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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
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:
QSAR calculation, based on generally accepted model (ECOSAR), supported by publication based on slightly different input data. Details reported in the QPRF document attached to background materials.
Qualifier:
according to
Guideline:
other: REACH guidance on QSARs, R.6, May 2008
GLP compliance:
no
Analytical monitoring:
not required
Test organisms (species):
other: Green alga (arbitrary species, QSAR calculation)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
ca. 7.9 mg/L
Basis for effect:
growth rate

Previous EC50 estimate (Bowmer et al., 1998, based on calculated log Pow of 3.168): 11 mg/L.

Recent EC50 estimate (ECOSAR, based on measured log Pow of 3.3): 7.9 mg/L.

The latter is adopted in a worst-case approach.

Validity criteria fulfilled:
yes
Conclusions:
The algal toxicity of 2-ethylhexyl-S-lactate is predicted by QSAR calculation to result in a 96-h ErC50 of 7.9 mg/L.
Executive summary:

The algal toxicity of 2-ethylhexyl-S-lactate was predicted by QSAR calculation (ECOSAR), resulting in a 96 -h ErC50 of 7.9 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-04 to 2019-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) No. 266/2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch number: 1802000003
CAS No.: 186817-80-1
Purity (certified): 98 %
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.
- Sampling method: At the start, samples were taken from one additional replicate of the test item concentration and the control and at the end samples from pooled replicates.
- Sample storage conditions before analysis: All samples were immediately extracted and the extracts were stored at room temperature if necessary. Until the start of the analysis the prepared samples were stored on an autosampler.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: Stock solution, media preparation: 80.0 mg test item/L were freshly prepared with dilution water. The solution was stirred for 10 minutes (room temperature, 1100 rpm) with a magnetic stirrer. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Preparation of the test item solutions: Out of the stock solution six concentrations were prepared in a geometric series with a dilution factor of √10: 0.253 – 0.800 – 2.53 – 8.00 – 25.3 – 80.0 mg/L, corresponding to the geometric mean measured test item concentration of: 0.136 – 0.417 – 1.53 – 5.48 – 20.5 – 64.1 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
- Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
- Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567–5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994)

ACCLIMATION
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): OECD TG 201 medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
33 mg CaCO3/L
Test temperature:
21.5 to 22.0 °C
pH:
7.73 to 9.41
Conductivity:
137 µS/cm
Nominal and measured concentrations:
Nominal: 0.253 - 0-800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L
Geometric mean measured test item concentration of: 0.136 – 0.417 – 1.53 – 5.48 – 20.5 – 64.1 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Fill volume: 100
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): n.a.
- Initial cells density: Nominal: approximately 5E+03–1E+04 cells/mL; actual: 5416 cells/mL
- Control end cells density: 1959999 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n.a.

GROWTH MEDIUM
- Standard medium used: yes (Nutrient medium Z according to LÜTTGE et al. (1994))

TEST MEDIUM / WATER PARAMETERS
- Ultrapure water: Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).
- Test medium: OECD TG 201 medium):
NH4Cl: 15 mg/L
MgCl2 × 6 H2O: 12 mg/L
CaCl2 × 2 H2O: 18 mg/L
MgSO4 × 7 H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3 × 6 H2O: 0.064 mg/L
Na2EDTA × 2 H2O: 0.1 mg/L
H3BO3: 0.185 mg/L
MnCl2 × 4 H2O: 0.415 mg/L
ZnCl2: 3.0E-03 mg/L
Na2MoO4 × 2 H2O: 7 x 10-3 mg/L
CoCl2 × 6 H2O: 1.5E-03 mg/L
CuCl2 × 2 H2O: 1.0E-05 mg/L
NaHCO3: 50 mg/L
pH: 8.1 ± 0.2

- Total organic carbon: < 2 mg C/L
- Alkalinity: 0.8 mmol/L
- Conductivity: 137 µS/cm
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: The pH-values at the start of exposure were measured in one additional replicate of each concentration level and the control. At the end of exposure, the pH-values were measured from pooled samples of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: mean value: 5577 lux
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Inhibition of yield, Inhibition of growth rate
- Determination of cell concentrations: The cell density was measured daily via chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentrations of 100 mg/L. A separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours were taken into account in the evaluation.
- The cell density was measured daily via chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentrations of 100 mg/L. A separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours were taken into account in the evaluation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10: 0.253 - 0-800 - 2.53 - 8.00 - 25.3 - 80.0 mg/L (nominal)

RANGE FINDING STUDY
-Test concentrations: 1, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
inhibition
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
inhibition
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
inhibition
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
inhibition
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks:
inhibition
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): 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 (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
- EC-values and statistical analyses: EC10-,EC20- and EC50- values with confidence intervals of yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

- The NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01. Levene’s test on variance homogeneity was done with a significance level of 0.01. Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for growth rate. Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for growth rate.Monotonicity of Concentration/Response was tested by non-parametric trend analysis with a significance level of 0.05 for yield.Step-down Jonckheere-Terpstra Test Procedure was performed with a significance level of 0.05 for yield.

