1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

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:

2017-11-17 to 2017-11-24

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

Version / remarks:

August 24, 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:

Expiry date: June 16, 2020

Analytical monitoring:

yes

Details on sampling:

- Method: In a first series three replicate samples (25 mL per replicate) were taken from each test concentration and six replicates (25 mL per replicate) from the control and sent to individual analysis.
- Sample storage conditions before analysis: At a temperature below – 15 °C. The first series of the samples taken was sent to individual analysis. The duplicate samples were kept separately as a reserve until the date of the issue of the Final Report. After the Final Report will be issued, the frozen samples will be discarded.
- Sampling frequency: day 0, 3, 5 and 7

Vehicle:

no

Details on test solutions:

- Method: 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 (according to OECD Series on Testing and Assessment No. 23). Appropriate amounts of test item were suspended in the dilution water (20 x AAP medium; see 4.4) separately for each concentration level in order to give the loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. The solutions were handled by ultrasonic bath for 10 minutes thereafter stirred for a period of 24 hours to achieve equilibrated concentration. The solutions will then be filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solutions will be freshly prepared in the testing laboratory just before introduction of the test organisms and at 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, and individual colonies consisted of 3 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:

other: 20X AAP Medium

Limit test:

no

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.

Hardness:

Not specified

Test temperature:

24 +/- 2 °C

pH:

7.77 - 8.56 for fresh solutions and from 8.32 - 8.74 for spent solutions during the test

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: : 6.25, 12.5, 25.0, 50.0 and 100 mg/L
Geometic mean measured: 0.058, 0.160, 0.286, 0.524 and 0.916 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: 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.
- Agitation: No
- Renewal of test solution:
November 17, 2017: Start of experiment, start of 1st renewal period
November 20, 2017: End of 1st renewal period , start of 2nd renewal period
November 22, 2017: End of 2nd renewal period , start of 3rd renewal period
November 24, 2017: End of 3rd renewal period, end of experiment
- Initital no. of fronds per vessel: 11
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituated fresh water

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: The pH of the medium was checked prior to use and readjusted when necessary
- Photoperiod: Continuous light
- Light intensity and quality: 6500 to 10000 lux, using cool-white fluorescent light tubes; test vessels were placed randomly on a tray. 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.

EFFECT PARAMETERS MEASURED
- Determination of frond number: Manual counting
- Determination of biomass: Dry weight

RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, 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 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, 50 and 100 mg/L were prepared by the aappropriate 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 and is excluded from the Statement of Compliance.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.916 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:

EC50

Effect conc.:

> 0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.916 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:

EC50

Effect conc.:

> 0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.916 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

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: 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:

For the determination of the LOEC and NOEC, the calculated mean growth rate and yield at the calculated test concentrations were tested on significant differences to the control value using analysis of variance (ANOVA) and Dunnett t-test (2-sided, α = 0.05) by SPSS PC+ software program. The data were checked for homogeneity of variance by Levene’s test.

Table 1: Calculation of geometric mean measured concentration of the test item in the test solutions

Nominal concentration (mg/L)	Measured concentration(mg/L)						Mean measured concentration (mg/L)
	1^strenewal period		2^ndrenewal period		3^rdrenewal period
	Start	End	Start	End	Start	End
Control	-	-	-	-	-	-	-
6.25 (WAF^*)	0.05^**	0.05^**	0.05^**	0.05^**	0.12	0.05^**	0.058
12.5 (WAF^*)	0.23	0.11	0.24	0.11	0.23	0.11	0.160
25 (WAF^*)	0.42	0.19	0.49	0.18	0.49	0.16	0.286
50 (WAF^*)	0.70	0.36	0.65	0.37	0.83	0.41	0.524
100 (WAF^*)	1.14	0.61	1.30	0.70	1.30	0.72	0.916

- not detected

* water accomdated fraction (OECD No. 23.)

** test concentrations measured < LOQ (Limit of quantification) were calculated as LOQ/2(OECD No. 23.)

Table 2: Growth rates (µ) and Percentage Inhibition of µ based on Frond Number

Concentration (mg/L)		Growth rate (µ) and % inhibition ofµ
Nominal	Measured	0–168 h(based on frond number)
		µ	% Iµ
Control	-	0.3644	-
6.25 (WAF^*)	0.058	0.3601	1.19
12.5 (WAF^*)	0.160	0.3579	1.78
25 (WAF^*)	0.286	0.3680	-0.97^**
50 (WAF^*)	0.524	0.3556	2.43
100 (WAF^*)	0.916	0.3642	0.06

