-7 (basic pH) . For this reason (according OECD GD No.23, chapter 7, and its different subsections 7.6 Coloured test chemicals and 7.8 Ion ised test chemicals and after consultation with sponsor) three preliminary tests were performed with different pH 4.0, 6.0 and 8.1 to determine the most toxic form.

All three preliminary tests were performed in a range of the test item nominal concentrations 0.1 – 5 mg·L-1. Samples for analytical determination were taken at the beginning and at the end of the test from the lowest test concentration (0.1 mg·L-1), the highest test concentration (5.0 mg·L-1) and from the highest test concentration without algae.
The analytical results showed, that the test item was not sufficiently stable in the test medium at conditions of the test. The reason is probably its photodegrading properties.
The preliminary test (pH 4.0) did not meet the validity criteria in biomass increase and variation coefficient for growth rate.
In the preliminary test with pH 8.1 slightly higher toxicity was observed for variable ,,growth rate,, and ,,yield,, than in the preliminary test with pH 6.0.

Based on the results of toxicity observed in the preliminary tests, pH 8.1 was chosen for the full test which was performed in a range of the test item nominal concentrations 0.1 – 50.0 mg·L-1. Samples for analytical determination were taken at the beginning, after 24 hours, 48 hours and at the end of the test.

Reason for concentration increase: low toxicity for the variable ,,Growth rate,, in the preliminary test. In order to be able to evaluate ErC50, it was necessary to include higher concentrations in the full test


Main test concentrations: 50, 18.5, 6.9, 2.6, 1.0, 0.4 and 0.1 mg·L-1
As reported in the section of the validation of the analytical method, due to the analytical conditions acidic pH (mobile phase has pH 3) the sampled test solutions were acidifed before the analyses and therefore it is possible to quantify the total amount of test substance but not the ratio of each single chemical species (cationic vs. carbinol).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): The Botanic Institute of the Czech Academy of Science, Třeboň

-Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

- Adaptation of algal culture: The pH of the test medium for algal pre-cultivation was adjusted to pH 6 (for the test with pH values 4.0. and 6.0).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

21.0 – 21.5 °C

pH:

7.6-8

Dissolved oxygen:

not reported

Conductivity:

conductivity of the deionised water: 1.39 µS·cm-1

Nominal and measured concentrations:

A (mg/l) B C D E F
Nominal 0h 24h 48h 72h geometric mean
50.0 45.1 26.8 25.4 19.8 27.92
18.5 17.0 12.8 12.3 10.3 12.89
6.9 6.0 5.5 5.3 3.1 4.83
2.6 2.3 2.1 1.8 1.5 1.90
1.0 0.87 0.86 0.85 0.68 0.81
0.4 0.36 0.38 0.41 0.27 0.35
0.1 0.16 0.16 0.16 0.15 0.16

Details on test conditions:

The test contained three parallel series of the test item concentrations and three controls without the test item. The volume of the test solution was 50 mL in each testing flask. The initial density of 5 000 cells per mL was used in the test. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber. The growth rates () and the yield (Y) and subsequently percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.

Volume of inoculum algae culture: 2.01 mL in 500 mL mixture
Lighting during the test: 7 445 – 7 450 lux
Exposition time: 72 hours
Volume of tested mixture: 50 mL
Initial concentration of algae culture: 5 000 cells in a 1 mL
Without aeration.
Agitation of algae culture by shaking

The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber. The growth rates () and the yield (Y) and subsequently percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 7.33 mg/L

95% CI:

> 4.75 - < 11.28

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.95 mg/L

95% CI:

> 1.34 - < 2.85

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The analytical determination of the test item concentrations was performed at the beginning of the test, after 24, 48 hours and at the end of the test in all test concentrations. The analytical results showed that the test item was not stable in the test medium under the conditions of testing, probably due to photodegradation

Geometric mean of measured concentrations were calculated from concentrations measured at the start, after 24, 48 hours and at the end of the test (72 hours). Geometric mean values were calculated using mathematical function ,,GEOMEAN,, by commercial software Microsoft Excel 2019.

Results with reference substance (positive control):

Results of reference test
The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference item, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 07.03. to 10.03. 2022 are the following:

72 hour – ErC50 = 0.77 mg·L-1 (95% confidence limit: 0.49 – 1.21 mg·L-1)

These results are taken from the final report No. 19-330, which is stored in the office of the head of Toxicity I group.

