2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-14 to 2019-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“.

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment No. 23

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 25 mg/L
- Sampling method: For determination of the test item concentrations, samples were taken from the test item solution and from the control at the start and at the end of each renewal period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). A test item suspension was prepared by adding an amount of 0.0405 g test item to 1620 mL test medium (20X AAP medium) in the first renewal period, 0.0401 g test item to 1604 mL test medium (20X AAP medium) in the second renewal period and 0.0404 g test item to 1616 mL test medium (20X AAP medium) in the third renewal period in order to give the loading rate of 25 mg test item/L. This test item solution was handled by ultrasonic bath for approximately 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material.
The test solution was freshly prepared in the testing laboratory at the start of each renewal period.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Species and Strain: Lemna gibba (G3)
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany
- Preculture: 7-10 days (7 days in this study) before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

22.3 – 24.8 °C in the climate chamber
22.9 – 24.1 °C in the test flask

pH:

7.78 - 8.82

Nominal and measured concentrations:

Nominal concentration: 25 mg/L
Measured concentration: 0.305 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
Beakers
- Type: Closed , covered by glass petri dishes.
- Material, size, fill volume: All-glass beakers; total capacity of 400 mL, volume of the test liquid in the vessels was 160 mL.
- Renewal rate of test solution: The test item solution (as well control solution) was renewed twice during the test (on days 3 and 5).
- Initial frond number: The initial frond number in the test cultures was 11. The number of colonies and fronds was identical in each test vessel.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20X AAP Medium was used

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, the pH was adjusted to 7.5 ± 0.1 with 1 N HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: Light intensity between 6500-10000 lux using fluorescent light tubes (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED
At the start of the test, frond numbers in the test vessels were recorded. The number and appearance of fronds of Lemna gibba were determined in each testing vessel during the 168-hour test on the 3rd, 5th and 7th days. No change in plant development, frond size or appearance was noted as well as additional observations of root length, significant features of the test media or other abnormalities. In addition to determinations of frond number during the test, effects of the test item on final biomass were also assessed based on determination of dry weight at the beginning and at the end of the study.

RANGE-FINDING STUDY
- Test concentrations:
100mg/L (WAF)
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP Preliminary Range-Finding Test (see table 1 in section 'Any other information on results incl. tables') and since the analytical method validation showed that the test item is poorly soluble, the main test was performed as a limit test using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 25 mg/L and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.305 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

Change in plant development during the test: No, the plants were healthy, there were no symptoms observed at the 3rd, 5th and 7th day of the experiment.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The date of the last study with reference item 3,5-Dichlorophenol was: 01 - 08 March 2019.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 6.177 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 8.617 mg/L, EydwC50 (7 day, yield based on dry weight): 5.668 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 7.144 mg/L.

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each treatment and at each replicate at the start, on the 3rd, 5th days and at the end of the test.
The doubling time of frond number in the control was calculated.
The average specific growth rates were calculated for the entire test period based on frond numbers and dry weights for each treatment and each parallel.
The yield was calculated based on frond numbers and dry weights for each treatment and each parallel.
Mean value of the dry weight and standard deviation were calculated for three independent frond number samples at the start and for Lemna cultures used in the test in each replicate at the end of the test.
For the determination of the LOEC and NOEC, the calculated growth rate and yield at the test concentration were tested on significant differences to the control value using Independent Samples T-Test (α = 0.05) by SPSS PC+ software program.

Preliminary Range-Finding Test

Table 1: Results of the Preliminary Range-finding Test

Nominal concentrations (mg/L)	Untreated control	WAF (100)
Average number of fronds (day 7)	95.33	86.50
Growth Rates (µ) (0-7d)	0.308	0.294
% Inhibition of µ (0-7d)	-	4.49

Analytical Results

For determination of the test item concentrations, samples were taken from the test concentration level and from the control at the start and at the end of each renewal period. The measured concentration values were not within ± 20 % of the nominal during the experiment, it was below LOQ or not detected. Therefore, test item concentration was calculated as the time-weighted mean of the measured concentrations (according to OECD No. 23). Calculation of the test concentration is given in the table below.

