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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:
2018-05-08 to 2018-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted on March 23, 2006; Annex 5 corrected: July 28, 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 100 mg/L (nominal)

- Sampling method: The samples (incl. control group) were analyzed undiluted due to a low water solubility with regard to the chemical structure. The test item concentration directly after preparation (0 hour) was < LOD (LOD = 0.003 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentration was performed during the study.
Vehicle:
yes
Remarks:
Reconstituted wtaer
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium (reconstituted water and test item) was freshly prepared. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green alga
- Strain: 61.81 SAG
- Source: Pflanzenphysiologisches Institut der Universität Göttingen", Germany

ACCLIMATION
- Acclimation period: 72-h
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
19.6 to 22.4 °C
pH:
7.88 to 8.34
Nominal and measured concentrations:
Nominal concentrations: 100.0 mg/L;
Measured Concentration: < LOD
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glass flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The light intensity measured immediately before start of the study was 7256 Lux ± 2.7% and 7329 Lux ± 3.1% at the end of the study.


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L
Reference substance (positive control):
no
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > 0.003 mg/L (LOD)
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > 0.003 mg/L (LOD)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: > 0.003 mg/L (LOD)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: > 0.003 mg/L (LOD)
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: > 0.003 mg/L (LOD)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: > 0.003 mg/L (LOD)
Details on results:
No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
For the reference item, potassium dichromate, the following EC values (Growth rate and Yield), NOEC and LOEC were determined:
72h EC50 of 0.86 mg/L (95 % CI: 0.78 - 0.94 mg/L) for growth rate
72h EC50 of 0.43 mg/L (95 5 CI: 0.41 -0.45 mg/L) for yield
72h NOEC: 0.4 mg/L
72 LOEC: 0.2 mg/L

Validity of the Test


In the control group, the biomass increased by a factor of 121.0 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of Variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 17.3 %. According to the OECD test guideline, the mean coefficient of Variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of Variation of the average specific growth rates in the replicates of the control group after 72 hours was 0.7 %. According to the OECD test guideline, the coefficient of Variation must not be higher than 7 %. Thus, the validity criterion was fulfilled.


 


Coefficient of Variation of the average specific growth rates in the replicates of the control:


 






































Control replicate no.



Section-by-section arithmetic means 0 – 72-h (1/d)



1



1.5783



2



1.6085



3



1.5936



4



1.5984



5



1.6064



6



1.6002



CV



0.7%



 


The mean of the replicate coefficients of Variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours:


 






































Control replicate no.



Coefficients of Variation (CV)



1



19.1%



2



12.0%



3



14.2%



4



18.3%



5



18.3%



6



22.1%



Mean



17.3%



 


Results


Algal Growth Measurement


Algal biomass (cell number) after 24, 48 and 72 hour exposure:


 












































































































































Nominal concentration [mg/L]



Biomass of algae (Cell number per mL*)



Control


 



 



0 hours



24 hours



48 hours



72 hours



1.0000



3.4470



21.0294



113.8590



1.0000



4.1247



20.5524



124.6590



1.0000



3.8430



19.8567



119.1870



1.0000



3.5460



19.6830



120.9240



1.0000



3.5874



19.5930



123.8580



1.0000



3.3147



18.7767



121.5810



Mean



1.0000



3.6438



19.9152



120.6780



SD



0.00000



0.29351



0.78801



3.88738



CV



0.0



8.1



4.0



3.2



100.0


 



 



0 hours



24 hours



48 hours



72 hours



1.0000



3.3570



19.0413



119.0880



1.0000



3.6063



19.8450



124.9110



1.0000



3.3597



19.7820



123.7950



1.0000



3.4983



20.8413



128.0070



1.0000



3.7197



20.8620



128.2770



1.0000



3.3003



19.0026



119.9340



Mean



1.0000



3.4736



19.8957



124.0020



SD



0.00000



0.16455



0.82110



3.89566



CV



0.0



4.7



4.1



3.1



*Cell number divided by factor 10000


 


Yield


Yield after 24, 48 and 72 hour exposure:


 
























































































































Nominal concentration [mg/L]



Yield



Control



 



