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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study meets criteria for Klimisch score 2 because it generally follows an internationally recognised method (EPA OTS 797.1050) and was carried out to Good Laboratory Practice. However, there was no analytical confirmation of the test concentrations. In addition, only 2 replicates per concentration were used and test vessels containing 1000 mg/l nominal WAF loadings were slightly cloudy at the start of the test and at 24 hours.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts
SOURCE: benzenesulfonic acid, C14-24 branched/linear alkyl derivative, calcium salt / benzenesulfonic acid, C14-24 branched/linear alkyl derivative, calcium salt
3. CATEGORY APPROACH JUSTIFICATION

Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
Remarks:
None reported
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Total organic carbon (TOC) measurements of initial (0 hours) high (1000 mg/L), low (100 mg/L) and control test solutions and at test termination (96 hours).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The 100, 300 and 1000 mg/L nominal loadings were prepared using sterile enriched algal growth media. Individual water accommodation fractions were prepared by stirring the vessel for 24 hours with a Teflon coated magnetic stir bar, which was settled for 1 hour, and removing the water phase for the toxicity test.
- Concentration of vehicle in test medium: 0, 100, 300 and 1000 mg/l nominal WAF loading rates
- Evidence of undissolved material: Test vessels containing 1000 mg/l nominal WAF loadings were slightly cloudy at the start of the test and at 24 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Cells taken from a log-growth phase in-house culture originally purchased from University of Texas at Austin alga collection


ACCLIMATION
- Acclimation period: At least 14 days prior to test
- Culturing media and conditions: Sterile enriched algal growth media
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
24.0°C at 0 hours, 23.9°C at 24 hours, 23.5°C at 48 hours, 23.3°C at 72 hours and 23.5°C at 96 hours.
pH:
Refer to Table 1
Dissolved oxygen:
Not reported
Salinity:
Not relevant
Nominal and measured concentrations:
Control, 100, 300 and 1000 mg/L WAF.

TOC levels were 2-8 mg/L in the control, 2-4 mg/L at 100 mg/L WAF, 2-3 mg/L at 300 mg/L WAF and 2-3 mg/L at 1000 mg/L WAF loading. TOC levels were not considered to be indicative of actual test material concentrations and results are therefore based on nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL stoppered with foam plugs to reduce the entry of dust randomly placed on an orbital shaker to approximately 100 cycles per minute
- Renewal rate of test solution: Solution was not renewed
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Two

GROWTH MEDIUM: Sterile enriched alga media adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Adjusted to pH 7.5
- Photoperiod: Constant light (24 hours per day)
- Light intensity: 47-50 µEin/m²second

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Visual determination by means of direct microscope examination with a hemocytometer

TEST CONCENTRATIONS
- Test concentrations: Control, 100, 300 and 1000 mg/L WAF
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Remarks on result:
other: 95% Fiducial Limits: 100-300 mg/L, calculated using the number of algal cells/ml
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
150 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Remarks on result:
other: 95% Fiducial Limits: 100-300, calculated using the average specific growth rate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Details on results:
The control response was satisfactory.

At 72-hours, cell density measurements were 80%, 62% and 70% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, cell density measurements were 70%, 66% and 88% of the control at 100, 300 and 1000 mg/L WAF, respectively.

At 72-hours, specific growth rate measurements were 95%, 90% and 92% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 93%, 92% and 97% of the control at 100, 300 and 1000 mg/L WAF, respectively.


- Exponential growth in the control: yes
- Observation of abnormalities:
- Unusual cell shape: No abnormalities
- Colour differences: No abnormalities
- Flocculation: No abnormalities
- Adherence to test vessels: No abnormalities
- Aggregation of algal cells: No abnormalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test vessels containing 1000 mg/l nominal WAF loadings were slightly cloudy at the start of the test and at 24 hours
Reported statistics and error estimates:
A parametric one-way analysis of variance (ANOVA) and Dunnett's test were used to calculate the no-observed effect level when data were normally distributed and a Kruskal-Wallis test was used when data were not normally distributed.

