Betaines, C12-16 (even numbered) -alkyldimethyl

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:

10th October - 13th October 2017

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)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

14 February 2017

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.5400, Algal Toxicity, Tiers I and II

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 0.01, 0.026, 0.064, 0.16, 0.4 and 1.0 mg/L
- Sampling method: Duplicate samples (~2-2 mL) were taken in glass tubes at the applied test concentration levels (each replicates) and from the control (three replicates only) at 24 hour intervals during the experiment.
- Sample storage conditions before analysis: After sampling, samples were frozen and kept approximately at -20°C prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A work solution with a concentration of 40.0 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath (~5 minutes). This work solution was diluted with OECD Medium to give the stock solution of 1.0 mg/L (nominal) (50.0 mL work solution was filled up with test media to 2000 mL).
The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.

ACCLIMATION
- Culturing media and conditions (same as test or not): Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

The temperature was in the range of 22.6 – 22.9 °C measured in the flask and between 22.0 and 23.0 °C measured within the climate chamber.

pH:

The range of the pH was 7.22 – 8.36 during the experiment.

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Main test:
Nominal: 0.01, 0.026, 0.064, 0.16, 0.4 and 1.0 mg/L.
Geometric mean measured: 0.004, 0.008, 0.032, 0.096, 0.306 and 0.852 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmyer flask
- Type (delete if not applicable): air-permeable stopper
- Material, size, headspace, fill volume: glass
- Aeration: no
- Initial cells density: 1E04 cells/mL test medium
- Control end cells density: mean = 69.33E04 cells/mL test medium after 72 h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: approx 8335 lux, spectral range 400-700 nm.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 0, 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study:

Nominal concentrations (mg/L)/ Average of cell number at 72 hours (x 1E04 cell/mL)

Untreated control/76.33; 0.1/44.50; 1/1.50; 10/1.00; 100/1.00

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.165 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.:

0.032 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.:

0.096 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.076 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Chuff cells were observed at the tested concentrations of 0.096, 0.306 and 0.852 mg/L (measured) during the experiment.

Results with reference substance (positive control):

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

See 'ther information' below.

Validity

The cell density in the control cultures increased by the factor of 69.33 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 12.73 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.80 %.

All validity criteria were met, therefore the study can be considered as valid.

Average Specific Growth Rates:

The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the concentration range 0.096– 0.852 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 0.032 mg/L (measured). The 72 h E_rC₅₀was determined [by Probit analysis (TOXSTAT software)] as 0.165 mg/L (95 % confidence limits: 0.143 – 0.190 mg/L).

Areas under the growth curves:

The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the concentration range of 0.032– 0.852 mg/L (measured), accordingly theNo Observed Effect Concentration (NOEC) determined as 0.008 mg/L (measured). The 72 h E_bC₅₀value was determined [by Probit analysis (TOXSTAT software)] as 0.070 mg/L (95 % confidence limits: 0.059 – 0.082 mg/L).

Yield:

The results of the statistical evaluation (based on Bonferroni t-Test; a=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value atthe tested concentration range of 0.032– 0.852 mg/L (measured), accordingly theNo Observed Effect Concentration (NOEC) determined as 0.008 mg/L (measured). The 72 h E_yC₅₀value was determined [by Probit analysis (TOXSTAT software)] as 0.076 mg/L (95 % confidence limits: 0.065 – 0.088 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

ErC50 = 0.165 mg/L (measured), NOErC = 0.032 mg/L (measured).

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours in a test performed according to OECD TG 201.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 0.096 – 0.852 mg/L (measured), therefore the NOErC was determined as 0.032 mg/L (measured) and LOErC was determined as 0.096 mg/L (measured). The ErC50 was determined as 0.165 mg/L (measured). The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 0.032 – 0.852 mg/L (measured). The NOEC was determined as 0.008 mg/L (measured); the LOEC was determined as 0.032 mg/L (measured).

Description of key information

72 -h ErC50 = 0.165 mg/L, NOErC = 0.032 mg/L (Pseudokirchneriella subcapitata; OECD TG 201).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.165 mg/L

EC10 or NOEC for freshwater algae:

0.032 mg/L

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours in a test performed according to OECD TG 201.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 0.096 – 0.852 mg/L (measured), therefore the NOErC was determined as 0.032 mg/L (measured) and LOErC was determined as 0.096 mg/L (measured). The ErC50 was determined as 0.165 mg/L (measured). The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 0.032 – 0.852 mg/L (measured). The NOEC was determined as 0.008 mg/L (measured); the LOEC was determined as 0.032 mg/L (measured).