Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OECD 201 GLP study carried out with some slight deviations: - The mean coefficient of variations of the intersection specific growth rate is 36%, while the limit of acceptance is 35% - The test item recrystallized at day 3. However, this was taken into account since time weighted geometrical mean concentrations were used for ECx calculations. The r² of the regression curve was 1, which demonstrate the perfect fitness of the data. Therefore, this study is considered as Klimisch 1.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
: see the rationale for reliability described above.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
Test item concentrations were determined at the beginning of the test and every 24h in all concentration groups (including control). For this purpose, two additional inoculated flasks at each test concentrations were added. One flask was used at 24h for test item determination; the second one was used at 48h. Before analytical analysis, cell density was determined and then filtered through a 0.45 µm nitrocellulose filter to remove algae.
The test item concentrations were determined under the analytical procedure presented below. Aliquots were removed from each prepared concentration at each observation time. Samples were then diluted in acetonitrile before analysis.
Vehicle:
no
Details on test solutions:
As the test item is a crystalline solid, it was placed in an incubator at 37°C in darkness until its complete melting then at 30°C before sampling, in order to get a homogeneous sample. The test medium was also placed under the same conditions.
A 30 mg/L stock solution was then prepared by dissolving 30 mg of test item in 1L of test medium. The substance was pipetted on a soluble strip which was introduced in a 1L volumetric flask. The solution was stirred at 30°C during 24h in darkness: the solution was found to be clear, no precipitation was observed (the strip was completely dissolved). Test solutions were prepared from dilution of this stock solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, CCAP 278/4 stock was obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK). Transfers of P. subcapitata were made regularly to provide suitable subcultures. The conditions used for culturing algae are described in Annex 2 of the OECD 201 guideline. A verification of the stock culture was performed before use by microscopic observation in order to ensure the absence of micro-organisms and deformed cells. Three or four days before the beginning of the study, a pre-culture was prepared by inoculating a sample of stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-culture was incubated under the same conditions as those used for the stock cultures. At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to get an initial cell density of 5000 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
n.d.
Test temperature:
23°C
pH:
See table 1 in the "other information on materials and methods" section
Dissolved oxygen:
See table 1 in the "other information on materials and methods" section
Salinity:
Around 0 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0 ; 0. 3 ; 0.9 ; 3 ; 9.4 ; 30 mg/L
Time weighted geometric concentrations: 0 ; 0.11 ; 0.25 ; 0.75 ; 2.85 ; 20.13 mg/L
See table 2 in the "other information on results" section.
Details on test conditions:
The test was conducted at the following nominal concentration: 0.3, 0.9, 2.9, 9.4 and 30 mg/L. Three vessels were prepared at each test concentration and six vessels for the control group. Each vessel was inoculated with 5000 cells/mL.
After 24 and 48h of incubation, aliquot of 200 µL were sampled from each inoculated test flask and pipetted into a microplate. After 72 h, the samples with high cell density were diluted to ¼ (50 µL + 150 µL of filtered dilution water) and pipetted into a microplate. Microplates were analysed using a flow cytometer (Guava easyCyte™ flow cytometer Merck Millipore). Time between sampling and measurement was approximately 15 – 30 min. Cell densities were determined using 96 wells single use microplates and a laser beam at 488 nm. The cytometer is calibrated every week using the Guava check kit. Before each measurement, settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than algae were used to assess the toxic effects.
The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell density was less than 500 cells/µL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore).
The pH was measured using a METTLER TOLEDO 345 pH meter in all test solutions, in non-inoculated flask at the beginning of the test and in inoculated flasks at the end of the test. Dissolved oxygen concentrations were measured with a WTW OXI 538 oxymeter meter in all test solutions, in non-inoculated flasks at the beginning of the test and in inoculated flasks at the end of the test.
Additional inoculated flasks were used to monitor the test item concentrations at 24 hours and 48 hours, in order to not disturb the flasks used for cell density measurement. The temperature in the incubator was continuously recorded throughout the test.
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Oxygen and pH variations stayed within the ranges recommended by the OECD 201 guideline. Cell multiplication factor was higher than 16. The mean coefficient of variations of the intersection specific growth rate is 36%, while the limit of acceptance is 35% but this did not affect the results.
ErC10 = 0.21 mg/L
ErC50 = 0.24 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? YES
- EC50: 0.64 mg/L
Reported statistics and error estimates:
Raw data were treated according to the recommendations of the OECD 201 guideline: the test item concentrations which inhibit 10% and 50% of the average specific growth rate (reported as ErC10 and ErC50) were determined by interpolation using the software Sigmaplot 12.0.

Table 2: Daily measurements of the test item concentration throughout the study

Test solution

(nominal conc. mg/L)

Measured concentrations (mg/L

 

0h

24h

48h

72h

Control

<DL

ND

ND

<DL

0.3

0.33

0.32

0.24

<DL*

0.9

0.99

0.78

0.71

<DL*

3.0

3.16

2.90

2.99

<DL*

9.4

9.98

9.67

10.3

0.005

30.0

33.0

32.1

31.1

2.02

< DL (0.00072 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.0024 mg/L): concentration lower than the Quantification Limit of the analytical method

ND: Not Determined

*: DL (0.00072 mg/L) was used for time weighted geometrical mean calculations

Table 3: Specific growth rate inhibition at the end of the experiment

Test solution

(nominal conc. mg/L)

Growth rate

Iµi (%)

Control

-

0.3

11.6

0.9

67.3

3.0

103.2

9.4

102.9

30.0

102.4

Validity criteria fulfilled:
yes
Conclusions:
A GLP OECD 201 study was conducted on undecylenic acid (also named 10-undecenoic acid, CAS 112-38-9) in July 2014. This study is quoted as Klimisch 1. Based on specific growth rate inhibition, EC50 = 0.24 mg/ L. EC10 = 0.21 mg/L
Executive summary:

A GLP OECD 201 study was conducted on undecylenic acid (also named 10-undecenoic acid, CAS 112-38-9) in July 2014. This study is quoted as Klimisch 1. Test item concentration was measured every day. At day 3, the test item recrystallised in the vessels and was not detected in the medium (<DL). Concentration calculations were then based on time weighted geometrical means, according to the formula exposed in the OECD guidance 23 on difficult substances. The probit regression analysis of specific growth rate inhibition against test item concentrations showed a r² of 1. Based on specific growth rate inhibition, EC50 = 0.24 mg/ L. EC10 = 0.21 mg/L

Description of key information

The effects of undecylenic acid to algae were determined in a laboratory test according to OECD Guideline 201 and GLP requirements.

The freshwater green alga Pseudokirchneriella subcapitata was exposed for 72-hours to a series of test concentrations. Based on the time weighted geometric mean measured concentrations, the 72-h ErC50 was 0.24 mg/L and the 72-h ErC10 was 0.21 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.24 mg/L
EC10 or NOEC for freshwater algae:
0.21 mg/L

Additional information