- Software: The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures. Calculations were carried out using software:
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Version 3.3.0, ToxRat Solutions GmbH

Cell densities are listed in Table 1. Evaluations of growth rate inhibition and yield inhibition are given in Table 2 (calculated based on cell density values). Section-by-section and average specific growth rate are given in Table 3.

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the geometric mean measured test item concentrations.

Table1:      Cell densities

Geometric mean measured test item concentration[mg/L]

Replicate no.

Cell density [cells/mL]

0 hours

24 hours

48 hours

72 hours

64.1

1

5416

4435

4384

2770

 

2

5416

6007

4369

3337

 

3

5416

5438

4732

2653

 

Mean

5416

5293

4495

2920

20.5

1

5416

9761

20419

96341

 

2

5416

11019

27030

130319

 

3

5416

9551

25813

106218

 

Mean

5416

10110

24421

110959

5.48

1

5416

21225

170027

1177304

 

2

5416

19185

156095

1179592

 

3

5416

19971

148102

1201450

 

Mean

5416

20127

158075

1186115

1.53

1

5416

27049

213448

2084463

 

2

5416

25156

209563

1641183

 

3

5416

27665

228645

1940078

 

Mean

5416

26623

217219

1888575

0.417

1

5416

27531

241436

1957160

 

2

5416

30788

244318

1935936

 

3

5416

28734

242147

1962320

 

Mean

5416

29018

242634

1951805

0.136

1

5416

26317

236568

1635098

 

2

5416

28495

242643

2013050

 

3

5416

32803

237970

1952930

 

Mean

5416

29205

239060

1867026

Control

1

5416

29182

239572

2110656

 

2

5416

25097

185771

1590607

 

3

5416

33324

287775

1980339

 

4

5416

26336

244771

2003511

 

5

5416

29943

255071

2119369

 

6

5416

29021

222972

1955512

 

Mean

5416

28817

239322

1959999

 

Table 2: Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration[mg/L]

Replicate no.

Growth rate [d–1]

Inhibition of growth rate [%]

Yield [cells/mL]

Inhibition of yield [%]

64.1

1

 

-0.224

100

 

-2646

100

 

2

 

-0.161

100

 

-2079

100

 

3

 

-0.238

100

 

-2763

100

 

Mean

(+)

-0.208

100

(+)

-2496

100

20.5

1

 

0.960

51

 

90925

95

 

2

 

1.06

46

 

124903

94

 

3

 

0.992

49

 

100802

95

 

Mean

(+)

1.00

49

(+)

105543

95

5.48

1

 

1.79

9

 

1171888

40

 

2

 

1.80

9

 

1174176

40

 

3

 

1.80

8

 

1196034

39

 

Mean

(+)

1.80

8

(+)

1180699

40

1.53

1

 

1.98

-1

 

2079047

-6

 

2

 

1.91

3

 

1635767

16

 

3

 

1.96

0

 

1934662

1

 

Mean

(-)

1.95

1

(-)

1883159

4

0.417

1

 

1.96

0

 

1951744

0

 

2

 

1.96

0

 

1930520

1

 

3

 

1.96

0

 

1956904

0

 

Mean

(-)

1.96

0

(-)

1946389

0

0.136

1

 

1.90

3

 

1629682

17

 

2

 

1.97

-1

 

2007634

-3

 

3

 

1.96

0

 

1947514

0

 

Mean

(-)

1.95

1

(-)

1861610

5

Control

1

 

1.99

 

 

2105240

 

 

2

 

1.89

 

 

1585191

 

 

3

 

1.97

 

 

1974923

 

 

4

 

1.97

 

 

1998095

 

 

5

 

1.99

 

 

2113953

 

 

6

 

1.96

 

 

1950096

 

 

Mean

 

1.96

 

 

1954583

 

 

Table 3: Section-by-section and average specific growth rates of the control group (0–72 hours)

Replicate No.

