Phenol, 4-[[4-(dimethylamino)phenyl]azo]-, 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:

21 February 2018 - 03 July 2018

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 (EC) No 761/2009, Annex VI, C.26

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentrations, two series of samples were taken from the testing concentrations and control at the start (prior to distributing of the test item solutions to the test vessels) and at the end of each renewal period. The first series of the samples taken was sent to individual analysis.
- Sample storage conditions before analysis: At a temperature below -15 °C.

Vehicle:

no

Details on test solutions:

- Method: Preparation of test solutions was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). Appropriate amounts of test item were dissolved in the dilution water (20X AAP medium) separately for each concentration level in order to give the appropriate loading rates of each test concentrations. The resulting test solutions was handled by ultrasonic bath for 10 minutes thereafter stirred rigorously for a period of approximately 24 hours to achieve an equilibrated concentration. These solutions were then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Untreated control group (20X AAP medium without test item) was included in the test. As the test item is proved not to be stable for 7 days in 20X AAP Medium, the test and control solutions were renewed twice during the test (on days 3 and 5). The test solutions and the control medium were distributed into the test vessels. The volume of the testing liquid was 160 mL/vessel. Sterile 400 mL glass beakers covered by glass petri dishes were used as testing vessels in the test groups and in the control.
- Controls: Positive control with the reference substance 3,5-dichlorophenol, negative control without the test item and the reference substance

Test organisms (species):

Lemna gibba

Details on test organisms:

- Species and strain: Lemna gibba (G3)
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany
- Justification of strain: Lemna gibba is a fast-growing species. The small size, the simple structure, vegetative reproduction and the short generation time makes it suitable and convenient for culturing and testing.
- Preculture: 7 to 10 days before testing, sufficient colonies were transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
- Initial frond number: The initial frond number in the test cultures was 11, and individual colonies consisted of 2 to 4 visible fronds. The number of colonies and fronds was identical in each test vessel.
- Replicates and controls: The test included three replicates at each test concentration and six replicates in the control group.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

24 +/- 2 °C

pH:

The pH was checked at the start and at the end of each renewal period of the test in the test concentration and the control. The pH of the control medium did not increase by more than 1.5 units during the test. It ranged from 7.67 to 8.58 for fresh test and control solutions and from 8.37 to 8.68 for spent test and control solutions.

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal loading rate: 0.1, 1, 10, 50 and 100 mg/L

Details on test conditions:

- Temperature: The cultures were maintained at a temperature in the range of 24 +/- 2 °C, which was checked at the beginning of the study and every 24 hours (in a surrogate flask filled with water in the climate chamber). In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber during the experimental period.
- pH: The pH was checked at the start and at the end of each renewal period in the test media of the test concentrations and the control. The pH of the control medium did not increase by more than 1.5 units during the test.
- Light intensity: The test vessels were placed randomly on a tray, where the continuous light intensity was between 6500 and 10000 lux. It was reached by using cool-white fluorescent light tubes. The differences in light intensity over the test area did not exceed +/- 15 % provided equal conditions for each test vessel. The light intensity was checked and recorded at least once during the test.
- Test units: All-glass beakers covered by glass petri dishes, with total capacity of approx. 400 mL were used. The volume of the test liquid in the vessels was 160 mL. Each test unit was uniquely identified with at least study code, test item concentration (or control) and replicate.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on frond number and dry weight

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on frond number and dry weight

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on frond number and dry weight

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on frond number and dry weight

Details on results:

PRELIMINARY STUDY
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. In the preliminary range-finding test a test item stock solution was prepared by adding 0.07 g of test item into 700 mL dilution water (20X AAP medium) to get the nominal concentration of 100 mg test substance /L. The stock solution was handled in ultrasonic bath for 10 minutes, stirred rigorously for 24 hours then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The further test item solutions of 0.1, 1, 10 and 50 mg/L were prepared by the appropriate dilution of the stock solution. Untreated control ran parallel in the test. The pre-test was performed with two replicates per test item treated group (containing 11 fronds in total per test vessel) for a period of 7 days. A concurrent control was run (with three replicates). The preliminary range-finding test was not performed in compliance with the GLP-Regulations.

