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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:
06 Mar 2017 to 10 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 2006, corrected July 2011
Principles of method if other than guideline:
Organisms were exposed to a Water Accommodated Fraction (WAF) / Water Soluble Fraction (WSF), because the test item is a UVCB and poorly soluble in water. No filtration step was included in the preparation of test solutions to remove the undissolved fracdtion of the test substance.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Fatty acids, C12-14, a-sulfo, disodium salts
- Lot/batch No.: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Arrival: 16 November 2016
- Carbon content: 44.7 %
- Stability: Stable
- Volatility: Not-volatile
- Appearance: Solid beige (powder)
- Expiry date: Oct 2017
- Storage conditions: Room temperature
Analytical monitoring:
yes
Details on sampling:
Four samples of ca. 8 mL were taken from each additional vessel (without test organisms in order to not disturb the TOC-analysis) of treatment E, F, G, H, I, J and NC at 0 h and 72 h for TOC-analysis. Of these 4 samples per treatment and time, 2 were analysed directly after sampling and 2 were stored as retain samples at ≤ -18 °C until finalization of the study.
Vehicle:
no
Details on test solutions:
The test solutions were prepared as Water Accommodated Fractions (WAFs). See 'Any other information on materials and methods incl. tables'. Afterwards, the test solutions were shaken for 24 h using an overhead shaker at room temperature in the dark. The test vessels were prepared with 48 mL of each test solution along with 2 mL algal inoculum suspension. Additional vessels for TOC analysis (NC, E, F, G, H, I and J) were prepared with 78.72 mL test solution along with 3.28 mL test medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Source: Culture Collection of Algae at the University of Goettingen
- Method of cultivation: The strain used for this study has been cultured at Hydrotox GmbH since May 2016. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium axenic conditions to keep it in exponential growth.
- Preparation of pre-cultures and inoculum: Before the start of the test, 1.315 mL of the algae stock suspension was diluted with 48.685 mL OECD TG 201 medium to obtain a concentration of 5E+04 cells/mL. This preculture was incubated for 4 d at 22.3 - 23.0 °C and 90.7 μE·m-2·s-1 ± 5.6 % continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in 104.615E+05 cells/mL. To obtain a nominal inoculum concentration of 1.75E+05 cells/mL, 3.345 mL pre-culture was diluted into 200 mL OECD TG 201 medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
1.63 °dH (mean of 3 measurements)
Test temperature:
22.4 - 22.8 °C
pH:
7.3 - 7.8
Conductivity:
160 - 261 μS/cm
Nominal and measured concentrations:
- Nominal loading levels: 0 (control), 0.1, 0.5, 1.0, 2.2, 5.0, 10, 25, 50, 100 and 220 mg/L (based on previous testing)
- Measured concentrations (calculated C-contents): 0 (control), 2.235, 4.470, 11.175, 22.350, 44.700 and 98.340 mg/L (6 highest concentrations). See 'Any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks, Schott, Mainz
- Fill volume: 100 mL
- Type: closed, sealed with cellulose stoppers
- Initial cells density: 1.75E+05 cells/mL
- Control end cells density: 3.925E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
OECD TG 201 medium according to OECD 201 (2006), Annex 3 (original medium of OECD TG 201, also according to ISO 8692). The following solutions were prepared:
- Solution A1: NH4Cl: 1.5 g; MgCl2 × 6 H2O: 1.2 g; CaCl2 × 2 H2O: 1.8 g; MgSO4 × 7 H2O: 1.5 g; KH2PO4: 0.16 g; dissolved in 1000 mL deionised water
- Solution A2: Fe(III)Cl3 × 6 H2O: 0.0640 g; Na2EDTA × 2 H2O: 0.1000 g; dissolved in 1000 mL deionised water
- Solution A3: H3BO3: 0.185 g; Mn(Cl2) × 4 H2O: 0.415 g; Na2MoO4 × 2 H2O: 0.007 g; ZnCl2: 0.003 g; CoCl2 × 6 H2O: 0.0015 g; CuCl2 × 2 H2O: 0.00001 g; dissolved in 1000 mL deionised water (for ZnCl2, CoCl2 and CuCl2 higher concentrated solutions were prepared and the correspondent volumes used for A3).
- Solution A4: NaHCO3: 50 g; dissolved in 1000 mL deionised water. Solutions A1 - A3 were autoclaved, solution A4 was sterile-filtered (pore diameter 0.22 μm).
- Other: A 10-fold concentrated medium was prepared by using 100 mL of stock solution A1 and 10 mL of stock solutions A2, A3 and A4, filling up to 1000 mL with ultrapure water and aerating for 45 min. The 1-fold concentrated medium was prepared by diluting 700 mL of the 10-fold concentrated medium with 6300 mL ultrapure water.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: 90.7 μE·m-2·s-1 ± 5.6 %