24 hours



48 hours



72 hours



2.4470



20.0294



112.8590



3.1247



19.5524



123.6590



2.8430



18.8567



118.1870



2.5460



18.6830



119.9240



2.5874



18.5930



122.8580



2.3147



17.7767



120.5810



Mean



2.6438



18.9152



119.6780



SD



0.29351



0.78801



3.88738



CV



11.1



4.2



3.2



100.0



 



24 hours



48 hours



72 hours



2.3570



18.0413



118.0880



2.6063



18.8450



123.9110



2.3597



18.7820



122.7950



2.4983



19.8413



127.0070



2.7197



19.8620



127.2770



2.3003



18.0026



118.9340



Mean



2.4736



18.8957



123.0020



SD



0.16455



0.82110



3.89566



CV



6.7



4.3



3.2



 


Mean Specific Growth Rate


Mean specific growth rate after 24, 48 and 72 hour exposure:


 
























































































































Nominal concentration [mg/L]



Mean specific growth rate



Control



 



24 hours



48 hours



72 hours



1.238



1.523



1.578



1.417



1.511



1.609



1.346



1.494



1.594



1.266



1.490



1.598



1.277



1.488



1.606



1.198



1.466



1.600



Mean



1.290



1.495



1.598



SD



0.0790



0.0198



0.0109



CV



6.1



1.3



0.7



100.0



 



24 hours



48 hours



72 hours



1.211



1.473



1.593



1.283



1.494



1.609



1.212



1.492



1.606



1.252



1.518



1.617



1.314



1.519



1.618



1.194



1.472



1.596



Mean



1.244



1.495



1.607



SD



0.0470



0.0206



0.0105



CV



3.8



1.4



0.7



 


Morphological Appearance


No morphological changes of the treated algae in comparison to the control algae were observed.


 


EC-Values


For the test item the following EC values were determined:


 


























Parameter (0-72h)



Growth rate



Yield



EC10


95% confidence interval



> 0.003 mg/L (nominal >100 mg/L)


n.d.



> 0.003 mg/L (nominal > 100 mg/L)


n.d.



EC20


95% confidence interval



> 0.003 mg/L (nominal > 100 mg/L)


n.d.



> 0.003 mg/L (nominal > 100 mg/L)


n.d.



EC50


95% confidence interval



> 0.003 mg/L (nominal > 100 mg/L)


n.d.



> 0.003 mg/L (nominal > 100 mg/L)


n.d.



 


Analytical results


Nominal and corresponding analytical concentrations of samples are given in the following table:


 


  



























Nominal test


item concentration [mg/L]



Analytical results



0h



Meas. Conc. [mg/L]



[%]



0



< LOD



-



100



< LOD



-



 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 for growth rate and yield were >0.003 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.
Executive summary:

The acute toxicity of the test item on the growth rate of the green algae species Pseudokirchneriella subcapitata was investigated in a GLP-study according to OECD TG 201 and EU method C.3. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells/mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and the test item concentration was analyzed via HPLC with UV detection. The test item concentration directly after preparation was < LOD (LOD: 0.003 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.


The following EC values (Growth rate and Yield) were determined:


72-h EC10 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


72-h EC20 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


72-h EC50 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


The 72h EC50 for growth rate and yield were > 0.003 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.

Description of key information

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 for growth rate and yield were > 0.003 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study (reference 6.1.5-1).

Key value for chemical safety assessment

Additional information

The acute toxicity of the test item on the growth rate of the green algae species Pseudokirchneriella subcapitata was investigated in a GLP-study according to OECD TG 201 and EU method C.3. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10 000 cells/mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and the test item concentration was analyzed via HPLC with UV detection. The test item concentration directly after preparation was < LOD (LOD: 0.003 mg/L) and, thus, could not be determined. Therefore, no further analysis of the test item concentrations was performed during the study. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.


The following EC values (Growth rate and Yield) were determined:


72-h EC10 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


72-h EC20 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


72-h EC50 (growth rate and yield): > 0.003 mg/L (nominal > 100 mg/L)


The 72h EC50 for growth rate and yield were > 0.003 mg/L (nominal > 100 mg/L) and, thus, could not be determined in this study.