Disodium [2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

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:

Experimental start date - 21 September 2016; Experimental completion date - 06 November 2016; Study completion date: 28 November 2016.

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

GLP compliance:

yes

Specific details on test material used for the study:

Identification: FAT 20013/D TE
Physical state/Appearance: Grey powder
Batch: 1502011 (China)
Purity: 69.5 %
Expiry Date: 03 August 2020
Storage Conditions: Approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

In order to determine the stability of the test item under test conditions, a sample of each test concentration was taken for chemical analysis on Day 2 (fresh media) and Day 5 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

Culture medium

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days. The test was conducted in glass conical flasks (500 mL). Two replicate flasks were prepared for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10 and 1.0 mg/L. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Test organisms (species):

Lemna minor

Details on test organisms:

A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

24 ± 1 ”C

pH:

6.5 - 7.4

Nominal and measured concentrations:

Nominal amounts of test item (100 and 32 mg) were separately dissolved in culture medium and the volume adjusted to 1 liter to give 100 and 32 mg/L test concentrations respectively from which a series of dilutions was made to give further test concentrations of 10, 3.2 and 1.0 mg/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: Conical flask
- Material, size, headspace, fill volume: Glass.
- Aeration: No
- Agitation: No
- Renewal rate of test solution (frequency/flow rate): On Days 2 and 5 the test solutions were renewed
- No. of colonies per vessel: Three
- No. of fronds per colony: Three
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): Two

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP).
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was recorded on Day 0 and Day 7.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the prepared culture medium will be adjusted, if necessary, to 6.5 ± 0.2 with either 1M HCl or NaOH.
- Photoperiod: 7 days continuation.
- Light intensity and quality: 7000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting
- Determination of biomass: dry weight
- Determination of frond area: No

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentration of 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 86 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

yield

Details on results:

Range-finding Test
The frond counts and percentage inhibition of growth values from the exposure of Lemna minor to the test item during the range-finding test were determined. The results showed no effect on growth at the test concentration of 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations on Day 2 (fresh media) (see Annex 3) showed measured test concentrations to range from 49 % to 84 % of nominal whilst concentrations of the corresponding expired media on Day 5 showed measured test concentrations to range from 62% to 77% of nominal. Analysis of the test preparations which had been incubated alongside the test for a period of 7 days showed measured test concentrations to range from 82% to 88% of nominal indicating that the test item was stable over the test duration. Examination of the data could find no cause for the low measured concentrations seen on Day 2 and Day 5.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 80% to 91% of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 66% and 79% of nominal respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Validation Criteria
The data show that the doubling time of the control cultures was 1.75 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at day 0: 9
Mean frond number in control cultures at day 7: 103

Growth Data Based on Frond Number
Numbers of fronds in each flask in the definitive test were determined and the average specific growth rates, yields and percentage inhibition values calculated.
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the frond number data

Average Specific Growth Rate
ErC10 (frond number) = 0.67 mg/L
ErC20 (frond number) = 3.7 mg/L
ErC50 (frond number) = 69 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.

Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.75 mg/L test concentrations (P≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 0.75 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.7 mg/L.

Yield
EyC10 (frond number) = 0.94 mg/L
EyC20 (frond number) =1.2 mg/L
EyC50 (frond number) = 3.9 mg/L
Where:
EyCx = the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out for the control and all test concentrations. There were no statistically significant differences between the control and 0.75 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 0.75 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.7 mg/L.

Growth Data Based on Dry Weight

The dry weight of Lemna minor in each flask in the definitive test was determined and average specific growth rates, yield and percentage inhibition values calculated. Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:

Average Specific Growth Rate

ErC10 (dry weight) = 1.2 mg/L
ErC20 (dry weight) = 2.5 mg/L
ErC50 (dry weight) = >86 mg/L*
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.

* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Statistical analysis of the average specific growth rate data was carried out for the control and all test concentrations. There were no statistically significant differences between the control and 0.75 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 0.75 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.7 mg/L.

Yield
EyC10 (dry weight) = 0.88 mg/L
EyC20 (dry weight) = 1.1 mg/L
EyC50 (dry weight) = 3.2 mg/L

Where:
EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and all test concentrations. There were no statistically significant differences between the control and 0.75 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 0.75 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 2.7 mg/L.

Results with reference substance (positive control):

See "Any other information on results incl. tables"

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981), and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955), were carried out on the average specific growth rate and yield data at 7 days for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999-2001).

Results with test substance.

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	69	0.75	2.7
	Dry Weight	>86*	0.75	2.7
Yield	Frond Number	3.9	0.75	2.7
	Dry Weight	3.2	0.75	2.7

* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50 % inhibition of growth rate.

Results with Positive control:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	3.4	0.625	1.25
	Dry Weight	3.0	0.625	1.25
Yield	Frond Number	1.8	0.625	1.25
	Dry Weight	1.4	0.625	1.25

The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Validity criteria fulfilled:

yes

Conclusions:

The average specific growth rate (frond number) EC50 was reported to be 69 mg/L and the corresponding NOEC was reported to be 0.75 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”.

Methods

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development. Chemical analysis was performed on random samples collected at Day 0 and Day 7.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 80 % to 91 % of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 66 % and 79 % of nominal respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Exposure of Lemna minor to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	69	0.75	2.7
	Dry Weight	>86*	0.75	2.7
Yield	Frond Number	3.9	0.75	2.7
	Dry Weight	3.2	0.75	2.7

Key value for chemical safety assessment

EC50 for freshwater plants:

69 mg/L

EC10 or NOEC for freshwater plants:

0.75 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”.

Methods

Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development. Chemical analysis was performed on random samples collected at Day 0 and Day 7.

Results

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 80% to 91% of nominal. Near nominal concentrations were obtained from the test preparations on Day 7 with the exception of the 1.0 and 3.2 mg/L test samples which saw a decline in measured concentrations to 66% and 79% of nominal respectively. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Exposure of Lemna minor to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	Measurement Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate	Frond Number	69	0.75	2.7
	Dry Weight	>86*	0.75	2.7
Yield	Frond Number	3.9	0.75	2.7
	Dry Weight	3.2	0.75	2.7