Table 2. Selenastrum capricornutum cell growth data

Concentration (mg/l)

Replicate

Number of cells/ml x103

0 hours

24 hours

48 hours

72 hours

96 hours

Control

1

10

18

123

826

1555

2

10

21

145

1008

1866

Mean

10

20

134

917

1711

% Control

-

-

-

-

-

100

1

10

15

18

680

1250

2

10

18

133

790

1145

Mean

10

17

126

735

1198

% Control

100

85

94

80

70

300

1

10

<10

120

506

1125

2

10

<10

107

638

1120

Mean

10

<10

114

572

1122

% Control

100

<50

85

62

66

1000

1

10

<10

132

654

1610

2

10

<10

125

632

1385

Mean

10

<10

129

643

1498

% Control

100

<50

96

70

88

Table 3. Average specific growth rate and percentage change from the control of Selenastrum capricornutum

Concentration

Average specific growth rate

Percentage change from the control

24 hours

48 hours

72 hours

96 hours

24 hours

48 hours

72 hours

96 hours

0 (control)

0.028

0.054

0.063

0.054

-

-

-

-

100

0.021

0.053

0.060

0.050

25

3

5

7

300

0.000

0.051

0.056

0.049

100

6

10

8

1000

0.000

0.053

0.058

0.052

100

2

8

3

Validity criteria fulfilled:
yes
Conclusions:
The test substance was not toxic to freshwater algae at loading rates up to and including 1000 mg/L nominal WAF
Executive summary:

In a 96-hour acute toxicity study, the cultures of Selenastrum capricornutum (now Pseudokirchneriella subcapitata) were exposed to test substance (115733-09-0) at nominal concentrations of 100, 300 and 1000 mg/l as WAF loadings under static conditions in accordance with the EPA OTS 797.1050. 

 

Daily cell counts were made visually by means of direct microscopic examination with a hemocytometer.

 

This study is classified as reliable with restrictions (Klimisch Code 2) and satisfies the guideline requirements for a toxicity study to aquatic algae and cyanobacteria.

  

Results Synopsis

 

Test Organism: Selenastrum capricornutum (now Pseudokirchneriella subcapitata)

Test Type: Static

 

At 72-hours cell density measurements in the treatments were 80%, 62% and 70% of the control at 100, 300 and 1000 mg/L, respectively.

At 96-hours cell density measurements in the treatments were 70%, 66% and 88% of the control at 100, 300 and 1000 mg/L, respectively.

At 72-hours, specific growth rate measurements were 95%, 90% and 92% of the control at 100, 300 and 1000 mg/L WAF, respectively.

At 96-hours, specific growth rate measurements were 93%, 92% and 97% of the control at 100, 300 and 1000 mg/L WAF, respectively.

72-hour and 96-hour Effective Loading Rate (EL50) = >1000 mg/L nominal WAF.

72-hour and 96-hour No Observed Effect Levels = 1000 mg/L nominal WAF based on both specific growth rate and cell density.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study meets criteria for Klimisch code 2 because it generally followed an internationally recognised method (EPA OTS 797.1050) and was carried out to Good Laboratory Practice. However, there was no analytical confirmation of the test concentrations. In addition, only two replicates per concentration were used and there was evidence of undissolved test material at all concentrations.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts
SOURCE: benzenesulfonic acid, mono C15-30 branched alkyl and di C11-13 branched and linear alkyl derivatives / benzenesulfonic acid, mono C15-30 branched alkyl and di C11-13 branched and linear alkyl derivatives..
3. CATEGORY APPROACH JUSTIFICATION

Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
Remarks:
None reported
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Total organic carbon (TOC) measurements of initial (0 hours) high (1000 mg/L), low (100 mg/L) and control test solutions and at test termination (96 hours).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The 100, 300 and 1000 mg/L nominal loadings were prepared using sterile enriched algal growth media. Individual water accommodation fractions were prepared by stirring the vessel for 24 hours with a Teflon coated magnetic stir bar, which was settled for 1 hour, and removing the water phase for the toxicity test.
- Concentration of vehicle in test medium: 0, 100, 300 and 1000 mg/l nominal WAF loading rates
- Evidence of undissolved material: Testing vessels containing 100 mg/l nominal WAF loading rates appeared cloudy at 24 and 48 hours. Test vessels containing 300 and 1000 mg/l nominal WAF loadings were cloudy throughout the entire test. Insoluble test material was observed to adhere to the test vessels at 300 and 1000 mg/l nominal WAF loading rates att 48, 72 and 96 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Cells taken from a log-growth phase in-house culture originally purchased from University of Texas at Austin alga collection


ACCLIMATION
- Acclimation period: At least 14 days prior to test
- Culturing media and conditions: Sterile enriched algal growth media
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
24.0°C at 0 hours, 24.2°C at 24 hours, 24.1°C at 48 hours, 24.0°C at 72 hours and 24.1°C at 96 hours
pH:
Refer to Table 1
Dissolved oxygen:
Not reported
Salinity:
Not relevant
Nominal and measured concentrations:
Control, 100, 300 and 1000 mg/L WAF.

