Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July - 17 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

23rd March, 2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“. Dated 24 August 2009

Version / remarks:

24 August 2009

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

EC Number:

291-833-0

EC Name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

Cas Number:

90480-94-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-aminophenol with 4-​(2-​naphthalenylamino)​phenol and sodium polysulfide

Test material form:

solid

Details on test material:

Test item: Leuco Sulphur Black 11
Appearance: black solid
CAS No: 90480-94-7
EC No: 291-833-0

Specific details on test material used for the study:

Date of production: 26 October 2016
Expiry date: 26 October 2021

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Based on the results of the preliminary experiment, the nominal concentration of 100 mg/L was investigated in the main study (limit test).
The measured recovery of the test item ranged from 0.750 to 0.776 mg/L for fresh solutions and from 0.617 to 0.661 mg/L for spent solutions. Concentrations
deviated more than 20 % from the nominal at the start and at the end of the renewal periods, therefore the geometric mean of the measured concentrations were calculated to determine exposure concentration.

Test solutions

Vehicle:

yes

Details on test solutions:

Method: 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). The following test item amounts were suspended in the appropriate amount of the dilution water (20X AAP Medium): 0.1509 g in 1509 mL in the first and third renewal periods and 0.1501 g in 1501 mL in the second renewal period. The loading rate of the test solutions was 100 mg test item/L at the start of each renewal period. The test suspension was handled in ultrasonic bath for 10 minutes thereafter stirred rigorously for a period of 24 hours to achieve an equilibrated test concentration. This test suspension 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 just before introduction of the test organisms at the start of each renewal period.
- Controls: Negative control (20X AAP medium was used as untreated control), positive control (with the reference substance 3,5-dichlorophenol)

Test organisms

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 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. The number of colonies and fronds was identical in each test vessel.
- Replicates and controls: The test included six replicates at test concentration and six replicates for the control group.
 

Study design

Test type:

semi-static

Water media type:

other: 20X AAP Medium

Limit test:

yes

Total exposure duration:

7 d

Remarks on exposure duration:

The study was performed over an exposure period of 7 days in 20X AAP Medium and at temperature in the range 24 ± 2 ºC, in six replicates, each containing 11 fronds/test vessel initially.

Test conditions

Test temperature:

The cultures were maintained at a temperature in the range of 24 ± 2 ºC (22.1 – 24.4°C in the climate chamber and 22.8 – 23.1 °C in the test vessels), 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 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 7.75 to 8.43 for fresh test and control solutions and from 8.46 to 8.68 for spent test and control solutions.

Nominal and measured concentrations:

Based on the results of the non-GLP Preliminary Range-Finding Test, the main test was a limit test performed using the WAF method including a loading rate of 100 mg/L and a concurrent control group.

Preparation of test solution was performed using the WAF method. The following test item amounts were suspended in the appropriate amount of the dilution water (20X AAP Medium): 0.1509 g in 1509 mL in the first and third renewal periods and 0.1501 g in 1501 mL in the second renewal period. The loading rate of the test solutions was 100 mg test item/L at the start of each renewal period.

Details on test conditions:

The test containers were kept during the test in a climate chamber with controlled environmental conditions.

Test type: Semi-static test. Based on the results obtained during analytical method validation (Study number: 805-100-3122) the test item is not stable for the duration of 7 days in 20X AAP medium. Therefore the test and control solution were renewed twice during the test (on days 3 and 5).
Temperature: The cultures were maintained at a temperature in the range of 24 ± 2 ºC (22.1 – 24.4°C in the climate chamber and 22.8 – 23.1 °C in the test vessels), 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 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 7.75 to 8.43 for fresh test and control solutions and from 8.46 to 8.68 for spent test and control solutions.
Light intensity: The test vessels were placed randomly on a tray, illuminated continuously at a light intensity between 6500-10000 lux (measured 7973 – 8123 lux, SD: 47 lux) using fluorescent light tubes (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the start of the test at the position occupied by test containers. The differences in light intensity between the measurement points (i.e. position of fronds) did not exceed ± 15 % and therefore provided equal conditions for each test culture.
Test units: All-glass beakers (total capacity of 400 mL) were used and were covered by glass petri dishes. The volume of the test liquid in the vessels was 160 mL. Each test unit was uniquely identified with study code, test item concentration (and control) and replicate.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.703 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.703 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 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 0.703 mg/L, based on water accommodated fraction of the total product

Effect concentrations 3

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.703 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

other: yield

Remarks on result:

other: no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba

Effect concentrations 4

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.703 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

other: yield

Remarks on result:

other: 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 0.703 mg/L, based on water accommodated fraction of the total product

Details on results:

Validity of the Test

The doubling time of frond number in the control was 1.80 days (less than 2.5 days). The validity criterion was within acceptable limit and therefore the
study can be considered as valid.

