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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2022 - 19 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken six times during exposure for the quality measurement of the test solution: three times a weekly measurement on a set of samples from the freshly prepared solution at 0-day, 10-day and 19-day, and the spent solution before the renewal at 3-day, 12-day and 21-day. Spent solution samples were centrifuged (3000 rpm, 10 min) to remove food.

Vehicle:

yes

Remarks:

N,N-dimethylformamide (DMF)

Details on test solutions:

The weighed test sample (0.00500 g) was dissolved in N,N-dimethylformamide (DMF), and it was filled up to 50 mL with DMF to prepare a 100 mg/L test item solution. The required volume of the test item solution was diluted with DMF to prepare a 20.0 mg/L stock solution, which was 10000 times of the test concentration. The required volume of the stock solution was added to dilution water while stirring under the water surface and furthermore stirred, and then used as the test solution. The preparation of the test solution was carried out at every renewal. Final concentration of DMF in vehicle control and exposure group was 100 µL/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Clone A
- Age at study initiation: young daphnids (<24-h old)
- Method of breeding: in-house culturing under the same conditions as used in the test
- Source: University of Sheffield, UK
- Feeding during test: Chlorella vulgaris was fed once a day at ration level between 0.1 and 0.2 mgC (content of organic carbon)/daphnid/day

Test type:

semi-static

Water media type:

freshwater

Remarks:

M4 (Elendt medium) according to OECD TG 211

Limit test:

yes

Total exposure duration:

21 d

Hardness:

245-248 mg CaCO3/L

Test temperature:

19.9-20.3 °C

pH:

7.7-8.3

Dissolved oxygen:

8.1-8.9 mg/L

Nominal and measured concentrations:

Nominal: 0.00200 mg/L
Measured concentrations: time-weighted mean 0.00203 mg/L. The endpoints were expressed based on the nominal concentration. See details in Table 1 in 'Any other information on results incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beaker, covered with transparent plastic lid, containing approx. 80 mL test solution
- Aeration: no
- Renewal rate of test solution: renewal of whole test solution three times every week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium, prepared according to OECD TG 211
- Culture medium different from test medium: no, same medium
- Intervals of water quality measurement: six times during exposure (3 times a weekly measurement of both freshly prepared and spent solutions), dissolved oxygen concentration, pH, water temperature and total hardness were measured. Additionally, continuous temperature monitoring in a separate test vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 17 µmol/m2/s

EFFECT PARAMETERS MEASURED
Survival or mortality of parental daphnids was observed daily. The number of living offspring produced by parent animal was counted daily from the appearance of the first brood to the end of the exposure. For parental animals, the size, swimming behavior, presence of abnormal appearance, distinction of female or male, presence of egg in brood chamber (time to production of first brood), and presence of ephippium or aborted brood were observed daily during exposure. Body length of surviving parent animals was measured after finishing the exposure with a stereomicroscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, limit test.
- Range finding study: a 48-h solubility test was performed (without daphnids), to confirm the solubility of the test item in dilution water (result: 0.00149 mg/L). A first preliminary test with a nominal concentration of 0.0002 mg/L showed that the test substance was stable in a 3-day period. A second preliminary 21-d study with nominal concentrations of 0.00200 and 0.000200 mg/L (geometric spacing factor of 10) was performed. Based on this preliminary test, a nominal concentration of 0.00200 mg/L, control and vehicle control in a semi-static test system was chosen for the definitive study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (acute reference test)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.002 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival, reproduction, body length, time to production of first brood

Duration:

21 d

Dose descriptor:

other: EC10, EC20, EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival, reproduction, body length, time to production of first brood

Details on results:

There were no statistically significant differences between the control and the vehicle control with regard to mean cumulative number of living offspring per parent and body length, therefore the pooled control group was used for statistical comparison with the exposure group.

See Table 2 for summary of the results.

Mortality of adult daphnids:
- No mortality was observed in the exposure group, the control group or the vehicle group.

Time to production of first brood:
- All test groups (exposure, control and vehicle control) produced their first brood on day 8.

