A mixture of: 2,4 -bis(N'-(4-methylphenyl)ureido)toluene; 2,6 -bis(N'-(4-methylphenyl)ureido)toluene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July to 27 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document, No.23 "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"

Version / remarks:

Second Edition, February 2019

Deviations:

not applicable

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Annex 5 corrected July 28, 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(2-methyl-1,3-phenylene)bis[3-(4-methylphenyl)urea]

Cas Number:

1187573-14-3

Molecular formula:

C23H24N4O2

IUPAC Name:

1,1'-(2-methyl-1,3-phenylene)bis[3-(4-methylphenyl)urea]

Constituent 2

Reference substance name:

1,1'-(4-methyl-1,3-phenylene)bis[3-(4-methylphenyl)urea]

Cas Number:

54390-87-3

Molecular formula:

C23H24N4O2

IUPAC Name:

1,1'-(4-methyl-1,3-phenylene)bis[3-(4-methylphenyl)urea]

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Lot Number: KY-ET 20181112
Purity of KY-ET > 99%
Impurity unknown component < 1%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: limit concentration
- Sampling method: approx 150 mL at the start of exposure sampled from the preparation container; approx 180 mL at end of exposure taken out with equal volume of the test solution from the test vessels in each test level
Algae were removed by centrifugation (3000 rpm, 10 min) from the samples taken at the end of exposure

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test sample (0.100 g) and medium (1000 mL) were mixed to produce 100 mg/L as the nominal concentration, and the mixture was stirred with a magnetic stirrer for 48 hours to prepare the test solution, and the test solution was divided into each test vessel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): at the start of exposure, test solution was a white suspension, and it was a greenish white suspension at the end of exposure due to the algal growth. The solution of control was colorless and clear at the start of exposure, and it was green at the end of exposure due to the algal growth.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source (laboratory, culture collection): laboratory
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Passage cultured under sterile conditions in the test facility

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

No information

Test temperature:

22.3 - 22.5 ºC

pH:

7.7 - 8.3

Dissolved oxygen:

No information

Nominal and measured concentrations:

WAF prepared at 100 mg/L gave a measured concentration of 0.00145 mg/L at the start and 0.00221 mg/L at the end of the exposure. The time weighted average was 0.0018 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flask (with gas-permeable Silicosen); 100 mL/test vessel
- Aeration: not indicated
- Initial cells density: The algae were counted in pre-culture incubated under the same conditions as the test for 3 days with a Particle Counter (Model COULTER Z2) and were added to test vessels to bring the initial cell concentration of 0.75 x 10^4 cells/mL. Related chlorophyll fluorescence value was 62.
- Control end cells density: 9400 (mean of 6 measurements)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM and TEST MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: Continuous illumination provided by alternative LED light to fluorescent lamp
- Light intensity and quality: 88-90 µmol/m^2 sec

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: biomass via chlorophyll fluorescence value every 24 hours after the start of exposure. The blank correction was conducted by measuring the value of blank solution in each test level.
- Chlorophyll measurement: Spectrophotofluorometer; Model RF-6000 (Shimadzu, Instrument No. PF-005)
- As stated in the OECD 201, alternative techniques like chlorophyll fluorescence can be used if a satisfactory correlation with biomass can be demonstrated over the range of biomass occurring in the test. The conversion from biomass to chlorophyl value is only reported at the start of the test (0.75 x 104 cells/mL (counting) = 62 Chlorophyl fluor value). The laboratory periodically checks the consistency of cell concentration and chlorophyll fluorescence values. The chlorophyll fluorescence values measured during the exposure period in this study are in the range where a correlation between cell density and chlorophyll fluorescence values has been observed. Although no direct conversion was made, even densities higher than those at the start of the exposure were sufficient.

TEST CONCENTRATIONS
- Test concentrations: Limit test at around the solubility in medium
- Results used to determine the conditions for the definitive study: The test concentration was decided from the results of the preliminary study. See attached

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

This study was conducted as a limit test in order to estimate the effect on the test organisms around the solubility of test item in medium.
As the results of preliminary tests, it was confirmed that the test solution containing the undissolved test item had no physical effect on the test organism. Therefore, the definitive study was conducted in suspension. As a result, no adverse effect on the test organisms was not found in the definitive study. Therefore, it was decided that the test item had no adverse effect on the test organism around the solubility of test item in medium. The test item concentration in the test solution at the end of exposure was higher than at the start of exposure. However, the TWA measured concentration of test solution (0.00180 mg/L) was around the solubility of test item in medium (0.00166 mg/L : measured value in preliminary study).

Results with reference substance (positive control):

An algae growth inhibition study of potassium dichromate with the test organisms was periodically conducted. The data (March 26 to March 29, 2019) with alternative LED light to fluorescent lamp is:
ErC50 (0-3d) = 1.1 mg/L
This value was within the stipulated range [mean +/- 2S.D. (0.61-1.4 mg/L (n=32, result with fluorescent lights)] to background data in this test facility.

Any other information on results incl. tables

Preliminary tests were conducted:

1. Solubility of test item in medium: WAF prepared from 100 mg/L, stirred for 48 hrs and after settlement for 1 hour, the middle layer was taken out and filtered with a membrane filter (PTFE, 0.45 µm pore size, Merck) by suction (filter was pre-treated with the test solution of about 50 mL). Measured concentration of soluble test item in medium was 0.00166 mg/L (mean of 3 values).

2. Preliminary study of effect on test organism: WAF prepared from 100 mg/L and stirred for 48 hrs, used as such as test solution. Preliminary study performed in triplicate, measurement by chlorophyll fluoresence. 72h-growth rate inhibition was 0.91%. The concentration of the test item in the test solutions was measured (at the start 0.002 mg/L, at the end 0.00255 mg/L). In addition, the concentration of the test item in the test solution without algae was also measured in order to confirm whether the test item was absorbed to algae or not (at the start 0.002 mg/L, at the end 0.00181 mg/L). No absorption to algae was obvious.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see overall remarks

Conclusions:

The EC50 based on growth rate was > 0.0018 mg/L. The NOEC based on growth rate was 0.0018 mg/L. No effects were observed at the maximum solubility of the substance in medium.

Executive summary:

An algae growth inhibition test was conducted in accordance with OECD 201 and GLP.
The organisms were exposed to a supersaturated solution (WAF of 100 mg/L after 48 hours stirring was used as such) and no effects on growth were observed. Analytical support showed that the test concentrations were in the neighborhood of the maximum solubility in medium (determined in preliminary study from the filtered WAF as 1.66 µg/L). The effect of conducting the test with a supersaturated solution was checked in the preliminary test: there was no effect of the supersaturation on the growth of algae.
The EC50 based on growth rate was > 0.0018 mg/L. The NOEC based on growth rate was 0.0018 mg/L. No effects were observed at the maximum solubility of the substance in medium.