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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 25, 2007 - June 28, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Rationale for read-across: 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 + 2Ca2SiO4 +9CaCO3 + 13H2O <-> 14Ca2+ + 2SiO2 + 9CO2 + 28OH- 100 mg “Reaction mass of limestone and dicalcium silicate” or 0.08 mmol sets free 2.24 mmol OH- 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 "Reaction mass of limestone and dicalcium silicate" 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 lime (chemical), hydraulic and calcium hydroxide. As such, it can be reasonably expected that the effect on pH of "Reaction mass of limestone and dicalcium silicate" is comparable to calcium hydroxide for a same application on a weight basis. Consequently, read-across from calcium hydroxide to "Reaction mass of limestone and dicalcium silicate" is justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No pH adjustment in test medium.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
No pH adjustment in test medium.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUPAC name: Calcium dihydroxide

Sampling and analysis

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.

Test solutions

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

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

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
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
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)

Results and discussion

Effect concentrationsopen allclose all
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.

Applicant's summary and conclusion

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.