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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Feb - 21 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 23, 2006; Annex 5 corrected July 28, 2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Dodecanoic acid, monoester with oxybis[propanediol]
EC Number:
619-220-9
Cas Number:
96499-68-2
Molecular formula:
not applicable, the substance is UVCB
IUPAC Name:
Dodecanoic acid, monoester with oxybis[propanediol]

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling: Samples for analysis were taken from all test concentration levels at the start of exposure, at 24 and 48 h and at the end of exposure.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.0585 g of the test item and 1300 mL of the test medium were mixed to prepare a stock solution with a nominal loading rate of 45.0 mg/L and stirred for 48 h. The test solutions were prepared by dilution of the stock solution with the appropriate amount of test medium.
- Eluate: no
- Differential loading: no
- Controls: blank control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater green algae
- Strain: ATCC22662
- Source: American Type Culture Collection, supplied on 30 June, 1995
- Method of cultivation: sterile conditions; temperature: 21-24 °C; light intensity: nominal 6923 lux; continuous illumination provided by alternative LED light to fluorescent lamp

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.1 - 22.5 °C
pH:
7.8 - 7.9
Nominal and measured concentrations:
nominal: Control, 1.00, 3.16, 10.0, 31.6 and 100 mg/L
measured: < detection limit, 0.310, 0.979, 3.02, 10.1 and 39.6 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilised 300 mL Erlenmeyer flasks with gas-permeable Silicosen
- Initial cells density: 0.75 x 10E04 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: according to guideline
- Culture medium different from test medium: same as test medium
- Intervals of water quality measurement: The pH value was measured at test start and end. The temperature was measured at test start, after 24 and 48 h and at test end.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 6769-7077 lux in wavelength range 400 - 700 nm; continuous illumination provided by alternative LED light to fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Biomass was measured every 24 hours after test start using chlorophyll fluorescence measurement (spectrofluorometer model RF-6000, Shimadzu).
- Chlorophyll measurement: yes, using a spectrofluorometer
- Other: Cell conditions were evaluated microscopically for each test concentration level at the end of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Two preliminary tests were performed.
Study 1: Nominal concentrations of 10.0 and 100 mg/L were chosen. Growth rate inhibition was 5.8% at the low concentration and > 100% at the high concentration.
Study 2: Nominal concentrations of 2.00, 16.0 and 50.0 mg/L were chosen. Growth rate inhibition was 1.0% at the lowest concentration, 31% at the intermediate concentration and > 100% at the highest concentration.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 12.4-14.9 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At 10.1 and 39.6 mg/L, some bloated cells were observed. Cells of other treatment solutions were the same as the control. For the control cells, the cell condition was normal.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At test start and end, the test solution of 39.6 mg/L was colourless and clear with some bubbles. All other test solutions were colourless and clear at test start. At test end all other test solutions, except 39.6 mg/L, showed green colour due to algal growth.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50 (72 h) = 1.3 mg/L
Reported statistics and error estimates:
Estimation of EC50: The percentage inhibition in each exposure level was plotted as a semi-logarithmic graph against the corresponding concentration. Linear regression analysis (least square method) was carried out using the data within the range showing linearity visually (100 and 31.6% levels) to estimate the EC50 and 95% confidence limits, which were estimated from the intercept of the regression line with the parallel drawn to the absciss at 50% inhibition. The EC50 was denoted as ErC50 based on growth rate.

Estimation of NOEC: Regarding growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. Then, one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test was used to determine the significant difference between the control and exposure levels. Concentration levels above the ErC50 were excluded for each analysis.

Any other information on results incl. tables

Table 1: Biomass values

Values are based on the measured value of the pre-culture.

Measured concentration [mg/L]

No.

Chlorophyll fluorescence value (relative value)

0 h

24 h

48 h

72 h

Control

A

43

200

950

3800

B

43

180

870

3500

C

43

190

840

3600

D

43

180

860

3700

E

43

190

830

3400

F

43

190

830

3200

Mean

43

190

860

3500

SD

0

7.0

44

230

0.310

A

43

200

980

4100

B

43

180

900

3500

C

43

190

850

3300

Mean

43

190

910

3600

SD

0

11

64

440

0.979

A

43

210

980

3600

B

43

200

920

3500

C

43

190

780

3300

Mean

43

200

890

3500

SD

0

12

100

150

3.02

A

43

210

930

3700

B

43

190

830

3500

C

43

200

950

3700

Mean

43

200

900

3600

SD

0

10

65

130

10.1

A

43

110

400

1500

B

43

130

430

1500

C

43

130

460

1700

Mean

43

120

430

1600

SD

0

9.9

31

100

39.6

A

43

14

1.2

1.6

B

43

13

2.0

2.0

C

43

13

2.7

3.9

Mean

43

13

2.0

2.5

SD

0

0.59

0.73

1.3

Table 2: Growth rate and growth inhibition rate

Measured concentration [mg/L]

No.

Growth rate 0-72 h

Growth inhibition rate [%]

Control

A

1.49

-

B

1.47

-

C

1.48

-

D

1.49

-

E

1.46

-

F

1.43

-

Mean

1.47

-

SD

0.0226

-

0.310

A

1.53

-3.8

B

1.47

0.16

C

1.45

1.2

Mean

1.48

-0.82

SD

0.0393

2.7

0.979

A

1.48

-0.54

B

1.47

0.14

C

1.45

1.4

Mean

1.46

0.34

SD

0.0147

1.0

3.02

A

1.49

-1.3

B

1.47

0.18

C

1.49

-1.1

Mean

1.48

-0.74

SD

0.0118

0.80

10.1

A

1.19

19

B

1.20

19

C

1.23

16

Mean

1.21

18

SD

0.0209

1.4

39.6

A

1.10

175

B

-1.03

170

C

-0.797

154

Mean

-0.977

166

SD

0.160

11

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 factor of 74

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 4.5%

 yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 1.5%

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.