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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

3 February 2010 to 9 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Uncoated nano CaCO3

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 - R6 pooled) and the 100% v/v saturated solution test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20 ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Information supplied by the Sponsor indicated that the test item had a water solubility of approximately 14 mg/L. Pre-study solubility work indicated that a testable solution of the test item could not be attained using conventional methods e.g. ultrasonication or high shear mixing. It was therefore considered that the use of a saturated solution method of preparation with a 48-Hour stirring period followed by filtration to remove any undissolved test item was a suitable method of preparation for the test item.

An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. An aliquot (1 litre) of the 100% v/v saturated solution was inoculated with 11.1 mL of algal suspension to give a test solution with a 0-Hour measured concentration of 14 mg/L.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No data

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

Control pH: 7.7-7.8
Test pH: 7.9

Nominal and measured concentrations:

Nominal concentration: 100% v/v saturated solution
Measured concentration: 14 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed - flasks were plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: Each flask contained 100 mL of solution
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.61 x 10^5 cells per mL. Inoculation of 1 litre of test medium with 11.1 mL of this algal suspension gave an initial nominal cell density of 4 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration (replicates): 6 replicate flasks
- No. of vessels per control (replicates): 6 replicate flasks
- Agitation: flasks were constantly shaken at approximately 150 rpm for 72 hours

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition:
NH4Cl: 15.0 mg/L
MgCl2.6H2O: 12.0 mg/L
CaCl2.2H2O: 18.0 mg/L
MgSO4.7H2O: 15.0 mg/L
KH2PO4: 1.60 mg/L
NaHCO3: 50 mg/L
H3BO3: 0.185 mg/L
MnCl2.4H2O: 0.415 mg/L
ZnCl2: 0.00300 mg/L
FeCl3.6H2O: 0.0640 mg/L
CoCl2.6H2O: 0.00150 mg/L
Na2MoO4.2H2O: 0.00700 mg/L
CuCl2.2H2O: 0.000010 mg/L
Na2EDTA.2H2O: 0.100 mg/L

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux intensity provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Chemical analysis of the 100% v/v saturated solution prepared for use in the range-finding test showed a dissolved test item concentration of 13 mg/L was obtained.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L were tested)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
- Any stimulation of growth found in any treatment: The growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item at a 0-Hour measured test concentration of 14 mg/L over the 72 Hour exposure period.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

The test concentration of 14 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 14 to 15 mg/L and so the results are based on the 0-Hour measured test concentrations alone.

There were no statistically significant decreases in growth rate or yield (P≥0.05), between the control and 14 mg/L test group and therefore the NOECs based on growth rate and yield were 14 mg/L.

Results with reference substance (positive control):

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:

ErC50 (0 – 72 h): 0.49 mg/L (it was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits)
EyC50 (0 – 72 h): 0.18 mg/L, 95% confidence limits 0.16 – 0.21 mg/L
NOEC based on growth rate: 0.0625 mg/L
NOEC based on yield: 0.0625 mg/L
LOEC based on growth rate: 0.125 mg/L
LOEC based on yield: 0.125 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

The pH values of the control cultures were observed to increase from pH 7.5 – 7.6 at 0 hours to pH 7.7 – 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Table 1:Daily Specific Growth Rates for the Control Cultures in the Definitive Test

-		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.045	0.047	0.062
	R2	0.036	0.037	0.081
	R3	0.041	0.042	0.075
	R4	0.040	0.053	0.067
	R5	0.044	0.051	0.060
	R6	0.042	0.031	0.080
	Mean	0.041	0.044	0.071

R₁- R₆= Replicates 1 to 6

 

 

Table 2: Inhibition of Growth Rate and Yield in the Definitive Test

0-Hour Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.051	-	1.58E+05	-
	R2	0.052		1.64E+05
	R3	0.053		1.79E+05
	R4	0.053		1.83E+05
	R5	0.052		1.63E+05
	R6	0.052		1.59E+05
	Mean	0.052		1.68E+05
	SD	0.001		1.09E+04
14	R1	0.056	[8]	2.26E+05	-
	R2	0.053	[2]	1.81E+05	-
	R3	0.056	[8]	2.15E+05	-
	R4	0.055	[6]	2.11E+05	-
	R5	0.058	[12]	2.47E+05	-
	R6	0.059	[13]	2.83E+05	-
	Mean	0.056	[8]	2.27E+05	[35]
	SD	0.002	-	3.47E+04	-

*In accordance with the OECD test guideline only the mean value for yield is calculated

R1 – R6 = Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

 

 

 

Validity criteria fulfilled:

yes

Remarks:

All validation criteria as specified in the OECD guideline were satisfied

Conclusions:

The effect of the calcium carbonate (nano) on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 14 mg/L. Correspondingly the No Observed Effect Concentration was 14 mg/L.
This study showed that there were no toxic effects at saturation.

Description of key information

The effect of the calcium carbonate (nano) on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 14 mg/L.  Correspondingly the No Observed Effect Concentration was 14 mg/L. This study showed that there were no toxic effects at saturation.