Methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 February 2017 and 07 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification (Trade Name): Lilial ME Anthranilate (Lilianth BHT):
Identification (IUPAC Name): methyl 2-((3-(4-(tert-butyl)phenyl)-2-methylprop-1-en-1-yl)amino)benzoate
Physical state/Appearance: Yellow liquid – observed to have formed a yellow, wet looking crystalline solid when opened for use

Analytical monitoring:

yes

Details on sampling:

Range finding test:
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive test:
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Two additional sample of the 100% v/v saturated solution test preparation were incubated alongside the test to provide samples for analysis at 24 and 48 hours. A sample of the 100% v/v saturated solution prepared at 0 hours containing no algal cells was incubated alongside the test to provide a sample for uninoculated analysis at 72 hours.

Vehicle:

no

Details on test solutions:

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.42 mg/L could be obtained using a saturated solution method of 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 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10 and 1.0% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (10.0 mL) to give the required test concentrations of 1.0, 10 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 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to give a 100% v/v saturated solution.
An aliquot (3 liters) of this saturated solution was inoculated with algal suspension (47.0 mL) to give the required test concentration of 100% v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 ºC.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 ºC until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.9 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Nominal and measured concentrations:

Range-finding test:
Nominal test concentrations of 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured concentration of 0.079 mg/L was obtained. A decline in measured concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.025 mg/L indicating that the test item was either unstable and/or was adsorbing to the algal cells present.

Definitive test:
nominal: 100% v/v saturated solution

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.20 mg/L. A decline in measured test concentrations to less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.025 mg/L was observed at 24, 48 and 72 hours. An additional sample prepared at 0 hours which contained no algal cells showed a measured concentration of less than the LOQ at 72 hours indicating that the decline in measured concentrations was attributable to instability of the test item rather than adsorption of the test item to the algal cells present.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0125 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentration was determined to be 0.049 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation. The control group was maintained under identical conditions but not exposed to the test item.

- Initial cells density:
5.00 x 10^3 cells per mL

- No. of vessels per concentration (replicates):
6

- No. of vessels per control (replicates):
6


GROWTH MEDIUM
- Standard medium used: yes
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

- Photoperiod:
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 24, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Definitive test:
Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.049 mg/L over the 72-Hour exposure period.
The test concentration of 0.049 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.
Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >0.049 mg/L
ErC20 (0 - 72 h): >0.049 mg/L
ErC50 (0 - 72 h): >0.049 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 0.049 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the control and 0.049 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.049 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 0.049 mg/L
EyC20 (0 - 72 h): >0.049 mg/L
EyC50 (0 - 72 h): >0.049 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05), between the control and 0.049 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.049 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 60 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 3.00 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.9 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	8.98E+03	4.24E+05	-	-
	R2	6.89E+03	4.18E+05
	Mean	7.93E+03	4.21E+05
1.0	R1	8.07E+03	4.59E+05	2	7
	R2	8.15E+03	3.29E+05
	Mean	8.11E+03	3.94E+05
10	R1	7.57E+03	3.97E+05	2	11
	R2	7.57E+03	3.54E+05
	Mean	7.57E+03	3.76E+05
100	R1	6.39E+03	3.91E+05	0	5

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.058		3.24E+05
	R2	0.058		3.22E+05
	R3	0.057		2.96E+05
	R4	0.057	-	3.05E+05	-
	R5	0.053		2.23E+05
	R6	0.057		3.02E+05
	Mean	0.057		2.95E+05
	SD	0.002		3.70E+04
0.049	R1	0.054	5	2.35E+05
	R2	0.059	[4]	3.43E+05
	R3	0.056	2	2.74E+05
	R4	0.051	11	1.94E+05
	R5	0.053	7	2.17E+05
	R6	0.059	[4]	3.34E+05
	Mean	0.055	3	2.66E+05	10
	SD	0.003		6.18E+04

 [ ]

*    In accordance with the OECD test guideline only the mean value for yield for is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were >0.049, >0.049 and 0.049 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at a nominal concentrations of 100 % v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test material 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 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 10000g for 30 minutes to produce a 100% v/v saturated solution of the test item.

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.20 mg/L. A decline in measured test concentrations to less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.025 mg/L was observed at 24, 48 and 72 hours. An additional sample prepared at 0 hours which contained no algal cells showed a measured concentration of less than the LOQ at 72 hours indicating that the decline in measured concentrations was attributable to instability of the test item rather than adsorption of the test item to the algal cells present.

Given this decline in measured test concentrations it was considered appropriate to estimate the results based on the geometric mean measured test concentration which was determined to be 0.049 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 and EC10 values based on the geometric mean measured test concentration of greater than 0.049 mg/L. The No Observed Effect Concentration was determined to be 0.049 mg/L.

This study showed that there were no toxic effects at saturation.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at a nominal concentrations of 100 % v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test material 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 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 10000g for 30 minutes to produce a 100% v/v saturated solution of the test item.

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 0.20 mg/L. A decline in measured test concentrations to less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.025 mg/L was observed at 24, 48 and 72 hours. An additional sample prepared at 0 hours which contained no algal cells showed a measured concentration of less than the LOQ at 72 hours indicating that the decline in measured concentrations was attributable to instability of the test item rather than adsorption of the test item to the algal cells present.

Given this decline in measured test concentrations it was considered appropriate to estimate the results based on the geometric mean measured test concentration which was determined to be 0.049 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 and EC10 values based on the geometric mean measured test concentration of greater than 0.049 mg/L. The No Observed Effect Concentration was determined to be 0.049 mg/L.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.049 mg/L

EC10 or NOEC for freshwater algae:

0.049 mg/L