Ethyl 4-dimethylaminobenzoate

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:

16 March 2017 to 06 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.
-Definitive test: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
-Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
-Based on this information the test material was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document, therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
-As a result of the trial the test material was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a 100 % v/v saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C
- 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 10^3 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 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.7 to 8.3

Nominal and measured concentrations:

Nominal test concentrations: 0.32, 1.0, 3.2, 10 and 32 % v/v saturated solution
0 hour measured test concentrations: 0.080, 0.29, 1.1, 4.9 and 15 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed- the flasks were plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 5.00 x10^3cells/ mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


TEST MEDIUM
- 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 25.5 mg/L, MgCl2.6H2O 12.16 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.6 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.186 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00327 mg/L, FeCl3.6H2O 0.160 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA.2H2O 0.30 mg/L.
-The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
-Constantly shaken at approximately 150 rpm for 72 hours.


EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Samples were taken at 24, 45 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 103 cells/mL) was taken as the starting cell density. To determine the potential effect of the test material 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.
- Water Quality Criteria: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.
-Test material concentrations were analytically verified.

TEST CONCENTRATIONS
- Range finding study: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution for a period of 72 hours.
- Results used to determine the conditions for the definitive study: yes
-Preparation of test material solutions: A nominal amount of test material (1100 mg) was dispersed in 11 litres 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 material was removed by filtration through a 0.2 µm Sartorius Sartopre filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2, 1.0 and 0.32 % v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 5.6 mL of algal suspension to give the required test concentrations of 0.32, 1.0, 3.2, 10 and 32 % v/v saturated solution.

DATA EVALUATION
-Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation: µ = (ln Nn – ln N1) / (tn – t1)
Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:
Ir = [(µc - µt) / µc] x 100
Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:
Y = Nn – N0
Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test
For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:
Iy = [(Yc – Yt)/ Yc ] x 100
Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

DETERMINATION OF ECx VALUES
-For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
-Where appropriate 9 5% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

STATISTICAL ANALYSIS
-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) was carried out on the growth rate and yield data after 72 hours 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).

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not exceed 35 %.
- The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits 2.3 to 3.4 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.71 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.96 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of yield

Remarks on result:

other: 95 % confidence limits: 0.74 to 1.2 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of yield

Details on results:

RANGE-FINDING TEST
-The results showed no effect on growth at the test concentration of 0.10 % v/v saturated solution. However, growth was observed to be reduced at 1.0, 10 and 100 % v/v saturated solution.
-Based on this information test concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution were selected for the definitive test.
-Chemical analysis of the test preparations at 0 hours (see Annex 5) showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.16 mg/L, to 5.3 mg/L. There was no significant change in the measured concentrations at 72 hours indicating that the test material was stable over the test duration.

DEFINITIVE TEST
-Verification of Test Concentrations: Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.16 to 15 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on 0-Hour measured test concentrations only. Where a measured concentration of less than the LOQ was obtained, a value of half the LOQ, i.e. 0.080 mg/L was substituted.
-A summary of the growth rate and inhibition of yield results can be seen in Table 1.
-Growth Data: From the data recorded, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72-Hour exposure period. The following results were determined from the data:
Inhibition of Growth Rate: ErC10 (0 - 72 h): 0.71 mg/L, ErC20 (0 - 72 h): 1.2 mg/L and ErC50 (0 - 72 h): 2.8 mg/L; 95% confidence limits 2.3 – 3.4 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 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, 0.080 and 0.29 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) based on growth rate was 0.29 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.1 mg/L.
-Inhibition of Yield: EyC10 (0 - 72 h): 0.28 mg/L, EyC20 (0 - 72 h): 0.44 mg/L and EyC50 (0 - 72 h): 0.96 mg/L; 95 % confidence limits 0.74 – 1.2 mg/L. Where: EyCx is the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out as for the growth rate data. There were no statistically significant differences between the control, 0.080 and 0.29 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) based on yield was 0.29 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.1 mg/L.

VALIDATION CRITERIA
-The following data show that the cell concentration of the control cultures increased by a factor of 143 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 10^5 cells per mL. Mean cell density of control at 72 hours: 7.14 x 10^5 cells/mL.
-The mean coefficient of variation for section by section specific growth rate for the control cultures was 16 % 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 3 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

OBSERVATION ON CULTURES
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.080, 0.29, 1.1 and 4.9 mg/L, however cell debris was observed to be present in the test cultures at 15 mg/L.

WATER QUALITY CRITERIA
-Temperature was maintained at 24 ± 1 °C throughout the test.
-The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.2 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

OBSERVATIONS ON TEST MATERIAL SOLUBILITY
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 0.080, 0.29 and 1.1 mg/L test cultures were observed to be green dispersions whilst the 4.9 and 15 mg/L test cultures were observed to be clear colourless solutions.

Results with reference substance (positive control):

-Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference material gave the following results: ErC50 (0 to 72 h): 1.4 mg/L; 95 % confidence limits 1.2 – 1.5 mg/L and EyC50 (0 to 72 h): 0.60 mg/L; 95 % confidence limits 0.52 – 0.69 mg/L.
-The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Table 1: A summary of the growth rate and inhibition of yield results

Response Variable	EC50 (mg/L)	95 % Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	2.8	2.3	to	3.4	0.29	1.1
Yield	0.96	0.74	to	1.2	0.29	1.1

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-Hour measured test concentrations: Growth rate EC50: 2.8 mg/L with 95 % confidence limits of 2.3 to 3.4 mg/L and yield inhibition EC50: 0.96 mg/L with 95 % confidence limits of 0.74 to 1.2 mg/L with a NOEC of 0.29 mg/L.

Executive summary:

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32 % v/v saturated solution (three replicate flasks per concentration) 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 (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.16 to 15 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on 0-Hour measured test concentrations only. Where a measured concentration of less than the LOQ was obtained, a value of half the LOQ, i.e. 0.080 mg/L was substituted.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-Hour measured test concentrations: Growth rate EC50: 2.8 mg/L with 95 % confidence limits of 2.3 to 3.4 mg/L and yield inhibition EC50: 0.96 mg/L with 95 % confidence limits of 0.74 to 1.2 mg/L with a NOEC of 0.29 mg/L.

Description of key information

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32 % v/v saturated solution (three replicate flasks per concentration) 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 (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.16 to 15 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on 0-Hour measured test concentrations only. Where a measured concentration of less than the LOQ was obtained, a value of half the LOQ, i.e. 0.080 mg/L was substituted.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-Hour measured test concentrations: Growth rate EC50: 2.8 mg/L with 95 % confidence limits of 2.3 to 3.4 mg/L, Growth rate EC10 = 0.71 mg/L, and yield inhibition EC50: 0.96 mg/L with 95 % confidence limits of 0.74 to 1.2 mg/L with a NOEC of 0.29 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.8 mg/L

EC10 or NOEC for freshwater algae:

0.71 mg/L