Acetamide, N-(2,4-dinitrophenyl)-, reaction products with p-phenylenediamine, sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

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:

02 August - 24 October 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:

23rd March, 2006

Deviations:

no

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

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)

Specific details on test material used for the study:

Date of production:04 April 2016
Expiry date: 04 April 2021

Analytical monitoring:

yes

Details on sampling:

In the first series six replicate samples (25 mL per replicate) were taken from the test concentration and six replicates (25 mL per replicate) from the control and sent to individual analysis. The second series of samples (150 mL of each test group/ sampling occasion) were kept separately as a reserve until the date of the issue of the Final Report at a temperature below – 15 °C. After the date of issuing the Final Report the frozen samples are discarded.

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. The following test item amounts were suspended in the appropriate amount of the dilution water: 0.1508 g in 1508 mL in the first renewal period and 0.1503 g in 1503 mL in the second and third renewal periods. 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 (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Gibbous duckweed
- Strain: G3
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany
- Preculture: 7 days 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.
- 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 three replicates at each test concentration and six replicates in the control group.

Test type:

semi-static

Water media type:

other: 20X AAP Medium

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

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.

Test temperature:

24 ± 2 °C

pH:

7.91 to 8.44 for fresh test and control solutions and from 8.36 to 8.62 for spent test and control solutions

Nominal and measured concentrations:

Nominal: 100 mg/L
Geometric mean measured: 0.175 mg/L

Details on test conditions:

- Test type: Semi-static test. Based on the results obtained during analytical method validation 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 – 23.5°C in the climate chamber and 22.9 – 23.0 °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 7.91 to 8.44 for fresh test and control solutions and from 8.36 to 8.62 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 7936 – 8120 lux, SD: 54 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.

RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, a semi-static preliminary range-finding test was conducted to determine the approximate toxicity of the test item. In the preliminary range-finding test a test item solution was prepared by adding 0.0529 g test item into 529 mL dilution water (20X AAP Medium) in the first renewal period, 0.0512 g test item into 512 mL dilution water (20X AAP Medium) in the second renewal period and 0.0503 g test item into 503 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 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 preliminary range-finding test was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.175 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.175 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.175 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.175 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Details on results:

Validity of the Test
The doubling time of frond number in the control was 1.85 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 – 23.5°C in the climate chamber; 22.9 – 23.0°C in the test vessels
pH values: 7.91 - 8.62
Light intensity: 8044 lux (mean value); SD: 54 lux

Aalytical 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 10 % of the lowest concentration of the calibration curve. In the treated group mean of the initial measured concentration was 0.317 mg/L, 0.294 mg/L and 0.313 mg/L at the start of the first, second and third renewal period, respectively. The test item concentration was measured to be lower than LOQ (0.2 mg/L) at the end of the first renewal period. Therefore, this test item concentration was calculated as LOQ/2 (according to OECD Series on Testing and Assessment No. 23). As the test item concentration was measured to be lower than LOD (0.1 mg/L) at the end of the second and third renewal periods, these test item concentrations were calculated as LOD (according to OECD Series on Testing and Assessment No. 23). The exposure concentration was calculated as the geometric mean of the start and end values during the study and determined to be 0.175 mg/L (equivalent to 100 mg/L nominal concentration).
 
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 0.175 mg/L (calculated) based on frond number and dry weight (Independent Sample T-test, 2-tailed, α=0.05). The 168-h NOEC was determined to be 0.175 mg/L, while the 168-h LOEC was determined to be greater than 0.175 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 0.175 mg/L (calculated) based on frond number and dry weight (Independent Sample T-test, 2-tailed, α=0.05). The 168-h NOEC was determined to be 0.175 mg/L, while the 168-h LOEC was determined to be higher than 0.175 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 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:

For determination of the NOEC and LOEC values Independent Sample T-test was used (2-tailed, α = 0.05).

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^*)	0.317	0.100^1)	0.294	0.100^2)	0.313	0.100^2)	0.175

-      not detected

*   water accommodated fraction (OECD No. 23.)

¹⁾    LOQ/2

²⁾    LOD

Growth Rates (µ) and Percentage Inhibition of µ based 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	-	0.375	-
100 (WAF^*)	0.175	0.379	-1.08^**

*   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	-	0.3903	-
100 (WAF^*)	0.175	0.4039	-3.50^**

*   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	-	140.50	-
100 (WAF^*)	0.175	144.83	-3.08^**

*   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	-	0.0082	-
100 (WAF^*)	0.175	0.0090	-10.59^**

*   water accommodated fraction (OECD No. 23.)

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*)	0.175	–	–	–	–	–	–

*   water accommodated fraction (OECD No. 23.)

**negative value means growth increase

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 0.175 mg/L, based on water accommodated fraction of total product.

Executive summary:

The toxicity of the test item to Lemna gibba was assessed in a semi-static study according to OECD Guideline 221, Commission Regulation (EC) No 761/2009, Annex VI, C.26 and GLP principles. As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solutions was performed using the WAF method (in accordance with OECD Series on Testing and Assessment No. 23). 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 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. The corresponding calculated concentration was 0.175 mg/L (based on total product). The test included six replicates and the control group with six replicates. 400 mL Petri dishes-covered glass beakers were filled with 160 mL testing solutions and two Lemna gibba colonies with four and one colony with three fronds were added to each vessel. Growth and morphological changes were assessed on days 3, 5 and 7. For determination of the test item concentrations (based on total product), samples were taken from the test solution and control at the start and at the end of each renewal period. Test item concentration was determined using UV/VIS spectrophotometric method. For the determination of the NOEC and LOEC values Independent Sample T-test was used (2 -tailed,α= 0.05). All validity criteria were met and therefore the study can be considered as valid.

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 0.175 mg/L, based on water acommodated 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 0.175 mg/L, based on water accommodated fraction of total product.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.175 mg/L