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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:
25 Augustr 2017 to 08 March 2018
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:
2006, including correction of 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
however, a suitable method of compound specific analysis could not be developed for the relevant concentration range of this study
Details on sampling:
Details on sampling
Duplicate samples were taken from the uninoculated control and each test group at 0 and 72 hours. All samples were stored frozen prior to analysis.Given that a suitable method of compound specific analysis could not be developed, analysis of the test preparations was carried out by TOC analysis.
Vehicle:
no
Details on test solutions:
A nominal amount of test item (32 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 32 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 3.2, 1.0, 0.32, 0.10 and 0.032 mg/L. An aliquot (500 mL) of each of the 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L stock solutions was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
An additional 200 mL of each test concentration containing no algal cells was incubated alongside the test to provide samples for TOC analysis at 0 and 72 hours.
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, 0.032, 0.10 and 0.32 mg/L test cultures were observed to be green dispersions. The 1.0 mg/L test cultures were observed to be light green dispersions whilst the 3.2 mg/L test cultures were extremely pale green almost clear and colorless dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
Not specified
Test temperature:
24 +/- 1 °C. The temperature within the incubator was recorded daily.
pH:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the test guideline.
Dissolved oxygen:
Not specified
Salinity:
Not applicable
Conductivity:
not specified
Nominal and measured concentrations:
nominal test concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L were used in the definitive test
Details on test conditions:
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10e4 to 10e5 cells/mL
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.61 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Solubility / Colour
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, 0.032, 0.10 and 0.32 mg/L test cultures were observed to be green dispersions. The 1.0 mg/L test cultures were observed to be light green dispersions whilst the 3.2 mg/L test cultures were almost clear and colorless dispersions.

Range-finding Tests
Based on the results obtained in the range-finding test, concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the initial experiment.

Initial Experiment
After 72 hours exposure significant inhibition of yield was observed to have occurred at 0.10 mg/L (41%). Furthermore there was not a concentration dose response at the two lowest test concentrati ons employed. Based on this, concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L were selected for the definitive test.

Definitive Test
Total Organic Carbon: TOC analysis of the test preparations at 0 and 72 hours showed measured carbon concentrations of less than the limit of quantification (LOQ), considered to be 1.0 mg C/L. Given that the test item contained 36.59% carbon, at the maximum concentration of 3.2 mg/L, a measured carbon concentration of around the LOQ (1.17 mg/L) was expected. As such the results obtained from analysis do not infer that no test item was present, just that any which was present, was at a level below that which the analytical instrumentation could detect.

Growth data: The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period. The Inhibition of Growth Rate was
ErC10 (0 to 72 hour) : 0.64 mg/L
ErC20 (0 to 72 hour) : 0.93 mg/L
ErC50 (0 to 72 hour) : 1.8 mg/L*
where ErCx is the test concentration that reduced growth rate by x%.
There were no statistically significant differences (P≥0.05), between the control, 0.032, 0.10 and 0.32 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.32 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 1.0 mg/L.

Yield: EyC10 (0 to 72 hour) : 0.15 mg/L
EyC20 (0 to 72 hour) : 0.26 mg/L
EyC50 (0 to 72 hour) : 0.61 mg/L; 95% confidence limits 0.50 to 0.75 mg/L
Where EyCx is the test concentration that reduced yield by x%.
No statistically significant differences (P≥0.05), were detected between the control, 0.032, 0.10 and 0.32 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05). Inspection of the data showed 26% inhibition of yield occurred at 0.32 mg/L which was considered to be significant. As such the NOEC based on yield was 0.10 mg/L. Correspondingly the LOEC based on yield was considered to be 0.32 mg/L.
Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
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
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
The following methods were used: One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Algal cells aggregated at concentrations equal or greater than 0.10 mg/L. Consequently, algal cell density measurements based on cell counts might have been unreliable.

Validity criteria fulfilled:
yes
Conclusions:
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 1.0 - 3.2 mg/L (nominal). The NOEC was determined as 0.32 mg/L (nominal) and the LOEC was determined as 1.0 mg/L (nominal). The 0-72 h yields were significantly different from that of the control group in the examined nominal concentration range of 0.32 – 3.2 mg/L (nominal). The NOEC was determined as 0.10 mg/L (nominal) and the LOEC was determined as 0.32 mg/L (nominal). Aggregated cells were observed to be present in the test cultures at concentrations equal or greater than 0.10 mg/L. Consequently, the algal cell density measurements which were based on cell counts might have been unreliable.
Executive summary:

