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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:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The test has been conducted according to OECD guidelines, but not in compliance with GLP. No measurements of test concentrations or dissolved oxygen were performed. The publication does not give sufficient experimental details in order to derive the results and to check whether the validity criteria are fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Principles of method if other than guideline:
No measurements of test concentrations or dissolved oxygen were performed.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIES-35
- Source (laboratory, culture collection): Independent Administrative Institution, National Institute for Environmental Studies, Japan
- Age of inoculum (at test initiation): no data
- Method of cultivation: under passage breeding, aseptically


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None.
Hardness:
No data
Test temperature:
23 ± 2ºC
pH:
No data.
Dissolved oxygen:
No data.
Salinity:
Not applicable.
Nominal and measured concentrations:
No data.
Details on test conditions:
TEST SYSTEM
- Test vessel: no data
- Initial cells density: ca. 1x E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS: OECD recommended medium

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: contineous
- Light intensity and quality: ca 4000 lux


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- 0, 5.5, 10, 18, 32, 56, and 100 mg/L
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The growth inhibition was 10.7% (growth rate) at the highest concentration 100 mg/L.
Results with reference substance (positive control):
No data.
Reported statistics and error estimates:
No data.
Validity criteria fulfilled:
not specified
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 12, 2000 - December 10, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At the start of exposure, 2 mL of the test solution was collected from 3 testing containers into 3 respective vials.
At the end of the exposure, 5 mL of the test solution was collected again from the 3 testing containers and centrfuged. Approx. 2.0 mL of the supernatant liquid was skimmed and was again collected into 3 respective vials. Methanol was added in an equal amount, and subsequently mounted on an LC-MS autosampler.
Vehicle:
no
Details on test solutions:
Test material was dissolved with a culture medium to prepare a 100 mg/L solution. It was sterilized by filtration to prepare the test material stock solution. The stock solution was added to a culture medium sterilized by filtration to prepare a test solution. The test solution was clear and colourless with no observed precipitation.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: ATCC-22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): an algae cultured for 3 days prior to the start of exposure.
- Method of cultivation: aseptically passage breeding


ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none, microscopically confirmed
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None.
Hardness:
Not measured.
Test temperature:
23 ± 2 ºC
pH:
7.2 - 7.8
Dissolved oxygen:
Not measured.
Salinity:
Not applicable.
Nominal and measured concentrations:
The ratio of the measurement to the set concentration (0.16 - 10 mg/L) was 86.1 - 97.5% at the beginning of the test and 68.3 - 83.1% at 72 hours. Although at 72 hrs, the actual measurements were only within ± 20% of the highest set concentrations, at these concentrations no significant effects were observed. Therefore, it is assumed valid to use nominal values to calculate the test results.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 300 mL glass triangular flask (with perforated silicon cap)
- Aeration:
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: measured using a Coulter Counter
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4000 - 5000 lux


TEST CONCENTRATIONS
- Ratio used for test concentrations: 2.3 (0.16, 0.36, 0.82, 1.89, 4.35 and 10 mg/L)
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: not mentioned in the report
- Results used to determine the conditions for the definitive study: not mentioned in the report
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The maximum Inhibition rate of 18.07% was observed at the highest concentration, 10 mg/L by biomass method, however EC50's could not be derived in this test by both biomass method and growth rate method.
At all concentrations, the inhibition rates were not significant different to the control, so LOEC's could not be derived.
Results with reference substance (positive control):
The growth inhibition concentration (EbC50) for 72 hr exposure was 0.41 mg/L
Reported statistics and error estimates:
Data analysis: regression analysis for EC50, Dunnett multiple comparison test, Bartlett test and ANOVA for NOEC.
Method of calculating mean measured concentrations: Geometric mean
Validity criteria fulfilled:
yes
Conclusions:
ECr50 (24-48h and 24-72h) > 10 mg/L
NOECr (24-48h and 24-72h) >= 10 mg/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October, 2008 to June , 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study has been performed according to OECD guidelines, but not in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
no
Remarks:
Since the study was not conducted under GLP, it has a klimisch 2 score for reliability.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of test material in the test solution was determined at the start (0hour) and termination (72hour) of exposure.
Newly prepared test solution was sampled from preparation vessel. The test solutions for analysis at termination (72hour) of exposure were mixed for six vessels set each concentration respectively.

