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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: highest concentration 2.5 mg/l and 0.16 mg/l

- Sampling method: The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analysed on the day of delivery. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analysed 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 two stock solutions of the test item with different pH value (4.5 and 7.8) were prepared in the dilution water for preliminary tests. The pH was adjusted with hydrochloric acid. The one stock solution of the test item with pH 7.8 was prepared in the dilution water for definitive test.
5 mg of the test item was weighed into 500 mL of the dilution water for the preliminary tests and for definitive test. The concentrations of solutions used in the preliminary and definitive test were obtained by dilution of the stock solution with dilution water.

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 Ionised test chemicals and after consultation with sponsor) two preliminary tests were performed with different pH to determine the most toxic form.

The preliminary tests were performed at pH 4.5 and 7.8 in a range of the test item nominal concentrations 0.1 – 5 mg·L-1 in a static design. Samples for analytical determination were taken at the beginning and at the end of the test from the highest concentration (5.0 mg·L-1) and the lowest concentration (0.1 mg·L-1 ) The analytical results showed, that the test item was stable in the dilution water at conditions of the test in the highest concentration (5.0 mg·L-1). In the lowest concentration (0.1 mg·L-1) the test item was not stable in the dilution water at conditions of the test. The reason is probably due to its photodegration properties.

In the preliminary test with pH 7.8 slightly higher toxicity was observed than in the preliminary test with pH 4.5.
On the basis of the preliminary tests results the definitive test was performed at pH 7.8 and in the dark. The definitive test was performed in a range of nominal concentrations from 0.04 mg·L-1 to 2.5 mg·L-1. The geometric concentration series of the test item with factor 2.0 was used for the test.
Main test concentrations: 2.5, 1.25, 0.63, 0.32, 0.16, 0.08 and 0.04 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna Straus
- Strain/clone:
- Justification for species other than prescribed by test guideline: the prescribed organism was used
- Age at study initiation (mean and range, SD): young daphnia born in 24h
- Weight at study initiation (mean and range, SD): not relevant
- Length at study initiation (length definition, mean, range and SD): not reported
- Source: internal breeding, obtained by aperiodic parthenogenesis
- Feeding during test: no feeding

The grow-up gravid females are separated into dilution water. Young daphnia born
in 24 hours are segregated to the handling container from which they entered the experiment. At handling a care should be taken of not to penetrate the air under animal’s shell.


ACCLIMATION
Adaptation: Young daphnia used for preliminary test with pH 4.5 were born into dilution water with pH 6.0.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test temperature:

Range of temperature: 19.0– 19.9 °C

pH:

7.7-7.8

Dissolved oxygen:

8.6-9.2 mg/l

Nominal and measured concentrations:

nominal concentrations tested : 0.16 and 2.5 mg/l
measured concentrations tested: 0.17 (0% ) and 3.8 (+ 3.6%) mg/l

Details on test conditions:

TEST SYSTEM
- Volume of test item solutions: 50 mL (least 2 mL per individual)
- Aeration: no aeration
- No. of organisms per vessel: 5 animals per replicate
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):


TEST MEDIUM / WATER PARAMETERS
The holding and culturing of the Daphnia is performed at conditions (light, temperature, medium) similar to those which are used in the test.
For holding and culturing of the daphnia the M4 medium was used (according to Annex 2
of the EU method C.2 and according to Annex 3 of the OECD TG 202).
For test the dilution water was used (according to Annex 2 of the EU method C.2 and according to Annex 3 of the OECD TG 202). Dilution water was prepared just before the test by admixing 2.5 mL of stored solutions A, B, C and D into 1 000 mL of deionized water.
Because culturing and test medium were different (M4 and dilution water), we included pre-test acclimation period: brood daphnids were maintained in dilution water for 48 hours prior to the start of the test.
Adaptation: Young daphnia used for preliminary test with pH 4.5 were born into dilution water with pH 6.0.


Dilution water
A. 117.6 g CaCl2·2H20 in 1L deionized water
(Batch No.: PP/2021/00741, exp.: 03/2023, Lach-Ner, s. r.o.)
B. 49.3 g MgSO4·7H2O in 1L deionized water
(Batch No.: PP/2018/08687, exp.: 04/2023, Lach-Ner, s. r.o.)
C. 25.9 g NaHCO3 in 1L deionized water
(Batch No.: PP/2019/11502, exp.: 09/2024, Lach-Ner, s. r.o.)
D. 2.3 g KCl in 1L deionized water
(Batch No.: PP/2021/08772, exp.: 06/2024, Lach-Ner, s. r.o.)

