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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:
2018-12-03 to 2018-12-10
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:
OECD Guideline No. 201 for Testing of Chemicals (2011)

Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Council Regulation (EC) No. 266/2016/Method C.3
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: Benzaldehyde
CAS No./EC No.: 100-52-7/202-860-4
Batch number: #1803-1
Purity (certified): > 99.5 % wt
Appearance: Clear, colorless to light yellow, liquid
Water solubility: 6.95 g/L at 25 °C
Relative density: 1.041 – 1.046 at 25 °C
Vapor pressure: 169 Pa at 25 °C
Expiry date: 2020-01-15
Analytical monitoring:
yes
Details on sampling:
Chemical Analysis of Test Item Concentrations

Determination of the test item: The samples were analysed under GLP with a GC-MS method, which has been implemented under non-GLP and documented finally in the GLP raw data.

Sampling schedule: All concentration levels and the control were analytically verified via GC-MS after 0 hours (start of exposure) and 72 hours (end of the exposure) with algae.

Sampling and pre-treatment: Separate replicates for the test item analysis after 0 hours and 72 hours were prepared in the original test vessels with algae at the start of the exposure and incubated under test conditions until analysis. The samples were stabilized immediately after sampling by extraction. The samples were preferably measured within one day after sampling.

Biological Parameters

Chlorophyll a-fluorescence: 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. No self-fluorescence was observed in the preliminary range finding test at the 100 mg/L test item solution.

Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Physical Chemical Properties

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.

Equipment
pH-Meter, pH 191 (WTW)
Hygro-thermograph (KLIMATHERM)
Digital Lux meter (TESTO)
Fluorometer: Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH) for determination of the cell density
Shaker, SM 25 A (BÜHLER)
Light microscope (ZEISS Lab 16)
Fluorescent tubes, OSRAM L 36 W/865, cool daylight (test), OSRAM L 30 W/840, cool white energy saver (culture)
Piston Stroke Pipettes (THERMO SCIENTIFIC, BRANDT)
Analytical balance, LA230-S (SARTORIUS)
Balance, PLS 1200-3A (KERN)


Vehicle:
no
Details on test solutions:
Test item: Benzaldehyde

Stock solution, media preparation: A stock solution with a nominal test item concentration of 50.0 mg/L was freshly prepared with dilution water prior to the start of exposure. The stock solution was agitated until the solution was visually clear.

Test concentrations (nominal): Seven concentration levels were tested in a geometrical series with (nominal) dilution factor of √10: 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0140 – 0.0235 – 0.0867 – 0.104 – 0.174 – 7.28 – 42.4 mg/L. The concentration levels are based on the results of a preliminary range finding test.

Control: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Reference Item: Potassium dichromate was tested as a reference item.

Replicates: Three replicates per concentration level and six for the control were tested. Separate replicates for each measuring time were prepared.

Test containers: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals

Test volume: 59 mL


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4

Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism: Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

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).

Preculture: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.

Initial cell density Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6442 cells/mL


Test type:
static
Test temperature:
21 – 24 °C
Details on test conditions:
Test method: Static procedure

Performance of the test: A static test was carried out. With regard to the volatility of the test item sealed glass flasks without headspace were used to reduce loss of test item.

Duration of the test: 72 hours

Replicates: Three replicates per concentration level and six for the control were tested. Separate replicates for each measuring time were prepared.

Test containers: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals

Test volume: 59 mL

Preculture: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.

Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6442 cells/mL

Application: Application was carried out by adding appropriate volumes of algae inoculum to the test solutions.

Incubation: The flasks were positioned randomly and repositioned daily.

Temperature (target): Nominal range: 21 – 24 °C, controlled at ± 2 °C

Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

Light intensity (target): Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime: 24 hours/day light

Light homogeneity (target): Within ± 15% over incubation area
Reference substance (positive control):
yes
Remarks:
Potassisum dichromate
Duration:
72 h
Dose descriptor:
EC50
Remarks:
growth rate
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Remarks:
growth rate
Effect conc.:
0.039 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
Yield
Effect conc.:
8.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Remarks:
Yield
Effect conc.:
0.021 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The toxicity of Benzaldehyde (Batch no.: #1803-1) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-12-03 to 2018-12-10 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A stock solution with a nominal concentration of 50.0 mg/L was prepared once prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was agitated until it was visually clear. Seven concentration levels were tested in a geometrical series with a spacing factor of √10: 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0140 – 0.0235 – 0.0867 – 0.104 – 0.174 – 7.28 – 42.4 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

At the start of the exposure the measured concentrations were in the range of 84 to 98% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 80% of the nominal test item concentrations.

Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28 (with headspace) and 2018-07-10 to 2018-07-13 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPS.

Cell Densities

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

42.4

1

6442

12487

18805

33087

2

6442

11385

18524

38019

3

6442

11596

19726

33561

Mean

6442

11823

19018

34889

7.28

1

6442

14438

60431

227201

2

6442

17819

55625

207966

3

6442

17636

51489

261122

Mean

6442

16631

55848

232096

0.174

1

6442

18626

55039

193548

2

6442

16710

55481

209972

3

6442

14813

57532

182367

Mean

6442

16716

56017

195296

0.104

1

6442

17201

54404

229326

2

6442

18346

58704

167571

3

6442

17479

50114

224470

Mean

6442

17675

54407

207122

0.0867

1

6442

23350

93598

330791

2

6442

22117

82178

354300

3

6442

21474

107321

339479

Mean

6442

22314

94366

341523

0.0235

1

6442

29000

119738

376940

2

6442

26846

122410

363311

3

6442

28010

127662

437786

Mean

6442

27952

123270

392679

0.0140

1

6442

29867

117713

403553

2

6442

25108

114570

393325

3

6442

25145

117226

412291

Mean

6442

26707

116503

403056

Control

1

6442

30371

124892

454916

2

6442

29418

136033

403900

3

6442

29341

133611

402584

4

6442

29517

134733

410578

5

6442

28137

127280

441907

6

6442

30746

125627

398513

Mean

6442

29588

130363

418733

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

Replicate

Growth Rate

Inhibition of Growth Rate

Yield

Inhibition of Yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

42.4

1

 

0.55

61

 

26645

94

2

 

0.59

57

 

31577

92

3

 

0.55

60

 

27119

93

Mean

(+)

0.56

60

(+)

28447

93

7.28

1

 

1.19

15

 

220759

46

2

 

1.16

17

 

201524

51

3

 

1.23

11

 

254680

38

Mean

(+)

1.19

14

(+)

225654

45

0.174

1

 

1.13

18

 

187106

55

2

 

1.16

17

 

203530

51

3

 

1.11

20

 

175925

57

Mean

(+)

1.14

18

(+)

188854

54

0.104

1

 

1.19

14

 

222884

46

2

 

1.09

22

 

161129

61

3

 

1.18

15

 

218028

47

Mean

(+)

1.15

17

(+)

200680

51

0.0867

1

 

1.31

6

 

324349

21

2

 

1.34

4

 

347858

16

3

 

1.32

5

 

333037

19

Mean

(+)

1.32

5

(+)

335081

19

0.0235

1

 

1.36

2

 

370498

10

2

 

1.34

3

 

356869

13

3

 

1.41

-1

 

431344

-5

Mean

(-)

1.37

2

(-)

386237

6

0.0140

1

 

1.38

1

 

397111

4

2

 

1.37

1

 

386883

6

3

 

1.39

0

 

405849

2

Mean

(-)

1.38

1

(-)

396614

4

Control

1

 

1.42

 

 

448474

2

 

1.38

 

 

397458

3

 

1.38

 

 

396142

4

 

1.38

 

 

404136

5

 

1.41

 

 

435465

6

 

1.37

 

 

392071

Mean

 

1.39

 

 

412291

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

Control

1

1.55

1.41

1.29

1.42

0.129

9.10

14.5

2

1.52

1.53

1.09

1.38

0.252

18.3

3

1.52

1.52

1.10

1.38

0.239

17.3

4

1.52

1.52

1.11

1.39

0.234

16.9

5

1.47

1.51

1.25

1.41

0.144

10.2

6

1.56

1.41

1.15

1.38

0.206

15.0

 

 

 

Mean

1.39

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

1.33

 

SD = Standard deviation  CV = Coefficient of variation

Physicochemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits in the definitive study. The test media were clear throughout the exposure phase.