TOC levels were non-detected (<1) to 3 mg/L in the control, 1-2 mg/L at 100 mg/L WAF, and 5-6 mg/L at 1000 mg/L WAF loading. TOC levels were not considered to be indicative of actual test material concentrations and results are therefore based on nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL stoppered with foam plugs to reduce the entry of dust randomly placed on an orbital shaker to approximately 100 cycles per minute
- Renewal rate of test solution: Solution was not renewed
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Two

GROWTH MEDIUM: Sterile enriched alga media adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Adjusted to pH 7.5
- Photoperiod: Constant light (24 hours per day)
- Light intensity: 47-50 µEin/m²second

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Visual determination by means of direct microscope examination with a hemocytometer

TEST CONCENTRATIONS
- Test concentrations: Control, 100, 300 and 1000 mg/L WAF
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
457 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Remarks on result:
other: 95% Fiducial Limits: 300-1000 mg/l
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Details on results:
The control response was satisfactory.

At 72-hours, cell density measurements were 133%, 75% and 64% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, cell density measurements were 93%, 77% and 52% of the control at 100, 300 and 1000 mg/L WAF, respectively.

At 72-hours, specific growth rate measurements were 108%, 92% and 88% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 98%, 95% and 86% of the control at 100, 300 and 1000 mg/L WAF, respectively

- Exponential growth in the control: yes
- Observation of abnormalities:
- Unusual cell shape: No abnormalities
- Colour differences: No abnormalities
- Flocculation: No abnormalities
- Adherence to test vessels: No abnormalities
- Aggregation of algal cells: No abnormalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Testing vessels containing 100 mg/l nominal WAF loading rates appeared cloudy at 24 and 48 hours. Test vessels containing 300 and 1000 mg/l nominal WAF loadings were cloudy throughout the entire test. Insoluble test material was observed to adhere to the test vessels at 300 and 1000 mg/l nominal WAF loading rates att 48, 72 and 96 hours
Reported statistics and error estimates:
A parametric one-way analysis of variance (ANOVA) and Dunnett's test were used to calculate the no-observed effect level when data were normally distributed and a Kruskal-Wallis test was used when data were not normally distributed.

Binomial non-linear interpolation methods were used to calculate EL50s.

Table 2. Selenastrum capricornutum cell growth data

Concentration (mg/l)

Replicate

Number of cells/ml x103

0 hours

24 hours

48 hours

72 hours

96 hours

Control

1

10

12

118

352

1412

2

10

20

128

388

1146

Mean

10

16

123

370

1279

% Control

-

-

-

-

-

100

1

10

14

196

398

1206

2

10

16

198

586

1166

Mean

10

15

197

492

1186

% Control

100

94

160

133

93

300

1

10

12

116

274

996

2

10

18

132

248

968

Mean

10

15

124

246

982

% Control

100

94

101

75

77

1000

1

10

10

48

226

540

2

10

<10

92

246

784

Mean

10

<10

70

236

662

% Control

100

<63

57

64

52

Table 3. Average specific growth rate and percentage change from the control of Selenastrum capricornutum

Concentration (mg/l)

Average specific growth rate

Percentage change from control

24 hours

48 hours

72 hours

96 hours

24 hours

48 hours

72 hours

96 hours

0 (Control)

0.020

0.052

0.050

0.050

-

-

-

-

100

0.017

0.062

0.054

0.050

14

-19

-8

2

300

0.017

0.052

0.046

0.048

14

0

8

5

1000

0.000

0.041

0.044

0.044

100

22

12

14

Validity criteria fulfilled:
yes
Conclusions:
The test substance was not toxic to freshwater algae at loading rates up to and including 1000 mg/L nominal WAF.
Executive summary:

In a 96 hour acute toxicity study, the cultures of Selenastrum capricornutum (now Pseudokirchneriella subcapitata) were exposed to test substance (71486-79-8) at nominal concentrations of 100, 300 and 1000 mg/L as WAF loadings under static conditions in accordance with the EPA OTS 797.1050.

