2-({2-[4-(dimethylamino)benzoyloxy]ethyl}(methyl)amino)ethyl 4-(dimethylamino)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:

20 November 2006 - 23 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EU Comission Directive 92/69/EEC 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

Ecological effects Test guidelines, April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test article name LFC 2098
Chemservice sample code AA26964
Declared purity (according to CoA) 100%
Chemical class : Aminoester
Chemical formula C23H31O4N3
Molecular weight 413
Preparation date 05/29/2006
Solubility in water Practically insoluble
Density Not available
Physical form at 20 °C Solid (powder)
Colour White
Storage conditions Room temperature, protect from light
Safety precautions Routinary hygienic procedures

Analytical monitoring:

yes

Details on sampling:

Sampled at 0h and 72 h
Duplicate samples of the test water and the control were taken at the indicated times from each concentration, but only one sample from the test water and the control were analysed, while the duplicates were stored to be analysed only if necessary.
Since the samples could not be analysed immediately after sampling, they were stored in at 4°C in the dark until the analysis day.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

The study was performed with the unicellular, fresh-water green algae PseudokirchnerielIa subcapitata, SeIe-IRSA, cultured in the Chemservice laboratory and originally obtained from the lRSA-CNR (Water Research Institute-Italian Research Council), Brugherio, Milano.
The algae have been cultured in a temperature controlled room at 24°C ± 2°C under continuous uniform illumination of approximately 6000 Lux in the spectral range 400-700 nm.
The culture medium was the same of the test medium; the stock culture were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of (302 and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

CaCl2 x 2 H20 18.0 mg/L

Test temperature:

The average room temperature was 21.4 ± 0.9 “C,with a maximum of 23.1 °C and a minimum of 19.4 °C.

pH:

7.60 - 7.96

Dissolved oxygen:

Not measured

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Time Nominal Measured (mg/L)
0 h Fortified 1 mg/L n.d
0 h Fortified 3.2 mg/L n.d
0 h Fortified 10 mg/L 0.038
0 h Fortified 31.6 mg/L 0.17
0 h Fortified 100 mg/L 0.598
72 h Fortified 1 mg/L n.d
72 h Fortified 3.2 mg/L n.d
72 h Fortified 10 mg/L n.d
72 h Fortified 31.6 mg/L 0.015
72 h Fortified 100 mg/L 0.175

Details on test conditions:

The test concentrations were chosen on the basis of the results of a non-GLP preliminary test and included five nominal concentrations in a geometric series, namely 10, 3.2, 10.0, 31.6 and 100 mg/L.
Prior to the start of the test, a culture medium stock solution of 100 mg/L has been prepared by direct weighting. Given the low water solubility of LPG 2098, the suspension was mixed by a magnetic stirrer for 48 hours to achieve the maximum solubilisation, then it was filtered by a filtering system. Adequate aliquots of the solution were then taken, diluted in the algal medium and manually shaken to obtain the test solutions.
A negative control without the test item was also run to check the acceptability of the algal population used in the assay.

Cell density was measured every 24 hours by fluorescent reading with spectrofluorophotometer in few millilitre samples taken from each test concentration replicate and from controls. The observed values were used to calculate the percentage inhibition of cell growth as biomass and as growth rate in comparison with control.
The EbCx and ErCx have been calculated by the mean of the CETIS elaboration software v1.026D, using a linear interpolation model.

The negative control was considered only to check that the growth of the algal population used in the test met the validity criteria.

The treatment vessels were identified by experiment number, group and replicate number.
All the described procedures have been conducted under a laminar flow bench to ensure the axenicity of the algal cultures.
For the test concentrations and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers have been used.
Three replicates for each concentration and six for control were prepared. At the start of the test, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain a starting cell density of around 10“ cells/ml.
The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 106 cells/ml.
The test was incubated in a temperature-controlled room, under continuous illumination and constant shaking.

Temperature: the ambient temperature under the lights was continuously measured by the mean of a data logger. It was in the range 19.4 - 23.1 °C. This represents a deviation from mentioned guidelines, but this had no negative impact on the test, given that no negative effect was observed in the control.

Light conditions: continuous illumination; light intensity was 6000-10000 lux.

Test duration: 72 hours

Test water: Reconstituted water according to OECD (Guideline 201, 2002)

Reference substance (positive control):

no

Remarks:

[State positive control substance]

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

51.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

13.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

85 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60.6 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 387 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

6.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

27.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21.7 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4 - 11.2 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

46.9 - 56.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10.3 - 16.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

79 - 90.5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50.4 - 70.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.1 - 6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8 - 16.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.1 - 8.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

23.6 - 32 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.7 - 26.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test item concentration has shown a large decrease compared to nominal values already short after the test solutions were prepared (0.2% to 0.6% at time 0), while a minor decrease occurred during the following days (to 0.05 - 0.50 % of the nominal after 72 hours). The very rapid concentration decline is likely due to the low water solubility of test item and to the filtering process used for the preparation of the test solutions.

