Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine, sodium polysulfide and sodium metabisulfite

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

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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, No. 23 (Second Edition), ENV/JM/MONO(2000)6/REV1, 8 February 2019

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the determination of the test item concentration, samples were taken from the test item treated group and from the control group (4 x 25 mL at the start and 4 x 25 mL at the end of the study). The samples were analysed using spectrophotometric method at 575 nm wavelength.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The preparation of test item solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). 0.0303 g of test item was suspended in 303 mL dilution water (ISO medium) in order to give the loading rate of 100 mg test item/L.
The solution was handled by ultrasonic bath for approx. 10 minutes thereafter was stirred for a period of 24 hours to achieve equilibrated concentration. The solution was then be filtrated through a membrane filter (0.45 μm; Thermo NALGENE® membrane*) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of test animals (start of experiment).

*Lot number: 1247803
Expiration date: January 2024

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
- Age of parental stock (mean and range, SD): Less than 24 hours old
- Sex: Female
- Feeding during test: no

ACCLIMATION
- Acclimation period: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

Test type:

static

Water media type:

other: ISO medium

Limit test:

yes

Total exposure duration:

48 h

Hardness:

249 mg/L (as CaCO3)

Test temperature:

18°C - 22°C

pH:

7.81 - 8.04

Dissolved oxygen:

7.47 - 8.59 mg/L

Salinity:

Not Applicable

Conductivity:

No data

Nominal and measured concentrations:

Main test:
Nominal: 100 mg/L
Mean measured: 27.95 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: glass, 50 mL, 20 mL, 40 mL.
- Aeration: No
- Renewal rate of test solution: Based on the results obtained during analytical method validation (Study number: 805-100-3260) the test item is stable for the duration of 48 hours in ISO Medium, therefore a static test was performed.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium, according to OECD 202

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness.
- Light intensity: 757 lux

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 are considered to be immobile. In addition to immobility any abnormal behaviour or appearance is reported.

RANGE FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, one non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
In static non-GLP preliminary test ten daphnids (divided into 2 replicates) in each test concentration and control were exposed for 48 hours.
The stock solution used in the test was prepared by mechanical dispersion. 0.0552 g of test item was dissolved in 552 mL dilution water (ISO medium) in order to obtain the test concentration of 100 mg/L. This stock solution was placed into an ultrasonic bath for approximately 10 minutes. The further test solutions were prepared by appropriate dilution of this stock solution.
The concentration levels used and results (48-h) of the preliminary range-finding test are summarised in the table 1 in the section ‘Any other information on materials and methods incl. tables’.
Based on the results of the non-GLP Preliminary Range-Finding Test, the main test was a limit test performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 100 mg test item/L and a concurrent control group.
The analytically measured test item concentrations were not within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the measured concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 27.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: The test item had no toxic effect up to concentration of 27.95 mg/L (mean measured based on total product)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

27.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on mean measured concetration of total product

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 27.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Based on mean measured concentration of total product

Details on results:

Validity of study:
Immobilisation was not observed in the control group and the dissolved oxygen concentration in control and test vessels was in the range of 7.47- 8.59 mg/L during the experiment (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results:
The quantification of the test item in the test solutions was performed by a previously validated analytical method. Samples were taken from the test concentration and the control at the start and at the end of the experiment and analysed by spectrophotometric method.
The mean measured concentration of the test item in the test solution was 28.0 mg/L at the start and 27.9 mg/L at the end of the test. The measured concentrations of the tested substance were not within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the mean measured concentration value. Test item was below Limit of Detection (LOD) in the control group.

Biological Results:
No immobilisation or any abnormal behaviour of test animals was observed in the control group and no immobilization or any abnormal behavior of the test animals was observed at the measured test concentration of 27.95 mg/L after 48 hours of exposure.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- Relevant effect levels: 24h EC50 = 1.58 mg/L (95% conf. limits: 1.34 – 1.85 mg/L)

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 of the test item were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Blue TBR had no toxic effects on the mobility of Daphnia up to the concentration of 27.95 mg/L (mean measured based on total product). The 48-h NOEC was determined to be 27.95 mg/L and the 48-h LOEC and the EC50 values were determined to be > 27.95 mg/L (mean measured) (directly from the raw data). All validity criteria were met. The results are based on the measured test item concentration (27.95 mg/L).

Executive summary:

The test substance was assessed using Daphnia magna in a 48-hour Acute Immobilisation Test according to OECD TG 202. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours. Based on results obtained in a non-GLP preliminary experiment a limit test was performed in which, the test animals were exposed to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration) for 48 hours in the test medium 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.

A static test was performed as the test item was previously shown to be stable in the ISO medium for 48 hours. The analytically measured test item concentration did not remain within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the geometric mean of the measured concentration (27.95 mg/L).

Healthy, young female daphnids with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to 100 mg/L (nominal) test item and in the control (ISO Medium). The immobilisation of the test animals was recorded 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges.

No immobilisation or any abnormal behaviour of test animals was observed in the control group and no immobilization or any abnormal behavior of the test animals was observed at the measured test concentration of 27.95 mg/L after 48 hours of exposure. All validity criteria were met and therefore the study can be considered as valid.

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Blue TBR had no toxic effects on the mobility of Daphnia up to the concentration of 27.95 mg/L (measured). All validity criteria were met. The NOEC value is equal to the mean measured test item concentration (27.95 mg/L) based on total product.

 

 

Description of key information

In the acute 48-hour Daphnia magna immobilisation study performed with the WAF (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L (total product, i.e. Blue TBR), the obtained results showed that the test item had no toxic effect on the test system. The 48-hour acute toxicity test on Daphnia magna, showed that the test item Blue TBR had no toxic effects on the mobility of Daphnia up to the concentration of 27.95 mg/L (measured). All validity criteria were met. The NOEC value is equal to the mean measured test item concentration (27.95 mg/L) based on total product.

Additional information

The test substance was assessed using Daphnia magna in a 48-hour Acute Immobilisation Test according to OECD TG 202. Young Daphnia were exposed to aqueous test media containing the test item Blue TBR for 48 hours.

Based on results obtained in a non-GLP preliminary experiment a limit test was performed in which the test animals were exposed to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration) for 48 hours in the test medium 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)).

A static test was performed as the test item was previously shown to be stable in the ISO medium for 48 hours. The analytically measured test item concentration did not remain within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the geometric mean of the measured concentration (27.95 mg/L).

Healthy, young female daphnids with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to 100 mg/L (nominal) test item and in the control (ISO Medium). The immobilisation of the test animals was recorded 24 and 48 hours after treatment. Environmental conditions were recorded. All measured values remained within acceptable ranges.

No immobilisation or any abnormal behaviour of test animals was observed in the control group and no immobilization or any abnormal behavior of the test animals was observed at the measured test concentration of 27.95 mg/L after 48 hours of exposure. All validity criteria were met and therefore the study can be considered as valid.

In this 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item Blue TBR had no toxic effects on the mobility of Daphnia up to the concentration of 27.95 mg/L (measured). All validity criteria were met. The NOEC value is equal to the mean measured test item concentration (27.95 mg/L) based on total product.