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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
Study period:
2018-04-23 to 2018-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 16GWFA037
- Expiration date of the lot/batch: 01.04.2019
- Purity test date: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: as the test item instantly hydrolyses on addition to water and titanium dioxide is produced as one of the degradation products, it was not possible to identify if the titanium measured originated from the parent substance or the degradate, and therefore the results obtained cannot be used to confirm the presence of test item in its parent form and are not stability indicating

- All other template details: Not reported
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control and test concentrations 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L
- Sampling method: Samples were taken from the solvent control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Two additional sample of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An initial solvent stock solution was prepared by dissolving 500 mg of test item in 10 mL of dimethylformamide with the aid of ultrasonication for approximately 5 minutes. A series of dilutions was made from this stock solution to give further solvent stock solutions of 160, 50, 16 and 5.0 mg/10 mL. An aliquot (100 µL) of each of the solvent stock solutions was separately dispersed in 1 liter of culture medium with the aid of magnetic stirring for approximately 10 minutes to give 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L stock solutions.
- Controls: negative control and solvent control (100 µL/L of dimethylformamide).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Other relevant information: Information provided by the Sponsor indicated the test item was not readily soluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Preliminary media preparation trials indicated that a dissolved test item concentration of approximately 3.5 mg/L was obtained from a solvent spike method of preparation indicating this to be the limit of water solubility of this item under test conditions

- All other template details: Not reported
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 (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): not reported
- Method of cultivation: not reported

- All other template details: Not reported
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 ºC
pH:
7.9 ± 0.2
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test concentrations (mg/L): 0.050, 0.16, 0.50, 1.6 and 5.0.
Geometric mean measured test concentrations (mg/L): 0.039, 0.11, 0.39, 1.1 and 3.4.

Despite extensive method development, a suitable substance specific method of analysis for this test item could not be developed. Consequently, verification of exposure concentrations was based on the measured levels of total dissolved titanium in the samples taken during the test, from which the measured levels of the test item were calculated based on its theoretical titanium content. However, as the test item instantly hydrolyses on addition to water and titanium dioxide is produced as one of the degradation products, it was not possible to identify if the titanium measured originated from the parent substance or the degradate, and therefore the results obtained cannot be used to confirm the presence of test item in its parent form and are not stability indicating.
Analysis of the test preparations at 0 hours showed measured titanium concentrations to range from 0.0056 to 0.57 mg Ti/L. Analysis of test preparations after each 24-Hour interval showed measured titanium concentrations to range from 0.0057 to 0.58 mg Ti/L at 24 hours, from 0.0058 to 0.55 mg Ti/L at 48 hours and from 0.0036 to 0.13 mg Ti/L at 72 hours.

The test items theoretical titanium concentration, equivalent test concentrations of 0.041 to 4.2 mg/L, 0.041 to 4.2 mg/L, 0.042 to 4.0 mg/L and 0.026 to 0.93 mg/L were obtained at 0, 24, 48 and 72 hours respectively.
Given the decline in measured test concentrations observed in the 72-Hour test preparations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations (calculated from the test items theoretical titanium concentration) in order to give a “worst case” analysis of the data.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Material, size, headspace, fill volume: 250 mL glass conical flasks filled with 100 mL of test medium
- Initial cells density: 5.0E+3 cells/mL for control and solvent control
- Control end cells density: 6.92E+5 cells/mL for control. 7.40E+5 cells/mL for solvent control
- 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
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the start and after 72 hours
- All other template details: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: The pH in the culture medium was adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: continuous
- Light intensity and quality: approximately 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: coulter counter after 24, 43, 72 hours
- Chlorophyll measurement: not reported

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1st range finding test: 0.50 and 5.0 mg/L. 2nd range finding test: 0.005, 0.050 and 0.50 mg/L.
- Results used to determine the conditions for the definitive study: The results of the initial range-finding test showed significant inhibition of growth occurred at both 0.50 and 5.0 mg/L and so a second range-finding test was conducted with nominal test concentrations of 0.0050, 0.050 and 0.50 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate an ErC50 value as at the limit of water solubility, no more than 26% inhibition of growth rate occurred.
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.84 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities after 72h
- Colour differences: at the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-hour test period all control, solvent control, 0.039 and 0.11 mg/L test cultures were observed to be green dispersions. The 0.39 mg/L test cultures were observed to be pale green dispersions while the 1.1 and 3.4 mg/L test cultures were observed to be extremely pale green dispersions.
- All other template details: Not reported
Results with reference substance (positive control):
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

NOEC growth rate: 0.25 mg/L
NOEC yield: 0.25 mg/L
LOEC growth rate: 0.50 mg/L
LOEC yield: 0.50 mg/L
Reported statistics and error estimates:
Not reported

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

 Response Variable  EC50 (mg/L)

 95% Confidence Limits

(mg/L)

 No Observed Effect Concentration

(NOEC)

(mg/L)

Lowest Observed Effect Concentration

(LOEC)

(mg/L) 

 Growth Rate  >3.4  *  0.39  1.1
 Yield  1.1  * 0.11 0.39

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Validity criteria fulfilled:
yes
Remarks:
Cell concentration of the control and solvent control cultures increased by a factor of 138 and 148 after 72 hours, respectively. The mean coefficient of variation for section for the control and solvent control cultures was 10% and and 12%, respectively
Conclusions:
There were no significant reduction in growth rate between the solvent control, 0.039, 0.11 and 0.39 mg/L test concentrations (P=0.05), however all other test concentrations were significantly different (P<0.05). Inspection of the data showed that while the 0.39 mg/L test group was not statistically significantly different from the solvent control, 19% inhibition of yield occurred which was considered to be significantly different. As such the NOEC based on yield was considered to be 0.11 mg/L. Correspondingly the LOEC based on yield was considered to be 0.39 mg/L.

Description of key information

A study according to OECD 201 is available. A suitable substance specific method of analysis for the test item could not be developed in the study. Consequently, verification of exposure concentrations was based on the measured levels of total dissolved titanium in the samples taken during the test, from which the measured levels of the test item were calculated based on its theoretical titanium content. However, the analytical method applied in the studies is not considered appropriate for the test item, thus the nominal concentrations are used for the chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:
5 mg/L
EC10 or NOEC for freshwater algae:
0.5 mg/L

Additional information