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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 – 25 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Anthracene oil
Cas Number:
90640-80-5
Molecular formula:
Not applicabel
IUPAC Name:
Anthracene oil
Test material form:
other: depending on composition (UVCB) solid or viscous liquid
Details on test material:
anthracene oil; anthracene oil ≤ 50 ppm BaP, AOL (anthracene oil low)
- substance type: organic
Specific details on test material used for the study:
- Name of test material (as cited in study report): anthracene oil (< 50 ppm BaP)
- Lot/batch No. of test material: ATE-No. 10442
- Stability under test conditions: WAF (water accommodated fraction) limited
- Storage condition of test material: room temperature, exclusion of light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Samples for TOC-analysis were taken at 0 h and 72 h.
- Measurements of the cell concentrations per mL: at time 24 h, 48 h and 72 h by optical densitometry at 525 nm

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) were prepared by stirring various amounts of the test item anthracene oil (AOL) in Alga-medium for 24 h with magnetic stirrers (open conditions). The WAFs were prepared in 2-L glass beakers. The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loading rates of AOL were weighed on microscope slides, that afterwards were placed into the glass beakers. The beakers were then filled with Alga-medium. Mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. To ensure this, the vortex developed at the surface by stirring was set at ~ 10 % of the water depth.
After stirring for 24 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation. The extracts gained with this method were clear.
- Pre-conditioning: The test vessels of the Definite Alga-Test were rinsed with either control media or WAFs for ca. 0.5 h before the definite start of the tests to facilitate adsorption of the test item to the glass walls of the beakers. Control media and WAFs were then discarded, and the test vessels were refilled again with control media and WAFs, respectively.
- Differential loading: control, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L, 25 mg/L, and 50 mg/L
- Controls: without test material (blank; Alga medium)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: : SAG 86.81
- Source: Albrecht von Haller Institut für Pflanzenwissenschaften der Universität Göttingen
- Age of inoculum (at test initiation): ca. 72 h
[Pre-culture: Incubation in 250 mL Erlenmeyer flasks at ± 25 °C on a shaker (80 - 120 rpm), ca. 72 h, ca. 10000 lux (Gro-Lux fluorescent tubes), permanent illumination]
- Method of cultivation: Sterile on agar tubes (OECD-Medium, 0.8 % Agar), transferred to a new agar tube every 2 months

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none

Test conditions

Hardness:
no data specified: Compositions of stock solutions given
Test temperature:
23.5 - 23.7 °C (25 ± 2°C)
pH:
initial (with algae): 7.9
final (with algae): 8.5 (control); 9.2 (1 mg/L); 8.8 (2.5 mg/L); 10.0 (5 mg/L); 10.2 (10 mg/L); 8.4 (25 mg/L); 8.3 (50 mg/L)
Salinity:
no data specified: Compositions of stock solutions given
Nominal and measured concentrations:
Nominal concentrations: control, 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L, 25 mg/L, and 50 mg/L
Measured concentration (as TOC):
Time [h] Control 2.5 mg/L 10 mg/L 50 mg/L loading
=================================================================
0 0.4 0.6 1.1 2.4 mgC/mL (TOC of WAFs)
72 1.6 1.9 3.8 1.9 mgC/mL (TOC of WAFs)
=================================================================
The oil-related TOC was about 0.2 - 0.3 mg C/L for 2.5 mg oil/L, 0.7 - 2.0 mg C/L for 10 mg oil/L, and 2.0 mg C/L for 50 mg oil/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL-Erlenmeyer
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: approx. 5000 cells/mL
- Control end cells density: approx. 155200 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted from deionised water and mineral stock solutions

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: permanent
- Light intensity and quality: approx. 10000 Lux (Gro-Lux fluorescent tubes)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: densitometrically at 525 nm (turbidity)
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 and 2.5
- Range finding study
- Results used to determine the conditions for the definitive study: based on results from daphnia test

