Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC/MS/MS

Details on sampling:

Control: at 72 hours
Test concentration/s (nominal): 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L at 0, 24, 48 and 72 hours

Vehicle:

no

Remarks:

mortar and pestle, ultrasonic batch and magnetic stirrer

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations
- test item was pulverized with a mortar and a pestle
- 200.6 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer
- pH was measured to be 9.4 and was adjusted to pH 7.7
- to produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.658 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus 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 to 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 Particle Counter Z2, Beckman Coulter.
- 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 the 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

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (= 22.5 CaCO3)

Test temperature:

21 - 24 °C

pH:

7.8 - 8.0

Nominal and measured concentrations:

nominal [mg/L]: 0.1, 0.32, 1.0, 3.2, 10, 32 and 100
Measured (geometric mean; Mannichbase 1, 2 and 3) [mg/L]: 0.05, 0.15, 0.48, 1.88, 4.49, 19.21, 50.12, 51.79

Details on test conditions:

Exposure conditions
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers test volume: 100 mL
- Culturing apparatus: Shaking incubator 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.
- Light intensity: A light intensity ranging from 60 to 120 µE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a Particle Counter Z2 (Beckman Coulter) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 7 test concentrations plus 1 control, 3 replicates per concentration, 3 replicates per control
- Initial cell density in the test cultures appro-ximately 5000 cells per millilitre
- additionally highest test concentration without algae
- Test item concentration/s: 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Method of administration: stock solution
- Duration of exposure: 72 hours
- Criteria of effects: item-induced inhibition of yield [y] and growth rate [r] of the algal population

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

63.2 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.:

4.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

19.2 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.:

21.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The results are expressed in terms of geometric mean measured concentrations as sum of Mannichbase 1 (2x substituted), 2 (3x substituted) and 3 (4x substituted). Effective concentrations of the sum of Mannichbase 1, 2 and 3 ranged from 63.1 % to 86.4 % of nominal values at 0 hours, 38.4 % to 67.4 % of nominal values at 24 hours, from 37.0 % to 65.0 % of nominal values at 48 hours and from 31.8 % to 58.5 % of nominal values at 72 hours.

Reported statistics and error estimates:

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.
Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-Test Procedure.

Validity criteria fulfilled:

yes

Remarks:

cell density in control increased by a factor of at least 16 in 72 h; mean coefficient of variation for section-by-section specific growth rates in control <=35 %; CV of average specific growth rates during whole test in replicate control cultures <= 7 %

Conclusions:

For HPP 12879-1 an EC50 of 63.2 mg/L and a NOEC of 4.5 mg/L were determined after 72 h towards algae under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of HPP 12879-1 on the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. It 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).

A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.1, 1, 10 and 100 mg/L.

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of HPP 12879-1 dissolved in dilution water. All test concentrations and control were performed in triplicate. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer. During the test a temperature range of 21 – 24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure.

The maintenance of the test item concentrations was proved by analytical measurements. In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using a replicate of the test concentrations and the control vessels.

In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, at all measuring points (0, 24, 48 and 72 hours) a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations.

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 (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-Test Procedure. The following values were determined after 72 h based on growth rate: EC50 = 63.2 mg/L, EC10 = 21.1 mg/L, NOEC = 4.5 mg/L and LOEC = 19.2 mg/L.

The 72 hour EC 10 and EC 50 were calculated by probit analysis. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

The results are expressed in terms of geometric mean measured concentrations as sum of Mannichbase 1, 2 and 3. Effective concentrations of the sum of Mannichbase 1, 2 and 3 ranged from 63.1 % to 86.4 % of nominal values at 0 hours, 38.4 % to 67.4 % of nominal values at 24 hours, from 37.0 % to 65.0 % of nominal values at 48 hours and from 31.8 % to 58.5 % of nominal values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of HPP 12879-1 on the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. It 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 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of HPP 12879-1 dissolved in dilution water. All test concentrations and control were performed in triplicate. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer. During the test a temperature range of 21 – 24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure.

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 (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-Test Procedure. The following values were determined after 72 h based on growth rate: EC50 = 63.2 mg/L, EC10 = 21.1 mg/L, NOEC = 4.5 mg/L and LOEC = 19.2 mg/L.

The 72 hour EC 10 and EC 50 were calculated by probit analysis. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

The results are expressed in terms of geometric mean measured concentrations as sum of Mannichbase 1, 2 and 3. Effective concentrations of the sum of Mannichbase 1, 2 and 3 ranged from 63.1 % to 86.4 % of nominal values at 0 hours, 38.4 % to 67.4 % of nominal values at 24 hours, from 37.0 % to 65.0 % of nominal values at 48 hours and from 31.8 % to 58.5 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

63.2 mg/L

EC10 or NOEC for freshwater algae:

4.5 mg/L