Daily cell counts were made visually by means of direct microscopic examination with a hemocytometer.

This study is classified as reliable with restrictions (Klimisch Code 2) and satisfies the guideline requirements for a toxicity study to aquatic algae and cyanobacteria.

Results Synopsis

 

Test Organism: Selenastrum capricornutum (now Pseudokirchneriella subcapitata)

Test Type: Static

 

At 72-hours cell density measurements in the treatments were 133%, 75% and 64% of the control at 100, 300 and 1000 mg/L, respectively.

At 96-hours cell density measurements in the treatments were 93%, 77% and 52% of the control at 100, 300 and 1000 mg/L, respectively.

At 72-hours, specific growth rate measurements were 108%, 92% and 88% of the control at 100, 300 and 1000 mg/L WAF, respectively.

At 96-hours, specific growth rate measurements were 98%, 95% and 86% of the control at 100, 300 and 1000 mg/L WAF, respectively

 

72-hour and 96-hour Effective Loading Rate (EL50) = >1000 mg/L

72-hour and 96-hour No Observed Effect Concentrations = 1000 mg/L based on both specific growth rate and cell density

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study meets criteria for Klimisch code 2 because it followed an internationally recognised method (EPA OTS 797.1050) and was carried out to Good Laboratory Practice. However, there was no analytical confirmation of the test concentrations. In addition, evidence of undissolved test material was noted at all test concentrations.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts
SOURCE: benzenesulfonic acid, mono and dialkyl derivatives, magnesium salt
3. CATEGORY APPROACH JUSTIFICATION

Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
Remarks:
None reported
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Total organic carbon (TOC) measurements of initial (0 hours) high (1500 mg/L), low (125 mg/L) and control test solutions and at test termination (96 hours).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The 125, 250, 500, 1000 and 1500 mg/L nominal loadings were prepared using sterile enriched algal growth media. Individual water accommodation fractions were prepared by stirring the vessel for 24 hours with a Teflon coated magnetic stir bar, which was settled for 1 hour, and removing the water phase for the toxicity test.
- Concentration of vehicle in test medium: 0, 125, 250, 500, 1000 and 1500 mg/l nominal WAF loading rates
- Evidence of undissolved material: All non-control test vessels were cloudy throughout the test
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Cells taken from a log-growth phase in-house culture originally purchased from University of Texas at Austin alga collection


ACCLIMATION
- Acclimation period: At least 14 days prior to test
- Culturing media and conditions: Sterile enriched algal growth media
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
24°C
pH:
Refer to Table 1
Dissolved oxygen:
Not reported
Salinity:
Not relevant
Nominal and measured concentrations:
Control, 100, 300 and 1000 mg/L WAF.

TOC levels were non-detected (<1) to 1 mg/L in the control, 2-3 mg/L at 125 mg/L WAF, and 5-6 mg/L at 1500 mg/L WAF loading. TOC levels were not considered to be indicative of actual test material concentrations and results are therefore based on nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL stoppered with foam plugs to reduce the entry of dust randomly placed on an orbital shaker to approximately 100 cycles per minute
- Renewal rate of test solution: Solution was not renewed
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): Three

GROWTH MEDIUM: Sterile enriched alga media adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were <1.0 and <10 mg/L, respectively.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Adjusted to pH 7.5
- Photoperiod: Constant light (24 hours per day)
- Light intensity: 47-50 µEin/m²second

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Visual determination by means of direct microscope examination with a hemocytometer

