4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

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 May 2016 and 08 September 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)

Remarks:

UK GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material:
PCR92X140707 (China)
- Expiration date of the lot/batch:
14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Approximately 4 °C 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

Vehicle:

yes

Remarks:

The culture medium will be prepared in reverse osmosis purified water. The pH of the prepared culture medium will be adjusted, if necessary, to 6.5 ± 0.2 with either 1M HCl or NaOH

Details on test solutions:

The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 2.44 mg/L could be obtained using a saturated solution method of preparation. The test concentration to be used in the initial experiment was 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% v/v saturated solution for a period of 7 days. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for the control and each test concentration. A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0 % v/v saturated solution. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Initial Experiment
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100 % v/v saturated solution to confirm that at the highest attainable concentration, no effect on growth was observed. This experiment was conducted using a static testing regime with no media renewal. The experiment was terminated on Day 4 due to a significant reduction in the number of fronds present in the 100 % v/v saturated solution test preparations. Given this it was considered appropriate to conduct the definitive using a test concentration range of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 50, 25, 12.5 and 6.25 % v/v saturated solution. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

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:

no

Total exposure duration:

7 d

Hardness:

Not reported

Test temperature:

24 ± 1 ”C

pH:

6.5 ± 0.2

Nominal and measured concentrations:

The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100 % v/v saturated solution.
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100 % v/v saturated solution to confirm that at the highest attainable concentration,
Based on the above, it was considered appropriate to conduct the definitive using a test concentration range of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution

Details on test conditions:

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 2.44 mg/L could be obtained using a saturated solution method of preparation. The test concentration to be used in the initial experiment was 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% v/v saturated solution for a period of 7 days. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for the control and each test concentration. A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0 % v/v saturated solution. 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. At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for 7 days. On Days 2 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 2, 5 and 7. 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.

Initial Experiment
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100 % v/v saturated solution to confirm that at the highest attainable concentration, no effect on growth was observed. This experiment was conducted using a static testing regime with no media renewal. The experiment was terminated on Day 4 due to a significant reduction in the number of fronds present in the 100% v/v saturated solution test preparations. Given this it was considered appropriate to conduct the definitive using a test concentration range of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.

Definitive Test
Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 50, 25, 12.5 and 6.25 % v/v saturated solution. Each of the prepared concentrations was 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, 3 and 5 (fresh media) and on Days 3, 5 and 7 (old media).

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.
On days 3 and 5 the test solutions were renewed.

Reference substance (positive control):

yes

Remarks:

3,5-dichlotophenol; assessed in a separate study between 18 May 2016 and 30 May 2016

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

time-weighted mean measured test concentration

Basis for effect:

other: Yield

Details on results:

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared 100 % v/v saturated solution test preparations on Days 0, 3 and 5 showed measured test concentrations of 1.0, 1.6 and 0.99 mg/L, respectively, were obtained. A decline in measured test concentrations was observed in the corresponding old or expired test preparations on Days 3, 5 and 7 to 0.17, 0.21 and 0.20 mg/L, respectively. Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentration in order to give a “worst case” analysis of the data.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.79 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 : 93

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) = >0.54 mg/L
ErC20 (frond number) = >0.54 mg/L
ErC50 (frond number) = >0.54 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, 6.25, 12.5, 25, 50 and 100 % v/v saturated solution 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 100 % v/v saturated solution (0.54 mg/L).

Yield
EyC10 (frond number) = >0.54 mg/L
EyC20 (frond number) = >0.54 mg/L
EyC50 (frond number) = >0.54 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, 6.25, 12.5, 25, 50 and 100 % v/v saturated solution test concentrations (P≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 100 % v/v saturated solution (0.54 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) = >0.54 mg/L
ErC20 (dry weight) = >0.54 mg/L
ErC50 (dry weight) = >0.54 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. There were no statistically significant differences between the control, 6.25 12.5, 25, 50 and 100% v/v saturated solution test concentrations (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 100 % v/v saturated solution (0.54 mg/L).

Yield
EyC10 (dry weight) = >0.54 mg/L
EyC20 (dry weight) = >0.54 mg/L
EyC50 (dry weight) = >0.54 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, 6.25, 12.5, 25, 50 and 100 % v/v saturated solution test concentrations (P ≥0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 100 % v/v saturated solution (0.54 mg/L).

Results with reference substance (positive control):

A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at con
centrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Lemna minor to the reference item gave the following results:

Response Variable Measurement Variable EC50 (mg/L) 95% Confidence Limits No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate Frond Number 3.4 3.1-3.8 0.625 1.25
Dry Weight 3.0 2.7-3.2 0.625 1.25
Yield Frond Number 1.8 1.6-2.2 0.625 1.25
Dry Weight 1.4 1.2-1.7 0.625 1.25
The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item

Response variable	Measurement variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
Average specific growth rate	Frond number	>0.54	0.54
	Dry weight	>0.54	0.54
Yield	Frond number	>0.54	0.54
	Dry weight	>0.54	0.54

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and No Observed Effect Concentration (NOEC) of FAT 40444/B was >0.54 and 0.54 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)”. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 2.44 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test and initial experiment, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development.

Chemical analysis of the freshly prepared 100% v/v saturated solution test preparations on Days 0, 3 and 5 showed measured test concentrations of 1.0, 1.6 and 0.99 mg/L respectively were obtained. A decline in measured test concentrations was observed in the corresponding old or expired test preparations on Days 3, 5 and 7 to 0.17, 0.21 and 0.20 mg/L respectively. Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted mean measured test concentration in order to give a “worst case” analysis of the data. Exposure of Lemna minor to the test item based on the time-weighted mean measured test concentration gave the following results:

 

Response variable	Measurement variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)
Average specific growth rate	Frond number	>0.54	0.54
	Dry weight	>0.54	0.54
Yield	Frond number	>0.54	0.54
	Dry weight	>0.54	0.54

Description of key information

EC₅₀ and NOEC of Disperse Red 092 calculated using average specific growth rate based on frond number was determined to be >0.54 and 0.54 mg/l (saturation concentration), respectively.

Key value for chemical safety assessment

EC50 for freshwater plants:

0.54 mg/L

EC10 or NOEC for freshwater plants:

0.54 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 according to the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”. Following a preliminary range-finding test and initial experiment, Lemna minor was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. Based on the findings of the study, EC₅₀ and NOEC calculated using average specific growth rate based on frond number was determined to be >0.54 and 0.54 mg/l (saturation concentration), respectively.