Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine, sodium polysulfide and sodium metabisulfite

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:

2019-05-31 to 2019-06-07 (start of experiemt to end of experiment)

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". Dated August 24, 2009

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, No. 23 (Second Edition), ENV/JM/MONO(2000)6/REV1, 8 February 2019

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: LS310517
- Expiration date: 31 May 2022

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentration, samples (50 mL per replicate) were taken from the testing concentration and from the control at the start (prior to distributing of the test solution to the test vessels), 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 0.1209 g test item to 1209 mL test medium (20X AAP medium) in the first renewal period, 0.1202 g test item to 1202 mL test medium (20X AAP medium) in the second renewal period and 0.1202 g test item to 1202 mL test medium (20X AAP medium) in the third renewal period in order to give the loading rate of 100 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
- 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:

22.4 – 24.7 °C in the climate chamber
22.9 – 23.1 °C in the flask

pH:

8.12 - 8.79

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured concentrations: 20.28 mg/L. Not within ± 20 % of the nominal during the experiment

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Material, size, fill volume: Glass, total capacity of 400 mL, fill with 160 mL test solution
- Type of cover: Glass petri dishes
- Aeration: No
- Renewal rate of test solution: The test solution (as well as 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 replicates per concentration (replicates): 6
- No. of replicates per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (20X AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source / preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Sterile test condition: yes
- Adjustment of pH: yes, the pH was adjusted to 7.5 ± 0.1 with 1 NM HCl
- Photoperiod: Continuous illumination
- Light intensity and quality: 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. In the test item treated group decrease of frond size was observed during the experiment and small amount of the test item was adsorbed on the roots and on the lower side of the leaves observed at the end of the test. 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 TEST
In the semi-static preliminary range-finding test, a test item solution was prepared by adding 0.0500 g test item into 500 mL dilution water (20X AAP Medium) in the first renewal period, 0.0502 g test item into 502 mL dilution water (20X AAP Medium) in the second renewal period and 0.0500 g test item into 500 mL dilution water (20X AAP Medium) in the third renewal period to get the nominal concentration of 100 mg test substance /L. The test solution was handled in ultrasonic bath for approx. 10 minutes, stirred rigorously for 24 hours then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test. The pre-test was performed with three 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 results are shown in the section “Any other information on results incl. tables”.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol is tested at least twice a year

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

20.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

20.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 20.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 20.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Change in plant development during the test: yes, in the test item treated group decrease of frond size was observed during the experiment and small amount of the test item was adsorbed on the roots and on the lower side of the leaves.

Results with reference substance (positive control):

-Results with reference substance valid? Yes
The date of the last with reference item 3,5-Dichlorophenol was: 11 - 18 October 2019.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 5.187 mg/L,
ErfnC50 (7 day, growth rate based on frond numbers): 7.624 mg/L, EydwC50 (7 day, yield based on dry weight): 5.136 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.497 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.

Results of the Preliminary Range-finding test

Nominal concentrations (mg/L)	Untreated control	100
Average number of fronds (day 7)	198.00	203.00
Growth Rates (µ) (0-7d)	0.413	0.416
% Inhibition of µ (0-7d)	-	-0.74

Remark: negative inhibition means growth stimulation

Based on the results of the non-GLP Preliminary Range-Finding Test (see above), the main test was a limit test performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L and a concurrent control group.

 

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. Therefore, test item concentration was calculated as geometric mean of the measured concentrations (according to OECD No. 23). Calculation of the test concentration is given in the table below:

Table: Calculation of the Test Concentration

Concentration (mg/L)	Mean of the Measured concentrations (mg/L) (n=6)
	First renewal period		Second renewal period		Third renewal period
	Start	End	Start	End	Start	End
Control	< LOD	< LOD	< LOD	< LOD	< LOD	< LOD
100(WAF^*)	25.6	14.4	27.2	16.5	29.2	14.4
Geometric mean	20.28

*           WAF = water accommodated fraction (OECD No. 23.); loading rate: 100 mg/L nominal

 

Growth rate

No significant inhibition of the average specific growth rate based on frond number in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on growth rate (frond number) was determined to be 20.28 mg/L, while the LOEC based on growth rate was determined to be > 20.28 mg/L.

Significant inhibition of the average specific growth rate based on dry weight in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on growth rate (dry weight) was determined to be < 20.28 mg/L, while the LOEC based on growth rate was determined to be < 20.28 mg/L.

 

Yield

No significant inhibition of the yield based on frond number in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on yield (frond number) was determined to be 20.28 mg/L, while the LOEC based on yield was determined to be > 20.28 mg/L.

Significant inhibition of the yield based on dry weight in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on yield (dry weight) was determined to be < 20.28 mg/L, while the LOEC based on yield was determined to be > 20.28 mg/L.

 

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.

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 Blue TBR had no toxic effects on the growth rate and yield based on frond number. The overall 7-day NOEC based on frond number was determined to be 20.28 mg/L (nominal 100 mg/L) and the overall 7-day LOEC based on frond number was determined to be > 20.28 mg/L (nominal > 100 mg/L). The overall 7-day NOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L) and the overall 7-day LOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L). All validity criteria were met. The results are based on the nominal and on geometric mean of the measured test item concentrations. Test solution was prepared by utilizing the water accommodation fraction (WAF) approach.

Executive summary:

The phytotoxicity of the test item Blue TBR 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 (168 hours) 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 100 mg/L were compared with that in the control in a limit test. The effects of Blue TBR 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 geometric 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 Blue TBR had no toxic effects on the growth rate and yield (based on frond number) but had significant toxic effect on the growth rate and yield (based on dry weight). The overall 7-day NOEC based on frond number was determined to be 20.28 mg/L (nominal 100 mg/L) and the overall 7-day LOEC based on frond number was determined to be > 20.28 mg/L (nominal > 100 mg/L). The overall 7-day NOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L) and the overall 7-day LOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L). All validity criteria were met.

Description of key information

In the 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Blue TBR had no toxic effects on the growth rate and yield (based on frond number).

The overall 7-day NOEC based on frond number was determined to be 20.28 mg/L (nominal 100 mg/L).

The overall 7-day LOEC based on frond number was determined to be > 20.28 mg/L (nominal > 100 mg/L).

All validity criteria were met. The results are based on the nominal and on geometric mean of the measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

20.28 mg/L

Additional information

The phytotoxicity of the test item Blue TBR 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 (168 hours) 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 100 mg/L were compared with that in the control in a limit test. The effects of Blue TBR 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 geometric 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 Blue TBR had no toxic effects on the growth rate and yield (based on frond number) but had significant toxic effect on the growth rate and yield (based on dry weight). The overall 7-day NOEC based on frond number was determined to be 20.28 mg/L (nominal 100 mg/L) and the overall 7-day LOEC based on frond number was determined to be > 20.28 mg/L (nominal > 100 mg/L). The overall 7-day NOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L) and the overall 7-day LOEC based on dry weight was determined to be < 20.28 mg/L (nominal < 100 mg/L). All validity criteria were met.