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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 May 2017 to 25 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
OECD Guideline 201 with GLP certificate

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Undecanolactone
EC Number:
254-399-3
EC Name:
Undecanolactone
Cas Number:
39282-36-5
Molecular formula:
C11H20O2
IUPAC Name:
undecanolactone
Specific details on test material used for the study:
CAS: 321735-36-8
Name: Meadowfoam lactone
Formula: Modified natural fatty acids (complex nature)
Density: 0.92 g/cubic cm
State: White waxy solid
Solubility: Insoluble in water, soluble in acetone, chloroform and hexane
Batch: 1383Q12001
Purity: not provided
Expiry date: 30 August 2017
Storage: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using Coulter Multisizer particle counter. Chemical analysis of the test preparations were made at 0 and 72 hours.

RANGE-FINDING TEST
Due to the low aqueous solubility and complex nature of the test item, the study medium was prepared as a water accommodated fraction (WAF) of the test item. Preliminary investigational work confirmed that stirring for a prolonged period did not produce significantly higher levels of dissolved test item in the WAF.

A range-finding test was undertaken to confirm loading rate for the definitive test.

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (8.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

DEFINITIVE TEST
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed. Samples were taken from the control and 100 mg/l loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.





Test solutions

Vehicle:
no
Details on test solutions:
A nominal amount of test item (200 mg) was added to a glass slide and suspended within 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. After filtration through a glass wool plug the aqueous phase was also passed through a sheet of filter paper to ensure as much undissolved test item was removed as possible. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test item present.

An aliquot (1 liter) of the 100 mg/L loading rate WAF was inoculated with algal suspension (8.4 mL) to give the required test concentration of 100 mg/L loading rate WAF. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms

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 in the laboratory under constant aeration and illumination at 21 ± 2 °C. 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 (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
no data
Test temperature:
24 +/- 1 degrees C
pH:
7.5
Dissolved oxygen:
no data
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
0 and 100 mg/l loading rates were used in the definitive test. Samples were analysed at 0 and 72 hours. Measured concentrations were below the limit of quantification (LOQ) of 0.12 mg/l. The laboratory reports that this does not infer that no test item was in solution, but that any dissolved test item was at a concentration of less than the LOQ. Given that the toxicity cannot be atributed to a single component or mixture of components, but to the test item as a whole, the results are reported based on nominal loading rates.
Details on test conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.98 x 105 cells per mL. Inoculation of 1 liter of test medium with 8.4 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were stored frozen prior to analysis.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate was used as the reference item at concentrations of 0.25, 0.5, 1, 2 and 4 mg/l.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Remarks:
Growth endpoint
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: water accommodated fraction
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: water accommodated fraction
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: water accommodated fraction
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: water accommodated fraction
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: water accommodated fraction
Results with reference substance (positive control):
ErC50 (0 to 72 hr) = 1.4 mg/l with 95% confidence limits of 1.2 to 1.5 mg/l
EyC50 (0 to 72 hr) = 0.6 mg/l with 95% confidence limits of 0.52 to 0.69 mg/l
Reported statistics and error estimates:
There were no statistically significant differences between the control and the 100 mg/l loading rate WAF samples.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

A study was performed to assess the effect of the test substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed the OECD Guideline No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.12 mg/l. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of >100 mg/l loading rate WAF. The No Observed Effect Loading Rate was 100 mg/l loading rate WAF.