The effect of the test item on algae was assessed in a GLP and guideline study (OECD testing guideline 201) using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours. A test item specific analytical method could not be develped for the relevant concentration range of this study. Therefore it was considered appropriate to calculate the results based on nominal test concentrations. All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.032 mg/L. However, aggregated cells were observed to be present in the test cultures at 0.10, 0.32, 1.0 and 3.2 mg/ L. The EC50(growth rate) was determined as 1.8 mg/L (95% confidence limits could not be determined because the values did not fit the models available for calculation), the EC50 (yield) was 0.61 mg/L (95% CI 0.50 - 0.75 mg/L). The NOEC (growth rate) was 0.32 mg/L, with the LOEC at 1.0 mg/L; the NOEC (yield) was 0.10 mg/L with the LOEC at 0.32 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
Phaeodactylum tricornutum (P. tricornutum) wild type 646 strain were grown in four different mediums as follows: control group (f/2 medium), CB[6] group (f/2 medium with 3 different concentrations of CB[6], CB[7] group (f/2 medium with 3 different concentrations of CB[7], and CB[8] group (f/2 medium with 2 different concentrations of CB[8]. The cell cultures were grown over 11 days and the concentration of algae cells was determinied by optical density (OD) which was measured at 730 nm by plate reader
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
wild type 646 strain
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
11 d
Test temperature:
room temperature
Nominal and measured concentrations:
Nominal concentrations tested: 100 µM, 200 µKm, 400 µM
Details on test conditions:
Algal cell cultures were grown over 11 days in sterile Nunc Cell Culture Treated EasYFlasks. Suspension cultures were maintained at room temperature and agitated using a rotating shaker at 20 RPM with constant light illumination. Aliquots of 200 µL were removed and pitpetted into Corning 3603 black-sided clear bottomed plates. Optical density measurements were made at 730 nm using a CLARIOstar Microplate Reader.
The f/2 medium was prepared according to Guillard and Ryther (1962).
Reference substance (positive control):
no
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
400 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The NOEC (11 days) of Cucurbit[6]uril towards Phaeodactylum tricornutum was 400 µM (ca. 400 mg/L)
Executive summary:

The growth rate of P. tricornutum in CB[6] added f/2 medium did not change when the concentration of CB[6] varied from 100 µM to 300 µM.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
Phaeodactylum tricornutum (P. tricornutum) wild type 646 strain were grown in four different mediums as follows: control group (f/2 medium), CB[6] group (f/2 medium with 3 different concentrations of CB[6], CB[7] group (f/2 medium with 3 different concentrations of CB[7], and CB[8] group (f/2 medium with 2 different concentrations of CB[8]. The cell cultures were grown over 11 days and the concentration of algae cells was determinied by optical density (OD) which was measured at 730 nm by plate reader
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Phaeodactylum tricornutum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
11 d
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
100 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 400 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
The NOEC (11 days) of CB[7] towards P. tricornutum was at 100 µM. The EC50 was higher than 400 µM.
Executive summary:

CB[7] reduced the growth rate of P. tricornutum in f/2 medium at concentrations of 200 and 400 µM. 100 µM were without effect.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
Phaeodactylum tricornutum (P. tricornutum) wild type 646 strain were grown in four different mediums as follows: control group (f/2 medium), CB[6] group (f/2 medium with 3 different concentrations of CB[6], CB[7] group (f/2 medium with 3 different concentrations of CB[7], and CB[8] group (f/2 medium with 2 different concentrations of CB[8]. The cell cultures were grown over 11 days and the concentration of algae cells was determinied by optical density (OD) which was measured at 730 nm by plate reader
GLP compliance:
no
Test organisms (species):
Phaeodactylum tricornutum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
11 d
Test temperature:
room temperature
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
200 µmol/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
There was no obvious growth rate change of P. tricornutum at 100 and 200 µM in f/2 medium. Undissolved CB[8] was found under the microscope.

Description of key information

Key values are taken from a GLP study performed in accordance with OECD test guideline 201, EU method C3 and US EPA procedure OCSPP 850.5400

Key value for chemical safety assessment

EC50 for freshwater algae:
1.8 mg/L
EC10 or NOEC for freshwater algae:
0.32 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 1.0 to 3.2 mg/L (nominal). The NOEC was determined as 0.32 mg/L (nominal) and the LOEC was determined as 1.0 mg/L (nominal). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 0.32 to 3.2 mg/L (nominal). The NOEC was determined as 0.10 mg/L (nominal) and the LOEC was determined as 0.32 mg/L (nominal).

The EC50 for growth rate was 1.8 mg/L, and for yield 0.61 mg/L. At any concentrations equal or greater 0.10 mg/L chuffed and aggregated cells were observed.

Exposure over 11 days to nominal concentrations of 100 mg/L or higher of CB[6], CB[7] or CB[8] had however no effects on the growth rate of Phaeodactylus tricornutum in an earlier, non-GLP study.