Disodium 1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

30 March 2016 to 05 Aug 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 92349/B
Physical state/Appearance: Blue solid
Batch: 26/2014 (Thailand)
Purity: 85.5 %
Expiry Date: 16 December 2019
Storage Conditions: Stored frozen at approximately -20 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test groups (replicates R1 - R3 pooled) on Day 0 (fresh media) and on Day 7 (old media) 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. A further sample of each test concentration was incubated alongside the test to provide samples for analysis on Day 7 which contained no lemna.

Vehicle:

no

Details on test solutions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture Medium
NaNO3 85 mg/L
KH2PO4 13.4 mg/L
MgSO4.7H2O 75 mg/L
CaCl2.2H2O 36 mg/L
Na2CO3 20 mg/L
H3BO3 1.0 mg/L
MnCl2.4H2O 0.20 mg/L
Na2MoO4.2H2O 0.010 mg/L
ZnSO4.7H2O 0.050 mg/L
CuSO4.5H2O 0.0050 mg/L
Co(NO3)2.6H2O 0.010 mg/L
FeCl3.6H2O 0.84 mg/L
Na2-EDTA.2H2O 1.40 mg/L

The culture medium will be prepared in reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP). The pH of the prepared culture medium will be adjusted, if necessary, to 6.5 ± 0.2 with either 1M HCl or NaOH.

Test organisms (species):

Lemna minor

Details on test organisms:

The test was carried out using Lemna minor. 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:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 °C.

pH:

7.0 to 7.3 on day 0
7.4 to 9.5 on day 7

Nominal and measured concentrations:

Range-finding test: Nominal: 1.0, 10 and 100 mg/L

Definitive Test: Nominal: 1.0, 3.2, 10, 65 and 100 mg/L

Details on test conditions:

Range-finding test:
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. The control group was maintained under identical conditions but not exposed to the test item. On Days 2 and 5 the test solutions were renewed.

Definitive test:
Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 65, 32 10, 3.2 and 1.0 mg/L.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 and Day 7.

Exposure Conditions
As in the range-finding test glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Each control and test flask was inoculated with 3 colonies of Lemna minor (total 9 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) for 7 days. A static testing regime was employed. The pH of each control and test flask was recorded on Day 0 and Day 7. The temperature and light intensity in the incubator were recorded daily.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Growth Data Based on Frond Number
Inspection of the data from Day 2 onwards showed that there was a significant reduction in growth in the 32 mg/L R2 test replicate. Observations made on the appearance on Day 5 and Day 7 indicated the presence of necrotic fronds, possibly caused by the colonies having been caught on the side of the vessel above the water line when the vessel was returned to the incubator after observations on Day 2. As the data for the remaining two replicates were consistent with each other, the reduction in growth seen was considered not to be test item related and as such this replicate was omitted from all calculations.

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) = 36 mg/L
ErC20 (frond number) = >100 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, 1.0, 3.2, 10 and 32 mg/L test concentrations (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 32 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 65 mg/L.

Yield
EyC10 (frond number) = 8.4 mg/L
EyC20 (frond number) = 23 mg/L
EyC50 (frond number) = >100 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, 1.0, 3.2, 10 and 32 mg/L test concentrations (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 32 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 65 mg/L.

Growth Data Based on Dry Weight
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) = 30 mg/L
ErC20 (dry weight) = 73 mg/L
ErC50 (dry weight) = >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 out for the control and all test concentrations. There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32 mg/L test concentrations (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 32 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 65 mg/L.

Yield
EyC10 (dry weight) = 8.1 mg/L
EyC20 (dry weight) = 20 mg/L
EyC50 (dry weight) = 91 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, 1.0, 3.2, 10, 32 and 65 mg/L test concentrations (P≥0.05), however all the 100 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect.

Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 65 mg/L. Correspondingly the “Lowest Observed Effect Concentration” (LOEC) was determined to be 100 mg/L.

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).

