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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:
26 Sep. - 09 Oct. 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares L 100; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 24106
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Analytical monitoring:
yes
Remarks:
determination of Total Organic Carbon (TOC)
Details on sampling:
- Samples for TOC-analysis: at 0 h and 72 h
- Measurements of the cell concentrations per mL: at 24 h, 48 h, and 72 h
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 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 rate of Novares TL 10 was weighed in a weighing scoop, that afterwards was placed into the glass beaker. 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 control media and 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.
- 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 (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


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
no data specified: compositions of stock solutions are reported
Test temperature:
23.7 - 24.8 °C (25 ± 2°C)
pH:
initial (with algae): 8.0
final (with algae): 10.6 (control); 10.8 (100 mg/L)
Salinity:
no data specified: compositions of stock solutions are reported
Nominal and measured concentrations:
Pre-test:
Time [h] Control 100 mg/L loading
==========================================
24 (stirring) 0.1 1.0 mg C/mL (TOC of WAFs)
48 (stirring) 0.4 1.2 mg C/mL (TOC of WAFs)
==========================================

Main test:
Time [h] Control 100 mg/L loading
==========================================
0 (48 h stirring) 0.6 0.8 mg C/mL (TOC of WAFs)
72 0.5 0.9 mg C/mL (TOC of WAFs)
==========================================
The TS-related TOC was = < 0.5 mg C/L for 100 mg TS/L, 95%-C.I. = 0.3 mg/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. 452900 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 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: not applicable (limit test)
- Range finding study
- Results used to determine the conditions for the definitive study: based on results from daphnia test
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- 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
- Observation of abnormalities (for algal test):
Observations 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.
- 100 mg/L: comparable to control
Reported statistics and error estimates:
standard deviations, coefficient of variance

Growth rates are listed below, from 0 - 24, 24 - 48, and 48 - 72 h (Table 1) and average 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. 4)

[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

20380

1.32

1.40

0.09

6.28

Control 2

22015

1.48

Control 3

18746

1.32

Control 4

22015

1.48

Control 5

22015

1.48

Control 6

18746

1.32

100

22015

18215

1.48

1.29

0.13

10.40

-5.73

8.31

100

18746

1.32

5.73

100

15518

1.13

19.21

100

15518

1.13

19.21

100

18746

1.32

5.73

100

18746

1.32

5.73

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

104379

92715

1.72

1.52

0.12

7.60

Control 2

100201

1.52

Control 3

83905

1.50

Control 4

87917

1.38

Control 5

91970

1.43

Control 6

87917

1.55

100

104379

96196

1.56

1.67

0.13

7.92

-2.71

-9.93

100

108598

1.76

-15.94

100

87917

1.73

-14.47

100

91970

1.78

-17.44

100

79934

1.45

4.29

100

104379

1.72

-13.32

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

492359

452938

1.55

1.58

0.06

3.91

Control 2

535389

1.68

Control 3

423947

1.52

Control 4

430603

1.59

Control 5

444037

1.57

Control 6

391290

1.49

100

610409

507575

1.77

1.65

0.15

9.03

-11.51

-4.38

100

665380

1.81

-14.45

100

457637

1.65

-4.16

100

444037

1.57

0.60

100

444037

1.71

-8.26

100

423947

1.40

11.51

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. 5)

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

492359

1.53

1.50

0.04

2.50

Control 2

535389

1.56

Control 3

423947

1.48

Control 4

430603

1.49

Control 5

444037

1.50

Control 6

391290

1.45

100

610409

1.60

1.53

0.06

4.17

-6.75

-2.29

100

665380

1.63

-8.66

100

457637

1.51

-0.35

100

444037

1.50

0.32

100

444037

1.50

0.32

100

423947

1.48

1.35

Validity criteria fulfilled:
yes
Remarks:
1) Biomass in controls increased 90.6x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 8.3% (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.5% (nom. value ≤ 7%).
Conclusions:
Water Accommodated Fractions (WAFs) of NOVARES L 100 caused no toxic effects on Desmodesmus subspicatus. A 72-h ErL50 and EyL50 of > 100 mg/L and a NOELR of 100 mg/L have been determined.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep. - 09 Oct. 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares TL 10; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 26697
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Analytical monitoring:
yes
Remarks:
determination of Total Organic Carbon (TOC)
Details on sampling:
- Samples for TOC-analysis: at 0 h and 72 h
- Measurements of the cell concentrations per mL: at 24 h, 48 h, and 72 h
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 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 rate of Novares TL 10 was weighed in a weighing scoop, that afterwards was placed into the glass beaker. 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 control media and 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.
- 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 (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


