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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:
From 11-SEP-2006 to 12-SEP-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an international test guideline and to GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Dicerium tricarbonate
EC Number:
208-655-6
EC Name:
Dicerium tricarbonate
Cas Number:
537-01-9
IUPAC Name:
dicerium tricarbonate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: triplicates samples from each test medium (loading rate = 100 mg/L and dilutions 1:2, 1:4, 1:8, 1:16, 1:32) and from the control were sampled just before the start of the test and after 24, 48 and 72 hours. However, the cerium concentration were determined only in control, in undiluted filtrate and dilutions 1:4 and 1:2, as the other dilutions were below the 72h-NOELR, and thus were not relevant for interpretation.
- Sampling method: data not available
- Sample storage conditions before analysis: immediately after sampling, the samples were filtered through glass fibre microfilters and acidified with 3% (v/v) nitric acid to stabilize the test item during the storage period. Then the samples were deep-frozen and stored at about -20°C until analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
an undiluted filtrate of a supersaturated dispersion with the loading rate of 152 mg/L(corresponding to the loading rate of 100 mg/L when corrected with the water content of the test item) and the dilutions 1:2, 1:4, 1:8, 1:16, 1:32 of the filtrate were tested . The dispersion with the loading rate of 152 mg/L was prepared by dispersing 268.6 mg of the test item in 1800 mL of test water. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 min and intense stirring. The dispersion was stirred on a magnetic stirrer at room temperature in the dark for seven days. After the stirring period, the dispersion was filtered through a membrane filter (0.45 µm). The undiluted filtrate with the maximum concentration of dissolved test item was used as the highest concentrated test medium. The test medium was prepared just before the start of the test.
- Differential loading: yes
- Controls: blank (test water without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany)
- Age of inoculum (at test initiation): the algal were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.
- Method of cultivation: under standardized conditions according to the test guidelines: the algae were cultivated and tested in synthetic test water (analytical grade salts dissolved in sterile purified water).


ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions: same as in the test
- Any deformed or abnormal cells observed: data not available

Study design

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

Test conditions

Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
23°C
pH:
At the start of the test, the pH values in the test media and the control was 7.9.
At the end of the test, pH values of 7.9 and 8.0 were measured.
Dissolved oxygen:
not measured
Salinity:
not applicable
Nominal and measured concentrations:
nominal concentrations: undiluted filtrate (loading rate: 152 mg/L), dilution 1:2 (loading rate 76 mg/L), dilution 1:4 (loading rate 38 mg/L), dilution 1:8 (loading rate 19 mg/L), dilution 1:16 (loading rate 9.5 mg/L), dilution 1:32 (loading rate 4.8 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50 mL, filled with 15 mL of algal suspension
- Aeration: no, but algal suspensions were continuously stirred by magnetic stirrers.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 5000 algal cells per mL of test medium
- Control end cells density: 280000 cells/mL
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates


GROWTH MEDIUM
- Standard medium used: yes, the algae were cultivated in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: data not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in the test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test medium was also recorded daily. The concentration of phosphate was determined in the test medium and the control at the start of the test and then daily until the end of the test using a photometric method (Merck Spectroquant phosphate test 1.14842.0001). Prior to the determination, the algal cells were removed by filtration trough glass fibre microfilters (GF/C Whatman with a maximum pore size of about 1.2 µm).


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: the measured light intensity was about 7000 Lux (mean value) and was achieved by fluorescent tubes installed about 35 cm above the test flasks.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), with at least two measurements per sample. The measurements were performed at least in duplicate. Inhibition of algal growth was determined from: (i) the area under the growth curves (AUC) (= biomass integral), (ii) the specific growth rates (µ), and (iii) the yield (Y)
- Other: in addition, after 72 hours exposure, a sample was taken from the control and from a test concentration (dilution 1:2). The shape and size of the algal cells were examined microscopically in these samples.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: not reported
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:
act. ingr.
Remarks:
corrected for its water content
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not determined
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
67 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
corrected for its water content
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 39-79
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
corrected for its water content
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
corrected for its water content
Basis for effect:
growth rate
Details on results:
BIOLOGICAL RESULTS
- Exponential growth in the control (for algal test): yes (in the control, the biomass increased by a factor of 56 over 72 hours)
- Observation of abnormalities (for algal test): no, the shape and size of the algal cells was not affected

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of test media. The test media of all test concentrations were clear solutions throughout the entire test period.

