4,4'-methylenebis(2,6-xylidine)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-12-11 to 2018-02-14

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

2017-01-10

Specific details on test material used for the study:

Not applicable.

Analytical monitoring:

yes

Remarks:

The purpose of the analytical study was to determine the actual concentrations of 4,4'-Methylenebis(2,6-dimethylaniline) by HPLC-DAD method.

Details on sampling:

The purpose of the analytical study was to determine the actual concentrations of 4,4'-Methylenebis(2,6-dimethylaniline) by HPLC method in samples taken from the test solutions used during the ecotoxicity test.
Single samples for analysis were taken from the control and all test concentrations at the start and at the end of the test (t=48h).

Vehicle:

no

Details on test solutions:

The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water was added. Then an excess of the test item (approx. 100 mg) was weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1 L of saturated stock solution and thereafter was closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the saturated aqueous phase was extracted via the drain port (the first 100 mL were discarded) and was filtered, since fine particles were observed in the water column. Samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary to obtain the required test concentrations into 200-mL volumetric flasks based on the measured concentration of the stock solution (˜18 mg.L-1). Each flask was inverted several times before filling the test tube (without headspace) to ensure adequate mixing and homogeneity of test solutions. After filling and introduction of daphnids the vessels were sealed immediately. The test solutions in test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

The total water hardness was approximately 250 mg.L-1 (as CaCO3).

Test temperature:

The temperature of the test medium was situated between 20.8 and 20.9°C throughout the test (average value: 20.9°C).

pH:

The pH of this solution was in the range of 6 to 9.
Start t=0h: 8.70 to 8.87
End t=48h: 8.61 to 8.77

Dissolved oxygen:

= 60% of the air-saturation value at the end of the test
Start t=0h: 8.52 to 8.70 mg/L
End t=48h: 8.00 to 8.31 mg/L

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal test concentrations: 1.0, 2.1, 4.2, 8.7 and 18.0 mg test item.L-1 (spaced by a factor of approximately 2.06).

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: None, a Static was performed (stability of the test item was demonstrated in closed and static conditions during the range-finding test)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At the beginning and at the end of the test from all treatment groups.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2.06
- Range finding study: ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal concentrations of 0.1, 1.0, 10.0 mg test item.L-1 and to a concentration close to the solubility limit of the test item in test water (filtered stock solution directly added into test tubes), and to a control.
- Results used to determine the conditions for the definitive study: after 24 hours of exposure, immobilisation rates were 10% at 10 mg.L-1, 40% at 15 mg.L-1 (max solubility); after 48 hours of exposure, immobilisation rate was 90% at 10 mg.L-1 and 100% at 15 mg.L-1. Based on the results of a range-finding test, the following nominal concentrations: .1.0, 2.1, 4.2, 8.7 and 18.0 mg test item.L-1 (this last concentration was expected to be close to the solubility of the test item in test water)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

15.681 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 13.047-19.743

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.885 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: ND

Details on results:

After 48 hours of exposure, immobilisation rates were 0% at 1.0, 2.1 and 4.2 mg test item.L-1, 45% at 8.7 mg test item.L-1, and 100% at 18.0 mg test item.L-1.
Therefore, the highest concentration without effect was 4.2 mg test item.L-1 and the lowest concentration with 100% immobilisation was 18.0 mg test item.L-1.

Results with reference substance (positive control):

On August 17, 2017 (most recent test), the 24h-EC50 was 0.92 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The 24 and 48-hour EC50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat .

Biological results:

Acute immobilisation of daphnids after 24 and 48 hours in the final test

Nominal concentration(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.0	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
2.1	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
4.2	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
8.7	1 2 3 4	5 5 5 5	0 1 0 0	5	2 1 3 3	45
18.0	1 2 3 4	5 5 5 5	3 3 4 3	65	5 5 5 5	100

Analytical results:

The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were reproducible. Indeed, analyses of samples taken from alltest concentrations at the start and the end of the test revealed that test item levels found were stable, with losses of test item < 20%.Moreover, since the concentrations of the test item were satisfactorily maintained within ± 20% of the nominal concentrations throughout the test, the results can therefore be based on nominal values.

Concentrations of the test item (mg.L^-1) in test water - Final test.

