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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 25, 2007 - June 28, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No pH adjustment in test medium.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
No pH adjustment in test medium.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Start test: duplicate samples of all solutions
End test: test solutions of 2 replicates were mixed (precipitate left behind). Additionally, the test solutions of two replicates of the 235 mg/L concentration were mixed with 1N HCL (0.625 ml/replicate) to redissolve any precipitated calcium. Duplicate samples were taken from controls and treated vessels, and from test vessels without algae at the highest test item concentration.
Storage samples: in brown glass bottles at ambient temperature in the dark.
Vehicle:
no
Details on test solutions:
Test item was weighed separately into a glass beaker. Afterwards the content of the respective beakers was transferred to a 1000 mL measuring flask and filled up with temperature adjusted test medium. Turbidity was noted at 138 mg/L and higher, so the test item was not completely dissolved at these concentrations. It is also possible that due to reaction of the test item with test medium components and CO2, poorly soluble precipitates were formed.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata (SAG 61.81) supplied by Institut für Pflanzenphysiologie, University Göttingen, D-37073 Göttingen.
Age of the pre-culture: 3 days
Number of cells per ml in the pre-culture before inoculating the test solution: 1515000
Number of cells per ml test solution at the beginning of the test: 5000
Algal medium pH 5.8
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
107 mg CaCO3/L
Test temperature:
22.2-23.9 degC
pH:
INITIAL:
control: 5.8
48.0 mg/l: 6.4
80.0 mg/l: 6.8
138.0 mg/l: 7.2
235.0 mg/l: 8.1
400.0 mg/l: 11.4
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal: 0 (control), 48.0, 80.0, 138.0, 235.0 and 400.0 mg/l.
Nominal and measured concentrations were approximately similar.
Details on test conditions:
Test vessels: 300 ml Erlenmeyer flasks covered by air-permeable stoppers
Test medium: Kuhl and Lorenzen (1964) algal medium
Amount of test solution per test vessel: 100 ml
Number of replicates per test item concentration: 3
Number of replicates in the control: 6
Number of replicates for stability check (highest concentration without algae): 2
Light cycle: 24/0 hours light/dark
Type of light: fluorescent tubes of universal white type (L58W/840)
Light intensity: mean value of six measurements: 8025 lx (equivalent to 4440 - 8880 lx)
Temperature: 22.8 ± 0.30 °C
Shaker: 100 ± 5 oscillations/min (the test vessels were placed randomly on the shaker)
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 250 µl of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results were converted into biomass concentration using a calibration curve.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
79.22 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
106.02 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
184.57 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Flocculation: with increasing concentrations precipitates formed over time to which algae adhered, leading to flocculation. Visible precipitates formed at 138 mg/L and above leading to only a marginal increase in pH. It was therefore concluded that the initial pH of the test medium was not directly related to the biologically relevant effects. The formation of precipitates is likely the result of the reaction between Ca(OH)2 and CO2 dissolved in the medium yielding poorly soluble CaCO3.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the measured Ca concentrations were much below the nominal concentrations, due to the reaction of the test item with CO2 to poorly soluble CaCO3, thus forming precipitates. However, measurement of Ca after acidification at the end of the test resulted in a recovery of 97.7% .
Results with reference substance (positive control):
Growth rate: EC50 (0-72h): 1.635 mg/L
Reported statistics and error estimates:
The biological results were evaluated statistically. Probit analysis was used to calculate EC values.
Validity criteria fulfilled:
yes
Remarks:
Mean biomass increase in the control cultures: 139,4. Mean coefficient of variation for section-by-section specific growth rates in the control cultures: 7,0%. Coefficient of variation of average specific growth rates during test period in replicate contr
Conclusions:
A clear concentration-response relationship was observed.
The pH of the medium at concentrations resulting in a considerable growth inhibition, was below 8 and the biological findings are therefore not attributed to the initial pH of the test solutions.
It was observed that, however, that with increasing test item concentrations precipitates were formed over time to which algae adhered, leading to their flocculation. The flocculation of algae is thus considered to be the predominant biologically relevant effect in this system test.
The recovery of the test item at the end of the test was below 80% of the nominal concentration. This can be explained since the test item is known to react with CO2 to calcium carbonate, which is poorly soluble in water leading to the formation of precipitates. However, after acidification, the test item recovery was 97,7% of the nominal concentration, which conforms the establishment of the target concentration
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
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)
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:
yes
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 (delete if not applicable): 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.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lime (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98.2%

