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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
yes
Remarks:
Light intensity range and temperature were outside the recommended range. However, all necessary validity criteria were met and therefore it was accepted that this deviation had no effect on the outcome of the test or the integrity of the results.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A0076


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15±10°C), protected from light.
- Stability under test conditions: Stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: Duplicate samples were taken at the start and end of the 72 hour exposure period. Samples were taken from remaining test media after filling test vessels for 0 hours and pooled replicate flasks for 72 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The measured amounts of test substance were added directly to dilution water. The solutions were stirred for 22 hours and 25 minutes and settled for 4 hours 15 minutes.
- Eluate: After settling the first 100ml (approximately) of aqueous phase was removed (avoiding all settled and floating material) and discarded after filtering through a Whatman 54 filter.

- Controls: Six control flasks containing sterilised deionised water with added nutrients according to the OECD 201 standard.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test concentrations remained clear and colourless at 24 and 48 hours and were all green except for 100 mg/l which was slightly green.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, OBAN, Argyll PA37 1QA, Scotland, United Kingdom
- Age of inoculum (at test initiation): From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 10 4
cells/ml.
- Method of cultivation: Temperature: 20.9 – 21.2oC, Illumination: 6080 - 7960 lux continuous white light, Orbiting: set to 200rpm.
Culture media: Deionised water with added nutrients according
to OECD 201

ACCLIMATION
- Acclimation period: form 20th February to 10th April 2018
- Culturing media and conditions (same as test or not): the same as test
- Any deformed or abnormal cells observed: all cells
within the control and all test concentrations appeared normal, no morphological abnormalities were observed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
20.9 – 24.7°C
pH:
7.14 – 8.11
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 0, 6.25, 12.5, 25, 50 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml conical flask
- Material, size, headspace, fill volume: Flasks were filled with 100 ml
- Aeration: None reported
- Initial cells density: 1 x 10 4 cells/ml
- Control end cells density: 49.2 x 10 4 cells/ml
- No. of organisms per vessel: Approximately 1 x 10 4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterilised deionised water with added nutrients according to the OECD 201 standard
- Culture medium different from test medium: no
- Intervals of water quality measurement: The temperature and light intensity (lux) within the incubator was recorded at the beginning of the study, after 24, 48 hours and at the end of the 72 hour test period. The pH and temperature were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-hour test period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continouous
- Light intensity and quality: 6030 - 7960 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:Cell densities were measured using a haemocytometer and microscope at 24, 48 and 72 hours (+- 2 h).
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes. Data from the preliminary test identified the 72-hour EC50 as being >100mg/l (by growth rate) and between 10-100mg/l (by yield).
Reference substance (positive control):
yes
Remarks:
potassium dichromate, tested in a separate study (16 - 19 January 2017)
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
33.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 6.6-49.1 mg/L
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
114 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 87.1- 256 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
12.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 3.8-19.4 mg/L
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
46.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 34.3 - 65.3 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Other: When observed visually, the control conical flasks appeared clear and colourless after 24 and 48 hours and green after 72 hours. All test concentrations remained clear and colourless at 24 and 48 hours and were all green except for 100mg/l which was slightly green.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.06 mg/L
- Other: ISO 8692:2012 quotes the 72-hour ErC50 as 1.19mg/l (SD =
0.27), the result obtained in the reference test should be
in the range 0.65 and 1.73mg/l (mean value ± 2 x standard deviation).
Reported statistics and error estimates:
Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EL50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”.

Table 1. Percent inhibition

Nominal concertation (mg/L)

Percent inhibition by growth rate

Percent inhibition by yield

0-48 h

0-72 h

0-48 h

0-72 h

6.25

1

1

2

5

12.5

2

5

3

16

25

18

8

41

25

50

60

16

82

48

100

39

45

68

84

Table 2. Concentration of azelaic acid determined by HPLC analysis

Nominal concentration (mg/L)

Concentration of azelaic acid (mg/L)

