Reaction mass ofDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN1}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-) andDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]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 - 30 March 2016; Experimental completion date - 12 August 2016; Study completion date - 13 October 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 (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: FAT 20037 TE; batch no.: 1501016 (China)
- Expiration date of the batch: 19 May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 ºC in the dark

OTHER SPECIFICS:
- Physical state/Appearance: Black solid
- Purity: 45 %

Analytical monitoring:

yes

Details on sampling:

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. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

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.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna minor
- Source: Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada.
- OTHER SPECIFICS: 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:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 ºC

pH:

Control: 6.7 - 10.2,
At 1 mg/L: 6.7 - 10
3.2 mg/L: 6.7 - 8.9
10 mg/L: 6.7 - 8.3
32 mg/L: 6.7 - 7.7
100 mg/L: 6.7 - 7.5

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: Glass, 500 mL.
- No. of colonies per vessel: 3 colonies
- No. of fronds: 9 fronds
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): three

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: The number of fronds present in each test and control culture was recorded on Days 0, 2, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.
- Determination of biomass: [dry weight] The dry weight of the fronds in each control and treatment group was determined on Day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C to constant weight.

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: Continious
- Light intensity and quality: 7000 lux.

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 7 days
- Results used to determine the conditions for the definitive study: The results showed no significant 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:

A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L. Exposure conditions for the positive control were similar to those in the definitive test.

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Any visual signs of phytotoxicity (abnormalities):
- Decrease in frond size: No
- Necrosis / chlorosis: No
- Sinking of fronds: On Day 7 - fronds lying very close together, loss of buoyancy and some fronds were submerged.

Results with reference substance (positive control):

The EC50 values for 3,5-dichlorophenol based on average specific growth rate, EC50 (frond number): 3.4 mg/L and EC50 (dry weight): 3.0 mg/L
The EC50 values based on yield, EC50 (frond number): 1.8 mg/L and EC50 (dry weight): 1.4 mg/L

Verification of Test Concentrations

Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 91 % to 99 % of nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 18 % of nominal at 1.0 mg/L, through to 99 % of nominal at 100 mg/L. Post-study stability work conducted at concentrations of 1.0 and 100 mg/L showed that the test item was stable under test conditions for a 7-Day period. As such the decline in measured concentrations observed in the definitive test was considered to be due to adsorption of the test item to the biomass present. It can therefore be considered that the lemna were exposed to nominal concentrations of the test item and as such the results have been calculated based on nominal test concentrations only.

Validation Criteria

The doubling time of the control cultures was 1.70 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: 117

Growth Data Based on Frond Number

Average Specific Growth Rate

ErC10 (frond number) = 1.9 mg/L

ErC20 (frond number) = 6.5 mg/L

ErC50 (frond number) = >100 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 1.0 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 rates calculated from frond numbers was 1.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 3.2 mg/L.

Yield

EyC10 (frond number) = 1.1 mg/L

EyC20 (frond number) = 1.6 mg/L

EyC50 (frond number) = 10 mg/L

Where: EyCx = the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out. There were no statistically significant differences (P≥0.05), between the control and 1.0 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 1.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 3.2 mg/L.

Growth Data Based on Dry Weight

Average Specific Growth Rate

ErC10 (dry weight) = 0.67 mg/L

ErC20 (dry weight) = 4.1 mg/L

ErC50 (dry weight) = 92 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. There were no statistically significant differences (P≥0.05), between the control and 1.0 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 1.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 3.2 mg/L.

Yield

EyC10 (dry weight) = 1.1 mg/L

EyC20 (dry weight) = 1.4 mg/L

EyC50 (dry weight) = 5.0 mg/L

Where: EyCx = the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out. There were no statistically significant differences (P≥0.05), between the control and 1.0 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 1.0 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 3.2 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (frond number) based on the average specific growth rate was determined to be >100 mg/L.

Executive summary:

A study was performed according to OECD test guideline 221 in a GLP certified laboratory, to assess the effect of the test item on the growth of the freshwater plant Lemna minor. 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. Analytical verification of concentration was conducted using HPLC method. Samples were drawn at the start and end of study for this purpose. Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 91 % to 99 % of nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 18 % of nominal at 1.0 mg/L, through to 99 % of nominal at 100 mg/L. Based on the findings of the study, the EC50 values for the test substance based on average specific growth rate were, EC50 (frond number): >100 mg/L, EC50 (dry weight): 92 mg/L, while the EC50 values based on yield were, EC50 (frond number): 10 mg/L, EC50 (dry weight): 5 mg/L.

Description of key information

A study was performed according to OECD test guideline 221 in a GLP certified laboratory, to assess the effect of the test item on the growth of the freshwater plant Lemna minor. 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. Analytical verification of concentration was conducted using HPLC method. Samples were drawn at the start and end of study for this purpose. Chemical analysis of the test preparations on Day 0 (fresh media) showed measured test concentrations to range from 91% to 99% of nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 18 % of nominal at 1.0 mg/L, through to 99 % of nominal at 100 mg/L. Based on the findings of the study, the EC50 values for the test substance based on average specific growth rate were, EC50 (frond number): >100 mg/L, EC50 (dry weight): 92 mg/L, while the EC50 values based on yield were, EC50 (frond number): 10 mg/L, EC50 (dry weight): 5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L