* water accomdated fraction (OECD No. 23)

** negative value indicates increase in comparison to the control

Table 3: Growth rates (µ) and Percentage Inhibition of µ based 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	-	0.36643	-
6.25 (WAF^*)	0.058	0.36461	0.50
12.5 (WAF^*)	0.160	0.36618	0.07
25 (WAF^*)	0.286	0.37824	-3.22^**
50 (WAF^*)	0.524	0.36381	0.72
100 (WAF^*)	0.916	0.36409	0.64

* water accomdated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

Table 4: 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	-	130.33	-
6.25 (WAF^*)	0.058	126.00	3.32
12.5 (WAF^*)	0.160	124.00	4.86
25 (WAF^*)	0.286	133.67	-2.56^**
50 (WAF^*)	0.524	121.67	6.65
100 (WAF^*)	0.916	130.33	0.00

* water accomdated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

Table 5: Yield (y) and Percentage Inhibition of Yield based on Dry Weight

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Calculated	0–168 h(based on dry weight)
		y	% Iy
Control	-	0.00691	-
6.25 (WAF^*)	0.058	0.00675	2.29
12.5 (WAF^*)	0.160	0.00683	1.13
25 (WAF^*)	0.286	0.00749	-8.37^**
50 (WAF^*)	0.524	0.00671	2.82
100 (WAF^*)	0.916	0.00673	2.58

* water accomdated fraction (OECD No. 23.)

** negative value indicates increase in comparison to the control

Table 6: 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	-	n	-	n	-	n	-
6.25 (WAF^*)	0.058	n	-	n	-	n	-
12.5 (WAF^*)	0.160	n	-	n	-	n	-
25 (WAF^*)	0.286	n	-	n	-	n	-
50 (WAF^*)	0.524	n	-	n	-	n	-
100 (WAF^*)	0.916	n	-	n	-	n	-

* water accomdated fraction (OECD No. 23)

Legend:

"n": the plants were healthy

-: no symptoms observed

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 7-d EC10, EC50 and LOEC of the test item for growth rate and biomass, respectively, were determined to be > 0.916 mg/L (geom. mean measured). The 7-d NOEC of the test item both for growth rate and biomass was determined to be 0.916 mg/L (geom. mean measured).

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). Appropriate amounts of test item were suspended in the dilution water (20 x AAP medium) separately for each concentration level in order to give the loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. The test included three replicates at each test concentration and six replicates in the control group.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.The exposure concentrations were calculated as the geometric mean of the test item concentrations measured (by UV/VIS) at the start and end of each renewal period during the study and determined as 0.058, 0.160, 0.286, 0.524 and 0.916 mg/L. As the lowest test concentration of 6.25 mg/L (nominal) was below limit of quantification (LOQ), it was calculated as LOQ/2 according to OECD No. 23. In result, the average specific growth rate and yield was not statistically significantly different from the control group at any observed test item concentration based on frond number and dry weight (Dunnett t-test, 2-sided,a=0.05). The 7-d EC10, EC50 and LOEC for growth rate and biomass, respectively, were determined to be > 0.916 mg/L (geom. mean measured). The 7-d NOEC for growth rate and biomass was determined to be 0.916 mg/L. All validity criteria were met and therefore the study can be considered as valid.

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 7-d EC10, EC50 and LOEC of the test item for growth rate and biomass, respectively, were determined to be > 0.916 mg/L (geom. mean measured). The 7-d  NOEC of the test item both for growth rate and biomass was determined to be 0.916 mg/L (geom. mean measured).

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.916 mg/L

Additional information

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). Appropriate amounts of test item were suspended in the dilution water (20 x AAP medium) separately for each concentration level in order to give the loading rates of 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L. The test included three replicates at each test concentration and six replicates in the control group. 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.The exposure concentrations were calculated as the geometric mean of the test item concentrations measured (by UV/VIS) at the start and end of each renewal period during the study and determined as 0.058, 0.160, 0.286, 0.524 and 0.916 mg/L. As the lowest test concentration of 6.25 mg/L (nominal) was below limit of quantification (LOQ), it was calculated as LOQ/2 according to OECD No. 23. In result, the average specific growth rate and yield was not statistically significantly different from the control group at any observed test item concentration based on frond number and dry weight (Dunnett t-test, 2-sided,a=0.05). The 7-d EC10, EC50 and LOEC for growth rate and biomass, respectively, were determined to be > 0.916 mg/L (geom. mean measured). The 7-d NOEC for growth rate and biomass was determined to be 0.916 mg/L. All validity criteria were met and therefore the study can be considered as valid.