Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

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:

21st July 2020 - 23rd July 2020

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

October 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test material concentration, four samples (4 x 5 mL) were taken from the test material treatments and control group at the start and at the end of the experiment. The samples taken were sent to individual analysis.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
The water-accommodated fraction approach was taken.
A supersaturated solution (100 mg/L nominal loading) was prepared by adding the test material (0.0300 g) to the test medium (300 mL) one day before the start of the experiment.
The test solution was handled by ultrasonic bath for 10 minutes and shaken for a period of 24 hours. The test solution was filtrated through a membrane filter (0.45 μm; Thermo Nalgene membrane).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Straus.
- Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), 2100 Gödöllő, Kotlán S. u. 3. Hungary.
- Breeding: The Daphnia were bred at the Test Facility
- Age of animals at beginning of study: < 24 h
- Sex: Female.
- Acclimatisation: Test animals were bred under similar conditions as tht used during the exposure period (holding water, temperature, background colour). Brood daphnids were maintained in dilution water at the test temperature for 48 hours prior to the start of the test.
- Food: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

218.95 mg/L

Test temperature:

The test temperature was in the range of 21.1 - 21.3°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.8 – 21.8°C.

pH:

8.05 - 8.24

Dissolved oxygen:

7.08 - 7.63 mg/L

Nominal and measured concentrations:

Nominal concentration:100 mg/L
Measured mean concentration: 0.381 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker (50 mL)
- Fill volume: 40 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per untreated control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO Medium, according to OECD 202) was used as dilution water. Separate stock solutions of individual substances were prepared in deionized water: CaCl2 x 2 H2O (11.76 g/L), MgSO4 x 7 H2O (4.93 g/L), NaHCO3 (2.59 g/L), KCl (0.23 g/L). The ISO Medium was prepared by adding 25 mL from each of four stock solutions up to one litre deionised water proportionally.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness).
- Light intensity: 644 lux at start of test.

EFFECT PARAMETERS MEASURED: Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. In addition to immobility, any abnormal behaviour or appearance was reported.

UNTREATED CONTROL PERFORMED: yes (ISO Medium without test material)

RANGE-FINDING STUDY
- Test concentrations: 0, 0.1, 1, 10, 50 and 100 mg/L (WAF)
- Results used to determine the conditions for the definitive study: yes.
In the preliminary range-finding test ten (2 x 5) Daphnids in each test concentration and in the control were exposed for 48 hours. Under the conditions of the preliminary test, the test material had no toxic effect on the daphnids up to the limit of its solubility level in the test medium, achieved via water-accommodated fraction. Therefore, a limit test was performed using only this concentration level and a control in order to demonstrate that the test material is not toxic to Daphnia magna up to at least the saturation concentration (100 mg/L nominal concentration).

Reference substance (positive control):

not required

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.381 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: no effects at the limit of solubility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.381 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: no effects at the limit of solubility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.381 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: no effects at the limit of solubility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: no effects at the limit of solubility

Details on results:

ANALYTICAL RESULTS
The test material was not detected in the untreated control group (i.e. no signal was detected at the start of the experiment and the signal intensities measured for the control samples were below limit of quantification at the end of the experiment). In the treated group the mean measured test material concentration was 0.405 mg/L at the start and 0.358 mg/L at the end of the experiment. The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 0.381 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test material (equivalent to 100 mg/L nominal concentration).

BIOLOGICAL RESULTS
No immobilization was observed either in the control group or the test material treated group.
The 48 h LOEC is higher than the solubility level of the test material in the test medium, which corresponds to the geometric mean measured concentration of 0.381 mg/L. The NOEC value is equal to the geometric mean measured test material concentration of 0.381 mg/L.

Results with reference substance (positive control):

For the evaluation of the quality of the Daphnia clone and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-5406) with reference item potassium dichromate was: 04 – 05 March 2020.
The 24h EC50 value was determined to be 1.12 mg/L (95% conf. limits: 1.00 – 1.76 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Reported statistics and error estimates:

A limit test was performed and no toxic effects were observed. therefore statistical analysis was not necessary. The NOEC and LOEC values were determined directly from the raw data.

Summary of the Biological Endpoints.

Endpoints	Concentration
48 h-NOEC	0.381 mg/L
48 h-LOEC	> 0.381 mg/L

Concentration based on the geometric mean measured concentration of the water-accommodated fraction of test material.

Concentrations Measured during the Experiment

Sample code, WAF mg/L	Measured T74S concentrations
	Start (July 21, 2020)	End (July 23, 2020)
	Prepared samples, mg/L	Test vessels, mg/L
100	0.421	0.371
	0.364	0.379
	0.464	0.314
	0.371	0.366
Mean with the 95% confidence intervals	0.405±0.075	0.358±0.046
Control (n=4)	not detected	< LOQ

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48 h NOEC was 0.381 mg/L. The 48 h LOEC was > 0.381 mg/L.

Executive summary:

The acute toxicity of the test material to Daphnia magna was assessed in a GLP study, conducted according to OECD Guideline for Testing of Chemicals, Section 2, No. 202, Council Regulation (EC) No 440/2008, Annex Part C, C.2, EPA Ecological Effects Test Guidelines, OCSPP 850.1010 and Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment No. 23.

Acute toxicity of the test material was assessed on Daphnia magna in a 48-hour acute immobilisation test. Young Daphnia were exposed to aqueous test media containing the test material for 48 hours in a static test.

A limit test was performed in which, the test animals were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

During the study, healthy young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 40 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. The immobilisation of the test animals was observed 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of the experiment. All measured values remained within the acceptable ranges.

No immobilisation was observed either in the test material treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Immobilisation was 0.0 % in the control group and the dissolved oxygen concentration during the test in control and test vessels was in the range of 7.08-7.63 mg/L, therefore all validity criteria were within acceptable limits and fulfilled by the test. Additionally no abnormal behavior or appearance of test animals was detected.

Under the conditions of the study the 48 h NOEC was 0.381 mg/L. The 48 h LOEC was > 0.381 mg/L.

Description of key information

Under the conditions of the study the 48 h NOEC was 0.381 mg/L. The 48 h LOEC was > 0.381 mg/L.

Additional information

The acute toxicity of the test material to Daphnia magna was assessed in a GLP study, conducted according to OECD Guideline for Testing of Chemicals, Section 2, No. 202, Council Regulation (EC) No 440/2008, Annex Part C, C.2, EPA Ecological Effects Test Guidelines, OCSPP 850.1010 and Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment No. 23. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Acute toxicity of the test material was assessed on Daphnia magna in a 48-hour acute immobilisation test. Young Daphnia were exposed to aqueous test media containing the test material for 48 hours in a static test.

A limit test was performed in which, the test animals were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

During the study, healthy young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 40 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile. The immobilisation of the test animals was observed 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of the experiment. All measured values remained within the acceptable ranges.

No immobilisation was observed either in the test material treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Immobilisation was 0.0 % in the control group and the dissolved oxygen concentration during the test in control and test vessels was in the range of 7.08-7.63 mg/L, therefore all validity criteria were within acceptable limits and fulfilled by the test. Additionally no abnormal behavior or appearance of test animals was detected.

Under the conditions of the study the 48 h NOEC was 0.381 mg/L. The 48 h LOEC was > 0.381 mg/L.