The recovery from the two lowest concentrations at the beginning and at the end of test period and from the nominal concentration of 10 mg/L after 72 hours was calculated rather than measured, being the test item level below the Limit Of Detection (LOD).
The biological results are, then, not referred to the nominal concentrations but to the median of the measured concentrations, as recommended by the OECD Guidance Document on testing of difficult substances (2000). Where the test item has been not detected, the final concentration may be taken as the limit of detection for the method (OECD Guidance Document on testing of difficult substances, 2000), which in this case is 5 µg/L .

Reported statistics and error estimates:

Details of the statistic analysis are reported in Annex B.

The 72-hour EC10, EC20 and EC50 with their 95% confidence limits of LFC 2098 to Pseudokirchneriella subcapitata, calculated on the basis of the actual concentrations, for both the end-point, are reported in the following table:

Endpoint	72 h EC10 (ug/L)	72 h EC20 (ug/L)	72 h EC50 (ug/L)
Biomass	7.4 (4.0-11.2)	13.2 (10.3-16.7)	60.6 (50.4-70.8)
Growth rate	51.7 (46.9-56.5)	85.0 (79.0—90.5)	>387

Corresponding to the nominal concentrations as follows:

Endpoint	72 h EC10 (ug/L)	72 h EC20 (ug/L)	72 h EC50 (ug/L)
Biomass	3.6 (0.1 - 6.0)	6.2 (4.1 - 8.3)	21.7 (16.7 - 26.1)
Growth rate	12.2 (8.0 - 16.2)	27.8 (23.6 - 32.0)	> 100.0

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of test item LFC 2098 has been tested on Pseudokirchnerie/Ia subcapitata. The EC10, EC20 and EC50 with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) for both the end-point after 72 hours, calculated based on the median of the measured exposure concentrations as follows:

Endpoint 72 h EC10 (ug/L) 72 h EC20 (ug/L) 72 h EC50 (ug/L)
Biomass 7.4 (4.0-11.2) 13.2 (10.3-16.7) 60.6 (50.4-70.8)
Growth rate 51.7 (46.9-56.5) 85.0 (79.0—90.5) >387


Corresponding to the nominal concentrations as follows:

Endpoint 72 h EC10 (ug/L) 72 h EC20 (ug/L) 72 h EC50 (ug/L)
Biomass 3.6 (0.1 - 6.0) 6.2 (4.1 - 8.3) 21.7 (16.7 - 26.1)
Growth rate 12.2 (8.0 - 16.2) 27.8 (23.6 - 32.0) > 100.0

Executive summary:

The influence of the test item LFC 2098 on the growth of the green algal species Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum, was investigated in a 72-hour full test according to the EU Commission Directive 92/69/EEC, C3, 1992, the OECD Guideline No. 201, 2002 and the OPPTS 850.5400, 1996.

For this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous test medium containing the test item at five nominal concentrations in a geometric series, namely 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L under defined conditions. Besides the five concentrations, a negative control without the test item was also prepared to check the acceptability of the algal inoculum.

The actual test concentrations were analytically measured at the beginning and at the end of the test. The test item concentration has shown a very large decrease compared to nominal values already short after being prepared (0.2% to 0.6% at time 0), while a minor decrease occurred during the following days (to 0.05—0.50/0 of the nominal after 72 hours).

Therefore, the biological results are not referred to the nominal concentrations but to thenmedian of the measured concentrations, according to the OECD Guidance Document on testing of difficult substances (2000).

Algal cell density was measured every 24 hours by fluorescent reading with spectrofluorophotometer in few millilitre samples taken from each test concentration replicate and from controls. The pH of the test medium at different concentrations of test item at the beginning of the test was in the range 7.60-7.96. Therefore, the pH of the test medium was not adjusted before the start of the test. The average room temperature was 21.4 ± 0.9 “C, with a maximum of 23.1 and a minimum of 19.4 °C.

In the negative control the cell density has increased on average by a factor of 261. This value complies with the validity criteria of the test, according to the mentioned guidelines.

In the test medium concentrations the algal biomass (b) after 72 hours of exposure was inhibited by a minimum of 4% for lowest concentration to a maximum of 78% for the highest concentration, compared with control flasks. The growth rate at the end of the test period was inhibited to a minimum value of 1% for the lowest concentration to a maximum of 36% for the nominal concentration of 100 mg/L compared with control flasks.

The 72—hour EC10, EC20 and EC50 with their 95% confidence limits of LFC 2098 to Pseudokirchneriella subcapitata, calculated on the basis of the actual concentrations, for both the end-point, are reported in the following table:

Endpoint	72 h EC10 (ug/L)	72 h EC20 (ug/L)	72 h EC50 (ug/L)
Biomass	7.4 (4.0-11.2)	13.2 (10.3-16.7)	60.6 (50.4-70.8)
Growth rate	51.7 (46.9-56.5)	85.0 (79.0—90.5)	>387

Corresponding to the nominal concentrations as follows:

Endpoint	72 h EC10 (ug/L)	72 h EC20 (ug/L)	72 h EC50 (ug/L)
Biomass	3.6 (0.1 - 6.0)	6.2 (4.1 - 8.3)	21.7 (16.7 - 26.1)
Growth rate	12.2 (8.0 - 16.2)	27.8 (23.6 - 32.0)	> 100.0

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

12.2 mg/L

Additional information

EC50 72 hour > 100 mg/L