Table 2: Calculation of the test Concentration

Concentration (mg/L)	Measured concentration (mg/L)
	First renewal period		Second renewal period		Third renewal period
	Start	End	Start	End	Start	End
Control (n=6)	<LOQ	not detected	not detected	<LOQ	<LOQ	<LOQ
25 (WAF^*)	1.000**	1.000**	1.000**	0.000***	1.000**	0.000***
	1.000**	1.000**	1.000**	1.000**	1.000**	0.000***
	1.000**	1.000**	1.000**	1.000**	1.000**	0.000***
	1.000**	1.000**	1.000**	1.000**	1.000**	0.000***
	1.000**	1.000**	1.000**	1.000**	1.000**	0.000***
	1.000**	1.000**	1.000**	1.000**	1.000**	0.000***
Mean	1.000	1.000	1.000	0.833	1.000	0.000
Time-weighted mean	0.305

* WAF = water accommodated fraction (OECD No. 23.); loading rate: 25 mg/L nominal

** LOQ = limit of quantification (2 μg/mL), measured as < LOQ, therefore test concentration calculated as LOQ/2 (OECD No. 23.)

*** not detected, therefore test concentration calculated as 0.000 mg/L

Growth rate

No significant inhibition of the average specific growth rate based on frond number and dry weight in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined usingIndependent Samples T-Test(α = 0.05). Accordingly, the NOEC based on growth rate was determined to be 0.305 mg/L, while the LOEC based on growth rate was determined to be> 0.305 mg/L.

Yield

No significant inhibition of the yield based on frond number and dry weight in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined usingIndependent Samples T-Test(α = 0.05). Accordingly, the NOEC based on yield was determined to be 0.305 mg/L, while the LOEC based on yield was determined to be> 0.305 mg/L.

Validity of the Test
The doubling time of frond number in the control was 1.78 days (less than 2.5 days). The validity criterion was within acceptable limit and therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Leuco Sulfur Blue 13 presscake had not any toxic effect on the test system.
The overall 7-day NOEC was determined to be 0.305 mg/L (nominal 25 mg/L).
The overall 7-day LOEC was determined to be > 0.305 mg/L (nominal > 25 mg/L).
All validity criteria were met. The results are based on the time-weighted mean of the measured test item concentrations.

Executive summary:

The toxicity of the test item on the freshwater aquatic plant Lemna gibba was assessed in a study according to OECD guideline 221. Exponentially growing cultures of Lemna gibba were exposed to the test item over a period of 7 days in a semi-static system under defined conditions. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach (according to OECD No. 23). Growth and inhibition of growth in the test solution of the nominal test item concentration of 25 mg/L were compared with that in the control in a limit test. The effects of the test item compared to the control plant development were demonstrated by the changes of average specific growth rates and yield (both calculated on the basis of frond number and dry weight). The analytically measured test item concentration was not within ± 20 % of the nominal during the test period therefore all biological results are based on the time-weighted mean of the measured test item concentrations.

In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item had not any toxic effect on the test system. The overall 7-day NOEC was determined to be 0.305 mg/L (nominal 25 mg/L).
The overall 7-day LOEC was determined to be > 0.305 mg/L (nominal > 25 mg/L). All validity criteria were met.

Description of key information

In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Leuco Sulphur Blue 13 presscake had not any toxic effect on the test system.

The overall 7-day NOEC was determined to be 0.305 mg/L (nominal 25 mg/L).

The overall 7-day LOEC was determined to be > 0.305 mg/L (nominal > 25 mg/L).

All validity criteria were met. The results are based on the time-weighted mean of the measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.03 mg/L

Additional information

The toxicity of the test item on the freshwater aquatic plant Lemna gibba was assessed in a study according to OECD guideline 221. Exponentially growing cultures of Lemna gibba were exposed to the test item over a period of 7 days in a semi-static system under defined conditions. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach (according to OECD No. 23). Growth and inhibition of growth in the test solution of the nominal test item concentration of 25 mg/L were compared with that in the control in a limit test. The effects of the test item compared to the control plant development were demonstrated by the changes of average specific growth rates and yield (both calculated on the basis of frond number and dry weight). The analytically measured test item concentration was not within ± 20 % of the nominal during the test period therefore all biological results are based on the nominal and on the time-weighted mean of the measured test item concentrations.

In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item had not any toxic effect on the test system. The overall 7-day NOEC was determined to be 0.305 mg/L (nominal 25 mg/L).
The overall 7-day LOEC was determined to be > 0.305 mg/L (nominal > 25 mg/L). All validity criteria were met.