TEST CONCENTRATIONS
- Test concentrations: Control, 125, 250, 500, 1000 and 1000 mg/L WAF
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
590 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Remarks on result:
other: 95% Fiducial Limits: 250-1000 mg/l
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
530 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Remarks on result:
other: 95% Fiducial Limits: 250-1000 mg/l
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
1 300 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Remarks on result:
other: 95% Fiducial Limits: 1000-1500 mg/l
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 500 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1 400 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Remarks on result:
other: 95% Fiducial Limits: 1000-1500 mg/l
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 500 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
1 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
cell number
Remarks on result:
other: Fiducial Limits: 500-1500 mg/l
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 1 500 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rates
Basis for effect:
growth rate
Details on results:
At 72-hours, cell density measurements were 97%, 100%, 101%, 95% and 35% of the controls at 125, 250, 500, 1000 and 1500 mg/l, respectively. At 96-hours, cell density measurements were 78%, 61%, 62%, 58% and 31% of the controls at 125, 250, 500, 1000 and 1500 mg/l, respectively. Logarithmic growth was observed at all treatments up to and including 1000 mg/L; i.e. average cell density measurements 1383-1860 x10E3 cells/mL. Therefore, the 96-hour NOELs were determined to be 1000 mg/L, although statistical analysis determined the NOELs to be 125 mg/L. However, the hypothesis test was biased towards the unusually high control growth (2383 x10E3 cells/mL) between 72-96 hours upon which test concentration growth was compared. This produced an erroneous measurement of the NOEL.

At 72-hours, specific growth rate measurements were 100%, 100%, 100%, 98% and 75% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 95%, 91%, 91%, 89% and 72% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively.

- Exponential growth in the control: yes
- Observation of abnormalities:
- Unusual cell shape: No abnormalities
- Colour differences: No abnormalities
- Flocculation: No abnormalities
- Adherence to test vessels: No abnormalities
- Aggregation of algal cells: No abnormalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All non-control test vessels were cloudy throughout the test
Reported statistics and error estimates:
A parametric one-way analysis of variance (ANOVA) and Dunnett's test were used to calculate the no-observed effect level when data were normally distributed and a Kruskal-Wallis test was used when data were not normally distributed.

Binomial non-linear interpolation methods were used to calculate EL50s.

Table 2. Selenastrum capricornutum cell growth data

Concentration (mg/l)

Replicate

Number of cells/ml x103

0 hours

24 hours

48 hours

72 hours

96 hours

0 (Control)

1

10

32

170

320

1690

2

10

38

290

940

2640

3

10

38

300

1000

2820

Mean

10

36

253

753

2383

% Control

-

-

-

-

-

125

1

10

38

220

820

1670

2

10

28

210

640

1950

3

10

34

150

720

1960

Mean

10

33

193

727

1860

% Control

100

92

76

97

78

250

1

10

22

230

700

1250

2

10

24

240

830

1540

3

10

20

200

730

1460

Mean

10

22

223

753

1450

% Control

100

61

88

100

61

500

1

10

16

160

770

1550

2

10

16

200

730

1650

3

10

28

180

770

1250

Mean

10

20

180

757

1483

% Control

100

56

71

101

62

1000

1

10

14

260

660

1390

2

10

<10

200

760

1540

3

10

<10

240

720

1220

Mean

10

<11

233

713

1383

% Control

100

<31

92

95

58

1500

1

10

14

80

300

740

2

10

16

50

160

660

3

10

<10

40

320

820

Mean

10

<13

57

260

740

% Control

100

<36

23

35

31

Table 3. Average specific growth rate and percentage change from the control of Selenastrum capricornutum

Concentration (mg/l)

Average specific growth rate

Percentage change from control

24 hours

48 hours

72 hours

96 hours

24 hours

48 hours

72 hours

96 hours

0 (Control)

0.053

0.067

0.060

0.057

-

-

-

-

125

0.050

0.062

0.060

0.054

6

7

0

5

250

0.033

0.065

0.060

0.052

38

3

0

9

500

0.029

0.060

0.060

0.052

45

10

0

9

1000

0.004

0.066

0.059

0.051

92

1

2

11

1500

0.011

0.036

0.045

0.045

79

46

25

21

Validity criteria fulfilled:
yes
Conclusions:
The test substance was not toxic to freshwater algae at loading rates up to and including 1000 mg/L nominal WAF
Executive summary:

In a 96 hour acute toxicity study, the cultures of Selenastrum capricornutum (now Pseudokirchneriella subcapitata) were exposed to test substance (71486-47-5) at nominal concentrations of 125, 250, 500, 1000 and 1500 mg/L as WAF loadings under static conditions in accordance with the EPA OTS 797.1050. 