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
no data specified: compositions of stock solutions are reproted
Test temperature:
23.7 - 24.8 °C (25 ± 2°C)
pH:
initial (with algae): 8.0
final (with algae): 10.6 (control); 10.7 (100 mg/L)
Salinity:
no data specified: Compositions of stock solutions are reported
Nominal and measured concentrations:
Pre-test:
Time [h] Control 100 mg/L loading
==========================================
24 (stirring) 0.3 0.2 mg C/mL (TOC of WAFs)
48 (stirring) 0.2 0.2 mg C/mL (TOC of WAFs)
==========================================

Main test:
Time [h] Control 100 mg/L loading
==========================================
0 (24 h stirring) 0.6 0.2 mg C/mL (TOC of WAFs)
72 0.5 0.5 mg C/mL (TOC of WAFs)
==========================================
The TS-related TOC was indistiguishable from the blank control.
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. 448000 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 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: not applicable (limit test)
- Range finding study
- Results used to determine the conditions for the definitive study: based on results from daphnia test
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(highest concentration tested)
Basis for effect:
other: biomass and growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: slightly from 48 - 72 h
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Observation of abnormalities (for algal test):
Observations 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.
- 100 mg/L: comparable to control
Reported statistics and error estimates:
standard deviations, coefficient of variance

Growth rates are listed below, from 0 - 24, 24 - 48, and 48 - 72 h (Table 1) and average 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. 4)

[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

20380

1.32

1.40

0.09

6.28

Control 2

22015

1.48

Control 3

18746

1.32

Control 4

22015

1.48

Control 5

22015

1.48

Control 6

18746

1.32

100

18746

19836

1.32

1.38

0.08

6.04

5.73

1.91

100

22015

1.48

-5.73

100

18746

1.32

5.73

100

22015

1.48

-5.73

100

18746

1.32

5.73

100

18746

1.32

5.73

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

104379

92715

1.72

1.52

0.12

7.60

Control 2

100201

1.52

Control 3

83905

1.50

Control 4

87917

1.38

Control 5

91970

1.43

Control 6

87917

1.55

100

91970

92005

1.59

1.54

0.09

5.78

-4.97

-1.35

100

96065

1.47

2.76

100

87917

1.55

-2.00

100

91970

1.43

5.64

100

100201

1.68

-10.63

100

83905

1.50

1.09

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

492359

452938

1.55

1.58

0.06

3.91

Control 2

535389

1.68

Control 3

423947

1.52

Control 4

430603

1.59

Control 5

444037

1.57

Control 6

391290

1.49

100

492359

548340

1.68

1.78

0.08

4.29

-5.93

-12.60

100

550063

1.75

-10.17

100

499428

1.74

-9.67

100

595075

1.87

-17.89

100

610409

1.81

-14.08

100

542705

1.87

-17.87

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. 5)

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

492359

1.53

1.50

0.04

2.50

Control 2

535389

1.56

Control 3

423947

1.48

Control 4

430603

1.49

Control 5

444037

1.50

Control 6

391290

1.45

100

492359

1.53

1.56

0.03

1.87

-1.97

-4.29

100

550063

1.57

-4.44

100

499428

1.53

-2.29

100

595075

1.59

-6.18

100

610409

1.60

-6.75

100

542705

1.56

-4.14

Validity criteria fulfilled:
yes
Remarks:
1) Biomass in controls increased 90.6x in 72 h (nom. >= 16x). 2) Mean var-coeff. for section-spec. growth rates: 8.3% (nom. ≤ 35%). 3) Var-coeff. of av. spec. growth rates for the whole period in replicate controls: 2.5% (nom. value ≤ 7%).
Conclusions:
Water Accommodated Fractions (WAFs) of NOVARES TL 10 caused no toxic effects on Desmodesmus subspicatus. A 72-h ErL50 and EyL50 of > 100 mg/L and a NOELR of 100 mg/L have been determined.

Description of key information

Toxicity to aquatic algae was investigated in two studies (limit tests) using two different types/technical products of NAF-AO. At the limit concentration of 100 mg/L, no inhibitory effects on algal growth could be observed in either test. No data is available for marine water invertebrates.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Based on differences in the technical production process, different types of NAF-AO can be obtained. Two of this technical products (Novares L 100 and TL 10) were tested in toxicity tests with algae according to OECD TG 202 performed under GLP conditions. Concentrations reported for the aqueous test media relate to loadings.

The aquatic toxicity of NAF-AO is known to be low as observed in aquatic toxicity studies with other organisms. Therefore, the toxicity tests to aquatic algae were performed as limit tests. Both test materials did not adversely affect the growth of algae at the limit test concentration of 100 mg/L (NOELR 100 mg/L).