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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:
May - November 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed under the TG201 guidance of 1984. The study does not provide a description how and if validity criteria from that guideline were met. Water solubility and vapor pressure have not been properly indicated. The pH deviation was not discussed as to relevance for the final results. The results based on final measured concentrations are not fully accurate, however acceptable for worst case. No CoA provided.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
pH increases > 1 unit
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: 0, 1.0, 2.2, 4.4, 9.6, 21, and 46 mg/L
- Sampling method: Composite samples of each test concentration were drawn by mixing identical volumes of the test solutions taken from the test vessels. They were taken immediately before exposure and after 72 hours exposure.
- Sample storage conditions before analysis: Samples were kept at -18°C to -22°C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 50.4 mg test substance were mixed and made up to 1000 in mL with water to prepare the stock solution.
Calculated amounts of the stock solution were added to the test media to produce the nominal concentrations
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source: Collection of algae cultures, Pflanzenphysiologisches Institut University, Nikolausberger Weg 18, D-3400 Gottingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:According to the OECD TG 201

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no deformed or abnormal cells reported.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
24 ± 1 °C
pH:
t = 0h 7.8
t = 72h 8.1 - 9.8
Nominal and measured concentrations:
Nominal: 0, 1.0, 2.2, 4.4, 9.6, 21, and 46 mg/L
Measured t= 0h: <0.1, 1.0, 1.7, 2.8, 7.8, 12.7, 42.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL erlenmeyer flasks
- Type: closed, cotton stoppers
- Fill volume: 50 mL test solution per flask
- Aeration: on lab-shaker
- Initial cells density: 9400 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to the OECD 201 guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the OECD 201 guideline
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: not reported
- Photoperiod: Continues illumination
- Light intensity and quality: Cold white fluorescent light, 120 µE/m2 sec (approx. 800 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell densities were measured at 24, 48 and 72 hours exposure on a "TOA" cell counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. factor 2
- Range finding study: not reported
- Test concentrations:
Nominal: 0, 1.0, 2.2, 4.4, 9.6, 21, and 46 mg/L.

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated result
Remarks:
95% confidence limit 6.3 - 9.0 mg/L, Slope 1.631
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated result (5% level)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated value (5% level)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: calculated value
Remarks:
95% confidence limit 2.6 - 4.0 mg/L, slope: 3.101
Details on results:
- Exponential growth in the control: yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance was homogeneously distributed in the test vessels at all test times and test concentrations except at test conc. 46 mg/L (nominal) where a small deposit was observed at the end of exposure.

The measured test substance concentrations at the end of exposure were 22 - 36% of the nominal concentrations, therefore the measured end concentrations were taken for calculation the EbC/ErC50.
measured conc 72h: < 0.1, <0.1, 0.8, 1.0, 2.1, 4.9, 13.9 mg/L

The pH increase exceeds the range specified in the guideline. The exceedence occurs in the concentration levels with the best growth and is considered a consequence of this growth, not affecting the conclusions of the study.
Reported statistics and error estimates:
the ECx values were calculated according to the maximum likelihood method, logit model (Mc Cullagh P. & Nelder, J.A, 1983)

Inhibition related to growth rate:

 measured conc (mg/L)

 

replicate

1

 

replicate

2

 growth rates *0.001 (u)

replicate

3

 

replicate

4

 

replicate

5

 

replicate

6

 Mean (u)  Inhibition% (0 -72h)

 blank

 76  78  78  77  77  77  77  -

 <0.1

 77  71  75        74  3.5

 0.8

 77  77  78        78  0

 1.0

 72  74  75        74  4.2

 2.1

 75  76  76        76  1.7

 4.9

 43  45  42        43  43.7

 13.9

 30  25  23        26  66.5
Validity criteria fulfilled:
not specified
Remarks:
however the exponential growth can be concluded (> 16x cell density increase). No conclusion as to 2006 validity criteria possible.
Conclusions:
Under the conditions of the test, the 72h-EC50 value was determined to be 7.5 mg/L (95% confidence limits 6.3 - 9.0 mg/L) based on the growth rate inhibtion.
The use of the measured concentrations at 72h is considered worst-case to conclude effect levels.
The substantial increase in pH (deviation to the guideline) is not considered to affect the results of the study, since it is likely the consequence of the growth of the algae.
Executive summary:

An acute aquatic toxicity test was performed according to OECD TG 201 (1984) in a static system and in compliance with GLP. Scenedesmus subspicatus was nominally exposed to 0, 1.0, 2.2, 4.4, 9.6, 21, and 46 mg/L. 3 replicates per test concentration and 6 replicates for the control were tested. Measured concentrations were <0.1, 1.0, 1.7, 2.8, 7.8, 12.7, 42.4 mg/L at t = 0h and <0.1, <0.1, 0.8, 1.0, 2.1, 4.9, and 13.9 mg/L) at 72 hours. The measured test substance concentrations at the end of exposure were 22 - 36% of the nominal concentrations, therefore the measured end concentrations were taken for calculation the EbC/ErC50. The 72h-EbC50 was determined to be 3.4 mg/mL (95% confidence interval 2.6 – 4.0 mg/L, slope 3.101) and the 72h-NOEbC was determined to be 0.8 mg/L. Furthermore the 72h-ErC50 was determined to be 7.5 mg/L (95% confidence interval 6.3 – 9.0 mg/L, slope 1.631) and the 72h-NOErC was determined to be 2.1 mg/L).

Description of key information

An acute aquatic toxicity test was performed according to OECD TG 201 (1984) in a static system and in compliance with GLP. Desmodesmus (Scenedesmus) subspicatus was nominally exposed to 0, 1.0, 2.2, 4.4, 9.6, 21, and 46 mg/L. 3 replicates per test concentration and 6 replicates for the control were tested. Measured concentrations were <0.1, 1.0, 1.7, 2.8, 7.8, 12.7, 42.4 mg/L at t = 0h and <0.1, <0.1, 0.8, 1.0, 2.1, 4.9, and 13.9 mg/L) at 72 hours. The measured test substance concentrations at the end of exposure were 22 - 36% of the nominal concentrations, therefore the measured end concentrations were taken for calculation the EbC/ErC50. The 72h-EbC50 was determined to be 3.4 mg/mL (95% confidence interval 2.6 – 4.0 mg/L, slope 3.101) and the 72h-NOEbC was determined to be 0.8 mg/L. Furthermore the 72h-ErC50 was determined to be 7.5 mg/L (95% confidence interval 6.3 – 9.0 mg/L, slope 1.631) and the 72h-NOErC was determined to be 2.1 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:
7.5 mg/L
EC10 or NOEC for freshwater algae:
2.1 mg/L

Additional information