The dilution water should have a pH 7.8  0.2, values measured in this study were 7.9 in the preliminary tests and 7.8 in the definitive test.
The content of sum Ca + Mg ions should have the value of 2.50  0.10 mmol·L-1, values measured in this study were 2.52 and 2.53 mmol·L-1 in the preliminary tests and
2.56 mmol·L-1 in the definitive test.
The dilution water for preliminary test with pH 4.5 was adjusted at pH 4.5. The pH was adjusted with hydrochloric acid.

Measured values meet the requirements.


OTHER TEST CONDITIONS
- Adjustment of pH: with hdrochloridric acid
- Photoperiod: dark in the main test
- Light intensity: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilisation

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 5.0, 2.5, 1.0, 0.5 and 0.1 mg·L-1
- Results used to determine the conditions for the definitive study: see table below

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.22 mg/L

95% CI:

> 0.17 - < 0.27

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

ca. 1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

ca. 0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance is periodically verified
in six-month period by testing with the reference item, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 09.05. to 13.05.2022 are the following:

24 hour – EC50 = 1.13 mg·L-1 (95% confidence limit: 0.94 – 1.47 mg·L-1)
48 hour – EC50 = 0.67 mg·L-1 (95% confidence limit: 0.58 – 0.76 mg·L-1)


b) Interlaboratory test data
From the result 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
48h – EC50 generated at 7 laboratories. The following reference range of values could be calculated from those data:

48 hour – EC50 = 0.50 – 1.16 mg·L-1


c) Method validity evaluation
The immobilisation of the test organism by the reference item (48 hour – EC50) obtained
from our last reference test meets the calculated range from the interlaboratory test.

Preliminary test at pH 4.5
Nominal concentration	mg·L^-1	5.0	2.5	1.0	0.5	0.1	Control
Number of mobile animals	24 hours/1^stseries	0	0	1	3	5	5
Number of mobile animals	24 hours/2^nd series	0	0	0	3	5	5
Number of mobile animals	24 hours/3^rd series	0	0	1	3	4	5
Number of mobile animals	24 hours/4^th series	0	0	2	4	4	5
Number of mobile animals	24 hours/total	0	0	4	13	18	20
Immobilisation %	24 hours	100	100	80	35	10	0
Number of mobile animals	48 hours/1^st series	0	0	0	3	3	5
Number of mobile animals	48 hours/2^nd series	0	0	0	1	3	5
Number of mobile animals	48 hours/3^rd series	0	0	0	1	3	5
Number of mobile animals	48 hours/4^th series	0	0	0	1	2	5
Number of mobile animals	48 hours/total	0	0	0	6	11	20
Immobilisation %	48 hours	100	100	100	70	45	0
Preliminary test at pH 7.8
Nominal concentration	mg·L^-1	5.0	2.5	1.0	0.5	0.1	Control
Number of mobile animals	24 hours/1^stseries	0	0	1	2	5	5
Number of mobile animals	24 hours/2^nd series	0	0	0	3	3	5
Number of mobile animals	24 hours/3^rd series	0	0	0	3	4	5
Number of mobile animals	24 hours/4^th series	0	0	2	3	3	5
Number of mobile animals	24 hours/total	0	0	3	11	15	20
Immobilisation %	24 hours	100	100	85	45	25	0
Number of mobile animals	48 hours/1^st series	0	0	0	0	3	5
Number of mobile animals	48 hours/2^nd series	0	0	0	1	2	5
Number of mobile animals	48 hours/3^rd series	0	0	0	1	3	5
Number of mobile animals	48 hours/4^th series	0	0	0	0	2	5
Number of mobile animals	48 hours/total	0	0	0	2	10	20
Immobilisation %	48 hours	100	100	100	90	50	0
Main test at pH 7.8
Nominal concentration	mg·L^-1	2.5	1.25	0.63	0.32	0.16	0.08	0.04	Control
Number of mobile animals	24 hours	0	0	0	4	4	5	5	5
	(1^stseries)
Number of mobile animals	24 hours	0	0	0	3	5	5	5	5
	(2^nd series)
Number of mobile animals	24 hours	0	0	1	4	4	5	5	5
	(3^rd series)
Number of mobile animals	24 hours	0	1	1	4	5	5	5	5
	(4^th series)
Number of mobile animals	24 hours/total	0	1	2	15	18	20	20	20
Immobilisation %	24 hours	100	95	90	25	10	0	0	0
Number of mobile animals	48 hours	0	0	0	1	3	5	5	5
	(1^st series)
Number of mobile animals	48 hours	0	0	0	2	4	5	5	5
	(2^nd series)
Number of mobile animals	48 hours	0	0	1	2	3	4	5	5
	(3^rd series)
Number of mobile animals	48 hours	0	0	0	1	4	4	5	5
	(4^th series)
Number of mobile animals	48 hours/total	0	0	1	6	14	18	20	20
Immobilisation %	48 hours	100	100	95	70	30	10	0	0