Results with reference substance (positive control):

For the evaluation of the quality of the Lemna gibba cultures and the experimental conditions, 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study number: 392-221-3267) with reference item 3,5-dichlorophenol was: 08 – 15 September 2017.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 3.980 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 6.053 mg/L, EydwC50 (7 day, yield based on dry weight): 2.906 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.206 mg/L.

Reported statistics and error estimates:

Statistical analysis of the results was performed by analysis of variance (ANOVA) and appropriate statistical procedure (α = 0.05) by SPSS PC+ software. The data were checked for homogeneity of variance by Levene’s test..

Table: Results of the preliminary range-finding test

Nominal concentrations [mg/L]	Untreated control	0.1	1	10	50	100
Average number of fronds (day 7)	107.33	106.00	108.50	108.00	104.50	104.00
Growth Rates (µ) [0-7d]	0.325	0.323	0.327	0.326	0.322	0.321
% Inhibition of µ [0-7d]	-	0.59	-0.48*	-0.28*	1.17	1.39

* Negative value means growth increase

Table: Calculation of Exposure Concentration

Nominal concentration (mg/L)	Measuredconcentration(mg/L)						Mean measuredconcentration (mg/L)
	1^strenewal period		2^ndrenewal period		3^rdrenewal period
	Start	End	Start	End	Start	End
Control	-	-	-	-	-	-	-
100 (WAF^*)	0.52	0.29	0.52	0.27	0.59	0.32	0.40

Table: Growth rates (µ) and Percentage Inhibition ofµbased on Frond Number

Concentration (mg/L)		Growth rate (µ) and % inhibition ofµ
Nominal	Mean Measured	0–168 h(based on frond number)
		µ	% Iµ
Control	-	0.307	-
100 (WAF*)	0.40	0.304	0.91

*   water accommodated fraction (OECD No. 23.)

Growth rates (µ) and Percentage Inhibition ofµbased on Dry Weight

Concentration (mg/L)		Growth rate (µ) and % inhibition ofµ
Nominal	Mean Measured	0–168 h(based on dry weight)
		µ	% Iµ
Control	-	0.35564	-
100 (WAF*)	0.40	0.35061	1.41

Table: Yield (y) and Percentage Inhibition of Yield based on Frond Number

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Mean Measured	0–168 h(based on frond number)
		y	% Iy
Control	-	83.17	-
100 (WAF*)	0.40	81.33	2.20

*   water accommodated fraction (OECD No. 23.)

Table: Yield (y) and Percentage Inhibition of Yield based on Dry Weight

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Mean Measured	0–168 h(based on dry weight)
		y	% Iy
Control	-	0.00544	-
100 (WAF*)	0.40	0.00523	3.95

Table: Symptoms, Changes of Lemna gibba Plants Observed during the Test

Concentration (mg/L)		3^rdday of the experiment		5^thday of the experiment		7^thday of the experiment
Nominal	Mean measured	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	-	–	–	–	–	–	–
100 (WAF*)	0.40	–	–	–	–	–	–

Validity criteria fulfilled:

yes

Conclusions:

In a Growth Inhibition Test with Lemna gibba according to OECD 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26 the test item had no toxic effect at aquatic saturation (i.e. limit test concentration). The overall LOEC from growth rate and yield is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.4 mg/L, based on water accommodated fraction of total product.

Executive summary:

The acute toxicity of the test item to Lemna gibba was determined in a 7-d semi-static test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26 using the WAF method (according to OECD Series on Testing and Assessment No. 23). The test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba; the overall LOEC is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.4 mg/L, based on water accommodated fraction of the total product.

Description of key information

In a Growth Inhibition Test with Lemna gibba according to OECD 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26 the test item had no toxic effect at aquatic saturation (i.e. limit test concentration). The overall LOEC from growth rate and yield is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.4 mg/L, based on water accommodated fraction of total product.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.4 mg/L

Additional information

In a Growth Inhibition Test with Lemna gibba, the effects of the test item is determined according to OECD Guideline 221 using the WAF method (according to OECD Series on Testing and Assessment No. 23). The study is conducted in a semi-static system over a period of 7 days with nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L. Based on the result the LOEC and NOEC were determined to be > 100 mg/L and 100 mg/L (nominal) respectively. All validity criteria were met and therefore the study can be considered as valid. All biological results are based on the mean measured test item concentrations of the total product.