Environmental Conditions

Temperature: 22.1 – 24.4°C in the climate chamber, 22.8 – 23.1°C in the test vessels
pH values: 7.75 - 8.68
Light intensity: 8050 lux (mean value); SD: 47 lux

Analytical Results

A saturated test item solution (limit concentration) and a concurrent control were included in the main test. The concentration of the test item was analytically determined (based on total product) at the start and at the end of each renewal period. The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the lowest concentration of the calibration curve). In the treated group mean of the initial measured concentration was 0.776 mg/L, 0.775 mg/L and 0.750 mg/L at the start of the first, second and third renewal period, respectively. The test item concentration was measured to be 0.658 mg/L, 0.617 mg/L and 0.661 mg/L at the end of the first, second and third renewal period, respectively. The exposure concentration was calculated as the geometric mean of the start and end values during the study and determined to be 0.703 mg/L (equivalent to 100 mg/L nominal concentration) according to OECD Series on Testing and Assessment No. 23.

Biological Results

All biological results are based on the calculated mean concentration (based on total product).

Growth Rate

The average specific growth rate was not statistically significantly different from the control group at the test concentration of 0.703 mg/L (calculated) based on frond number and dry weight (Independent Sample T-test, 2-tailed, =0.05). The 168-h NOEC was determined to be 0.703 mg/L, while the 168-h LOEC was determined to be greater than 0.703 mg/L (solubility level of the test item in the test medium).

Yield

The yield was not statistically significantly different from the control group at the test concentration of 0.703 mg/L (calculated) based on frond number and dry weight (Independent Sample T-test, 2-tailed, =0.05). The 168-h NOEC was determined to be 0.703 mg/L, while the 168-h LOEC was determined to be higher than 0.703 mg/L (solubility level of the test item in the test medium).

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: 392-221-3834) with reference item 3,5-dichlorophenol was: 25 May – 01 June 2018. Endpoints of this study were: ErfnC50 (7 day, growth rate based on frond numbers): 7.831 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 7.722 mg/L, EyfnC50 (7 day, yield based on frond number): 5.000 mg/L, EydwC50 (7 day, yield based on dry weight): 5.609 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 replicate.
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 colonies used in the test in each replicate at the end of the test.
For the determination of the LOEC and NOEC, the calculated mean growth rate and yield at the calculated test concentration were tested on significant differences to the control value using Independent Sample T-test by SPSS PC+ software program (α = 0.05). The data were checked for homogeneity of variance by Levene’s test.

Any other information on results incl. tables

Summary of the Biological Endpoints

Endpoints for response variables (0-7 d)		Concentration(mg/L) (based on mean measured concentration)
Growth Rate (based on frond number and dry weight)	NOEC	0.703
	LOEC	> 0.703
Yield (based on frond number and dry weight)	NOEC	0.703
	LOEC	> 0.703

Results of the Preliminary Range-finding Test

Nominal concentrations (mg/L)		Untreated control		100 (WAF^*)
Average number of fronds (day 7)		187.33		179.00
Growth Rates (µ) (0-7d)		0.405		0.398
% Inhibition of µ (0-7d)		-		1.60

*Water accommodated fraction

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^*)	0.776	0.658	0.775	0.617	0.750	0.661	0.703

-      not detected

*   water accommodated fraction (OECD No. 23.)

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	-	0.384	-
100 (WAF^*)	0.703	0.395	-2.79^**

*   water accommodated fraction (OECD No. 23.)

**negative value means growth increase

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	-	0.4456	-
100 (WAF^*)	0.703	0.4596	-3.14^**

*   water accommodated fraction (OECD No. 23.)

**negative value means growth increase

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	-	151.00	-
100 (WAF^*)	0.703	163.50	-8.28^**

*   water accommodated fraction (OECD No. 23.)

**negative value means growth increase

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	-	0.0098	-
100 (WAF^*)	0.703	0.0108	-10.13^**

*   water accommodated fraction (OECD No. 23.)

**negative value means growth increase

 

Symptoms, Changes ofLemna gibbaPlants 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*)	0.703	–	–	–	–	–	–

*   water accommodated fraction (OECD No. 23.)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a Lemna sp. Growth Inhibition Test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26, the 7-d EC50 and the NOEC of the test item was > 0.703 mg/L (mean measured concentration; > 100 mg/L in nominal).

Executive summary:

The influence of the test item on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7‑day static test, based on the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test” (2006) with nominal concentrations of 100 mg product/L (limit test). Based on the results the LOEC and NOEC for the test item were determined to be > 100 mg/L and 100 mg/L (nominal concentration, corresponding to the mean measured concentration of 0.703 mg/L), respectively.

All validity criteria were met and therefore the study is considered as valid. All biological results are based on the mean measured test item concentrations of the total product.