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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 to 29 March 2018
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)
Deviations:
yes
Remarks:
The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13 and 14 October 2014 / Date of signature: 08 April 2015
Specific details on test material used for the study:
- Storage condition of test material: The test item was stored in a closed vessel dark and dry at 20 +/- 5°C, keep under inert gas
- Vapour pressure: 0.61 Pa at 20°C, 0.81 at 25°C: determined in LAUS study 16081102G925, OECD 104.
- Water solubility: 176 ± 11 g/L at 20.0 ± 0.3°C: determined in LAUS study 17011604G910 OECD 105).
- Henry's Law Constant: The substance has a low volatility according to Lyman et al., 1982 (H = 0.00043 Pa.m3/mol).
Analytical monitoring:
yes
Details on sampling:
The concentration of the test item in the test vessels was measured at the start, after 8h, 24h and 48h incubation and at the end of the test (72h).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 1000.2 mg/L of test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: one blank control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Unicellular freshwater green alga.
- Genus: Desmodesmus
- Species: subspicatus
- SAG Strain Number: 86.81
- Taxonomic position: Chlorophyta - Chlorophyceae
- Origin and Culture: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
No data
Test temperature:
21.2 – 21.9 °C
pH:
The pH of the blank control should not fluctuate by more than 1.5 units.
The change was 1.5 units in the blank control (from 7.6 to 9.1)
Dissolved oxygen:
No data
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentrations: 10 / 32 / 100 / 320 / 1000 mg/L
- Measured concentrations: 0.17 / 0.28 / 15.8 / 149.8 / 671.3 mg/L (see Table 6.1.5/2 in "Any other information on results incl. tables")
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Initial cells density: 2.4 *103 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes (the algal medium corresponds to the OECD TG 201 medium)
To avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL of Stock Solution IV (sodium hydrogen carbonate solution (50 g/L) was added to the major amount of algal medium (e.g. 90 - 99 % of the desired end volume). Then, pH was adjusted to 7.3 and the solution was filled up to 1000 mL.
Deviations from the nominal weighted loads were less than 5%.

OTHER TEST CONDITIONS
- Sterile test conditions: yes (stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration)
- Adjustment of pH: yes
- Light intensity and quality: 5000 Lux during the test
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no
- Other: At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 / 32 / 100 / 320 / 1000 mg/L nominal concentration. The concentrations to be tested based on non GLP pre-tests
- Non-GLP pre-tests: Non-GLP pre-tests were performed to determine the toxicity of the test item. In a pre-test the concentrations 0.1, 10 and 1000 mg/L were tested. No inhibition was ob-served at 10 mg/L (slight stimulation of algal growth at 0.1 and 10 mg/L). Complete inhibition of algal growth was observed at 1000 mg/L. Analytical determination of the test item was performed at t = 0h, 8h, 24h, 48h and 72h in test solutions with and without algae. The measured concentration in the solutions with and without algae was in a similar same range.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
194.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Normal and healthy appearance of the algae observed in the control and at all tested concentrations except at 1000 mg/L where no cells were visible.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 72h-ErC50 = 0.90 mg/L (95% CI: 0.77 - 1.06 mg/L); 72h-EyC50 = 0.42 mg/L (95% CI: 0.35 - 0.51 mg/L)
The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software. Calculation was performed with Microsoft Excel® and ToxRat® Professional, version 3.2.0.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

