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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06. Feb. 2006 - 23. Feb. 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the limit test singular samples were taken from both the test concentration and the blankcontrol.
Frequency : at t=O h and t=72 h
Volume : 20ml
Storage : Samples were stored in a refrigerator until analysis.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and the end of the test period. Additionally, singular reserve samples of 20 ml were taken for possible analysis. If not used, these samples were stored in a refrigerator for possible analysis until delivery of the final report with a maximum of three months.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The batch of FORMAMIDOPROPYLBETAINE tested was a clear yellowish liquid with a purity of
40.1 % and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a solution ofl 00 mg/l applying 10 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species : Selenastrum capricornutum, strain: NIVA CHLl
Source : In-house laboratory culture
Reason for selection : This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture : 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 in a climate room at a temperature of 23 ± 2°C.
Light intensity : 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium : M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Pre-culture : 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2 x 10 4 cells/ml (range-finding test) or 1 x 10 4 cells/ml (limit test). The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium : M2; according to the ISO-Standard, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation.
Test solutions : After preparation of test solutions , volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23 - 24.2 °C
pH:
8.2 - 9.9
Nominal and measured concentrations:
Limit test: 100 mg/L nominal
Measured: 0 h 93.4 mg/L, 72 h: 96.6 mg/L
Details on test conditions:
FORMAMIDOPROPYLBETAINE : 100 mg/l, the regulatory limit concentration
Controls : Test medium without test substance or other additives (blank-control)
Replicates : 6 replicates of the test concentration, 6 replicates of the blank-control, 1 replicate of the test concentration without algae
Test duration : 72 hours
Test type : Static
T est vessels : 100 ml, all-glass, containing 50 ml of test solution
Medium : M2
Cell density : An initial cell density of 1 x 10 4 cells/ml.
Illumination : Continuously using TLD-Iamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 79, to 100 µE.m-2.s-1.
Incubation : Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.
Reference substance (positive control):
yes
Remarks:
reference test with potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Results with reference substance (positive control):
Under the conditions of the reference study with Selenastrum capricomutum, potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher. The EC50 for cell growth inhibition (EbC50: 0-72h) was 0.63 mg/l with a 95% confidence interval ranging from 0.46 to 0.87 mg/l. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the EbC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ErC50: 0-72h) was 1.3 mg/I with a 95% confidence interval ranging from 0.98 to 1.6 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ErC50: 0-72h for the present batch corresponds with this range.

Mean cell densities (x 104cells/ml) during the limit test:

 

Concentration Test substance1[mg/L]

Exposure time (hours)

Blank-control

0

24

48

72

100

1.0

10.5

56.3

174.1

 

1.0

11.0

50.4

197.3

1Test substance IS FORMAMIDOPROPYLBETAINE

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Selenastrum capricornutum, no inhibition of cell growth or reduction of growth rate was recorded at the regulatory limit concentration. Therefore, the NOEC for both cell growth inhibition and growth rate reduction was 100 mg/I. The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ERC50: 0-72h) was beyond 100 mg/I.
Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with FORMAMIDOPROPYLBETAINE.

The study procedures described in this report were based on the ISO International Standard 8692, 2004. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992 and the OECD guideline No. 201,1984. The batch of FORMAMIDOPROPYLBETAINE tested was a clear yellowish liquid with a purity of 40.1%. The water solubility of FORMAMIDOPROPYLBETAINE at 20.2 ± 0.6°C was determined to be > 2160 g/l (NOTOX Project 453915).

Based on the results of a preceding range-finding test, a limit test was performed. Exponentially growing algal cultures were exposed to a nominal FORMAMIDOPROPYLBET AINE concentration of1 00 mgll and a blank-control. The initial cell density was 1 x 10 4 cells/ml and the total test period was 72 hours. Samples for confirmation of actual exposure concentrations were taken at the start and the end of the test period. Analysis of the sample taken at the start of the test showed that the test concentration was in agreement with nominal (99%) and remained stable during the test period (101 % of initial). Therefore, the NOEC and EC50-values were based on the nominal test concentration. The study generally met the acceptability criteria prescribed by the protocol and was considered

valid. No inhibition of cell growth or reduction of growth rate was recorded at the regulatory limit concentration. Therefore, the NOEC for both cell growth inhibition and growth rate reduction was 100 mg/l. The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond 100 mg/l.

Description of key information

The NOEC for both cell growth inhibition and growth rate reduction was 100 mg/l. The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond 100 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

Selenastrum capricornutum, Fresh Water Algal Growth Inhibition Test with FORMAMIDOPROPYLBETAINE.

The study procedures described in this report were based on the ISO International Standard 8692, 2004. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.3, 1992 and the OECD guideline No. 201,1984. The batch of FORMAMIDOPROPYLBETAINE tested was a clear yellowish liquid with a purity of 40.1%. The water solubility of FORMAMIDOPROPYLBETAINE at 20.2 ± 0.6°C was determined to be > 2160 g/l (NOTOX Project 453915).

Based on the results of a preceding range-finding test, a limit test was performed. Exponentially growing algal cultures were exposed to a nominal FORMAMIDOPROPYLBET AINE concentration of1 00 mgll and a blank-control. The initial cell density was 1 x 10 4 cells/ml and the total test period was 72 hours. Samples for confirmation of actual exposure concentrations were taken at the start and the end of the test period. Analysis of the sample taken at the start of the test showed that the test concentration was in agreement with nominal (99%) and remained stable during the test period (101 % of initial). Therefore, the NOEC and EC50-values were based on the nominal test concentration. The study generally met the acceptability criteria prescribed by the protocol and was considered valid. No inhibition of cell growth or reduction of growth rate was recorded at the regulatory limit concentration. Therefore, the NOEC for both cell growth inhibition and growth rate reduction was 100 mg/l.The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ErC50: 0-72h) was beyond 100 mg/l.