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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:
from 2017-05-08 to 2017-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2015-12
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 4.6, 10.0, 22.0, 46.0, 100.0 mg/L
- Sampling method: duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled. The concentrations of the test substance were determined in one of the duplicate samples from all test concentrations from both sampling times (0 and 72 hours). From the control samples, one of the duplicate samples was analyzed per sampling time.
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (10 mL acetonitrile per 10 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were frozen (at -20 ± 5 °C). Based on pre-experiments for investigation of the storage stability (non-GLP IES Study 20170154), the test item was found to be stable in the test water under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solution: The highest nominal concentration of 100 mg/L was prepared by completely dissolving 108.16 mg of test item in 1080 mL of test water using ultrasonic treatment for 2 minutes and intense stirring for 15 minutes at room temperature in a closed flask
Test solutions: This highest test concentration was diluted with test water to prepare the test media of the lower test concentrations
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany).
- Method of cultivation: The algae were cultivated at IES Laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
24 °C
pH:
7.5 (Start); 7.7 - 7.9 (End)
Nominal and measured concentrations:
fresh:
nominal: 4.6, 10.0, 22.0, 46.0, 100.0 mg/L; measured: 3.97, 8.71, 20.2, 42.0 85.7 mg/L (Sampling Day/Sampling Age [0 d / 0 h])
aged:
nominal: 4.6, 10.0, 22.0, 46.0, 100.0 mg/L; measured: 3.99, 9.04, 17.7, 39.0, 82.2 mg/L (Sampling Day/Sampling Age [3 d / 72 h])
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed (glass stopper)
- Material, size, headspace, fill volume: glass, no headspace, 60 mL
- Aeration: not specified
- 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, the algae were cultivated and tested in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 since a closed test system was applied. The concentration of NaHCO3 was increased by 235 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the pH of the test media as constant as possible.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test water was adjusted to 7.5 with a 1 M sodium hydroxide solution.
- Photoperiod: Continous illumination
- Light intensity and quality: 66 μE s-1 m-2, the light intensity over the incubation area was within a ± 15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations:fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Test concentrations: 1st study concentrrations: 0.1, 1.0, 10.0, 100.0 mg/L; 2nd study concentratios: 0.05, 0.5, 5.0, 50.0 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate in the reference test IES Study Number 20170077 was 1.0 mg/L (May 2017) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92-1.5 mg/L).
Reported statistics and error estimates:
The 95 % confidence intervals were calculated if possible by Probit Analysis using linear maximum likelihood regression.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-tes and Welch t-test with Bonferroni-Holm-adjustment, where appropriate. Statistical analysis was performed using ToxRat Professional®.

VALIDITY

The values for the validity criteria of the test were calculated by the statistic program ToxRat Professional®. The test was valid since the following performance criteria (according to the OECD 201) were met.

- In the control, the biomass increased by a factor of 181 over 72 hours. (Criterion: increase by at least a factor of 16 within three days).

- The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 22 %. (Criterion: must not be higher than 35 %).

- The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.7 %. (Criterion: must not be higher than 7 %).

Average Growth Rates (μ)

Nominal

Test Item

Concentration [mg/L]

Average Growth Rate μ (day-1) and Inhibition of μ (Ir)

0 – 24 h

0 – 48 h

0 – 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.327

0.0

1.710

0.0

1.732

0.0

4.6

1.275

3.9

1.704

0.3

1.722

0.6

10.0

1.192

10.2

1.684

1.5

1.709

1.3

22.0

1.233

7.1

1.664

2.7

1.693*

2.3

46.0

1.136*

14.4

1.633*

4.5

1.658**

4.3

100.0

0.973*

26.7

1.355*

20.8

1.417**

18.2

* Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

** Mean value statistically significantly lower than in the control (according to a Welch t-test one-sided smaller, α = 0.05).