Table 6.1.5/1: Cell numbers

Nominal Conc. in mg/L

Replicate

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

1

2380

9700

62640

484000

2

2380

9480

64580

439520

3

2380

9780

63920

457800

4

2380

9920

63000

470400

5

2380

9280

60280

431860

6

2380

9600

55540

421580

Mean

2380

9627

61660

450860

SD

0

227

3339

23980

10

1

2380

8320

39960

319160

2

2380

8920

35160

308320

3

2380

7680

37800

217980

Mean

2380

8307

37640

281820

SD

0

620

2404

55552

32

1

2380

9140

51740

287740

2

2380

11880

54420

290220

3

2380

11780

53680

252840

Mean

2380

10933

53280

276933

SD

0

1554

1384

20902

100

1

2380

9340

50440

105000

2

2380

9480

46600

106220

3

2380

10260

60140

106380

Mean

2380

9693

52393

105867

SD

0

496

6978

755

320

1

2380

7060

34780

63680

2

2380

7920

35980

71840

3

2380

8160

34500

66260

Mean

2380

7713

35087

67260

SD

0

578

786

4171

1000

1

2380

5620

7900

8800

2

2380

6620

6820

6540

3

2380

6320

5740

6700

Mean

2380

6187

6820

7347

SD

0

513

1080

1261

Table 6.1.5/2: Measured concentrations

Nominal
Concentrations

Measured Concentrations

mg/L

t = 0 h

t = 8 h

t = 24 h

t = 48 h

t = 72 h

Geometric mean measured concentrations

mg/L

% of nominal

mg/L

% of nominal

mg/L

% of nominal

mg/L

% of nominal

mg/L

% of nominal

mg/L

Blank control

n.d.

-

n.d.

-

n.d.

-

n.d.

-

n.d.

-

-

10

10.76

108

6.27

63

n.d.

-

n.d.

-

n.d.

-

0.17

32

35.03

109

24.42

76

n.d.

-

n.d.

-

n.d.

-

0.28

100

108.16

108

96.02

96

29.07

29

1.65

2

2.00

2

15.8

320

353.68

111

333.01

104

220.32

69

96.03

30

30.24

9

149.8

1000

1078.48

108

1033.07

103

825.96

83

442.28

44

335.03

34

617.3

n.d.: Not determinable

At the start, after 8 h, 24 h and 48 h incubation and at the end of the test (72h), the content of the test item in the test solutions was determined using HPLC. The test item was not stable during this study. The measured concentrations lay between 108 % and 111 % of the nominal concentrations at the beginning of the test and between 0 % and 34 % of the nominal concentrations at the end of the test.

Therefore, the determination of the results was based on the geometric mean of the measured concentrations. Since the test item was not detectable in the two lowest concentrations after 24 h incubation, the half of the LOD/LOQ (0.025 mg/L / 2 = 0.013 mg/L) was used to calculate the geometric mean for these treatments. Geometric mean is calculated by multiplication of the n participating concentrations and taking the nthroot.

Table 6.1.5/3: Inhibition of growth rate

Nominal Concentration in mg/L

Geometric mean in mg/L

Growth Rate

(0-72h) [day-1]

% inhibition

Growth rate (0-72h)

Blank control

--

Mean

1.75

0

SD

0.02

10

0.17

Mean

1.59

9.2

SD

0.07

32

0.28

Mean

1.58

9.3

SD

0.03

100

15.8

Mean

1.27

27.6

SD

0.00

320

149.8

Mean

1.11

36.3

SD

0.02

1000

671.3

Mean

0.37

78.7

SD

0.05

Validity criteria fulfilled:
yes
Remarks:
Increase factor biomass in the blank control = 189 (>16 in 72h, valid); Mean coefficient of variation of daily growth rates = 18% (<35%, valid); Coefficient of variation of average growth rate during the whole test period = 1% (<7%, valid)
Conclusions:
Based on the results of this study, the 72h-ErC50 and 72h-ErC10 values were determined at 194.03 and 7.20 mg/L, respectively, for growth rate, based on the geometric mean measured concentrations.
Executive summary:

This study was performed according to OECD Guideline 201 and EU Method C.3 with GLP statement in order to evaluate the toxicity of the registered substance towards Desmodesmus subspicatus. This freshwater green alga was chosen as a typical species of phytoplankton. The study was performed using 5 concentrations ranging from 10 to 1000 mg/L. Incubation time was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed in all tested concentrations.

 

At the start, after 8h, 24h and 48h incubation and at the end of the test (72h), the content of the test item in the test solutions was determined using HPLC.

The test item was not stable during this study. The measured concentrations lay between 108 % and 111 % of the nominal concentrations at the beginning of the test and between 0 % and 34 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. Since the test item was not detectable in the two lowest concentrations after 24 h incubation, the half of the LOD/LOQ (corresponding to 0.013 mg/L) was used for calculation of the geometric mean for these treatments.