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: The chlorophyll fluorescence was measured in each test vessel after 0 h, 24 h, 48 h and 72 h. Possible disturbance of the fluorescence measurement by slight turbidity caused by the test item was checked by additional measurements.
- Cell morphology: At the end of the test, the appearance of the algae was examined microscopically and noticeable deviations were documented.
- Turbidity: The turbidity of test solution I as well as NC was measured after stirring for 1 h and the turbidity test solutions A - J as well as NC after shaking for 24 h using a spectrophotometer at 400 nm,600 nm and 860 nm (recommended in DIN EN 27027 and ISO 7027). NC (test medium) as blank/zero value was subtracted from the values in the test item treatments.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE FINDING STUDY
A preliminary test without GLP was performed before start of this GLP-study. Nominal loading rates of 1, 10 and 100 mg/L test item were tested and resulted in inhibition of 1.0, -64.3 and 95.5 % for biomass and 0.7, -12.0 and 68.0 % for growth rate, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
116 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CL: 93.59 - 142.28
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
67.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CL: 57.00 - 78.80
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
54.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: CL: 23.63 - 124.66
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
27.69 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: CL: 13.84 - 55.40
Details on results:
- Growth rate/biomass: Growth rate was inhibited from a loading rate of 50 mg/L (8.8%) and increased to 30.5% at 100 mg/L and 97.1% inhibition at 220 mg/L. Inhibition of biomass showed similar results. See 'Any other information on results incl. tables'.
- Morphology: The morphology of the algal cells in the test item treatments A - G (0.1 - 22 mg/L loading) as well as in the control showed no obvious abnormality. In the test item treatments H - J (50 - 220 mg/L loading), isolated point-shaped cells were observed.
- Fluorescence disturbance: As the fluorescence of the mixtures of NC and treatment I showed no relevant deviation from the calculated mean of NC and treatment I individual measurements (mean deviation of 3.5 %, which is lower than the variability between replicates with a mean relative standard deviation of 5.0 %), a disturbance of the fluorescence measurement by turbidity caused by the test item and thereby influence on the test results, is not likely or rather very low and, therefore, negligible.
Results with reference substance (positive control):
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany, Lot no.: MKBF2111V, expiration date: January 2017). The recent quality testing was performed in July 2016 which resulted in an ErC50 of 0.99 mg/L (CL 95 %: 0.89 - 1.10 mg/L) and, therefore, is within the recommended range of 0.6 - 1.03 mg/L. The EbC50 was 0.52 mg/L (CL 95 %: 0.38 - 0.74 mg/L).
Reported statistics and error estimates:
With the statistical software ToxRat 3.2.1 (ToxRat Solutions GmbH, Alsdorf), ELRx and LOELR/NOELR for biomass and growth rate were determined.

