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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:
2012-03-26 to 2012-05-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding studyStability - A sample of each loading rate WAF was taken for analysis at 0 and 72 hours. All samples were stored at approximately -20°C prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.Definitive testSamples taken from control (Replicates R1 - R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. All 0-hour samples were stored at approximately -20°C prior to analysis.Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Validation of mixing periodPre-study investigational work was performed to determine if stirring for a prolonged period produced significantly higher levels of total organic carbon (TOC), as an indicator of soluble organic substances, inthe WAF.A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for 95 hours. After a 1-hour standing period, the mixtures were removed by siphon and samples taken for TOC.Range-finderAmounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give 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. A wide bore glass tube covered at one end with Nesco film 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. The aqueous phase or 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 were performed on the WAFs showed no microdispersions or undissolved test item present.An aliquot (500 mL) of each loading rate WAF was separately inoculated with algal suspension (10 mL) to give the required concentrations of 10 and 100 mg/L loading rate WAFDefinitive testAmounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2, 2 and 2 litres of culture medium to give 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively.The test concentrations were prepared in the same manner as for the range finder.An aliquot (1 L) of each loading rate WAF was separately inoculated with algal suspension (5.7 mL) to give the required concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rates mg/L loading rate WAF
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- 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.- Method of cultivation: periodic replenishment of culture medium. Maintained under constant aeration and illumination at 21°CACCLIMATION- Culturing media and conditions (same as test or not): noPrior to the start of the test sufficient master culture was added t approximately 100 mL volumes of culture media contained in conical flasks to give a cell density of approximately 10E03 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°C until the algal density was approximately 10E04 - 10E05 cells/mL- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not stated
Test temperature:
23-25°C
pH:
7.8 - 8.1
Dissolved oxygen:
Not stated
Salinity:
Not applicable
Nominal and measured concentrations:
Definitive test:Nominal: 1.0, 3.2, 10, 32 and 100 mg/L loading rates
Details on test conditions:
TEST SYSTEM- Test vessel: 250 mL glass conical flasks- Type (delete if not applicable): closed- Aeration: no- Initial cells density: 8.81E05 cells/mL- No. of organisms per vessel: 5 x 10E03 cells/mL- No. of vessels per concentration (replicates): 3- No. of vessels per control (replicates): 6GROWTH MEDIUM- Standard medium used: yesTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: reverse osmosis purified deionised water (Elga Optima 15+)- Culture medium different from test medium: noOTHER TEST CONDITIONS- Adjustment of pH: no (Adjustment of medium prior to test)- Photoperiod: 24 hours- Light intensity and quality: ca. 7000 lux from white lighting (380 - 730 nm)EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: electronic particle counterTEST CONCENTRATIONS- Range finding study- Test concentrations: 10 and 100 mg/L loading rate WAFs- Results used to determine the conditions for the definitive study:yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (0.25, 0.5, 1.0, 2.0 and 4.0 mg/L)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: loading rate WAF
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
92 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: loading rate WAF
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: loading rate WAF
Details on results:
Range finder:The results showed a flat response in terms of inhibition of growth at 10 and 100 mg/L loading rate WAF.Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were chosen for the definitive test.Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations showed measured test concentrations of 0.2 and 0,54 mg/L respectively were obtained at 0 hours whilst concentrations of 0.20 and 0.15 mg/L were obtained at 72 hours.Definitive test:The growth rate and yield were inhibited by the presence of the test item.Growth rate:EL10 (0-72h) = 92 mg/L loading rate WAFEL20 (0-72h) >100 mg/L loading rate WAFEL50 (0-72h) >100 mg/L loading rate WAFStatistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control , 1.0, 3.2 and 10 mg/L loading rate WAFs (P>=0.05), however, all other loading rates were significantly different (P<0.05) and, therefore the No Observed Effect Loading Rate based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on growth rate was 32 mg/L loading rate WAF.Yield:EL10 (0-72h) = 2.6 mg/L loading rate WAFEL20 (0-72h) = 14 mg/L loading rate WAFEL50 (0-72h) >100 mg/L loading rate WAFStatistical analysis was carried out as before. There were no statistically significant differences between the control , 1.0, 3.2 and 10 mg/L loading rate WAFs (P>=0.05), however, all other loading rates were significantly different (P<0.05) and, therefore the No Observed Effect Loading Rate based on yield was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on yield was 32 mg/L loading rate WAF.All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in either the control or test cultures at 1.0 and 3.2 mg/L loading rate WAFs, however cell debris was observed to be present in the test cultures at 10, 32 and 100 mg/L loading rate WAF.ValidationThe cell concentration of the control cultures increased by a factor of 115 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.Mean cell density of control at 0 hours: 4.86E03 cells per mLMean cell density of control at 72 hours: 5.56E05 cells per mLThe mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.The coefficient of variation for average specific growth rate for the control cultures over the test period (0-72h) was 3% and hence satisfied the validation criterion in the OECD Guideline which states that this must not exceed 7%.Physicochemical measurements:Validation of mixing period: There was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.Temperature was maintained at 23 - 25°C throughout the test. The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.1 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 Guidelines.Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, at 1.0 mg/L loading rate WAF to 1.3 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 hours in the range of less than the LOQ at 1.0 mg/L loading rate WAF to 0.72 mg/L at 100 mg/L loading rate WAF. Given that 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.
Results with reference substance (positive control):
- Results with reference substance valid: Yes- EC50:ErC50 (0-72h) = 1.4 mg/L (95% CI = 1.2 - 1.7 mg/L)EyC50 (0-72h) = 0.59 mg/L (95% CI = 0.53 - 0.65 mg/L)- Other:NOEC growth = 0.25 mg/LNOEC yield = 0.25 mg/LLOEC growth = 0.5 mg/LLOEC yield = 0.5 mg/L
Reported statistics and error estimates:
See above.

See attached document

Validity criteria fulfilled:
yes
Conclusions:
The EL50 (72h) based on inhibition of growth rate was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on growth rate was 32 mg/L loading rate WAF.
Executive summary:

Test Guidance

OECD Guideline No 201 and EC Method C.3

Method

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneiella subcapitata was exposed to WAFs of the test item over a range of nominal loadinfrates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results.

The EL50 (72h) based on inhibition of growth rate was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on growth rate was 32 mg/L loading rate WAF.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, at 1.0 mg/L loading rate WAF to 1.3 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 hours in the range of less than the LOQ at 1.0 mg/L loading rate WAF to 0.72 mg/L at 100 mg/L loading rate WAF.

Given that 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.

Description of key information

The EL50 (72h) based on inhibition of growth rate was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate  based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on growth rate was 32 mg/L loading rate WAF (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

Test Guidance

OECD Guideline No 201 and EC Method C.3

Method

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneiella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results.

The EL50 (72h) based on inhibition of growth rate was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate based on growth rate was 10 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate based on growth rate was 32 mg/L loading rate WAF.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, at 1.0 mg/L loading rate WAF to 1.3 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 hours in the range of less than the LOQ at 1.0 mg/L loading rate WAF to 0.72 mg/L at 100 mg/L loading rate WAF.

Given that 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.