Benzyldimethylamine

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:

2010-05-18 till 2010-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Principles of method if other than guideline:

The purpose of this method was to determine those concentrations which caused a 10 % and a 50 % adverse effect (= EC 10, EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum test concentration of 100 mg/L or at the limit of water solubility, respectively.
A range-finding (non-GLP) test preceded the main test and provided information about the range of concentrations which were used in the main test.
In the main test, the algae were exposed to the test item added to dilution water at a range of concentrations for a period of 72 hours. Defined
concentrations of the test item led to a certain inhibition of algal yield and growth rate at the end of the 72 hour study period.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples from the biological test were diluted with water if necessary and were extracted with diethylether after addition of internal standard

Sampling schedule:
Control : at 0 and 72 hours
Test concentrations : at 0 and 72 hours

The samples were analysed immediately.

Vehicle:

no

Details on test solutions:

- direct weighing
- 100 mg of the test item in 1 litre of dilution water
- The pH was mesured to be 9.4 and was adjusted to pH 7.9.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation: Exponentially-growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 – 120 µE. x m-2 x s-1 (measured in the range 400 to 700 nm
using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density
measurements were made using a microcell- counter, Sysmex F300, Digitana.
- Preparation of pre-cultures : Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for a test were taken from an exponentially-growing pre-culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Hardness:

1.3 °dH (= 22.5 mg/L CaCO3)

Test temperature:

21 °C to 24 °C +/- 2 °C,

pH:

7.8 - 10.3

Nominal and measured concentrations:

- Test item concentration/s (nominal): 0.032, 0.1, 0.32, 1.0, 3.2, 10 and 32 mg/L

Details on test conditions:

Exposure conditions
- Test vessels: 300 mL Erlenmeyer flasks with glas stoppers
- Culturing apparatus: Light chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and continuous uniform
illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was
incubated under the same conditions as the test flasks.
- Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 µE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, was used. Light intensity was checked before start of the study.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test
flask for measurements.
- Experimental design: 7 test concentrations plus 1 control
3 replicates per concentration, 3 replicates per control
Initial cell density in the test cultures approximately 5000 cells per millilitre
- Test item concentration/s: 0.032, 0.1, 0.32, 1.0, 3.2, 10 and 32 mg/L
- Method of administration: stock solution
- Duration of exposure: 72 hours
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95 % confidence limits: 0.99 -1.81 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95 % confidence limits: 0.10 - 0.38 mg/L)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (95 % confidence limits: 0.51 - 0.61 mg/L)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (95 % confidence limits: 0.20 - 0.30 mg/L)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.24 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.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- The software tool ToxRatPro Version 2.10 were not able to calculate a NOEC [r] and NOEC [y], due to insufficient variability among the counts in a Microcell counter. In agreement with existing regulatory practice, the ErC10 and the EyC 10 were used to calculate the NOEC [r] and NOEC [y].
- The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 71.3 - 100.0 % of nominal
values at 0 hours, and from 67.2 - 83.6 % of nominal values at 72 hours, respectively.

Growth Rate of Desmodesmus subspicatus (%inhibition after 72h)

Treatment (mg/L)	Mean	Std. Dev.	n	% inhibition
control	1.175	0.0000	3	0.0
0.026	1.175	0.0000	3	0.0
0.088	1.155	0.0209	3	1.8
0.221	1.155	0.0000	3	1.7
0.816	0.768	0.0000	3	34.7
2.615	0.231	0.0000	3	80.3
8.381	0.231	0.0000	3	80.3
25.702	0.231	0.0000	3	80.3

Table Analysis (HPLC values)

Test item concentration (mg/L)	HPLC values (mg/L)
	0h	72h
control	<0.094	<0.094
0.032	0.027	0.025
0.1	0.100	0.078
0.32	0.228	0.215
1.0	0.836	0.796
3.2	2.729	2.505
10	8.403	8.360
32	25.308	26.102

Validity criteria fulfilled:

yes

Remarks:

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. With the current test it was found to be 34.0. The test fulfils this validity criterion.

Conclusions:

The acute toxcity of benzyldimethylamine to Algae (Desmodesmus subspicatus) was tested according to EU method C.3 'Alga Inhibition Test' (2009) yielding an ErC50(72 h) of 1.34 mg/L and ErC10(72 h) of 0.24 mg/L (Currenta, 2010).

Executive summary:

A study was performed to assess the adverse effects of benzyldimethylamine on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of benzyldimethylamine dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration. The following values were determined:

ErC 50* (0-72 h) = 1.34 mg/L

ErC 10* (0-72 h) = 0.24 mg/L

NOEC [r]* (ta 0.05) = 0.24 mg/L#

# The software tool ToxRatPro Version 2.10 was not able to calculate a NOEC [r], due to insufficient variability among the counts in a Microcell counter. In agreement with existing regulatory practice, the ErC10 was used to calculate the NOEC [r].

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 71.3-100.0% of nominal values at 0 hours, and from 67.2-83.6% of nominal values at 72 hours, respectively.

Description of key information

The acute toxcity of benzyldimethylamine to Algae (Desmodesmus subspicatus) was tested according to EU method C.3 'Alga Inhibition Test' (2009) yielding an ErC50(72 h) of 1.34 mg/L and NOEC(72 h) of 0.24 mg/L (Currenta, 2010).

Key value for chemical safety assessment

EC50 for freshwater algae:

1.34 mg/L

EC10 or NOEC for freshwater algae:

0.24 mg/L

Additional information

A study was performed to assess the adverse effects of benzyldimethylamine on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of benzyldimethylamine dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration. The following values were determined:

ErC 50* (0-72 h) = 1.34 mg/L

ErC 10* (0-72 h) = 0.24 mg/L

NOEC [r]* (ta 0.05) = 0.24 mg/L#

# The software tool ToxRatPro Version 2.10 was not able to calculate a NOEC [r], due to insufficient variability among the counts in a Microcell counter. In agreement with existing regulatory practice, the ErC10 was used to calculate the NOEC [r].

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 71.3-100.0% of nominal values at 0 hours, and from 67.2-83.6% of nominal values at 72 hours, respectively.