Based on the results of this study, the 72h-ErC50 and 72h-ErC10 values were determined at 194.03 and 7.20 mg/L, respectively, for growth rate, based on the geometric mean measured concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2017-12-15
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:
1. SOFTWARE
iSafeRat® HA-QSAR toolbox

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.8

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1C(=O)C(=C(O1)C)O
The experimentally log KOW value of 0.95 measured at 20°C has been given as input to determine the water solubility with iSafeRat® holistic HA-QSAR v1.8 (= 31.1 g/L), which was then given as an input to the model.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not applicable
Remarks:
(QSAR model)
Principles of method if other than guideline:
The acute toxicity to aquatic algae was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 72-hour ErC50 tests on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 72-h ErC50 for algae and is valid within the applicability domain defined in the QMRF.
GLP compliance:
no
Specific details on test material used for the study:
- Melting point: 79°C (EFSA; 2015)
- log Kow: 0.95 (Chen Y., Sidisky L.M.; 2011)
- Water solubility: 31.1 g/L (KREATiS prediction)
- Mode of Action: polar narcosis (MOA 2)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Not applicable
Test organisms (species):
other: algae spp.
Details on test organisms:
Not applicable
Test type:
other: QSAR method
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
Not applicable
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The temperatures varied from approximately 20 to 25 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the ErC50 values found in experimental data.
pH:
The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.
Dissolved oxygen:
The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).
Salinity:
Not applicable.
Nominal and measured concentrations:
The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.
Details on test conditions:
Not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
123 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 72.3 - 210 mg/L
Details on results:
iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water
solubility and algal growth inhibition tests for 8 alkyl-/alkoxy-phenols (i.e. polar narcotic compounds).
The substance falls within the applicability domain of the model and was therefore reliably predicted for its acute toxicity (72h-ErC50) to algae.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not determinable

No additional information

Validity criteria fulfilled:
yes
Conclusions:
The substance falls within the applicability domain of the model and was therefore reliably predicted for its acute toxicity to algae. The 72h-ErC50 value was determined at 123 mg/L (95% CL: 72.3 - 210 mg/L).
Executive summary:

A QSAR prediction was performed to assess the inhibition of growth to algae of the registered substance. This QSAR model has been validated to be

compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 201. The criterion predicted was the ErC50 (Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question (MOA 2, polar narcosis). This QSAR is based on validated data for a training set of 8 chemicals derived from 72 -hour ErC50 test on algae, for which the concentrations of the test substance had been determined by chemical analyses over the test period.

The substance falls within the applicability domain of the model and was therefore reliably predicted for its acute toxicity to algae. The 72h-ErC50 value was determined at 123 mg/L (95% CL: 72.3 - 210 mg/L).

Description of key information

OECD guideline 201, key study, GLP, validity 1 :

72h-ErC50 (Desmodesmus subspicatus) = 194.03 mg/L based on geometric mean measured concentrations.

72h-ErC10 (Desmodesmus subspicatus) = 7.20 mg/L based on geometric mean measured concentrations;

Key value for chemical safety assessment

EC50 for freshwater algae:
194.03 mg/L
EC10 or NOEC for freshwater algae:
7.2 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, one experimental study and one QSAR prediction are available.

The experimental study (LAUS, 2018), assessed as the key study, was performed according to OECD Guideline 201 and EU Method C.3 with GLP statement in order to evaluate the toxicity of the registered substance towards Desmodesmus subspicatus. This freshwater green alga was chosen as a typical species of phytoplankton. The study was performed using 5 concentrations ranging from 10 to 1000 mg/L. Significant inhibition of algal growth was observed up to 72 hours in all tested concentrations. The test item was not stable during this study. The measured concentrations lay between 108 % and 111 % of the nominal concentrations at the beginning of the test and between 0 % and 34 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. Based on the results of this study, the 72h-ErC50 and 72h-ErC10 values were determined at 194.03 and 7.20 mg/L, respectively, for growth rate, based on the geometric mean measured concentrations.

The QSAR prediction (KREATiS, 2017; model: iSafeRat® holistic HA-QSAR v1.8), assessed as a supporting information, was performed to assess the inhibition of growth to algae of the registered substance. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 201. The growth inhibition of algae was determined using a validated QSAR for the Mode of Action in question (MOA 2, polar narcosis). This QSAR is based on validated data for a training set of 8 chemicals derived from 72 -hour ErC50 test on algae, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The substance falls within the applicability domain of the model and was therefore reliably predicted for its acute toxicity to algae. The 72h-ErC50 value was determined at 123 mg/L (95% CL: 72.3 - 210 mg/L). This prediction supports the experimental result obtained on the registered substance.