Yield (Y)

Nominal

Test Item

Concentration [mg/L]

Yield Y (x 10E4) and Inhibition of Y (Iy)

0 – 24 h

0 – 48 h

0 – 72 h

Y

Ir [%]

Y

Ir [%]

Y

Ir [%]

Control

2.01

0.0

21.16

0.0

128.43

0.0

4.6

1.86

7.1

20.89

1.3

124.71

2.9

10.0

1.64

18.1

20.03

5.3

119.94*

6.6

22.0

1.74

13.4

19.25*

9.0

114.05*

11.2

46.0

1.51*

24.6

18.00*

14.9

102.55*

21.1

100.0

1.21*

39.9

10.10*

52.3

49.54*

61.4

* Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

Table 3: Section-by-Section Growth Rates

Nominal

Test Item

Concentration [mg/L]

Section-by-Section Growth Rates (day-1) and Inhibition of the growth rates (Ir)

0 – 24 h

24 – 48 h

48 – 72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.327

0.0

2.094

0.0

1.77

0.0

4.6

1.275

3.9

2.133

-1.9

1.757

1.1

10.0

1.192

10.2

2.177

-4.0

1.759

1.0

22.0

1.233

7.1

2.095

-0.1

1.751

1.4

46.0

1.136

14.4

2.130

-1.7

1.708

3.9

100.0

0.973

26.7

1.736

17.1

1.541

13.3

Results for Test Samples

Sampling Day/ Sample Age

Nominal Concentration of
Test Item
cnom

Measured Concentration
 of Test Item

x

Sample Preparation Factor

F

Determined Concentration of Test Item

c

% of Nominal Concentration

[d/h]

[mg/L]

[mg/L]

[mg/L]

[%]

0/0

Control

n.d.

2

< LOQ

n.a.

(fresh)

4.6

1.98

2

3.97

86

 

10

4.36

2

8.71

87

22

10.1

2

20.2

92

 

46

21.0

2

42.0

91

 

100

42.9

2

85.7

86

3/72

Control

1.01 (a)

2

2.02 (a)

n.a.

(aged)

4.6

2.00 (b)

2

3.99

87

 

10

4.52 (b)

2

9.04

90

22

8.31 and 9.39 (b)

2

17.7 (c)

80

 

46

19.5 (b)

2

39.0

85

 

100

41.1 (b)

2

82.2

82

Validity criteria fulfilled:
yes
Conclusions:
In a 72- hour Algal Growth Inhibition Test according to OECD Guideline 201, a ErC50 > 100 mg/L and a ErC10 of 48 mg/L was determined.
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata, was investigated in a 72 -hour static test according to the OECD Guideline 201. As the test substance is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled (without headspace, 60 mL) with test medium, minimizing the air-space in the flasks and to avoid losses of test item by evaporation.The nominal test item concentrations tested were 4.6, 10, 22, 46 and 100 mg/L in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 since a closed test system was applied. Additionally, a control (test water without test item) was tested in parallel. The measured concentrations of the test substance in the test media of the test concentrations of 4.6 to 100 mg/L were between 86 and 92 % of the nominal values at the start of the test and 80 and 90 % at the end of the test. The test substance was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The test item had a significant inhibitory effect on the growth rate of the algae after the test period of 72 hours. A ErC50 > 100 mg/L and a ErC10 of 48 mg/L was determined.

Description of key information

In a 72 - hour Algal Growth Inhibition Test according to OECD Guideline 201, an ErC50 > 100 mg/L and a EC10 = 48.0 mg/L was determined

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
48 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2017). To avoid losses of the test item by evaporation the test test was performed using glass stoppered Erlenmeyer flasks (closed system) completely filled (without headspace, 60 mL) with test medium, minimizing the air-space in the flasks.The nominal test item concentrations tested were 4.6, 10, 22, 46 and 100 mg/L Additionally, a control was tested in parallel. The measured concentrations of the test substance in the test media of the test concentrations of 4.6 to 100 mg/L were between 86 and 92 % of the nominal values at the start of the test and 80 and 90 % at the end of the test. Therefore, the biological results were related to the nominal concentrations of the test item. The 72 -h-EC50 > 100 mg/L and the 72 -h-EC10 of 48.0 mg/L based on growth rate were > 100 mg/L and = 48.0 mg/L, respectively.