3. ANALOGUE APPROACH JUSTIFICATION
In the environment, lime substances rapidly dissociate or react with water. These reactions, together with the equivalent amount of hydroxyl ions set free when considering 100mg of the lime compound (hypothetic example), are illustrated below:
Ca(OH)2 <-> Ca2+ + 2OH-
100 mg Ca(OH)2 or 1.35 mmol sets free 2.70 mmol OH-
Ca(OH)2 + Ca2SiO4 + CaCO3 + 3 H2O <-> 4Ca2+ + SiO2 + CO2 + 8OH-
100 mg lime (chemical) hydraulic or 0.27 mmol sets free 2.16 mmol OH-
It has to be noted that CO32- is not expected to directly release two hydroxyl ions under most environmental conditions (depends on CO2 concentrations and pH) and this is therefore a worst case assumption.
From these reactions it is clear that the effect of lime (chemical) hydraulic will be caused either by calcium or hydroxyl ions. Since calcium is abundantly present in the environment and since the effect concentrations are within the same order of magnitude of its natural concentration, it can be assumed that the adverse effects are mainly caused by the pH increase caused by the hydroxyl ions. Furthermore, the above mentioned calculations show that the base equivalents are within a factor 2 for hydraulic lime (chemical) and calcium hydroxide. As such, it can be reasonably expected that the effect on pH of lime (chemical) hydraulic is comparable to calcium hydroxide for a same application on a weight basis. Consequently, read-across from calcium hydroxide to lime (chemical) hydraulic is justified.

4. DATA MATRIX
Source: No studies available
Target: 007): ErC50(72h) = 184.57 mg Ca(OH)2/L , nominal NOErC(72h) = 48 mg Ca(OH)2/L (Pseudokirchneriella subcapitata)
Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
79.22 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
106.02 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
184.57 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Ca(OH)2
Basis for effect:
growth rate

Description of key information

Klimisch 1 study (Egeler et al. (2007): growth rate of Pseudokirchneriella subcapitata: nominal EC50(72h) = 184.57 mg Ca(OH)2/L , nominal NOEC(72h) = 48 mg Ca(OH)2/L

Klimisch 1 study; 72-h ErC50 > 14 mg/L (measured, 100 %v/v saturated solution nominal); NOErC was 14 mg/L (measured, 100 %v/v saturated solution nominal) (Vryenhoef and Mullee, 2010)

Key value for chemical safety assessment

EC50 for freshwater algae:
184.57 mg/L
EC10 or NOEC for freshwater algae:
48 mg/L

Additional information

The acute toxicity to algae of calcium dihydroxide was assessed in a study performed according to OECD TG 201 under GLP (Egeler et al, 2007). In this study Pseudokirchneriella subcapitata were exposed to calcium dihydroxide at nominal concentrations of 0, 48, 80, 138, 235 and 400 mg/L for 72 hours. A clear concentration-response relationship was observed. The pH of the medium at concentrations resulting in a considerable growth inhibition was below 8 and the biological findings are therefore not attributed to the initial pH of the test solutions. It was observed that, however, that with increasing test item concentrations precipitates were formed over time to which algae adhered, leading to their flocculation. The flocculation of algae is thus considered to be the predominant biologically relevant effect in this system test. The recovery of the test item at the end of the test was below 80% of the nominal concentration. This can be explained since the test item is known to react with CO2 to calcium carbonate, which is poorly soluble in water leading to the formation of precipitates. However, after acidification, the test item recovery was 97.7% of the nominal concentration, which conforms the establishment of the target concentration. The 72-h ErC50 was 184.57 mg/L and the 72-h NOErC was 48 mg/L.

The acute toxicity to algae of calcium carbonate (nano) was assessed in a study performed according to OECD TG 201 under GLP (Vryenhoef and Mullee, 2010). In this study Desmodesmus subspicatus (green algae) were exposed to a 100 %v/v saturated solution of calcium carbonate (measured concentration = 14mg/L). No toxic effects were noted at the concentration tested. Hence, the 72 h ErC50 for calcium carbonate (nano) was found to be >14 mg/L (measured, 100 %v/v saturated solution nominal) and the NOErC was 14 mg/L (measured, 100 %v/v saturated solution nominal). The concentration of calcium carbonate (nano) that might cause acute toxicity is therefore greater than the maximum solubility of calcium carbonate in water.

Based on the results of the studies performed on calcium dihydroxide and calcium carbonate, it may be concluded that the acute toxicity to algae of grades of lime (chemical) hydraulic containing up to 40% calcium carbonate will be driven by the calcium dihydroxide content and that the results available for calcium dihydroxide represent the worse-case for all grades of lime (chemical) hydraulic.