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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:
Experimental part of study performed in period from 2013-09-05 to 2013-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out according to recommended EU method in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
see section Overall remarks
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Copper phtalocyanine monosulfonated and disulfonated
EC Number:
700-870-8
Molecular formula:
C32H15N8Cu(HSO3)n where n=1.3-1.6
IUPAC Name:
Copper phtalocyanine monosulfonated and disulfonated
Constituent 2
Reference substance name:
Copper phtalocyanine mono- and disulphonated
IUPAC Name:
Copper phtalocyanine mono- and disulphonated
Constituent 3
Reference substance name:
Hysperse 12
IUPAC Name:
Hysperse 12
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Hysperse 12
- Molecular formula:C32H15N8Cu(SO3H)1.4x2.5H2O
- Molecular weight: 733.33
- Substance type: technical product
- Physical state: powder
- Lot/batch No.: 01
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Limit test 100 mg/L
- The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test.
- The samples for analysis were taken from the concentration 100 mg/L and from the concentration 100 mg/L without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. all samples were stored at laboratory temperature.The analytical results showed that the test substance was stable in test medium under the conditions of testing.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION OF TEST MEDIUM
Water with conductivity smaller than 5 microS/cm was used for the preparation of solutions. All other chemicals used were of analytical grade.
According folowing recipes were prepared four basic stock solutions:
Solution A: NH4Cl 1.5 g + MgCl2.6H2O 1.2 g + CaCl2.2H2O + MgSO4.7 H2O 1.5 g + KH2PO4 0.16 g + water up to 1000 mL
Solution B: FeCl3.6 H2O 64 mg + Na2EDTA.2 H2O + water up to 1000 mL
Solution C: H3BO3 185 mg + MnCl2.4H20 415 mg + ZnCl2 3.0 mg + CoCl2.6H2O 1.5 mg + CuCl2.2H2O 0.01 mg + Na2MoO4.2H2O + water to 1000 mL.
Solution D: NaHCO3 50 g + water up to 1000 mL.

Test medium: 10 mL solution A + 1 mL solution B + 1 mL solution C + 1 mL solution D + water up to 1000 mL.
The solutions were keep in the dark at the temperature of 4+/- 1 °C in fridge. The temperature was checked every day.

PREPARATION AND APPLICATION OF TEST SOUTIONS

STOCK SOLUTION
The stock solution of the test substance was prepared in the test medium. 100 mg of the substance was weighed into 1000 mL of the medium for limit test. because the test substance was poorly soluble for dilution was used ultrasound for 45 minutes.

LIMIT TEST
Testing mixtures were prepared by dosing appropriate volumes of stock solution of the tested substance and inoculum into volumetric flasks. The test contained six parallel series of the test substance concentration and six controls without the test substance. The volume of the test solution was 25 mL in each testing flask.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Brinkmann 1953/SAG 86.81
- Source: The collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3 days before the start of the test. Inoculum culture was kept 3 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

ACCLIMATION
- Acclimation period: no
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no

Test conditions

Test temperature:
22.5 - 23.0 grad. C

pH:
7.6-7.8 control
7.6-7.7 100 mg Hysperse 12/L

Nominal and measured concentrations:
Loading rate: 100 mg/L (limit test)
Nominal concentration 100 mg/L - determined concentration at 0 hours 95.7 mg/L
72 hours 95.1 mg/L - change (0 - 72 hours) -6.3 %
Nominal concentration 100 mg/L (without algae) - determined concentration at 0 hours 98.6 mg/L
72 hours 99.0 mg/L - change (0 - 72 hours) + 4.0 %
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmayer flasks with stopper venting air
- Type: open
- Material: glass
- Aeration: without

- Initial cells density:
- Control end cells density: 5000 cells/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: The water with conductivity 0.295 mS/m was used for the preparation of solutions. All chemicals used were of analytical-grade.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Lighting: under continuous white light, 6850 lux to 6900 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: microscopically, Burker´s counting chamber
- Chlorophyll measurement: no

TEST CONCENTRATIONS
Limit test at 100 mg Hysperse 12/L


Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: NOECr
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOECy
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: not observed
- Colour differences: not observed
- Flocculation: not mentioned
- Adherence to test vessels: not mentioned
- Aggregation of algal cells: not mentioned
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: test concentrations were below solubility of test substance
Results with reference substance (positive control):
Reference test
The sensitivity of the test species and correctness of test performance were periodically verified on a six-month period by testing the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7, carried out in period from 19. 4. 2013 to 26. 4. 2013 were the following:
72 hour – ErC50 = 0.95mg/L (95% confidence limit: 0.75 – 1.15 mg/L)

The given results for the ErC50 was convenient to range of the results of the interlaboratory tests. The results of the last interlaboratory test arranged by CSlab s. r. o. in 2012 for potassium dichromate for 72h – ErC50, generated at 7 laboratories were :
72 hour – ErC50 = 0.53 – 1.23 mg/L

The evaluation of the last interlaboratory test for potassium dichromate was performed only according growth rate. The results for 72h - EyC50 are not to be compared. The evaluation of the last interlaboratory test arranged by CSlab, spol. s r. o. in 2012 for potassium dichromate was performed only according to growth rate. This fact does not serious problem because the data based on yield aro not used for the classification and risk assessment.
Reported statistics and error estimates:
The values of of ErC50 and EyC50 were calculated by means of software “Toxicita” (product of Water Research Institute, Ostrava, Czech Republic).
The determination of NOECr values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).
The loading rates of the test substance were used for all evaluations and results.

Any other information on results incl. tables

Percentual reduction of growth rate and percentual inhibition of cell growth – limit test

Loading rate

mg/L

Growth rate

Yield

Mean growth

u

Reduction

%

Mean yield

Y

Inhibition

%

100

Control

1.03

1.28

19.2

0.0

106458

228333

53.4

0.0

>

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Test quality criteria according to guideline:
1. Biomass in the control cultures should have increased exponentially by a factor of at least 16 within 72-hour test period.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35 %.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests.
The test quality criteria at point 1, 2 and 3 were fulfilled.
Executive summary:

The test substance - Hysperse 12 - was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV.

 

The limit test was performed with nominal concentration 100 mg/L. The analytical results showed, that the test substance was sufficiently stable in the test medium at the conditions of the test.

Based on no toxicity of the test substance found in limit test, the study was terminated.

Test results:

72 hour – ErC50 >100 mg/L      

72 hour – EyC50 <100 mg/L     

72 hour – NOECr  <100 mg/L     

72 hour – NOECy < 100 mg/L