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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:

06 - 23 Apr 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Due to the nature of the test item, analytical measurement is not possible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

temperature exceeded 23 ±2 °C

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: No.23-Guidance document on aquatic toxicity testing of difflcult substances and mixtures

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KlNGDOM, UK GLP Monitoring Authority

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

Sampling and analysis

Analytical monitoring:

no

Remarks:

Due to the nature of the test item, analytical measurement is not possible.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: For each nominal concentration the required amount of homogenised sample was added to 500 mL of culture medium in an aspirator. The aspirators were mixed for 48 h at 21 to 23 °C and then allowed to separate for four hours. The aqueous phase, avoiding all settled and floating material, was drawn off and filtered using Whatman No. 1 filter paper. The resultant solution was used as the test exposure solution.
- Controls: test medium without test material

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa, lnstitute of Freshwater Ecology Windermere Laboratory
- Age of inoculum (at test initiation): Exponential phase
- Method of cultivation: The tests and culture were performed in deionised water with added nutrients. Before use the water was sterilised by autoclaving at 120 °C for 30 min. Sterile nutrient stock solutions were then added and the pH value adjusted to 8.0 ± 0.2 to obtain the culture medium.

ACCLIMATION
- Culturing media and conditions: Temperature: 23 ± 2 °C, Illumination: 6000 - 10000 lux continous white light, Shaking: 200 rpm

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.3 - 26.5 °C (26.5 °C was measured at test end in test and control treatments)
temperature exceeded 23 ± 2 °C

pH:

7.1 - 7.9 (Control)
7.1 - 7.6 (Test item concentrations)

Nominal and measured concentrations:

Control, 10, 18, 32, 56, 100 mg/L (nominal loading rate)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 250 mL conical glass flask; 100 mL test solution, 150 mL headspace
- Initial cells density: 1 x 10E+04 cells/mL
- Control end cells density: 141.1 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-ionised,flltered and sterilised by autoclaving at 120 °C for 30 min.
- Culture medium different from test medium: Same medium
- Intervals of water quality measurement: At test start and test end.

OTHER TEST CONDITIONS
- Photoperiod: Continously
- Light intensity and quality: 6000 to 10000 lux white light

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell density was determined after 24, 48 and 72 h exposure periods. The cell counts were made using a haemocytometer and microscope. Cells, which showed abnormalities were included in the count but also reported in the observation chapter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Range finding study
- Test concentrations: Control, 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Percent of inhibition by growth rate 4, 3, 3 and 21% at 0.1, 1, 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: not possible to calculate 95% confidence limits

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: not possible to calculate 95% confidence limits

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: not possible to calculate 95% confidence limits

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All cells appeared normal, no cells were observed that showed any morphological abnormalities.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (72 h): 0.84 mg/L for growth rate (historical data: ErC50 (72 h): 0.86 mg/L)

Reported statistics and error estimates:

The statistical evaluation was caclulated with the program ToxCalcTMVersion 5.0 “Comprehensive Toxicity Data Analysis and Database software.

Any other information on results incl. tables

Biological Results

Table 1: Cell numbers (mean initial cell density: 1 x 10E+04 cells/mL)

Loading rate	Replicate	Cell density measurements (cells/mL x 10E+04)
[mg/L]		24 hours	48 hours	72 hours
0	1	5.0	21.3	130.7
	2	5.7	26.7	139.0
	3	5.0	17.7	126.0
	4	4.3	21.3	154.0
	5	7.3	29.7	159.7
	6	6.0	18.3	137.0
	Mean	5.6	22.5	141.1
10	1	4.3	17.0	95.0
	2	5.3	17.7	153.3
	3	5.3	15.0	88.7
	Mean	5.0	16.6	112.3
18	1	4.0	15.3	92.7
	2	7.0	19.0	90.0
	3	5.3	15.3	74.0
	Mean	5.4	16.5	85.6
32	1	7.7	17.0	80.7
	2	6.7	17.0	139.7
	3	4.0	16.7	68.7
	Mean	6.1	16.9	96.4
56	1	5.3	11.0	116.3
	2	5.0	15.0	89.7
	3	4.3	10.7	72.7
	Mean	4.9	12.2	92.9
100	1	4.0	16.0	109.0
	2	3.3	16.3	90.7
	3	5.3	12.3	88.3
	Mean	4.2	14.9	96.0

 Table 2: Inhibition of growth rate

Loading rate	Percent inhibition by growth rate
[mg/L]	0 - 48 h	0 – 72 h
10	9	5
18	10	10
32	9	9
56	20	9
100	13	8

 

Table 3: Endpoints ELx and NOELR values by growth rate (based on the nominal loading rate)

Periode of exposure	ErLx value
[h]	ErL10	ErL20	ErL50
0 to 48 h	>100 mg/L*	>100 mg/L*	>100 mg/L*
0 to 72 h	>100 mg/L*	>100 mg/L*	>100 mg/L*
NOELR	≥100 mg/L (0 – 72 hours)

* Not possible to calculate 95% confidence limits

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	141	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	16.78	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.94	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.