Table: Inhibition of biomass

Nominal test item loading rate [mg/L]

Inhibition of biomass [%]

24 h

48 h

72 h

NC

--

--

--

0.1

-15.9

25.7

2.8

0.5

10.7

18.9

-8.9

1

-18.9

-22.4

-58.3

2.2

-30.2

-36.8

-59.7

5

-18.9

-36.8

-74.1

10

-23.0

-35.4

-41.9

25

-24.7

-26.5

-33.0

50

-8.9

29.0

28.6

100

20.2

73.0

79.0

220

69.1

96.5

99.8

Table: Inhibition of growth rate

Nominal test item loading rate [mg/L]

Inhibition of biomass [%]

24 h

48h

72h

NC

--

--

--

0.1

-11.5

8.4

-0.1

0.5

5.9

6.5

-2.0

1

-10.1

-6.0

-9.7

2.2

-13.5

-8.4

-9.0

5

-17.1

-12.9

-14.0

10

-10.4

-8.2

-6.7

25

-10.7

-5.8

-5.1

50

-0.7

12.9

8.8

100

5.4

35.8

30.5

220

59.7

84.3

97.1

ADDITIONAL TEST RESULTS

Growth rate:

- LOELR: 50.00 mg/L

- NOELR: 22.00 mg/L

Biomass:

- LOELR: 50.00 mg/L

- NOELR: 22.00 mg/L

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
05 Jan 2017 to 10 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 2006, corrected July 2011
Principles of method if other than guideline:
Organisms were exposed to a Water Accommodated Fraction (WAF) / Water Soluble Fraction (WSF), because the test item is a UVCB and poorly soluble in water.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Fatty acids, C12-14, a-sulfo, disodium salts
- Lot/batch No.: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Arrival: 16 November 2016
- Carbon content: 44.7 %
- Stability: Stable
- Volatility: Not-volatile
- Appearance: Solid beige (powder)
- Expiry date: Oct 2017
- Storage conditions: Room temperature
Analytical monitoring:
yes
Details on sampling:
Four samples of ca. 5 mL were taken from each additional vessel (without test organisms in order to not disturb the TOC-analysis) of treatment A, B, C, D, E, F and NC at 0 h and 72 h for TOC-analysis. Of these 4 samples per treatment and time, 2 were analysed directly after sampling and 2 were stored as retain samples at ≤ -18 °C until finalization of the study.
Vehicle:
no
Details on test solutions:
The test solutions were prepared as Water Soluble Fractions (WSFs) by adding the appropriate amounts of the test item to the correspondent volume of test medium and shaking for 24 h using an overhead shaker at room temperature in the dark. See 'Any other information on materials and methods incl. tables'. The WSFs were filtered through a fibre glass filter with a retaining range till 0.6 μm. The filter was prepared by rinsing with purified water and preconditioning with ca. 200 mL WSF (which was discarded), to reduce adsorption of the test item. It was prevented from falling dry during the filtration process. The test vessels were prepared with 48 mL of each test solution along with 2 mL algal inoculum suspension. Additional vessels for TOC analysis were prepared with 67.2 mL test solution along with 2.8 mL test medium for each concentration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Source: Culture Collection of Algae at the University of Goettingen
- Method of cultivation: The strain used for this study has been cultured at Hydrotox GmbH since May 2016. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium axenic conditions to keep it in exponential growth.
- Preparation of pre-cultures and inoculum: Before the start of the test, 1.23 mL of the algae stock suspension was diluted with 48.77 mL OECD TG 201 medium to obtain a concentration of 5E+04 cells/mL. This pre-culture was incubated for 4 d at 22.3 - 22.9 °C and 73.2 μE·m-2·s-1 ± 5.6 % continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in 103.368E+05 cells/mL. To obtain a nominal inoculum concentration of 1.75E+05 cells/mL, 2.540 mL pre-culture was diluted into 150 mL OECD TG 201 medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
1.43 °dH (mean of 3 measurements)
Test temperature:
22.4 - 23.2 °C
pH:
7.1 - 7.7
Conductivity:
164.0 - 252 μS/cm
Nominal and measured concentrations:
- Nominal loading levels: 0 (control), 6.26, 12.5, 25, 50, 100 and 200 mg/L (based on range-finding study)
- Measured loading levels (as calculated C-contents): 0 (control), 2.798, 5.588, 11.175, 44.700 and 89.400 mg/L, See 'Any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks, Schott, Mainz
- Fill volume: 100 mL
- Type: closed
- Initial cells density: 1.75E+05 cells/mL
- Control end cells density: 3.925E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
OECD TG 201 medium according to OECD 201 (2006), Annex 3 (original medium of OECD TG 201, also according to ISO 8692). The following solutions were prepared:
- Solution A1: NH4Cl: 1.5 g; MgCl2 × 6 H2O: 1.2 g; CaCl2 × 2 H2O: 1.8 g; MgSO4 × 7 H2O: 1.5 g; KH2PO4: 0.16 g; dissolved in 1000 mL deionised water
- Solution A2: Fe(III)Cl3 × 6 H2O: 0.0640 g; Na2EDTA × 2 H2O: 0.1000 g; dissolved in 1000 mL deionised water
- Solution A3: H3BO3: 0.185 g; Mn(Cl2) × 4 H2O: 0.415 g; Na2MoO4 × 2 H2O: 0.007 g; ZnCl2: 0.003 g; CoCl2 × 6 H2O: 0.0015 g; CuCl2 × 2 H2O: 0.00001 g; dissolved in 1000 mL deionised water (for ZnCl2, CoCl2 and CuCl2 higher concentrated solutions were prepared and the correspondent volumes used for A3).
- Solution A4: NaHCO3: 50 g; dissolved in 1000 mL deionised water. Solutions A1 - A3 were autoclaved, solution A4 was sterile-filtered (pore diameter 0.22 μm).
- Other: A 10-fold concentrated medium was prepared by using 100 mL of stock solution A1 and 10 mL of stock solutions A2, A3 and A4, filling up to 1000 mL with ultrapure water and aerating for 45 min. The 1-fold concentrated medium was prepared by diluting 700 mL of the 10-fold concentrated medium with 6300 mL ultrapure water.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: 90.7 μE·m-2·s-1 ± 5.6 %
- Rotation: test vessels rotated around 2 rpm during incubation

