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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2020-02-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to attached QMRF and QPRF Documents
Qualifier:
according to guideline
Guideline:
other: REACH guidance on QSARS R.6
Principles of method if other than guideline:
Calculation of toxicity in Algae. Software used: ECOSAR 1.11 (EPISUITE) for chemical class "Vinyl/Allyl Ethers"
GLP compliance:
no
Specific details on test material used for the study:
SMILES : O(CCO)CC=C
Analytical monitoring:
not required
Test organisms (species):
other: Algae
Water media type:
freshwater
Remarks on exposure duration:
acute and chronic exposure (duration not specified)
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
796.73 mg/L
Remarks on result:
other: QSAR predicted value: The substance is within the applicability domain of the model

Values used to Generate ECOSAR Profile:

Log Kow: -0.061    (EPISuite Kowwin v1.68 Estimate)

Wat Sol: 4.54E+005 (mg/L, EPISuite WSKowwin v1.43 Estimate)

Conclusions:
Using ECOSAR v1.11 the toxicity 96-h EC50 value for Algae was determined to be 796.73 mg/L. The substance is within the applicability domain of the model.
Executive summary:

Using ECOSAR v1.11 the toxicity 96-h EC50 value for Algae was determined to be 796.73 mg/L. The substance is within the applicability domain of the model.

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-11-09 to 2020-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
01 March 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Adopted 23rd of March, 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For the determination of the test item concentration, six replicate samples (6 x 10 mL) were taken from each test concentration and six replicates (6 x 10 mL) from the control at the start and at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution used in the test was prepared by mechanical dispersion without using any solubilising agent. The test solution of 100 mg/L nominal was prepared by dissolving an amount of 0.0704 g test item in 704 mL OECD medium. After the formulation procedure algal cells were immediately introduced into the test solution (start of experiment).

- Controls: dilution water (OECD Medium)

- Evidence of undissolved material: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 2 – 4 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: none
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
The measured temperature in the flask was in the range of 22.3 – 22.6 °C and within the climate chamber between 21.7 – 23.6 °C.
pH:
8.39 - 8.86
Dissolved oxygen:
not specified
Salinity:
not applicable
Conductivity:
not specified
Nominal and measured concentrations:
Nominal conc.: 100 mg/L
Measured conc.: The mean of the analytically measured test item concentrations was 102.5 mg/L at the start and 102.4 mg/L at the end of the experiment.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume, volume filled with 100 mL of test medium
- Type: closed, flasks were covered with air-permeable stoppers
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD Medium, according to OECD 201)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: 7140 (SD: 76) lux.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1, 10, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP Preliminary Range-Finding Test, the test item has no toxic effect on algae up to at least 100 mg/L (limit test concentration), therefore the main study was performed using the single nominal concentration of 100 mg/L in a limit test. Untreated control ran parallel in the test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
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
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no effects observed
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The 72h ErC50: 0.70 mg/L (95 % confidence limits: 0.56 – 0.86 mg/L)
The 72h EyC50: 0.33 mg/L (95 % confidence limits: 0.24 – 0.43 mg/L)

Validity Criteria of the Study

- Cell density in the untreated control cultures increased by a factor of more than 16 (by a factor of 34.33) within 72 hours. This corresponds to a specific growth rate of 1.17 day-1.

 

 -The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0 - 1, 1 - 2 and 2 - 3, for 72 h-tests) in the control cultures did not exceed 35 %, it was 23.21 %.

- CV for section-by-section growth rate day 0 - 1: 25.28 %

- CV for section-by-section growth rate day 1 - 2: 26.98 %

- CV for section-by-section growth rate day 2 - 3: 17.38 %

 

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test.

- CV for average specific growth rate day 0 - 3: 6.14 %

All validity criteria were met therefore, the study can be considered as valid.

 

Analytical Results

The mean of the analytically measured test item concentrations was 102.5 mg/L at the start and 102.4 mg/L at the end of the experiment. That means 102.5 % of the nominal concentration at the start and 102.4 % of the nominal concentration at the end of the test. As the measured test item concentrations remained within ± 20 % of the nominal during the experiment, the biological results are based on the nominal test item concentration. The test item was not detected in the control samples (signal intensities measured were below the quantification limit).

 

Biological Results

Biological results are based on the nominal test item concentrations.

Growth Inhibition: The results of the statistical evaluation (Independent Sample Test (α = 0.05)) showed that the 0 - 72 h average specific growth rate and yield were not statistically significantly different from the untreated control at the test concentration of 100 mg/L.

Cell Density in Control: The cell density in the control has increased from nominal N = 1 x 10E4 cells/mL at the start of the test (0 hours) to 34.33 x 10E4 cells/mL at the end of the test (72 h).

Shape of the Algal Cells: No morphological abnormalities were observed in the control and in the test concentration of 100 mg/L during the test.