Concentrations of Azelaic Acid (mg/L) at the

following sampling times

Percentage recoveries of Azelaic Acid at the

following sampling times

0 hrs

72 hrs

0 hrs

72 hrs

6.25

2.97

5.235

5.022

175.934

168.766

12.5

5.95

6.964

5.775

117.012

97.041

25

11.9

12.724

11.662

106.905

97.983

50

23.8

25.458

22.005

106.945

92.439

100

47.6

52.939

48.934

111.192

102.781

  Table 3. Concentration of lithium determined by ICP-MS analysis

Nominal concentration (mg/L)

Nominal concentration of lithium (mg/L)

Concentrations of lithium (mg/L) at the

following sampling times

 Percentage recoveries of Lithium (%)

0 hrs

48 hrs

Mean measured

0 hrs

48 hrs

Mean measured

6.25

0.26

0.202

0.207

0.205

77.785

79.587

78.686

12.5

0.52

0.365

0.46

0.412

70.127

88.446

79.286

25

1.0

0.731

0.807

0.769

73.133

80.734

76.934

50

2.1

1.589

1.645

1.617

75.659

78.312

76.985

100

4.2

2.768

3.507

3.137

65.906

83.494

74.700

 

Validity criteria fulfilled:
yes
Remarks:
The biomass in the control increased by a factor of 49.2 (should be at least 16). The variation coefficient for the 72h period was 4.18% (must not exceed 7%) and for each time period was 16.71% (must not exceed 35%).
Conclusions:
Based on nominal concetrations, the 72 hour EL(r)50 and EL(y)50 were 114 mg/L and 46.4 mg/L, respectively. The 0 to 72-hour NOELR(r) was 25 mg/L and the 0-72 hour LOELR was 50 mg/L.
Executive summary:

A study was performed to assess the effects of the test item, The reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide,

on the growth of the green alga Pseudokirchneriella subcapitata (Crane 2019). The method followed was designed to be compatible with the OECD guideline 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Pseudokirchneriella subcapitata were exposed to a control and five test concentrations for 72 hours, under constant illumination and shaking. Samples of the algae were removed daily and cell concentrations determined for each control and treatment group, using a a haemocytometer and microscope.

All validity criteria for the definitive test were met. The test concentrations were confirmed by analysis of azelaic acid and lithium in the test solutions at 0 and 72 hours. The results indicate that the measured concentrations of azelaic acid remained within 80-120% of nominal except 6.25 mg/L test concentration which had a mean recovery of 172.350%. This is considered to be due to this concentration being close to the limit of quantification. For lithium, the results indicate that recovered concentrations of lithium were within 65.906- 88.446% of nominal.

The results were based on nominal concentrations only.

Based on nominal concetrations, the 72 hour EL(r)50 and EL(y)50 were 114 mg/L and 46.4 mg/L, respectively. The 0 to 72-hour NOELR(r) was 25 mg/L and the 0-72 hour LOELR was 50 mg/L.

The study was conducted according to the OECD guideline and is GLP-compliant therefore is reliable without restirctions and is considered as suitable as a key study for this endpoint.

Description of key information

Based on nominal concentrations, the 72 hour EL(r)50 and EL(y)50 to the green algae, P. subcapitata, were 114 mg/L and 46.4 mg/L respectively. The 0 to 72-hour NOELR(r) was 25 mg/L and the 0-72 hour LOELR was 50 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
114 mg/L
EC10 or NOEC for freshwater algae:
33.1 mg/L

Additional information

A study was performed according to the OECD guideline 201 to assess the effects of the test item, the reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide,

on the growth of the green alga Pseudokirchneriella subcapitata (Crane 2019).

All validity criteria for the definitive test were met. The test concentrations were confirmed by analysis of azelaic acid and lithium in the test solutions at 0 and 72 hours.

Based on nominal concetrations, the 72 hour EL(r)50 and EL(y)50 were 114 mg/L and 46.4 mg/L, respectively. The 0 to 72-hour NOELR(r) was 25 mg/L and the 0-72 hour LOELR was 50 mg/L.

The study was conducted according to the OECD guideline and is GLP-compliant therefore is reliable without restirctions and is considered as suitable as a key study for this endpoint.