 

Determined concentrations at the definitive test – pH 7.8
Nominal concentration mg/l	Measured concentration mg/l	Measured concentration mg/l
	0 hours	48 hours
2.5	2.6	2.7
0.16	0.17	0.17

Validity criteria fulfilled:

yes

Conclusions:

The analogue substance was tested for short-term toxicity to daphnia according to OECD 202. Under the experimental conditions the EC50, 48 h is equal to 0.22 mg/l.

Executive summary:

The analogue substance was tested for short-term toxicity to daphnia according to OECD 202. The substance is an ionic dye that shows two different chemical structures depending on the pH of the solution, i.e. the target substance and its carbinol base. Following the OECD 23 , preliminary tests at different pH were performed for assessing the most toxic design for the main test and the relavant test substance concentrations. After the dose range finding the pH equal to 7.8 was selected with a range of concentrations from 0.04 to 2.5 mg/l (nominal concentrations) spacing with a factor of 2. Moreover, due to potential photodegradation occuring at the lowest test concentration, the main test was performed in the dark. The analytical monitoring of the test concentrations was performed by HPLC-MS with measured concentrations within the 20% of the nominal values. Five animals of the species Daphnia Magna per replicate and four replicate per concentrations were tested and after 48h the mobility of the organisms was recorded. Under the experimental conditions the EC50,48 h resulted equal to 0.22 mg/l.

Description of key information

test item	year of the study	target/RA	purity	guidelines	results	notes	Data owner
Basic Green 4 acetate	2022	RA	89% without solvent	OECD 202/Short-term invertebrates	Daphnia Magna, static, pH 7.7-7.8 EC50, 48h ca 0.22 mg/l (nominal, mobility)	analytical monitoring performed via LC-MS
Basic Green oxalate	2007		no details	similar to OECD 202	Daphnia Magna, static LC50, 48h ca 4.65 mg/l (nominal, mobility)		Aquatic ecotoxicity of two dyes and three antiparasitics of importance in aquaculture on employing Daphnia magna.
Basic Green 4 oxalate	1998		no details	no details	Daphnia Magna LC50, 24h, 0.81 mg/l	only final results available	Dystar, report date 1998-05-26
Basic Green 4 oxalate (2:3)	1998		no details	no details	Palaemonetes Kadiakensis LC50, 96h, 1.9 mg/l
Basic Green 4 Chloride	1998		no details	no details	Palaemonetes Kadiakensis LC50, 96h, 0.81 mg/l
Basic Green 4 X	1988		no details	similar to OECD 202	Daphnia Magna, semistatic, pH 8.1-8.4 EC50, 48h, 0.81 mg/l EC100, 48h, 1.1 mg/l (nominal, geometric mean)	Dystar	Dystar, report# 41 A/88, Life cycle-Test mit Wasserfloehen Daphnia magna EC50 Immobilisierung und EC50 Reproduktion.
Basic Green X	1993			not reported-Handbook information	Daphnia Magna EC50, 48h, 0.13-0.29 mg/l	only final results available	Burchmore and Wilinson, pubblication
Basic Green 4 Chloride	1977		no details	no details	ostracod (Cypridopsis sp.) and freshwater prawn (Palaemonetes kadiakensis). LC50, 96h 1.9-3.45 mg/l	only final results available	Bills T. D., Marking L.L, Chandler J.H.J., pubblication

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

0.22 mg/L

Additional information

The substance is an ionic dye that shows two different chemical structure based on the pH of the ecotox medium. After preliminary testing, the short term toxicity to aquatic invertebrates was tested at pH 7.8 in the dark, with HPLC-MS as analytical monitoring.

The OECD202 was performed on analogue substance 1, whose purity is within the boundary composition of the target substance.