- Sampling method:
- Sample storage conditions before analysis:
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test material standard stock solution of 8000 mg/L was prepared. The weighed test material (purity:98.7%):161.2mg was dissolved in N,N-dimethyl formamide by 20mL of volumetric flask. The 10 μL of standard stock solution was added to 100 mL of test water to prepare the 0.80 mg/L of test solution.
- Controls: Solvent control was prepared to make same concentration of DMF solvent in test solution as 0.100 mL/L (10 μL DMF/100 mL test water).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethyl formamide
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokircheneriella subcapitata (Selenastrum capricornutum)
- Strain: strain NIES-35
- Source (laboratory, culture collection): supplied from National Institute for Environmental Study, Japan.
- Age of inoculum (at test initiation): no data
- Method of cultivation: aseptically under passage - breeding


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None.
Hardness:
No data.
Test temperature:
23 ±2 ºC
pH:
8.3 - 8.6
Dissolved oxygen:
Not determined.
Salinity:
Not applicable.
Nominal and measured concentrations:
The test material concentration in test solution was determined at the start and 72 hours after of exposure. The test material concentration was 0.71mg/L (nominal concentration: 0.80 mg/L) at the start of exposure, the ratio to nominal concentration was 89%. And the test material concentration was 0.69mg/L (nominal concentration: 0.80mg/L) at the terminal of exposure, the ratio to nominal concentration was 86%. As the measured values of test material concentration in test solution was within ±20% of the nominal concentration, the nominal concentration was used for calculation of the test result.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL conical flask (glass) attached with a porous silicon plug
- Initial cells density: 1.00×E4 cell/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS: A medium prescribed in the OECD Guidelines for Testing of Chemicals was prepared and used for test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4000 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter
- Chlorophyll measurement: no
- Other:


TEST CONCENTRATIONS
The maximum soluble concentration 0.8 mg/L was applied as test concentration in this limit test, according to preliminary test which concentration did not inhibit the growth.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): The cell concentration for the control proliferated to 210 fold at 72 hours of culture, showed the appropriate growth more than 16 fold was observed, so this test was confirmed as acceptable.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
The two-sided test was applied to comparison with the control test because of the homoscedasticity was observed for the examination of mean growth rate (0-72hr) by Bartlett analysis of variance.
Validity criteria fulfilled:
yes
Conclusions:
The growth inhibition concentration ErC50(0-72hr) derived from growth inhibition rate was more than 0.8mg/L.
No significant difference was observable at the maximum soluble concentration 0.8 mg/L compared with that of the control, so NOECr (0-72hr) was determined as >= 0.8mg/L.

Description of key information

Data on main components:

C16MES: 72h ECr50 > 10 mg/L nominal (measured values within +/- 20% of nominal)

C16MES: 72hr NOECr >= 10 mg/L nominal (measured values within +/- 20% of nominal)

Key value for chemical safety assessment

Additional information

The chemical safety assessment of both components will be performed separately.

Key value for C16MES: ECr50 > 10 mg/L; NOEC >= 10 mg/L

Supporting data from similar substance with shorter chain length:

C14MES: 72h EC50 > 100 mg/L nominal (not measured)

C14MES: 72h NOEC = 56 mg/L nominal (not measured)

The combination toxicology cannot be used to determine one single key value for chemical safety assessment of the reaction mass - indeed, the proportion of both components that enter the environment (after the STP) is not deemed to be the same as the proportion of the 2 components before the STP