 

Daily cell counts were made visually by means of direct microscopic examination with a hemocytometer.

 

 This study is classified as reliable with restriction (Klimisch Code 2) and satisfies the guideline requirements for a toxicity study to aquatic algae and cyanobacteria.

  

Results Synopsis

 

Test Organism: Selenastrum capricornutum (now Pseudokirchneriella subcapitata)

Test Type: Static

 

At 72-hours cell density measurements in the treatments were 97%, 100%, 101%, 95% and 25% of the control at 125, 250, 500, 1000 and 1500 mg/L respectively. 

At 96-hours cell density measurements in the treatments were 78%, 61%, 62%, 58% and 31% of the control at 125, 250, 500, 1000 and 1500 mg/L respectively.

At 72-hours, specific growth rate measurements were 100%, 100%, 100%, 98% and 75% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively.

At 96 -hours, specific growth rate measurements were 95%, 91%, 91%, 89% and 72% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively

 

72-hour Effective Loading Rate (EL50) = 1400 mg/L and >1500 mg/L based on cell density and specific growth rate, respectively.

 

96-hour Effective Loading Rate (EL50) = 1100 mg/L and >1500 mg/L based on cell density and specific growth rate, respectively.

 

72-hour and 96-hour No Observed Effect Loading Rates (NOELR) = 1000 mg/l based on both specific growth rate and cell density.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study meets criteria for Klimisch score 2 because it follows and internationally recognised method (OECD 201) and was carried out to Good Laboratory Practice. However, there was no analytical confirmation of the test concentrations.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE CATEGORY APPROACH
The read across follows Scenario 5 - Qualitatively and quantitatively similar effects are caused by a common compound, which is formed from all category members (as described in the 2017 Read-Across Assessment Framework document).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
TARGET: Benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts
SOURCE: Sulphonic acids, petroleum, calcium salts
3. CATEGORY APPROACH JUSTIFICATION

Linear and non-linear or branched alkylbenzene sulfonates are anionic surfactants with molecules characterized by a hydrophobic (apolar) and a hydrophilic (polar) group. As a group of chemicals, they are generally mixtures of closely related isomers and homologues. Each molecule contains an aromatic ring sulfonated at the para position and attached to either a linear or a branched alkyl chain at any position except the terminal carbons. The sulfonate group is a common functional group present in each of the category members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. The cation components of the chemicals (e.g. calcium, magnesium, sodium, or barium) are not expected to contribute significantly to the toxicity.
4. DATA MATRIX
See Read Across document attached to CSR
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations: 0 and 100 mg/l nominal WAF loading rate
Vehicle:
no
Details on test solutions:
PREPARATION OF CULTURE MEDIUM
Refer to Table 1.


PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 250 mg of test material was added to the surface of 2.5 litres of culture medium to give a 100 mg/l loading rate. The test material was stirred by a magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. Stirring was conducted for 24 hours. The mixture was then allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF.
- Evidence of undissolved material: Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland


ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions: Cultures were maintained in the laboratory by periodic replenishment of culture media described in Table 1 at a temperature of 20 ± 1°C under constant illumination (intensity of approximately 7000 lux) and constant aeration.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
24 ± 1°C
pH:
Refer to Table 2
Dissolved oxygen:
Not reported
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass conical flask containing 100 ml of test preparation, plugged with polyurethane foam bungs and incubated in an INFORS Multitron(R) Version 2 Incubator and constantly shaken at approximately 150 rpm
- Renewal rate of test solution: Test material was not renewed
- Initial cells density: 100000 cells/ml
- Control end cells density: 383000 cells/ml
- No. of vessels per concentration (replicates): Six (Two in the range-finding study)
- No. of vessels per control (replicates): Three (Two in the range-finding study)


GROWTH MEDIUM
- Detailed composition if non-standard medium was used: Refer to Table 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis deionised water
- Total organic carbon: Not reported


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Culture media was adjusted to pH 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity: Approximately 7000 lux


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Coulter(R) Multisizer Particle Counter


