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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:
18 April - 20 May 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Analytics show deviation from initial conc> 20% despite closed vessels. Results based on first 24h. No CoA, pH > 1.5 units difference during test.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
yes
Remarks:
Only effect concentration at 24h were derived due to technical impossibilities and lack of detectable levels of test substance at 72h
Principles of method if other than guideline:
Closed system was used according to the recommendations of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000) and the International Standard ISO 14442 (1999)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 0550609421036
- Expiration date of the lot/batch: December 2009
- Purity test date: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In closed containers at room temperature. Preferred storage temperature: between 15-25 °C. (At RCC: At room temperature at about 20 °C, away from direct sunlight.)
- Stability under test conditions: not provided

Aggregate state / physical form at room temperature: Liquid
Color: Colorless
Vapor pressure: 3 Pa (at 20 °C); 50.8 Pa (at 25 °C)
Density: 825 kg/m3 (at 20 °C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
at the start and at the end of the study period (t=0h and t=72h).

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled.
Additionally, one flask of test medium of the dilution 1:10 was incubated without algae under test conditions in order to document possible adsorption of the test item onto the algae.
Immediately after sampling, the samples were diluted 1:1 (v/v) with acetonitrile in order to stabilize the samples during the storage period.
All samples were stored at about -20 °C until analysis. In pre-experiments (non-GLP), the test item proved to be stable in the test water under these storage conditions.
The concentrations of the test item Methyl nonyl ketone were determined in the duplicate test medium samples from the dilutions of 1:32, 1:10, 1:3.2 and the undiluted filtrate. The samples from the dilution of 1:100 were not analyzed, since this concentration was below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Due to the limited solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared by mixing 109.2 mg of the test item into 1090 mL of test water using intense stirring. The stirring vessel was completely filled and sealed by a glass stopper. The dispersion was stirred for 24 hours at room temperature in the dark in order to dissolve a maximum amount of the test item in the dispersion.
After the stirring period, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of the test item during filtration. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test.

Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing with modifications according to the International Standard ISO 14442 [2]. The modifications were made to improve the growth conditions for the algae in a closed test system.
The concentration of NaHCO3 in the test water was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer were added to keep the pH in the test media during the test period as constant as possible.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata, (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany). The algae were cultivated in RCC’s laboratories under standardized conditions according to the test guidelines

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Only effect concentration at 24h were derived due to technical impossibilities and lack of detectable levels of test substance at 72h

Test conditions

Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 23-24 °C and illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The mean measured light intensity at the level of the test solutions was approximately 7700 Lux (range: 7100 to 8100 Lux, measured at nine places in the experimental area).
pH:
At the start of the test, the pH measured in the treatments was 8.2. At the end of the test, pH values of 8.1 to 10.2 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their fast growth in the closed test system
Nominal and measured concentrations:
A saturated solution (filtrate) and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the saturated solution were tested. Additionally, a control was tested in parallel (test water without test item).
The enlarged spacing factor of 3.2 between the test concentrations was chosen, as the concentration-effect relationship was flat according to the results of the range-finding test. Thus, a wide concentration range had to be tested.
Details on test conditions:
50-mL Erlenmeyer flasks were used per replicate. Each test flask was filled with approximately 60 mL algal suspension and sealed with a glass stopper to prevent loss of the volatile test item. The test flasks were labeled with the RCC study number and all necessary additional information to ensure unique identification. During the test, the test solutions were continuously stirred by magnetic stirrers.

The test design included three replicates per test concentration and six replicates of the control.
The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM). The initial cell density corresponded to 1.1 x 103 relative fluorescence units. The conversion factor between algal cell density and fluorescence signal was therefore 9.0 cells/mL per fluorescence unit.
A static test design in a closed test system was applied. The duration of the test was 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 1.8-2.0
Key result
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 0.72-0.86
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI 1.2-1.6
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Biological results:
During the test period of 72 hours, the highest inhibitory effect on the growth of the algae was determined during the first day of the test. A lag phase was determined in the algal cultures exposed to the test item. During the second and third day of the test, a recovery of the algal growth was determined in the test item treatments with the exception of the highest test concentration in which the algal growth was completely inhibited. The lag phase in the exposed algal cultures may indicate recovery after initial toxic stress or reduced exposure due to loss of the test item (although the test was performed in a closed test system).
Therefore, the algal growth inhibition determined during the first day of the test was taken into account for the evaluation of the study and the biological results were related to the initial measured concentrations of the test item in the test media.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the dilution of 1:3.2 and the algal cells in the control. The test item did not affect the shape and size of the algal cells up to at least this concentration.

Analytical results:
The average concentrations found in the unaged treatment samples from day 0 were 0.384 mg/L (Dilution 1:32), 1.41 mg/L (Dilution 1:10), 3.62 mg/L (Dilution 1:3.2) and 12.0 mg/L (Undiluted filtrate). The concentrations in the aged treatment samples from day 3 could not be reliably determined.

Validity criteria:
In the control the biomass increased by a factor of 242 over 72 hours (>16). The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, during 72 hours was 17%. (< 35%). The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.3%. (< 7%).
Results with reference substance (positive control):
The result of the latest positive control test performed in 2008 showed a sensitivity within the historical range of the RCC laboratory.(72-hour EC50 for the growth rate: 1.20 mg/L (RCC Study No. B83755), range of the 72-hour EC50 for the growth rate from 2000 to 2008: 0.71–1.74 mg/L).
Reported statistics and error estimates:
The EC10, EC20 and EC50 values for the inhibition of growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis. For the determination of the LOEC and NOEC, growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see details on results. The concentration could not be kept >= 80% of initial during the course of the study
Conclusions:
Based on the first day of the study, the NOEC for biomass and growth rate was determined to be 0.38 mg/L and the EC50 for the inhibition of the growth rate and biomass were 1.9 mg/L and 1.4 mg/L, respectively. The ErC10 was determined at 0.79 mg/L.
This study is considered acceptable although it does not fulfil the validity criteria which states the concentration of test substance should be 80% of initial
concentration during the test.
Executive summary:

The influence of the test item Methyl nonyl ketone on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the EU Commission Directive 92/69/EEC, C.3 (1992). As the test item is a volatile substance, the test was performed in a closed test system.

The following treatments were tested in parallel to a control: Dilution 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate (saturated solution).

At the start of the test, the analytically determined concentrations of the test item in the test media were 0.38 mg/L (dilution 1:32), 1.4 mg/L (dilution 1:10), 3.6 mg/L (dilution 1:3.2) and 12 mg/L (undiluted filtrate). At the end of the test, the concentrations of the test item in the test media could not be determined.

Based on the first day of the study, the NOEC for biomass and growth rate was determined to be 0.38 mg/L and the EC50 for the inhibition of the growth rate and biomass were 1.9 mg/L and 1.4 mg/L, respectively. The ErC10 was determined at 0.79 mg/L.