Nominal concentration (mg test item.L^-1)	Start (t=0h)		End (t=48h)		Relative loss to initial value (t=0h - t=48h) (%)	Geometric mean measured concentrations
	Meas. Conc (mg.L^-1)	%	Meas. Conc (mg.L^-1)	%		mg.L^-1	%
Control	Abs.	N.A.	Abs.	N.A.	N.A.	N.A.	N.A.
1.0	1.00	100	1.02	102	-2	1.01	101
2.1	2.19	104	2.03	97	7	2.11	100
4.2	4.52	108	4.08	97	10	4.29	102
8.7	8.79	101	8.48	97	4	8.63	99
18.0	17.99	100	17.49	97	3	17.74	99

N.A.: not applicable

Abs. = Absence: concentrations below the LOQ (0.02 mg.L^-1) and the LOD (0.01 mg.L^-1).

% = Percent of expected nominal concentration in test item.

Water quality parameters and environmental conditions throughout the test:

pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:= 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.8 and 20.9°C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

pH-values during the final test

	Nominal concentration(mg test item.L^-1)
	Control	1.0	2.1	4.2	8.7	18.0
Start t=0h	8.70	8.80	8.80	8.82	8.83	8.87
End t=48h	8.61	8.61	8.69	8.72	8.73	8.77

  

Dissolved oxygen concentrations (mg.L^-1) during the final test 

	Nominal concentration(mg test item.L^-1)
	Control	1.0	2.1	4.2	8.7	18.0
Start t=0h	8.70	8.60	8.59	8.54	8.52	8.53
End t=48h	8.31	8.10	8.00	8.07	8.08	8.02

All these dissolved-oxygen concentrations correspond to values > 60% of the air-saturation value.

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [0₂] Dissolved oxygen concentration at the end of the test was = 60% of the air-saturation value in controls and test vessels.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item 4,4'-Methylenebis(2,6-dimethylaniline) to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 value estimated was 8.855 mg test item.L-1.

Executive summary:

A study was performed to assess the acute toxicity of test item 4,4'-Methylenebis(2,6-dimethylaniline) to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266). The criterion measured was the EC₅₀ (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 1.0, 2.1, 4.2, 8.7 and 18.0 mg.L^-1and to a control. The immobility of the daphnids was determined in a closed static 48 -hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine if concentrations of the test item were maintained.

The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). Thus, the evaluation of the effects on daphnids was based on the nominal concentrations. After 48 hours of exposure, immobilisation rates were 0% at 1.0, 2.1 and 4.2 mg test item.L^-1, 45% at 8.7 mg test item.L^-1, and 100% at 18.0 mg test item.L^-1.

The toxic effect of test item4,4'-Methylenebis(2,6-dimethylaniline)to the freshwater invertebrateDaphnia magnawas investigated in a closed static test. Under the experimental conditions andbased on nominal concentrations,the 48-hour EC₅₀value estimated was 8.855 mg test item.L^-1

Results synopsis:

Test: Toxic effect of 4,4'-Methylenebis(2,6-dimethylaniline) to Daphnia magna according to OECD guideline No. 202.

48h-EL50: 8.855 mg/L (95% Confidence limits: 13.047 - 19.743 mg/L)

Description of key information

The toxic effect of test item 4,4'-Methylenebis(2,6-dimethylaniline) to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under the experimental conditions and based on nominal concentrations, the 48-hour EC₅₀value estimated was 8.855 mg test item.L^-1

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.855 mg/L

Additional information

A study was performed to assess the acute toxicity of test item4,4'-Methylenebis(2,6-dimethylaniline)toDaphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphniasp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266). The criterion measured was the EC₅₀(Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution ofthe test item over 48 hoursat the required nominal testconcentrations1.0, 2.1, 4.2, 8.7 and 18.0mg.L^-1and to a control.The immobility of the daphnids was determined in a closed static 48 -hour test by visual observation after 24 and 48 hours. Samples taken fromthe control and all test concentrationswere analysed at the startand the end of the testin order to determine if concentrations of the test item were maintained.

The test item levels were found to be relatively stable throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). Thus, the evaluation of the effects on daphnids was based on the nominal concentrations.After 48 hours of exposure, immobilisation rates were 0% at 1.0, 2.1 and 4.2mg test item.L^-1, 45% at 8.7mg test item.L^-1, and 100% at 18.0mg test item.L^-1.

The toxic effect of test item4,4'-Methylenebis(2,6-dimethylaniline)to the freshwater invertebrateDaphnia magnawas investigated in a closed static test. Under the experimental conditions andbased on nominal concentrations,the 48-hour EC₅₀value estimated was 8.855 mg test item.L^-1