TEST CONCENTRATIONS
- Test concentrations: 0 and 100 mg/l nominal WAF loading rate
- Range finding study
- Test concentrations: 0, 10 and 100 mg/l nominal WAF loading rate
- Results used to determine the conditions for the definitive study: No effects on growth were noted at 10 and 100 mg/l nominal WAF loading rates
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities were detected in any of the control or test cultures at 72 hours
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and end of the mixing period and after the 1 hour settlement period, the 100 mg/l WAF loading rate was observed to have formed a clear, colourless media column with test material floating at the media surface. Microscopic examination of the WAF showed there to be no globules/micro-dispersions of the test material. At the start of the test, all control and test cultures were observed to be clear, colourless solutions. After the 72-hour test period, all control and test cultures were observed to be green dispersions.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was conducted using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There were no statistical differences (P >=0.05) between the control and 100 mg/l WAF loading rate. Therefore, the NOEL was 100 mg/l nominal WAF loading rate.

Table 2: Effects of sulphonic acids, petroleum, calcium salts on growth of Scenedesmus subspicatus

Nominal loading rate (mg/l)

Replicate

pH

Cell density (cells/ml)

AUC at 72 hours

% Inhibition

Growth rate (0-72 hours)

% Inhibition

0 hours

72 hours

0 hours

24 hours

48 hours

72 hours

Control

1

7.2

7.6

9.46 x103

5.76 x104

1.77 x105

3.76 x105

9.48 x106

-

0.051

-

2

7.2

7.6

9.89 x103

5.76 x104

1.90 x105

3.92 x105

3

7.2

7.6

9.49 x103

5.31 x104

1.47 x105

3.82 x105

Mean

-

-

9.61 x103

5.61 x104

1.71 x105

3.83 x105

100

1

7.2

7.6

9.97 x103

5.96 x104

2.28 x105

4.14 x105

1.19 x107

[26]

0.052

[2]

2

7.2

7.6

1.02 x104

6.62 x104

2.30 x105

3.97 x105

3

7.2

7.6

9.80 x103

7.06 x104

2.22 x105

4.95 x105

4

7.2

7.6

1.10 x104

7.55 x104

2.31 x105

4.77 x105

5

7.2

7.6

1.03 x104

7.95 x104

2.33 x105

4.43 x105

6

7.2

7.6

9.78 x103

7.29 x104

2.33 x105

4.38 x105

Mean

-

-

1.02 x104

7.07 x104

2.29 x105

4.44 x105

AUC: Area under the curve

[Increase in growth compared to the control]

Comparison of areas under the growth curves

The area under the curve is taken to be an index of growth and was calculated using the following formula:

A=N1- N0x t1+N1+ N2– 2N0x (t2- t1) +Nn-1 + Nn – 2N0x (tn– tn-1)

         2                       2                                           2

where

A = area

N0= cell concentration at the start of the test

N1= cell concentration at t1

Nn= cell concentration at tn

t1= time of first measurement (hours from start)

tn= time at nth measurement (hours from start)

Percentage inhibition of growth at each loading rate (IA) was calculated by comparing the area under the test curve (At) with that under the control curve (Ac) using the following equation:

IA =Ac- Atx 100

         Ac

Comparison of growth rates

The average maximum growth rate (µ) for each culture was calculated from the straight section of the growth curve using the following equation:

µ =lnNn- lnN1

         tn- t1

where

µ = average maximum growth rate

N0= cell concentration at the start of test

Nn= cell concentration at tn

t0= time at first measurement (0 hours)

tn= time of nth measurement (hours from the start)

Executive summary:

In a 72 hour toxicity study, the cultures of the green algae (Scenedesmus subspicatus CCAP 276/20) were exposed to sulphonic acids, petroleum, calcium salts at nominal concentrations of 0 and 100 mg/l nominal WAF loading rate under static in accordance with the guideline OECD Guideline 201 (Alga, Growth Inhibition Test). The EL50based biomass was >100 mg/l and the EL50based on specific growth rate was >100 mg/l. There were no compound related phytotoxic effects.

