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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:
22 Jan - 25 Jan 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:
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

Reference
Name:
Unnamed
Type:
Constituent

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: The treatment solutions were prepared as water accomodated fractions (WAFs). The test sample and medium were mixed to prepare each nominal concentration. After stirring for 48 hours, the suspensions were filtered with a membrane filter (0.45 µm pore size). The filtrates were used as test solutions.
- Eluate: no
- Differential loading: yes
- Controls: blank control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of exposure, the test solution at 3.56 mg/L was colourless and clear. The test solutions at 8.16 mg/L to 74.3 mg/L levels were white in a concentration dependent manner.

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:
22.8 - 23.0 °C
pH:
7.8 - 7.9
Nominal and measured concentrations:
nominal: Control, 6.3, 13, 25, 50 and 100 mg/L
measured: < detection limit, 3.56, 8.16, 18.2, 33.6 and 74.3 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterilised 300 mL Erlenmeyer flasks with gas-permeable Silicosen; fill volume: 100 mL
- Initial cells density: 0.75 x 10E04 cells/mL
- Control end cell density: 98 x 10E04 cells/mL (mean of six replicates)
- 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
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 6692-7000 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: Cells were counted using a particle counter (model COULTER Z2).
- Other: Cell conditions were evaluated microscopically for each test concentration level at the end of exposure..

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: Nominal concentrations of 1.00, 10.0, 30.0 and 100 mg/L were chosen. Growth rate inhibitions of -1.5, 0.11, 2.8 and 2.4% in increasing order of test concentrations were recorded.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 74.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI was not determined
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI was not determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
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 all exposure levels, the cells were in the same condition as the control. The cells of the control showed no abnormalities.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of exposure, the test solution at 3.56 mg/L was colourless and clear. The test solutions at 8.16 to 74.3 mg/L levels were green due to algal growth.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (72 h) = 1.3 mg/L (mean ± 2 S.D.: 0.61 - 1.4 mg/L)
Reported statistics and error estimates:
Estimation of EC50: The EC50 was estimated as "> highest test concentration" since inhibition rates of >50% were not obtained at any exposure levels.

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.

Any other information on results incl. tables

Table 1: Biomass

Measured concentration [mg/L]

No.

Cell concentration (x 10E04 cells/mL)

0 h*

24 h

48 h

72 h

Control

A

0.75

4.5

24

99

B

0.75

4.6

23

110

C

0.75

4.8

23

92

D

0.75

4.7

24

98

E

0.75

4.9

23

98

F

0.75

4.9

23

98

Mean

0.75

4.7

23

98

SD

0

0.16

0.42

4.1

3.56

A

0.75

4.8

24

85

B

0.75

4.6

24

84

C

0.75

5.0

23

83

Mean

0.75

4.8

23

84

SD

0

0.17

0.40

1.1

8.16

A

0.75

4.7

22

81

B

0.75

4.3

22

83

C

0.75

4.7

22

82

Mean

0.75

4.6

22

82

SD

0

0.20

0.38

1.0

18.2

A

0.75

4.8

21

60

B

0.75

4.7

22

67

C

0.75

4.5

22

69

Mean

0.75

4.6

22

66

SD

0

0.18

0.49

4.6

33.6

A

0.75

4.7

22

71

B

0.75

4.8

23

69

C

0.75

4.8

22

70

Mean

0.75

4.7

22

70

SD

0

0.074

0.44

1.2

74.3

A

0.75

4.6

22

72

B

0.75

4.5

21

72

C

0.75

5.0

23

78

Mean

0.75

4.7

22

74

SD

0

0.22

0.69

3.7

* values at 0 h ware based on the pre-culture

Table 2: Growth rate and growth inhibition rate

Measured concentration [mg/L]

No.

Growth rate 0-72 h

Growth inhibition rate [%]

Control

A

1.63

-

B

1.65

-

C

1.60

-

D

1.63

-

E

1.62

-

F

1.63

-

Mean

1.63

-

SD

0.0140

-

3.56

A

1.61

0.68

B

1.61

0.94

C

1.61

1.2

Mean

1.61

0.92

SD

0.00385

0.24

8.16

A

1.56

3.9

B

1.57

3.4

C

1.57

3.6

Mean

1.57

3.6

SD

0.00423

0.26

18.2

A

1.46

10

B

1.50

7.9

C

1.51

7.1

Mean

1.49

8.4

SD

0.0240

1.5

33.6

A

1.52

6.7

B

1.51

7.3

C

1.51

6.9

Mean

1.51

7.0

SD

0.00561

0.35

74.3

A

1.52

6.5

B

1.52

6.3

C

1.55

4.7

Mean

1.53

5.8

SD

0.0165

1.0

Table 3: Measured concentrations of the test item in test solutions

Nominal concentration

Measured concentration [mg/L]

Percentage of measured concentration versus that at each preparation [%]

At test start

24 hours

48 hours

At test end

Geometric mean

Control

n.d.

n.d.

n.d.

n.d.

-

6.3

4.55

3.59

(78.9)

3.34

(73.4)

3.09

(68.0)

3.56

13

9.62

8.45

(87.8)

7.91

(82.2)

6.87

(71.4)

8.16

25

20.4

18.2

(89.6)

18.5

(90.8)

15.8

(77.4)

18.2

50

35.8

33.5

(93.5)

34.2

(95.7)

30.4

(84.9)

33.6

100

80.2

72.1

(89.8)

76.0

(94.7)

69.7

(86.8)

74.3

Table 4: 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 123

 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%

 13%

 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%.

 0.86%

 yes

Applicant's summary and conclusion

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