 

Table 1: Algal Cell Number (x 10E4 cell/mL) during the Test Period of 72 hours and Yield

Concentration
[mg/L]

 

Number of Cells (× 104/mL)

Yield

0 h

24 h

48 h

72 h

Control

 

1

2

11

40

39

 

1

2

9

41

40

 

1

3

7

40

39

 

1

2

9

31

30

 

1

2

6

30

29

 

1

3

8

24

23

Mean

1.00

2.33

8.33

34.33

33.33

SD

0.0

0.5

1.8

7.0

7.0

100

 

1

2

10

25

24

 

1

2

9

30

29

 

1

2

8

45

44

 

1

3

9

24

23

 

1

2

10

31

30

 

1

3

8

41

40

Mean

1.00

2.33

9.00

32.67

31.67

SD

0.0

0.5

0.9

8.5

8.5

 

Table 2: The Specific Growth Rates during the Test Period of 72 hours

Concentration
[mg/L]

 

Growth Rates (µ)

0 – 24 h

0 – 48 h

0 – 72 h

Control

 

0.0289

0.0500

0.0512

 

0.0289

0.0458

0.0516

 

0.0458

0.0405

0.0512

 

0.0289

0.0458

0.0477

 

0.0289

0.0373

0.0472

 

0.0458

0.0433

0.0441

Mean

0.0345

0.0438

0.0489

SD

0.0087

0.0044

0.0030

100

 

0.0289

0.0480

0.0447

 

0.0289

0.0458

0.0472

 

0.0289

0.0433

0.0529

 

0.0458

0.0458

0.0441

 

0.0289

0.0480

0.0477

 

0.0458

0.0433

0.0516

Mean

0.0345

0.0457

0.0480

SD

0.0087

0.0021

0.0035

 

Table 3: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Concentration
[mg/L]

Growth Rates (µ) and % Inhibition of µ

0 – 24 h

0 – 48 h

0 – 72 h

µ

%

µ

%

µ

%

Control

0.0345

-

0.0438

-

0.0489

-

100

0.0345

0.0

0.0457

-4.4

0.0480

1.7

 

Table 4: Yield (y) and Percentage Inhibition of y during the Test Period

Concentration
[mg/L]

Yield (y) and % Inhibition of y

0 – 72 h

y

%

Control

33.33

-

100

31.67

5.0

 

Table 5: The Section-by-section Specific Growth Rates in the Control Cultures

Section-by-Section Growth Rate

Expressed in hours

0 - 24 h

24 - 48 h

48 - 72 h

0.0345

0.0531

0.0590

Expressed in days

0 - 1 day

1 - 2 day

2 - 3 day

0.8283

1.2733

1.4159

 

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC10, EC20, EC50 values as well as the 72-h LOEC value based on growth rate and yield were determined to be > 100 mg/L. The 72-h NOEC for growth rate and yield was determined to be 100 mg/L.
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) over an exposure period of 72 hours in a closed static test system according to OECD 201 under GLP conditions. Based on the results of a preliminary test, the test item has no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed at 100 mg/L including a concurrent control. The purpose of the analytical part was to verify the concentration of the test item in the test medium. The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentration. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. The alga cell concentration was approximately 10,000 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. In the test no morphological effects were observed in the 100 mg/L test item concentration. For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year. The 72-h ErC50 of the reference substance is determined to be 0.70 mg/L (95 % confidence limits: 0.56 – 0.86 mg/L). The validity criteria of the test was met. Biological results are based on the nominal concentration of test item. In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had not any inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 values as well as the 72-h LOEC value based on growth rate and yield were determined to be > 100 mg/L. The 72-h NOEC for growth rate and yield was determined to be  100 mg/L.


 

Description of key information

The 72-h EC10, EC20, EC50 values as well as the 72-h LOEC value based on growth rate and yield were determined to be > 100 mg/L. The 72-h NOEC for growth rate and yield was determined to be 100 mg/L. 

Key value for chemical safety assessment

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

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) over an exposure period of 72 hours in a closed static test system according to OECD 201 under GLP conditions. Based on the results of a preliminary test, the test item has no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed at 100 mg/L including a concurrent control. The purpose of the analytical part was to verify the concentration of the test item in the test medium. The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentration. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. The alga cell concentration was approximately 10,000 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. In the test no morphological effects were observed in the 100 mg/L test item concentration. For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year. The 72-h ErC50 of the reference substance is determined to be 0.70 mg/L (95 % confidence limits: 0.56 – 0.86 mg/L). The validity criteria of the test was met. Biological results are based on the nominal concentration of test item. In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had not any inhibitory effects on the growth of Raphidocelis subcapitata. The 72-h EC10, EC20, EC50 values as well as the 72-h LOEC value based on growth rate and yield were determined to be > 100 mg/L. The 72-h NOEC for growth rate and yield was determined to be  100 mg/L.


The short-term toxicity on aquatic algae was also estimated using ECOSAR v1.11. The 96h EC50 was determined to be > 100 mg/L.