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: The chlorophyll fluorescence was measured in each test vessel after 0 h, 24 h, 48 h and 72 h. Possible disturbance of the fluorescence measurement by slight turbidity caused by the test item was checked by additional measurements.
- Cell morphology: At the end of the test, the appearance of the algae was examined microscopically and noticeable deviations were documented.
- Other, turbidity: The turbidity of test solution I as well as NC was measured after stirring for 1 h and the turbidity test solutions A - J as well as NC after shaking for 24 h using a spectrophotometer at 400 nm, 600 nm and 860 nm (recommended in DIN EN 27027 and ISO 7027). NC (test medium) as blank/zero value was subtracted from the values in the test item treatments.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

RANGE FINDING STUDY
A preliminary test without GLP was performed before start of this GLP-study. Nominal loading rates of 1, 10 and 100 mg/L test item were tested and resulted in inhibition of 1.0, -64.3 and 95.5 % for biomass and 0.7, -12.0 and 68.0 % for growth rate, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Remarks on result:
other: not determined
Remarks:
see 'Any other information on results incl. tables'
Details on results:
- Inhibition of growth rate / yield (biomass): Growth rate inhibition of 8.9% was observed from the lowest test concentration (6.26 mg/L) but decreased to -1.4% at 12.5 mg/L, whereafter it increased to 11.2% at 25 mg/L. After, the growth rate was inhibited in a dose-respondent manner up to 98.1% inhibition at 200 mg/L. Inhibition of yield (biomass) showed similar results. Since no NOELR could be derived the test was repeated, see 'Any other information on results incl. tables'
- Morphology: The morphology of the algal cells in the test item treatments A - C (6.26 – 25 mg loading/L) as well as in the control showed no obvious abnormality. In the test item treatments D – F (50 – 200 mg loading/L), isolated point-shaped cells were observed.
Results with reference substance (positive control):
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany, Lot no.: MKBF2111V, expiration date: January 2017). The recent quality testing was performed in July 2016 which resulted in an ErC50 of 0.99 mg/L (CL 95 %: 0.89 – 1.10 mg/L) and, therefore, is within the recommended range of 0.6 - 1.03 mg/L. The EbC50 was 0.52 mg/L (CL 95 %: 0.38 - 0.74 mg/L).