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: NOELR = No-observable effect loading rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EL = effective loading
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass (cell yield)
Remarks on result:
other: LOELR = lowest observable effect loading rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL100
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not specified
- Any stimulation of growth found in any treatment: from 24 to 72 h at 5 and 10 mg/L
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not applicable
- Effect concentrations exceeding solubility of substance in test medium: yes; effect concentrations are nominal values (loadings) with WAFs in the range of ca. 0.3 to ca. 2 mg TOC/L (corresponding nominal test material loadings from 2.5 to 50 mg/L)
- Observation of abnormalities (for algal test): by microscopy at the end of the testing period of 72 h:
Control: Appearance of alga cells was normal, cell proliferations could be observed regularly.
1 - 10 mg/L: Comparable to control.
25 mg/L: Appearance comparable to control, lower number of cells, only a few cell proliferations could be observed.
50 mg/L: Appearance comparable to control, very low number of cells, cell proliferation could not be observed.
Reported statistics and error estimates:
No data: due to high fluctuation of cell number in single replicate tests at the low concentrations, statistics were not considered reasonable and reliable.

Any other information on results incl. tables

Growth rates are listed below, from 0 - 24, 24 - 48, and 48 - 72 h (Table 1) and average growth rate from 0 - 72 h (Table 2):

Table 1: Loading rates of the Test item, cells/mL in the test flasks at 24, 48 and 72 h with mean value, section-by-section average specific growth rate [μ] during observation times 0-24 h, 24-48 h and 48-72 h, mean value of the average specific growth rate [μ], standard deviation and coefficient of variation Vc [%], inhibition [%] and mean value of inhibition of the average specific growth rate [%] during observation times 0-24 h, 24-48 h and 48-72 h (Rep. Tab. 3)

[Note: Negative inhibition means increased growth, apparently stimulation]

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

24 h

Control 1

18746

18746

1.32

1.32

0.00

0.00

Control 2

18746

1.32

Control 3

18746

1.32

1

15518

15518

1.13

1.13

0.00

0.00

14.3

14.3

1

15518

1.13

14.3

1

15518

1.13

14.3

2.5

15518

14456

1.13

1.06

0.13

12.6

14.3

20.1

2.5

12332

0.90

31.7

2.5

15518

1.13

14.3

5

12332

13394

0.90

0.98

0.13

13.6

31.7

25.9

5

15518

1.13

14.3

5

12332

0.90

31.7

10

15518

15518

1.13

1.13

0.00

0.00

14.3

14.3

10

15518

1.13

14.3

10

15518

1.13

14.3

25

6083

7118

0.20

0.33

0.24

71.4

85.2

74.8

25

9187

0.61

54.0

25

6083

0.20

85.2

50

6083

6083

0.20

0.20

0.00

0.00

85.2

85.2

50

6083

0.20

85.2

50

6083

0.20

85.2

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

48 h

Control 1

46053

55830

0.90

1.08

0.17

15.7

Control 2

64463

1.24

Control 3

56975

1.11

1

68269

68282

1.48

1.48

0.06

3.79

-36.9

-36.9

1

64463

1.42

-31.6

1

72116

1.54

-42.0

2.5

72116

64559

1.54

1.49

0.04

2.53

-42.0

-38.1

2.5

53293

1.46

-35.3

2.5

68269

1.48

-36.9

5

72116

72171

1.77

1.69

0.07

4.12

-63.2

-55.9

5

79934

1.64

-51.5

5

64463

1.65

-52.9

10

96065

90757

1.82

1.76

0.15

8.47

-68.5

-62.6

10

76004

1.59

-46.9

10

100201

1.87

-72.4

25

22015

24222

1.29

1.24

0.21

16.9

-18.9

-14.8

25

25325

1.01

6.27

25

25325

1.43

-31.8

50

6083

7118

0.00

0.14

0.24

173

100

87.3

50

9187

0.41

61.9

50

6083

0.00

100

Loading rate

[mg/L]