Mean cumulative number of living offspring:
- The mean cumulative number of living offspring per parent for 21 days in the control, the vehicle control and pooled control were 143, 140 and 141, respectively. The mean cumulative number of living offspring per parent for 21 days in the exposure level was 148, which was 105% of that in the pooled control. This was not statistically significantly different.
- There were no dead offspring or aborted eggs in any of the tested groups.

General condition of parent animal:
- No abnormal appearance or ephippia were observed in any of the tested groups.

Body length of parent animals:
- The mean body length of surviving parent animal in the control, vehicle control and the exposure level were 4.80, 4.73 and 4.80 mm, respectively, and there was no significant difference between the pooled control and the exposure level. The mean body length in the exposure level was 101% of that in the pooled control.

Water quality parameters (pH, oxygen concentration and temperature) were in accordance with the OECD 211 guideline.

Endpoints:
The NOEC values for survival, reproduction, body length and time to first brood production were all ≥0.002 mg/L. EC10, EC20 and EC50 values were not statistically calculated but can be considered to be >0.002 mg/L.

Results with reference substance (positive control):

48-h reference test with potassium dichromate (April 2022). 48-h EC50: 0.70 mg/L (within the stipulated range (mean±2SD: 0.047-1.3 mg/L, n=14) of background data in the test facility)

Reported statistics and error estimates:

Statistical analysis of cumulative number of living offspring per parent animal and the body length of parent animals was performed by Student t-test in Microsoft Excel.

Table 1: measured concentrations of test item in test solutions

Nominal concentration (mg/L)	Measured concentration (mg/L) (Percentage of measured concentration versus nominal concentration, %)
	0-day New	3-day Old	10-day New	12-day Old	19-day New	21-day Old	Time-weighted mean
Control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	-
Vehicle control	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	-
0.00200	0.00215 (107)	0.00205 (102)	0.00204 (102)	0.00200 (100)	0.00199 (99.4)	0.00194 (97.2)	0.00203 (101)

LOQ: 0.000520 mg/L

 

Table 2: Summary of results

Nominal concentration (mg/L)	Mean time to production of first brood (days)	Mean cumulative number of living offspring at 21 days (SD)	Mean cumulative number of dead offspring at 21 days	Mean body length at 21 days in mm (SD)
Control	8.0	143 (11.3)	0	4.80 (0.06)
Vehicle control	8.0	140 (11.1)	0	4.73 (0.11)
0.00200	8.0	148 (13.6)	0	4.80 (0.09)

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The overall NOEC value (based on survival, reproduction, body length, time to first brood production) was ≥0.002 mg/L (nominal concentration).

Executive summary:

A 21-day chronic toxicity test in Daphnia magna (1 daphnid per replicate, 10 replicates per group) was conducted under semi-static conditions with KY-ET at a nominal limit concentration of 0.002 mg/L, including a dilution water control and a vehicle control (DMF).

The measured concentrations during renewal intervals was 99.4-107% of nominal in fresh solutions and 97.2-102% of nominal in spent solutions. Endpoints were based on the nominal concentration. Water quality parameters (pH, oxygen concentration and temperature) were in accordance with the OECD 211 guideline. No mortality occurred in the control, vehicle control or the exposure group. Mean number of living offspring per adult was 143, 140 and 148 for the control, vehicle control and exposure group, respectively. There were no statistically significant effects on survival, reproduction, body length or time to first brood production. No abnormal behaviour was observed in any of the test groups.

Under semi-static conditions, the 21-day overall NOEC value of KY-ET to Daphnia magna based on survival, reproduction, body length and time to first brood production was ≥0.002 mg/L (nominal concentration). The EC₁₀, EC₂₀ and EC₅₀ values for all parameters were >0.002 mg/L (nominal concentration).

The study is considered valid and reliable without restriction.

Description of key information

The overall NOEC value (based on survival, reproduction, body length, time to first brood production) was ≥0.002 mg/L (nominal concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 0.002 mg/L