Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and 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:

March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 761 2009. Annex VI. C.26: ,Lemm sp. Growth Inhibition Test"

Version / remarks:

August 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, Paris

Version / remarks:

February 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5 mg/L
- Sampling method: For determination of the test item concentration, samples (50 mL from each replicate) were taken from the testing concentration and control in duplicates 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
- 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). A test item suspension was prepared by adding an amount of 0.0032 g test item to 1640 mL test medium (20X AAP medium) in the first renewal period. 0.0083 g test item to 1660 mL test medium (20X AAP medium) in the second renewal period and 0.0081 g test item to 1620 mL test medium (20X AAP medium) in the third renewal period in order to give the loading rate of 5 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 before the start of each renewal period.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Species and strain: Lemna gibba (G3)
- 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.
- Source: Friedrich Schiller Universitat, 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 of 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 vessels

pH:

7.78 - 8.82

Nominal and measured concentrations:

Nominal: 5 mg/L
Measured concentrations: Not within ± 20 % of the nominal during the test period. For details see section "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Material, size, fill volume: Glass, total capacity of 400 mL, filled with 160 mL test solution
- Type of cover: Covered by glass petri dishes
- Aeration: No
- Renewal rate of test solution: The test 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

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

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

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: 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. Any 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
In the preliminary range-finding test a test item solution was prepared by adding 0.0051 g test item into 1020 mL dilution water (20X AAP Medium) in the first renewal period. 0.0050 g test item into 1000 mL dilution water (20X AAP Medium) in the second renewal period and 0.0052 g test item into 1040 mL dilution water (20X AAP Medium) in the third renewal period to get the nominal concentration of 5 mg test substance/L. The test 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. 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.:

0.121 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.:

0.121 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.121 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.:

> 0.121 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Changes in plant development observed 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):

The date of the last study with reference item 3,5-Diclilorophenol 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 colonies used in the test in each replicate at the end of the test.
It was not necessary to analyse the average specific growth rate and yield in control and in treated group by statistical procedure, because there was no significant inhibition of growth rate and yield. Therefore, LOEC and NOEC values were given directly from the raw data.

Table 1: Results of the Preliminary Range-finding Test

Nominal concentrations (mg/L)	Untreated control	WAF (5)
Average number of fronds (day 7)	198.00	192.33
Growth Rates (µ) (0-7d)	0.413	0.408
% Inhibition of µ (0-7d)	-	1.22

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 loading rate of 5 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 concentrations were not within ± 20 % of the nominal during the experiment. Therefore the results are based on the geometrical mean measured test item concentration.

Table 2: Calculation of Test Concentration

Nominal concentration (mg/L)	Measured concentration (mg/L)
	First renewal period		Second renewal period		Third renewal period
	Start	End	Start	End	Start	End
Control	<LOD
5 (WAF*)	<LOD	0.207	0.249	<LOD	<LOD	<LOD
	<LOD	<LOD	0.174	<LOD	<LOD	<LOD
	<LOD	<LOD	0.216	<LOD	<LOD	<LOD
	<LOD	<LOD	0.270	<LOD	<LOD	<LOD
	<LOD	<LOD	0.244	<LOD	<LOD	<LOD
	0.173	<LOD	0.280	<LOD	<LOD	<LOD
Mean	0.112	0.118	0.239	0.100	0.100	0.100
Geaometric mean	0.121**

LOD = Limit of detection (0.1 µg/mL)

* WAF = water accommodated fraction (OECD No. 23.)

* * For calculation of geometrical mean concentration, values for < LOD were set to LOD (0.1 µg/mL), (OECD No. 23.)

Growth rate

No significant inhibition of the average specific growth rate was observed in the test item treated group. Therefore the NOEC and LOEC values were determined from the raw data. Accordingly the NOEC based on growth rate was determined to be 0.121 mg/L, while the LOEC based on growth rate was determined to be > 0.121 mg/L.

Yield

No significant inhibition of the yield was observed in the test item treated group, therefore the NOEC and LOEC values were determined from the raw data. Accordingly the NOEC based on yield was determined to be 0.121 mg/L, while the LOEC based on yield was determined to be > 0.121 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 Sulphur Brown 46 had no toxic effects on the test system up to the limit of solubility.
The 7-day NOEC was determined to be 0.121 mg/L (nominal 5 mg/L).
The 7-day LOEC was detenmned to be > 0.121 mg/L (nominal > 5 mg/L).
All validity criteria were met. The results are based on geometrical mean measured test item concentration. Test solution was prepared by utilizing the water accommodated fraction (WAF) approach.

Executive summary:

The phytotoxicity of the test item, Leuco Sulphur Brown 46, 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 5 mg/L were compared with that in the control in a limit test. The effects of Leuco Sulphur Brown 46 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 were not within ± 20 % of the nominal during the test period therefore all biological results are based on the geometrical mean of the measured test item concentration. In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Leuco Sulphur Brown 46 had no toxic effects on the test system. The 7-day NOEC was determined to be 0.121 mg/L (nominal 5 mg/L). The 7-day LOEC was determined to be > 0.121 mg/L (nominal > 5 mg/L). All validity criteria were met. The results are based on geometrical mean measured test item concentration.

Description of key information

In a 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Leuco Sulphur Brown 46 had no toxic effects on the test system up to the limit of solubility.

The 7-day NOEC was determined to be 0.121 mg/L (nominal 5 mg/L).

The 7-day LOEC was detenmned to be > 0.121 mg/L (nominal > 5 mg/L).

All validity criteria were met. The results are based on geometrical mean measured test item concentration. Test solution was prepared by utilizing the water accommodated fraction (WAF) approach.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.121 mg/L

Additional information

The phytotoxicity of the test item, Leuco Sulphur Brown 46, 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 5 mg/L were compared with that in the control in a limit test. The effects of Leuco Sulphur Brown 46 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 were not within ± 20 % of the nominal during the test period therefore all biological results are based on the geometrical mean of the measured test item concentration. In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Leuco Sulphur Brown 46 had no toxic effects on the test system. The 7-day NOEC was determined to be 0.121 mg/L (nominal 5 mg/L). The 7-day LOEC was determined to be > 0.121 mg/L (nominal > 5 mg/L). All validity criteria were met. The results are based on geometrical mean measured test item concentration.