Interlaboratory test data
From the results of the last interlaboratory test arranged by CSlab spol. s r.o. (accredited provider of proficiency testing) in 2019 we have obtained data for potassium dichromate
72h – ErC50 generated at 7 laboratories. The following reference range of values could be calculated from those data:

72 hour – ErC50 = 0.57 – 1.33 mg·L-1

These results are taken from the report PT/TX/1/2019, which is stored in the office of the head of Toxicity I group.

Reported statistics and error estimates:

The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat Professional Version 3.3.0.
The determination of NOEC values was done by statistical software ToxRat Professional Version 3.3.0.

Nominal, determined and geometric mean of measured concentrations – full test
Nominal	Determined concentration	Determined concentration	Determined concentration	Determined concentration	Geometric mean of measured
concentration	0 hours	24 hours	48 hours	72 hours	concentration
(mg·L^-1)	(mg·L^-1)	(mg·L^-1)	(mg·L^-1)	(mg·L^-1)	0 – 72 hours
					(mg·L^-1)
50.0	45.1	26.8	25.4	19.8	27.92
18.5	17.0	12.8	12.3	10.3	12.89
6.9	6.0	5.5	5.3	3.1	4.83
2.6	2.3	2.1	1.8	1.5	1.90
1.0	0.87	0.86	0.85	0.68	0.81
0.4	0.36	0.38	0.41	0.27	0.35
0.1	0.16	0.16	0.16	0.15	0.16

 

Percentual reduction of mean growth rate and percentual inhibition of mean yield – full test
test concentrations/ mg·L-1	growth rate		yield
	Mean	Reduction	Mean		Inhibition
	m	%	Y		%
27.92	0.07	94.6	1250		99.6
12.89	0.36	73.9	9583		96.8
4.83	0.93	32.0	76250		74.3
1.90	1.05	23.2	111667		62.4
0.81	1.16	15.1	157500		47.0
0.35	1.24	9.6	199167		33.0
0.16	1.29	5.7	234583		21.0
Control	1.37	0.0	297083		0.0

Validity criteria fulfilled:

yes

Conclusions:

The substance was tested for toxicity to aquatic algae following OECD201. Under the experimental conditions the ErC50, 72h is ca. 7.33 mg/l (geometric mean) based on growth rate.

Executive summary:

The test item was tested for growth inhibition on algae Desmodesmus subspicatus according to OECD TG 201.

The test item is ionised chemical and coloured dye that shows different forms at different pH, namely EC 255-288-2 (acidic pH) and its carbinol base EC 208-109-7 (basic pH). For this reason (according to OECD GD No.23, chapters 7.6 and 7.8 colured and ionised test chemicals and after consultation with the sponsor), the preliminary tests with three different pH (pH 4.0, 6.0 and 8.1) were performed to determine the most toxic form of the test item.  All three preliminary tests were performed in a range of the test item nominal concentrations 0.1 – 5 mg·L^-1. Samples for analytical determination were taken at the beginning and at the end of the test from the lowest test concentration (0.1 mg·L^-1), the highest test concentration (5.0 mg·L^-1)  and from the highest test concentration without algae.

Based on the results of toxicity observed in the preliminary tests with pH 4, 6 and 8.1, pH 8.1 was chosen for the full test and  was performed in a range of the test item nominal concentrations 0.1 – 50.0 mg·L^-1. Samples for analytical determination were taken at the beginning, after 24 hours, 48 hours and at the end of the test.

The analytical results showed, that the test item was not sufficiently stable in the test medium at conditions of the test. The reason is probably due to its photodegrading properties.

The guideline specifies that if evidence is available to demonstrate that the concentration of the test item in the full test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the full test, then the results can be based on nominal or measured initial values. In this study, as specified above, the concentrations of test item have not been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the full test, so the real measured concentrations of test item should be used for the results evaluation in a form of geometric means

Under the experimental conditions the ErC50, 72h is ca. 7.33 mg/l (geometric mean) based on growth rate.

Description of key information

test item		year of the study	target/RA	purity	guidelines	results	notes	Reference
Basic Green 4 acetate	water solubility: 6.65 g/l no melting, only decomposition	2022	RA	89% without solvent	OECD 201	Algae, static, pH 7,6-8 ErC50, 72h, ca. 7.33 mg/l (measured, growth rate)	analytical monitoring performed via LC-MS	Basic Green 4 Acetate  Alga, Growth Inhibition Test
Basic Green 4 oxalate	water solubility: 26.7 g/l no melting, only decomposition	1998		no details	no details	Algae ErC50, 72h, ca. 3.6 mg/l	only final results available	Dystar, report date 1998-05-13
Basic Green 4 oxalate (2:3)		1998	RA	no details	no details	Algae ErC50, 72h, ca. 3.6 mg/l	only final results available