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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:
Between 11 March 2010 and 26 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.
GLP compliance:
yes (incl. certificate)
Remarks:
Date for Inspection: 15/09/2009 Date for signature: 26/11/2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Lanolin Fatty Acids
Description: brown waxy solid
Batch number: 0000371886
Date received: 8 February 2010
Expiry date: 21 September 2010
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
100 mg/l loading rate WAF test group

- Sampling method:
Samples
were taken from the uninoculated control and 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I 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. 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 em 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 was removed by middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.

- Eluate:
Not applicable
- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
No

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

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 10E3 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 deg C until the algal cell density was approximately 10E4 - 10E5 cells/ml.

ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Any deformed or abnormal cells observed:
None recorded.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not recorded.
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
7.3 - 7.8
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
Dissolved oxygen:
Not recorded.
Salinity:
freshwater used
Nominal and measured concentrations:
Range-finding Test: 10 and 100 mg/l (nominal)

Definitive Test: 100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 100ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: 10E3 cells/ml

- Control end cells density: algal cell density was approximately 6.24 10E5 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 10E4- 10E5 cells/ml.

- No. of vessels per concentration (replicates):
two replicate flasks per concentration.

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes
For the purpose of the definitive test, the test material was dissolved directly in culture medium. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

TEST MEDIUM
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/I 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. 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 em 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 was removed by middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/I loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (12.5 ml) to give the required test concentration of 100 rng/l loading rate WAF.

Total Organic Carbon (TOC) analysis was performed on the uninoculated test preparations at 0 and 72 hours.



OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and three flasks each completely filled were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.21 x 10E5 cells per ml. Inoculation of 1 litre of test medium with 11 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were sealed with ground glass stoppers 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 taken at 0, 24, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 


- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS

- Range finding study:
10 and 100 mg/l loading rate WAF.

-Definitive Test
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 100 mg/l.

The control group was maintained under identical conditions but not exposed to the test material.

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
• The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and yield
Remarks on result:
other: 95% CL not stated
Details on results:
- Exponential growth in the control (for algal test): yes

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test material solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour settlement period the WAF was observed to have formed a clear colourless media column with large lumps of test item floating at the media surface.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

- Any stimulation of growth found in any treatment:
None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None recorded

Growth rate Data
The growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/I.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*20(0 - 72 h)           : >100 mg/l loading rate WAF
ErL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where ErL*xis the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/I loading rate WAF test group using a Student's t-test 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). There were no statistically significant differences (P ≥0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/iloading rate WAF.

* EL =Effective Loading Rate

Inhibition of yield
EyL*10(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*20(0 - 72 h)           : >100 mg/l loading rate WAF
EyL*50(0 - 72 h)           : >100 mg/l loading rate WAF

where EyL*xis the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as indicated above. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P ≥0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/iloading rate WAF.
Results with reference substance (positive control):
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:

ErC50 (0 - 72 h) : 0.49* mg/l
EyC50 (0 - 72 h) : 0.18 mg/I, 95% confidence limits 0.16 - 0.21 mg/I

No Observed Effect Concentration (NOEC) based on growth rate : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l

Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal range for this reference material.

* 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.
Reported statistics and error estimates:
A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/I loading rate 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).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

* EL = Effective Loading Rate
Executive summary:

Introduction. A study was performed to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) 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. Exposure of Desmodesmus subspicatus to the test item gave EL*50 values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Therefore, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

* EL =Effective Loading Rate