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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:
01 Mar - 14 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(EC) No. 761/2009
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of certificate: 19.11.2018 (The Department of Health of the Government of the United Kingdom)

Test material

Constituent 1
Reference substance name:
Reaction product of castor oil with glycerol
EC Number:
949-117-7
Molecular formula:
not applicable, substance is UVCB
IUPAC Name:
Reaction product of castor oil with glycerol
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- A nominal amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 100000 to 1000000 cells/mL.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Study design

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

Test conditions

Test temperature:
24 ±1 ºC
pH:
7.7 - 8.8
Nominal and measured concentrations:
- Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
- Measured (0 h): 0.861, 2.61, 8.83, 101 mg/L
- Measure (72 h): 1.15, 3.98, 10.9, 36.8, 116 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Type (delete if not applicable): open
- Fill volume: 100 mL
- Aeration: none

- Initial cells density: 5000 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: reverse osmosis purified deionized water was used to prepare culture medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was determined at initiation of the test and after 72 hours exposure, temperature was recorded daily, appearance of test media was recorded daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: warm white lighting (380 to 730 nm), intensity approximately 7000 lux
- constantly shaken at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 24, 46 and 72 hours using an electronic particle counter
- Other: Shape and size of the algal cells was inspected microscopically and any abnormalities recorded by removing a sample from each test and control culture (replicates pooled) at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: growth rate was observed to be reduced at 10 and 100 mg/L, significant effects on yield were seen at all test concentrations.
Reference substance (positive control):
yes
Remarks:
Positive control test with potassium dichromate was performed twice a year

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 13 - 19 mg/L (95% confidence limits)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- It was not possible to calculate a NOEC based on yield as significant inhibition of yield was observed to have occurred at all test concentrations employed.
- After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/L, however, no intact cells were observed to be present in the test cultures at 32 or 100 mg/L.
- At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72 Hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be pale green dispersions, the 32 mg/L test cultures were observed to remain as clear colorless solutions and the 100 mg/L test cultures were observed to have formed slightly hazy dispersions.
Results with reference substance (positive control):
-The positive control was conducted between 16 - 19 July 2019 with concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg potassium dichromate/L
- ECr50 (0 - 72 h): 1.3 mg/L; ECy50 (0-72 h): 0.55 mg/L
- Results from the positive control with potassium dichromate were within the normal ranges for this reference item
Reported statistics and error estimates:
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). EC50 values were then determined from the equation for the fitted line.Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
-All statistical analyses were performed using the SAS computer software package (SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc, Cary, NC, USA.).

Any other information on results incl. tables

Table 1. Mean Inhibition of Growth Rate and Yield

Nominal Concentration
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL x 104)

0 - 72 Hour

% Inhibition

0 - 72 Hour

% Inhibition

Control

0.073

-

9.72E+05

-

1.0

0.071

3

8.15E+05

16

3.2

0.071

3

8.04E+05

17

10

0.051

30

1.96E+05

80

32

0.014

80

9.28E+03

99

100

0.010

87

5.88E+03

99

 

Table 2. Cell densities of all treatments

Nominal Concentration

(mg/L)

Cell Densities*(cells per mL x 104)

24 Hours

46 Hours

72 Hours

Control

R1

2.68

14.9

99.0

R2

2.16

14.3

76.5

R3

2.67

15.3

102

R4

2.68

14.2

98.8

R5

3.32

18.1

95.2

R6

2.74

16.0

115

Mean

2.71

15.5

97.7

1.0

R1

2.53

13.9

79.4

R2

2.16

12.3

82.8

R3

2.51

12.5

83.8

Mean

2.40

12.9

82.0

3.2

R1

2.09

10.8

75.3

R2

2.50

12.9

85.8

R3

2.21

12.4

81.7

Mean

2.26

12.0

80.9

10

R1

2.12

4.61

21.5

R2

2.03

3.60

18.8

R3

1.60

2.96

20.0

Mean

1.92

3.72

20.1

32

R1

2.66

1.01

1.90

R2

2.67

0.901

1.34

R3

2.17

0.859

1.04

Mean

2.50

0.923

1.43

100

R1

3.85

1.87

1.69

R2

3.71

1.24

0.971

R3

2.91

1.24

0.601

Mean

3.49

1.45

1.09

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from3 counts for each of the replicate flasks

 

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 195

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%

9%

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%.

3%

 

 

Yes


 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See Table 3 in "any other information on results incl tables"