 

Environmental Conditions

Geometric mean measured test item concentration

pH-values

[mg/L]

Start; 0 hours

End; 72 hours

42.4

8.24

8.31

7.28

8.25

8.05

0.174

8.24

8.56

0.104

8.21

8.80

0.0867

8.18

9.16

0.0235

8.15

9.16

0.0140

8.16

9.14

Control

8.19

9.10

Room temperature [°C]:

min.: 22.5

max.: 23.5

mean value: 23.0

 

Light intensity
[lux]

n = 23

mean value: 5744

range of the measured values: 5025 to 6261 equalling -12.5 to 9.00%

CV [%]: 6.84%

CV = Coefficient of variation                                        n = number of measuring points

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of Benzaldehyde and the control were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS.

At the start of the exposure the measured concentrations were in the range of 84 to 98% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 80% of the nominal test item concentrations. The geometric mean measured concentrations are 42.4 – 7.28 – 0.174 – 0.104 – 0.0867 – 0.0235 – 0.0140 mg/L.

Measured Concentration of Benzaldehyde in the Definitive Test with Algae

Sampling date

Fresh Media,
0 hours

Old Media,
72 hours

 

Nominal test item concentration
[mg/L]

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Geometric mean measured concentration1)

[mg/L]

 

Benzaldehyde

50.0

 44.8

90

40.1

80

42.4

15.8

 14.8

94

3.58

23

7.28

5.00

4.75

95

0.00640*

0

0.174

1.58

1.41

89

0.00774*

0

0.104

0.50

0.419

84

0.0179

4

0.0867

0.158

0.138

87

< LOQ

-

0.0235

0.050

0.0490

98

< LOQ

-

0.0140

Control

< LOQ

-

< LOQ

-

< LOQ

Meas. conc.= measured concentration of the test item, dilution factors taken into account

% = percent of the nominal measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.008 mg/L of the test item)

1) = Values < LOQ taken into account with ½ LOQ (0.004 mg/L of the test item)

* = < LOQ but > 70% LOQ

NOEC, LOEC, ECx-values ofBenzaldehyde(0 - 72 hours)

                  (based on the geometric mean measured test item concentrations, with 95% confidence intervals)

 

Growth Rate Inhibition
[mg/L]

NOEC

0.0235

LOEC

0.0867

ErC10

0.0389 (0.0258 – 7.62)

ErC20

9.70 (5.79 – 13.3)

ErC50

33.1 (28.2 – 39.5)

 

Yield Inhibition

 [mg/L]

NOEC

0.0235

LOEC

0.0867

EyC10

0.0205 (0.0141 – 0.0275)

EyC20

0.0293 (0.0208 – 0.0419)

EyC50

8.05 (0.120 – 16.4)

 

EC50-Values of the Reference Item based on nominal concentrations[mg/L], (0-72 hours)

 

with Headspace

without Headspace

Valid Range

(average ± 3 x SD)

 

Growth Rate Inhibition

ErC50

0.799

0.244

0.744±0.626

95 % confidence interval

0.707 - 1.30

0.175 - 0.350*

 

Yield Inhibition

 

EyC50

0.607

0.236

0.409±0.208

 

95 % confidence interval

0.404 - 0.694

0.175 - 0.350*

 

SD = Standard deviation

*   = The highest concentration level without statistical significance and the lowest concentration level with statisical significance were used as confidence limits.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l.
Executive summary:

The acute toxicity of benzaldehyde to Pseudokirchneriella subcapitata was studied in a 72-hour toxicity study according to OECD 201. The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l.

Description of key information

The toxicity of benzaldehyde to Pseudokirchneriella subcapitata was studied in a 72-hour toxicity study according to OECD 201. The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l (mean measured concentration). An EC10 (growth) of 0.0389 mg/l and EC10 (yield) of 0.0205 mg/l were also reported.

Supporting studies showed 8-day NOEC (TGK) values of 20 mg/L (Bringmann and Kuhn, 1976) and 34 mg/l (Bringmann and Kuhn 1977; Bringmann and Kuhn, 1980). The outcome of a QSAR (ECOSAR) predicted an EC50 of 23 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
8.05 mg/L
EC10 or NOEC for freshwater algae:
0.021 mg/L

Additional information