REASONING FOR LACK OF DATA EVALUATION

No further data evaluation was performed. The collected data was not sufficient to determine adequately verified No Observed Effect Loading Rates (NOELRs). The test was repeated within the scope of a new study under ''worst case''-conditions, i.e., without filtration of the stock and test solutions. The repeat test was conducted using a larger concentration range (10 different loading rates). Herby, a superior grade study is available. The results of both studies are comparable.

Table: Inhibition of biomass and growth rate after 72 h exposure

Nominal test item loading rate [mg/L]

Inhibition at 72 h

Yield [%]

Growth rate [%]

NC

--

--

6.26

33.0

8.9

12.5

-9.9

-1.4

25

35.7

11.2

50

75.5

30.0

100

93.3

55.4

200

99.9

98.1

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'

Description of key information

ErC50 = 116 mg/L (Pseudokirchneriella subcapitata)

ErC10 = 67.02 mg/L (Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
116 mg/L
EC10 or NOEC for freshwater algae:
67.02 mg/L

Additional information

Two algae studies were performed according to OECD 201 with GLP. Since the test item is an UVCB and poorly soluble in water, algae were exposed to Water Accommodated Fractions (WAF) / Water Soluble Fractions (WSF):  Some inconclusive results being not statistically significant, lead to no clear No Observed Effect Loading Rates (NOELRs) in the first study. Therefore, the test was repeated with slight modifications: a) no filtration step was included in the preparation of test solutions to remove the undissolved fraction of the test substance and b) the range of the test concentrations was extended. Especially in the low concentration range between 0.1 - 10 mg/l additional measuring points were integrated. Therefore, the second study can be regarded as “worst case” and was considered as the key study.

In the key study (OECD 201 incl. GLP (BASF SE, 2017)), freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal rates of 0 (control), 0.1, 0.5, 1.0, 2.2, 5.0, 10, 25, 50, 100 and 220 mg/L for 72 hours under static conditions. The measured TOC-concentrations in the test item treatments were 94.1 - 123.2 % of the nominal TOC-concentration at 0 h and 84.7 - 99.1 % after 72 h exposure. All results refer to nominal test item loading rates. The algal biomass and growth rate were determined after 0 h, 24 h, 48 h and 72 h by the parameter chlorophyll fluorescence.

Inhibition of growth rate was observed from approximately 50 mg/L (8.8 %) and increased in a dose-dependent manner up 220 mg/L (97.1%). Biomass was also inhibited from approximately 50 mg/L (28.6%), and increased in a dose-dependent manner up to 99.8% at 220 mg/L. Based on these results, the 72-h ErL50 and 72-h EbL50 were determined at 116 and 54.88 mg/L. The 72-h ErL10 and 72-h EbL10 were determined at 67.02 and 27.69 mg/L, respectively.

 

In the study indicated as supporting (OECD 201 incl. GLP (BASF SE, 2017)), organisms were exposed under similar conditions. Exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal rates of 0 (control), 6.26, 12.5, 25, 50, 100 and 200 mg/L for 72 hours under static conditions. The measured TOC-concentrations in the test item treatments were 83.7 - 102.1 % of the nominal TOC-concentration at 0 h and 54.9 - 88.2 % at 72 h exposure. The algal biomass and growth rate were determined after 0 h, 24 h, 48 h and 72 h by the parameter chlorophyll fluorescence.

Since slight inconsistencies in the growth rate were determined being statistically insignificant, no adequately verified No Observed Effect Loading Rates (NOELRs) could be determined: e.g. growth rate was positively influenced (8 %, statistically insignificant) at 6.26 mg/l and at 12.5 mg/l no influencing effect was observed. In order to clarify, if inconsistencies were caused by artefacts or by effects of the test substance the test was repeated as described previously.