Cells / mL

Cells / mL

Mean value

Average Specific

growth rate

[µ]

Average Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation Vc [%]

Inhibition of Average Specific
growth rate

[%]

Inhibition of Average Specific

growth rate

Mean value

[%]

72 h

Control 1

121502

155179

0.97

1.02

0.05

5.29

Control 2

176835

1.01

Control 3

167200

1.08

1

134777

153236

0.68

0.81

0.14

17.1

33.2

20.9

1

167200

0.95

6.43

1

157730

0.78

23.2

2.5

162444

157826

0.81

0.90

0.21

23.7

20.3

11.6

2.5

167200

1.14

-12.3

2.5

143834

0.75

26.8

5

282272

269455

1.36

1.32

0.04

3.23

-34.0

-29.4

5

287978

1.28

-25.8

5

238115

1.31

-28.3

10

335108

341365

1.25

1.33

0.10

7.78

-22.7

-30.6

10

323078

1.45

-42.1

10

365907

1.30

-27.2

25

12332

28938

  0.00 *

0.26

0.23

88.4

100

74.8

25

35503

0.34

66.8

25

38979

0.43

57.7

50

3021

3021

   0.00 **

0.00

0.00

-

100

100

50

3021

   0.00 **

100

50

3021

   0.00 **

100

Table 2: Loading rate of the Test item, cells/mL in the test flasks at 72 h, average specific growth rate [μ] over the whole testing period 0-72 h, mean value of the average specific growth rate [μ], standard deviation and coefficient of variation Vc [%], inhibition [%] and mean value of inhibition [%] of the average specific growth rate [μ] (Rep. Tab. 4)

Loading rate

[mg/L]

Cells / mL

Average

Specific

growth rate

[µ]

Average

Specific

growth rate

[µ]

Mean value

Standard deviation

Coefficient of variation

Vc [%]

Inhibition of Average

Specific

growth rate

[%]

Inhibition of Average

Specific

growth rate

Mean value

[%]

72 h

Control 1

121502

1.06

1.14

0.07

5.92

Control 2

176835

1.19

Control 3

167200

1.17

1

134777

1.10

1.14

0.04

3.26

3.74

0.10

1

167200

1.17

-2.56

1

157730

1.15

-0.86

2.5

162444

1.16

1.15

0.03

2.32

-1.72

-0.82

2.5

167200

1.17

-2.56

2.5

143834

1.12

1.83

5

282272

1.34

1.33

0.03

2.62

-17.87

-16.4

5

287978

1.35

-18.45

5

238115

1.29

-12.90

10

335108

1.40

1.41

0.02

1.52

-22.88

-23.4

10

323078

1.39

-21.81

10

365907

1.43

-25.45

25

12332

0.30

0.55

0.21

39.00

73.62

52.1

25

35503

0.65

42.72

25

38979

0.68

39.99

50

3021

  0.00 *

0.00

0.00

-

100

100

50

3021

  0.00 *

100

50

3021

  0.00 *

100

* The measurements of the cell number by optical densitometry at 525 nm gave values quite below the initial cell concentration of 5000 cells/mL. Because of the uncertainty of the measurements in this lower absorbance range, the average specific growth rate [µ] has been assigned zero.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1) Biomass in controls increased 42x in 72 h (limit: ≥ 16x). 2) Mean var-coeff. for section-spec. growth rates: 10.3 % (limit: ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 1.7 % (limit: ≤ 7%).
Conclusions:
Due to the nature of the test substance (UVCB), the fraction dissolved in water (WAF) is different for different loadings (nominal concentrations). Therefore, aqueous toxicity was determined based on loadings.
In an alga growth inhibition test according to OECD TG 201 with the test substance anthracene oil, a NOELR (no-observable effect loading rate) and an EL50 (effective loading) of 14 mg/L and 25 mg/L, respectively, were determined. The test conditions in this study were open (open test vessels).