Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid

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:

5 April 2001 - 11 April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

7 June 1984

Deviations:

yes

Remarks:

The pH in the blank-control increased by more than 1.5 unit, but this was related to a relative high rate of algal growth. Hence, this deviation did not affect the validity criterion for algal growth.

GLP compliance:

yes

Specific details on test material used for the study:

50 % aqueous solution

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the final test samples were taken from each concentration before introduction of algae at the start of the exposure for possible analysis. At the end of the exposure, the replicates incubated with and without algae were pooled at each concentration
- Sample storage conditions before analysis: all samples were transferred to the Analytical Chemistry department directly after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of test solutions started with a stock solution of 200 mg/l in the range-finding test and 50 mg/l in the final test, applying 12-l 5 minutes of magnetic stirring. The stock in the range-finding test was diluted to 20 mg/l in M2 medium. The lower test concentrations were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 ml were added to each replicate of the respective test
concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10E4 cells/ml.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornufum
- Strain: NIVA CHLl
- Source (laboratory, culture collection):
- Age of inoculum (at test initiation):
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 (+/-) 2 °C.


ACCLIMATION
- Acclimation period: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10E4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/L (24 mg CaCO3/L

Test temperature:

22-23 °C

pH:

7.9-9.1

Nominal and measured concentrations:

0.06, 0.10, 0.18, 0.30, 0.50, 0.80, 1.3 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- fill volume: 100 mL
- Initial cells density: 1.0E4 cells/mL
- Control end cells density: 145.9E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium according to the ISO standard
- Intervals of water quality measurement: no information

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt (Sylvania), with a light intensity within the range of 60 to 120 µE*mE-2-*sE-1, not varying by more than 20%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cat-y 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank.

TEST CONCENTRATIONS
- Range finding study: Algae were exposed to a range of 0.02 to 20 mg/l, increasing by a factor 10.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- EC50 (cell grow inhibition): 0.83 mg/L; EC50 (growth rate reduction): 1.6 mg/L

At the start of the test, the actual test concentrations were in agreement with the nominal substance concentrations based on all three components separately, except for the two lowest concentrations 0.06 and 1.0 mg/L, where the measured concentrations were higher than nominal. This was probably caused by the lower reliability of the apparatus at these lower levels.

At the end of the 72-hour period the measured concentration had not decreased by more than 20 % based on the responses of the first peak. However, the other two components were less stable, especially the component linked to the third peak, where responses decreased to levels below detection at nominal concentrations below 0.80 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.15 mg/l with a 95 % confidence interval ranging from 0.07 to 0.33 mg/L.
The EC10 for cell growth inhibition (EBC10: 0-72h) was 0.05 mg/l with a 95 % confidence interval ranging from 0.02 to 0.11 mg/L.
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.41 mgll with a 95 % confidence interval ranging from 0.17 to 0.99 mg/L.
The EC10 for growth rate reduction (ERC10: 0-72h) was 0.13 mg/l with a 95 % confidence interval ranging from 0.06 to 0.32 mg/L.
The NOEC for cell growth inhibition was 0.06 mg/L (William’s test, Tukey test and Bonferroni test, p>O.O5).
The NOEC for growth rate reduction was 0.06 mg/L (William’s test).

Executive summary:

The toxicity of the substance as a 50 % aqueous solution towards the algae Pseudokirchneriella subcapitata was asessed in a test according to OECD guideline 201. After a range-finding test, a final test was performed exposing exponentially growing algal cultures to the substance at concentrations of 0.06, 0.10, 0.18, 0.30, 0.50, 0.80 and 1.3 mg/l, and a blank-control. The initial cell density was lE4 cells/ml. The total test period was 72 hours. Samples for determination of actual exposure concentrations were taken at the start and the end of the test period. Quantitative analysis of the substance was performed by Liquid Chromatographic method

with Mass Spectrometric detection (LC-MS-MS). The test substance was extracted from the samples by Solid Phase Extraction (SPE) and eluted from the SPE columns using methanol. Quantification was based on three separate responses. Recovery experiments showed different accuracy’s for recovery of the three separate components. At the start of the test, the actual test concentrations were in agreement with the nominal substance concentrations based on all three components separately, except for the two lowest concentrations 0.06 and 1.0 mg/L, where the measured concentrations were higher than nominal. This was probably caused by the lower reliability of the apparatus at these lower levels. At the end of the 72-hour period the measured concentration had not decreased by more than 20 % based on the responses of the first peak. However, the other two components were less stable, especially the component linked to the third peak, where responses decreased to levels below detection at nominal concentrations below 0.80 mg/l.

Under the conditions of the present study with Selenastrum capricomutum the substance reduced growth rate’ of this fresh water algae species significantly at nominally 0.1 mg/L and higher.

The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.15 mg/l with a 95 % confidence interval ranging from 0.07 to 0.33 mg/L. EC50 based on a.i: 0.075 mg/L

The EC10 for cell growth inhibition (EBC10: 0-72h) was 0.05 mg/l with a 95 % confidence interval ranging from 0.02 to 0.11 mg/L. EC10 based on a.i.: 0.025 mg/L

The EC50 for growth rate reduction (ERC50: 0-72h) was 0.41 mgll with a 95 % confidence interval ranging from 0.17 to 0.99 mg/L. EC50 based on a.i: 0.2 mg/L

The EC10 for growth rate reduction (ERC10: 0-72h) was 0.13 mg/l with a 95 % confidence interval ranging from 0.06 to 0.32 mg/L. EC10 based on a.i.: 0.065 mg/L.

The NOEC for cell growth inhibition was 0.06 mg/L (William’s test, Tukey test and Bonferroni test, p>O.O5). NOEC based on a.i: 0.03 mg/L.

The NOEC for growth rate reduction was 0.06 mg/L (William’s test). NOEC based on a.i.: 0.03 mg/L.

Description of key information

Growth rate : NOEC (72h) 0.03 a.i. mg/L; ErC10 (72h) = 0.065 mg/L; ErC50 (72h) = 0.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.2 mg/L

EC10 or NOEC for freshwater algae:

0.065 mg/L