Specific growth rate [d-1]

Mean (0–72 hours)

SD

±

CV [%]

Mean CV [%]

section-by-section

0–24 hours

24–48 hours

48–72 hours

1

1.68

2.11

2.18

1.99

0.266

13.4

13.6

2

1.53

2.00

2.15

1.89

0.321

16.9

3

1.82

2.16

1.93

1.97

0.173

8.8

4

1.58

2.23

2.10

1.97

0.343

17.4

5

1.71

2.14

2.12

1.99

0.243

12.2

6

1.68

2.04

2.17

1.96

0.255

13.0

 

 

Mean

1.96

 

 

 

 

 

SD ±

0.04

 

 

 

 

 

CV [%]

1.79

 

 

 

SD = Standard deviation CV = Coefficient of variation

 

Validity criteria fulfilled:
yes
Conclusions:
The 72-h ErC10 of Pseudokirchneriella subcapitata exposed to 2-ethylhexyl-S-lactate was determined to be 6.18 (5.43–7.02) mg/L (geometric mean of measured concentrations).
Executive summary:

In a 72-hour acute toxicity study, cultures of Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 were exposed to 2-ethylhexyl-S-lactate at measured concentrations of 0, 0.136, 0.417, 1.53, 5.48, 20.5, and 64.1 mg/L (geometric mean measured) under static conditions in accordance with OECD test guideline 201. The initially measured concentrations of 2-ethylhexyl-S-lactate were in the range of 90 % to 105 % and by the end of exposure after 72 hours in the range of 28 % to 72 % of nominal. All effect values given are based on the geometric mean measured concentrations of 2-ethylhexyl-S-lactate.

The NOEC and EC50 values based on growth rate were 1.53 and 21.0 mg/L, respectively. The % growth inhibition in the treated algal culture as compared to the control ranged from 1 to 100 %.

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algal growth inhibition test.

Results synopsis

Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4

Test type: Static

Growth rate inhibition

72-h NOEC: 1.53 mg/L

72-h ErC10: 6.18 mg/L (95 % C.I.: 5.43–7.02 mg/L)

72-h ErC50: 21.0 mg/L (95 % C.I.: 20.2–21.8 mg/L)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
results derived from a (Q)SAR model, with limited documentation / justification
Qualifier:
no guideline followed
Principles of method if other than guideline:
Structure-activity-relationship (SAR)
SARs were used calculating the toxicity. A reliable SAR from the scientific literature for non-polar narcosis is provided for comparison.
GLP compliance:
no
Analytical monitoring:
not required
Vehicle:
no
Test organisms (species):
Scenedesmus capricornutum
Test type:
other: SAR
Water media type:
freshwater
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
SAR prediction
Details on results:
SAR was derived from the following test data:
log EbC50 = -0 77 (log Kow) + 0.84
n = 7
r² = 0.79

ErC50 lactates:
log E.C50 = - 0.97 (log Kow) + 1 .83
n = 7
r = 0.93

ErC50 non-polar narcotics
log ErC50 = - 0.92 (log Kow) + 1 .40
n = 5
r² = 0.97

Source: Calamari, C., S. Galassi, F. Setti and M. Vighi (1983). Toxicity of selected chlorobenzenes to aquatic organisms. Chemosphere, 12, No. 2, 253-262.Where S. capricornutum is concerned, the ErC50 (reproduction based on cell counts) for the alkyl lactate esters is remarkably similar to a SAR for non-polar narcotic substances (chloro benzenes, re-calculated by Clements & Nabholz, 1984 with data from Calamari et al., 1983).
Validity criteria fulfilled:
not specified
Conclusions:
The predicted 72 to 96-h ErC50 for S. capricornutum equals 11 mg/L.
Executive summary:

Based on a SAR prediction the ErC50 for Selenastrum capricornutum was calculated to equal 11 mg/L.

Description of key information

In a study according to OECD test guideline 201 under GLP, the inhibition of 2-ethylhexyl-S-lactate to the growth of green algae resulted in a 72-h ErC50 of 21.0 mg/L, a 72-h ErC10 of 6.18 mg/L, and a NOErC of 1.53 mg/L in R. subcapitata. These effect values are expressed as geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
21 mg/L
EC10 or NOEC for freshwater algae:
6.18 mg/L

Additional information

QSAR calculation of the algal toxicity of 2-ethylhexyl-lactate (ECOSAR, 2015) gave a 96-h ErC50 of 7.9 mg/L.

Furthermore, in a publication by Bowmer et al. (1998) a SAR prediction of 11 mg/L is presented for the ErC50 in Selenastrum capricornutum (recent name: Raphidocelis subcapitata).

These results are considered as supportive data only, since constituting theoretical calculations, suffering from limited reporting of validity criteria.