Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

EC Number:

916-899-6

Molecular formula:

not available

IUPAC Name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling, method and storage: Analytical data are required by the guidelines for verification of test item concentrations as well as the stability of the test item over the entire test period. Analytical samples were taken at 0 hours (initial value) from fresh test solutions, after 2, 5 and 7 days from aged test solutions from all test item concentrations (100, 31.3, 9.77, 3.05, 0.954, 0.298, 0.0931mg/L) and control. For each sampling also a retain sample was taken.
Samples were taken from pooled replicates. 1 mL test medium were taken for each sample set.
All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution was prepared and further dilutions prepared with test medium.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Lemna gibba G 3
- Strain: Alismatales: Araceae
- Source (laboratory, culture collection): aseptic clones from the cultivation of the testing facility
- Age of inoculum (at test initiation):
- Method of cultivation: primarily cultured

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

23.5 – 24.5 °C

pH:

7.48 – 7.58

Nominal and measured concentrations:

See table

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel:Test vessels were 250 mL glass beakers each containing ~ 150 mL test solution to allow the plants to grow without touching the sides of the glass vessels. The test vessels were covered with a glass plate. Test vessels were randomised during the test.
- Agitation: no
Colonies consisting of 2 - 4 fronds were transferred from the inoculum culture into the test vessels containing a total of 12 fronds, each.
4 replicates of each concentration, six replicates for the control

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The medium used for the test was 20xAAP Medium for Lemna gibba (according to OECD 221, composition see Appendix B). The pH was 7.5 ± 0.1 after 1 hour.

OTHER TEST CONDITIONS
Test procedure: Static test
Duration: 7 days
Temperature: 23.5 – 24.5 °C
pH-value of fresh solutions: 7.48 – 7.58
Light intensity (mean): 7660 lux
Light tubes: T8-fluorescent tubes, length 900 mm, LT30W/865 daylight
Exposure to light: Continuously
Number of fronds: 12 per test vessel

EFFECT PARAMETERS MEASURED:
Frond numbers in each test vessel were determined at the start of the test. Frond numbers and the appearance of the colonies were checked on t = 0, 2, 5 and 7 days as well as any change in plant development, frond size, necrosis and additional observations of test media or other abnormalities.
The number of fronds and dry weight (biomass) were tabulated together with the concentrations of the test item for each measurement occasion.
At the end of the test all the fronds from each test vessel were dried at 60°C for about 48 hours. 6 x 12 fronds from the test culture were dried to represent the dry weight at test start.
Means of frond numbers for each test item concentration at each observation time were calculated. Means of dry weight for each test item concentration at day 7 were calculated. The specific growth rate (µ) for frond number and dry weight was calculated as the logarithmic increase for each replicate for control and treatments
Means of frond numbers for each test item concentration at each observation time were calculated. Means of dry weight for each test item concentration at day 7 were calculated. The specific growth rate (µ) for frond number and dry weight was calculated as the logarithmic increase for each replicate for control and treatments
The percentage inhibition of growth rate (% IR) was calculated as the difference between the mean growth rate of the control (µC) and the mean growth rate in the treatments (µT)
The mean doubling time (Td) of the culture for the control and treatments were calculated
Effects on yield are determined on the basis of total frond numbers and dry weight in each vessel at the start and end of the test. For the test concentrations, a mean value for yield was calculated. The mean percent inhibition in yield was calculated for each treatment group

Reference substance (positive control):

yes

Remarks:

3.5 Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.954 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.694 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

31.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Details on results:

Significant inhibitory effects were determined for yield of frond numbers at test item concentrations of 3.05 mg/L (nominal) and 2.32 mg/L (actual) and above. 67.4 % of inhibition of yield of frond numbers was observed in the highest test item concentration.
Significant inhibitory effects were determined for growth rate of frond numbers at test item concentrations of 9.77 mg/L (nominal) and 8.70 mg/L (actual) and above. 35.6 % of inhibition of growth rate of frond numbers was observed in the highest test item concentration.
Significant inhibitory effects were determined for yield of dry weight at test item concentrations of 3.05 mg/L (nominal) and 2.32 mg/L (actual) and above. 76.7 % of inhibition of yield of dry weight was observed in the highest test item concentration.
Significant inhibitory effects were determined for growth rate of dry weight at test item concentrations of 9.77 mg/L (nominal) and 8.70 mg/L (actual) and above. 40.2 % of inhibition of growth rate of dry weight was observed in the highest test item concentration.
On day 2 and 7 shortened root were observed at 0.298 mg/L and above.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Routine testing with 3,5-dichlorophenol (97%) resulted in a LOEC of 1.28 mg/L (nominal) and a NOEC of 0.512 mg/L (nominal)

Reported statistics and error estimates:

The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate of dry weight and yield of dry weight. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for all parameters). The EC10, 20, 50-values were determined by probit analysis following Gompertz distribution. Only concentrations within a clear dose response relation were used for calculations for yield and growth rate of dry weight. The evaluation of data was performed by SAS® (2002-2010). The calculation of the EC50 for growth rate of dry weight and fronds was not indicated since the inhibition was below 50 % at the highest test item concentration and hence the database was weak for probit analysis.

Any other information on results incl. tables

Please see attachment

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item was determined to be 9.954 mg/L based on inhibition of growth rate of frond numbers and growth rate of dry weight. The LOEC of the test item was determined to be 3.05 mg/L based on inhibitory effects on the yield of frond numbers and yield of dry weight. The corresponding EC10, EC20 and EC50 values were determined to be 0.694 mg/L, 3.16 mg/L and 31.1 mg/L respectively.

Executive summary:

The OECD 221 static test was performed to determine the effects of the test item on the growth of the duckweed Lemna gibba after 7 days of exposure.

Significant inhibitory effects were determined for yield of frond numbers and yield of dry weight at test item concentrations of3.05mg/L (nominal) and2.32mg/L (actual) and above. For growth rate of frond numbers and growth rate of dry weight significant inhibitory effects were determined at9.77mg/L (nominal) and 8.70mg/L (actual) and above. The overall LOEC was therefore determined to be 3.05 mg/L (nominal) and 2.32 mg/L (actual), the corresponding NOEC was set at 0.954 mg/L (nominal) and 0.706 mg/L (actual).

The EC10-value for yield of frond numbers was determined to be 0.694 mg/L (nominal) and 0.513 mg/L (actual). The EC10-value for growth rate of frond numbers was determined to be 5.55 mg/L (nominal) and 4.35 mg/L (actual). The EC10-value for yield of dry weight was determined to be 0.327 mg/L (nominal) and 0.252 mg/L (actual). The EC10-value for growth rate of dry weight was determined to be 3.14 mg/L (nominal) and 2.55 mg/L (actual).

The EC20-value for yield of frond numbers was determined to be 3.16 mg/L (nominal) and 2.67 mg/L (actual). The EC20-value for growth rate of frond numbers was determined to be 22.9 mg/L (nominal) and 18.9 mg/L (actual). The EC20-value for yield of dry weight was determined to be 1.54 mg/L (nominal) and 1.23 mg/L (actual). The EC20-value for growth rate of dry weight was determined to be 14.5 mg/L (nominal) and 12.1 mg/L (actual).

The EC50-value for yield of frond numbers was determined to be 31.1 mg/L (nominal) and 32.2 mg/L (actual), the EC50-value for yield of dry weight was determined to be 16.1 mg/L (nominal) and 13.4 mg/L (actual). Due to an inhibition of growth rate of frond numbers and growth rate of dry weight below 50 % the database was weak for probit analysis, hence the EC50-value was considered to be >100 mg/L (nominal) and >82.0 mg/L (actual).