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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:
03/01/2013 - 22/02/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study comparable to guideline under GLP

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
A study was performed to assess the effect of the test item on the growth of the green algaa Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for the Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

The test was performed on a water accommodated fraction (WAF)
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the UK, 30 November 2012

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Substance type : UVCB
Physical state : viscous liquid
Purity : 100%
Storage condition of test material : room temperature in the darkuid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations : at 0 and 72 hours : Pre-test 10 and 100 mg/L; Definitive test : 1.0, 3.2, 10, 32 and 100 mg/L
- Sample storage : - 20 degrees C

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Due to the low water solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). This is an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000) whereby organisms are exposed to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water.

Following a preliminary range finding test,Pseudokirchnerella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 degrees C.

The culture medium was stirred for 23 hours and the mixtures were then allowed to stand for 1 hour. The aqueous phase of WAF was removed by mid-depth siphoning. An aliquot (1 L) of each of the loading WAFs was separately inoculated with the algal suspension (9.6 mL) to give the required test concetrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchnerella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchnerella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata strain
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium maintained in the laboratory under constant aeration and illumination at 21 +/- 1 degrees C.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
24 +/- 1 degrees C
pH:
7.2 - 7.7
Nominal and measured concentrations:
Pre test:
Nominal: 10 and 100 mg/L
Measured: < limit of quantification (0 h) - 0.00508 mg/L (72 h) and 9.08 mg/L (0 h) - 0.122 mg/L (72 h)

Definitive test :
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured: for 100 mg/L: 7.04 mg/L (0 h) - 0.0399 mg/L (72 h)
Measured TWA: for 100 mg/L 3.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Fill volume: 100 mL
- Cell density (initial): 1xE03 cells/ml, the flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150rpm) and constant illumination at 24 +/- 1 degrees C until the algal cell density was approximately 1xE04 - 1xE05 cells/ml.
No. of vessels per concentration (replicates): 6 for the control and 3 for each treatment group.
Source water: dionized reverse osmosis

OTHER TEST CONDITIONS
- Illumination: Continuous
- Light intensity: 7000 lux
- Shaken: 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations at 0, 24, 46 and 72 hours using a Coulter Multisizer Particle Counter
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Details on results:
The growth rate and yield of Pseudokirchnerella subcapitata was not affected by the presence of the test item over th 72-Hour exposure period.

Inhibition of growth rate :

ErL10 (0-72 h): > 100 mg/L loading rate WAF
ErL20 (0-72 h): > 100 mg/L loading rate WAF
ErL50 (0-72 h): > 100 mg/L loading rate WAF

Inhibition of yield :

ErL10 (0-72 h): > 100 mg/L loading rate WAF
ErL20 (0-72 h): > 100 mg/L loading rate WAF
ErL50 (0-72 h): > 100 mg/L loading rate WAF

No Oberved Effect Loading Rate (NOEL) based on yield was 100 mg/L loading rate WAF.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
ErC50 (0-72 h) : 1.1 mg/L
EyC50 (0-72 h) : 0.70 mg?l

NOEC (growth rate): 0.50 mg/L
NOEC (yield): 0.25 mg/L
LOEC (growth rate): 1.0 mg/L
LOEC (yield): 0.50 mg/L

Any other information on results incl. tables

1. Cell Densities and Percentage Inhibition of Growth from the Range-finding test:

Nominal Loading rate (mg/L) Cell densities (cells per mL) Cell densities (cells per mL)
0 hours 72 hours 0 hours 72 hours
Control R1 5.92E+03 8.46E+05
R2 5.73E+03 6.76E+05
Mean 5.82E+03 7.61E+05
10 R1 5.50E+03 5.38E+05 6 28
R2 5.78E+03 5.56E+05
Mean 5.64E+03 5.47E+05
100 R1 5.26E+03 5.51E+05 9 35
R2 6.02E+03 4.47E+05
Mean 5.64E+03 4.99E+05

2. Inhibition of Growth rate and Yield in the Definitive Test:

Nominal Loading rate (mg/L) Growth rate
(cells/mL/hour)
Yield
(cells per mL)
0 - 72 h % inhibition 0 - 72 h % inhibition
Control R1 7.00E-02 7.43E+05
R2 6.60E-02 5.84E+05
R3 6.70E-02 6.22E+05
R4 6.90E-02 7.23E+05
R5 6.80E-02 6.65E+05
R6 7.00E-02 7.54E+05
Mean 6.80E-02 6.82E+05
SD 2.00E-03 6.95E+04
1 R1 7.20E-02 [6] 8.81E+05
R2 7.10E-02 [4] 8.56E+05
R3 7.10E-02 [4] 8.00E+05
Mean 7.10E-02 [5] 8.46E+05 [24]
SD 1.00E-03 4.12E+04
3.2 R1 6.90E-02 [1] 6.96E+05
R2 6.80E-02 0 6.71E+05
R3 7.30E-02 [7] 9.35E+05
Mean 7.00E-02 [3] 7.67E+05 [13]
SD 3.00E-03 1.46E+05
10 R1 7.20E-02 [6] 8.53E+05
R2 7.10E-02 [4] 8.30E+05
R3 7.30E-02 [7] 9.52E+05
Mean 7.20E-02 [6] 8.78E+05 [29]
SD 1.00E-03 6.46E+04
32 R1 7.10E-02 [4] 8.09E+05
R2 7.00E-02 [3] 7.87E+05
R3 7.30E-02 [7] 9.68E+05
Mean 7.10E-02 [5] 8.55E+05 [25]
SD 2.00E-03 9.87E+04
100 R1 7.20E-02 [6] 9.19E+05
R2 7.30E-02 [7] 9.21E+05
R3 6.90E-02 [1] 7.16E+05
Mean 7.10E-02 [5] 8.52E+05 [25]
SD 2.00E-03 1.18E+05

R1 -R6: Replicates 1 to 6

[xx]: Increase in growth compared to controls

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The ERC50 for algae is > 100 mg/L as loading rate and the NOEL is found to be 100 mg/L.
Executive summary:

The ERC50 for algae is > 100 mg/L as loading rate and the NOEL is found to be 100 mg/L (measured TWA 3.5 mg/L).