 

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

 

Results Synopsis

 

Test Organism: Scenedesmus subspicatus CCAP 276/20

Test Type: Static

 

72 hr NOELR (biomass and specific growth rate): 100 mg/l WAF loading rate

72 hr EC50(biomass): >100 mg/l WAF loading rate

72 hr EC50(growth rate): >100 mg/l WAF loading rate

Description of key information

Aquatic algae studies performed according to EPA OTS 797.1050 or OECD TG 201 on source substances are available for read across purposes. In each of these studies, the 96 hour Effective Loading Rate was equivalent to the highest concentrations tested.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Aquatic algae studies on read across source substances were performed according to EPA OTS 797.1050 or OECD TG 201. In each of these studies, the 96 hour Effective Loading Rate was equivalent to the highest concentrations tested. A series of 96-hour acute toxicity algal studies were performed on several substances using cultures of Selenastrum capricornutum (now Pseudokirchneriella subcapitata) (Ward, 1994). The first algal exposure was to test substance CASN 115733-09-0 at nominal concentrations of 100, 300 and 1000 mg/L as WAF loadings under static conditions in accordance with the EPA OTS 797.1050.  Daily cell counts were made visually by means of direct microscopic examination with a hemocytometer. At 72-hours cell density measurements in the treatments were 80%, 62% and 70% of the control at 100, 300 and 1000 mg/L, respectively. At 96-hours cell density measurements in the treatments were 70%, 66% and 88% of the control at 100, 300 and 1000 mg/L, respectively. At 72-hours, specific growth rate measurements were 95%, 90% and 92% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 93%, 92% and 97% of the control at 100, 300 and 1000 mg/L WAF, respectively. The 72-hour and 96-hour Effective Loading Rate (EL50) was >1000 mg/L nominal WAF. The 72-hour and 96-hour No Observed Effect Levels (NOEL) was 1000 mg/L nominal WAF based on both specific growth rate and cell density.

The next substance tested in the same study was CAS 71486-79-8 (Ward, 1994). Nominal concentrations of 100, 300 and 1000 mg/L as WAF loadings under static conditions were performed on green algae in accordance with the EPA OTS 797.1050. At 72-hours cell density measurements in the treatments were 133%, 75% and 64% of the control at 100, 300 and 1000 mg/L, respectively. At 96-hours cell density measurements in the treatments were 93%, 77% and 52% of the control at 100, 300 and 1000 mg/L, respectively. At 72-hours, specific growth rate measurements were 108%, 92% and 88% of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 98%, 95% and 86% of the control at 100, 300 and 1000 mg/L WAF, respectively. The 72-hour and 96-hour Effective Loading Rate (EL50) was >1000 mg/L and the 72-hour and 96-hour No Observed Effect Concentrations (NOEL) was 1000 mg/L based on both specific growth rate and cell density.

The final substance used in the study by Ward (1994) was CASN 71486-47-5. Green algae were dosed at nominal concentrations of 125, 250, 500, 1000 and 1500 mg/L as WAF loadings under static conditions in accordance with the EPA OTS 797.1050. At 72-hours cell density measurements in the treatments were 97%, 100%, 101%, 95% and 25% of the control at 125, 250, 500, 1000 and 1500 mg/L respectively. At 96-hours cell density measurements in the treatments were 78%, 61%, 62%, 58% and 31% of the control at 125, 250, 500, 1000 and 1500 mg/L respectively. At 72-hours, specific growth rate measurements were 100%, 100%, 100%, 98% and 75% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively. At 96-hours, specific growth rate measurements were 95%, 91%, 91%, 89% and 72% of the control at 125, 250, 500, 1000 and 1500 mg/L WAF, respectively. The 72-hour Effective Loading Rate (EL50) was 1400 mg/L and >1500 mg/L based on cell density and specific growth rate, respectively. The 96-hour Effective Loading Rate (EL50) = 1100 mg/L and >1500 mg/L based on cell density and specific growth rate, respectively. The 72-hour and 96-hour No Observed Effect Loading Rates (NOEL) were 1000 mg/l based on both specific growth rate and cell density.

In a 72 hour toxicity study, the cultures of the green algae (Scenedesmus subspicatus) were exposed to sulphonic acids, petroleum, calcium salts at nominal concentrations of 0 and 100 mg/L nominal WAF loading rate under static in accordance with the guideline OECD Guideline 201 (Alga, Growth Inhibition Test) (Mead, 2005). The 72 hour EC50 based on biomass was >100 mg/L WAF loading rate and the EC50 based on specific growth rate was >100 mg/L WAF loading rate. There was no compound related phytotoxic effects. The 72-hour NOEL (biomass and specific growth rate) was 100 mg/L WAF loading rate.