Range-finding test

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, and at the request of the Sponsor test concentrations of 1.0, 3.2, 10, 32, 65 and 100 mg/L were selected for the definitive test. Chemical analysis of the freshly prepared test samples on Day 2 showed near nominal concentrations were obtained. Whilst near nominal concentrations were obtained from the 10 and 100 mg/L test samples in the aged tests samples on Day 5 and Day 7, measured concentrations of 75 % and 77 % of nominal were obtained from the 1.0 mg/L test sample. In order to determine whether the decline in measured concentration was due to adsorption of the test item to the lemna and/or instability, additional samples were prepared on Day 0 during the definitive test which contained no lemna. These samples were incubated alongside the test prior to analysis on Day 7.

Verification of Test Concentrations

Chemical analysis of the test preparation on Day 0 (fresh media) showed measured test concentrations to range from 97 % to 101 % of nominal. Analysis of the test preparations on Day 7 (old media) showed near nominal concentrations were obtained for all test preparations with the exception of the 1.0 mg/L test preparation where a measured concentration of 66 % of nominal was obtained. Analysis of additional test samples prepared on Day 1 and incubated alongside the test which contained no lemna showed a similar pattern suggesting that the decline in measured concentration observed in the 1.0 mg/L test sample was due to instability rather than adsorption of the test item to the lemna present. The decline in measured concentration observed in the 1.0 mg/L test preparations was considered to have had no adverse effect on the outcome of the test given that this concentration was below the No observed Effect Concentration (NOEC). As such it was considered appropriate to calculate the results based on nominal test concentrations only.

Validation Criteria

The following data show that the doubling time of the control cultures was 1.56 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: 172

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and NOEC based on average specific growth rate (frond number) was found to be greater than 100 mg/L and 32 mg/L, respectively.

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, 65 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.

Results

Chemical analysis of the test preparation on Day 0 (fresh media) showed measured test concentrations to range from 97 % to 101 % of nominal. Analysis of the test preparations on Day 7 (old media) showed near nominal concentrations were obtained for all test preparations with the exception of the 1.0 mg/L test preparation where a measured concentration of 66 % of nominal was obtained. Analysis of additional test samples prepared on Day 1 and incubated alongside the test which contained no lemna showed a similar pattern suggesting that the decline in measured concentration observed in the 1.0 mg/L test sample was due to instability rather than adsorption of the test item to the lemna present. The decline in measured concentration observed in the 1.0 mg/L test preparation was considered to have had no adverse effect on the outcome of the test given that this concentration was below the No observed Effect Concentration (NOEC). As such it was considered appropriate to calculate the results based on nominal test concentrations only.

Exposure of Lemna minor to the test item based on the nominal test concentrations gave the following results:

Response Variable	Measurement Variable	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Average Specific Growth Rate	Frond Number	>100	32	65
	Dry Weight	>100	32	65
Yield	Frond Number	>100	32	65
	Dry Weight	91	65	100

Description of key information

The EC50 following 7-days exposure of Lemna minor to the test item, was determined to be (response variable: average specific growth rate, measurement variable: frond number) >100 mg/L, while the NOEC and LOEC were 32 and 65 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

32 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, 65 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.

Results

Chemical analysis of the test preparation on Day 0 (fresh media) showed measured test concentrations to range from 97 % to 101 % of nominal. Analysis of the test preparations on Day 7 (old media) showed near nominal concentrations were obtained for all test preparations with the exception of the 1.0 mg/L test preparation where a measured concentration of 66% of nominal was obtained. Analysis of additional test samples prepared on Day 1 and incubated alongside the test which contained no lemna showed a similar pattern suggesting that the decline in measured concentration observed in the 1.0 mg/L test sample was due to instability rather than adsorption of the test item to the lemna present. The decline in measured concentration observed in the 1.0 mg/L test preparation was considered to have had no adverse effect on the outcome of the test given that this concentration was below the No observed Effect Concentration (NOEC). As such it was considered appropriate to calculate the results based on nominal test concentrations only.

Exposure of Lemna minor to the test item based on the nominal test concentrations gave the following results:

Response Variable	Measurement Variable	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Average Specific Growth Rate	Frond Number	>100	32	65
	Dry Weight	>100	32	65
Yield	Frond Number	>100	32	65
	Dry Weight	91	65	100