PHOSPHATE CONCENTRATIONS
The inhibition of algal growth was presumably caused by a secondary effect, the complexation of the essential algal nutrient phosphate by the test item. The measured concentration of phosphate in the undiluted filtrate of the dispersion stirred for seven days was much lower than in the test water. A statistically significant decrease of the phosphate concentration was determined at the loading rates of 50 and 100 mg/L. Thus, the growth inhibition determined at these loading rates may have been caused by depletion of phosphate in the test medium.

ANALYTICAL MONITORING
At the start of the test, the concentrations of cerium measured in the dilutions 1:4, 1:2 and in the undiluted filtrate were 1, 2 and 4 µg/L, respectively corresponding to 1.6, 3.3 and 6.6 µg cerium carbonate/L (i.e. test item without water content). After 24, 48 and 72 hours test duration, the measured concentrations of cerium in these media were below thelimit of quantification of 1 µg cerium/L (corresponding to 1.6 µg cerium carbonate/L).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-hr EC50 for the growth rate = 1.0 mg/L (acceptance range : 0.44-1.16 mg/L) (potassium dichromate)
Reported statistics and error estimates:
The EL10 and EL50 values for the different growth parameters and their 95% confidence intervals were calculated as far as possible by Probit Analysis.
For the determination of the LOELR and NOELR, the calculated mean AUC, the mean growth and the mean yield at the test concentrations were compared to the corresponding control values by multiple Dunnett-tests.

Any other information on results incl. tables

Table 1: Algal cell density during the 72-hour test period

Dilution
(Loading rate) *

 

Density of algal cells
(cell number x 10,000/mL) after

 

 

24 h

48 h

72 h

Control

m

2,4

6,4

28,0

 

s

0,2

0,5

1,3

 

n

6

6

6

Dilution 1:32

m

2,4

6,4

27,2

(3,1 mg/L)

s

0,3

0,5

1,8

 

n

3

3

3

Dilution 1:16

m

2,3

6,5

25,9

(6,2 mg/L)

s

0,3

0,9

1,2

 

n

3

3

3

Dilution 1:8

m

2,1

6,3

24,7

(12 mg/L)

s

0,2

0,3

3,1

 

n

3

3

3

Dilution 1:4

m

2,5

6,6

26,9

(25 mg/L)

s

0,2

0,5

1,7

 

n

3

3

3

Dilution 1:2

m

2,2

6,4

23,6

(50 mg/L)

s

0,2

0,2

1,8

 

n

3

3

3

Undiluted

m

2,6

6,5

11,1

filtrate

s

0,2

0,3

0,5

(100 mg/L)

n

3

3

3

m: mean value ; s: standard deviation ; n: number of flasks

*: loading rate corrected for the water content of the test item

Table 2: Areas under the Growth Curves (AUC) and percentage inhibition of AUC (IAUC) during the test period

Dilution

Loading rate #

(mg/L)

Areas under the growth curves AUC

And % inhibition of AUC

0-24 h

0-48 h

0-72 h

AUC

IAUC(%)

AUC

IAUC(%)

AUC

IAUC(%)

Control

-

0.95

0.0

4.83

0.0

21.52

0.0

1:32

3.1

0.93

1.3

4.79

0.8

21.04

2.2

1:16

6.2

0.90

4.8

4.78

1.1

20.45

5.0

1:8

12

0.78

17.2

4.44

8.0

19.43

9.7

1:4

25

0.98

-3.1

4.98

-3.0

21.18

1.6

1:2

50

0.86

9.3

4.66

3.5

19.13*

11.1

Undiluted filtrate

100

1.05

-11.0

5.08

-5.1

13.35*

38.0

 

#: loading rate corrected for the water content of the test item

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in the control (according to Dunnett’s tests, one-sided, alpha = 0.05)

Table 3: Growth rates r and percentage inhibition of r (Ir) during the test period

Dilution

Loading rate #

Growth rate r and % inhibition of r

0-24 h

0-48 h

0-72 h

mg/L

r (1/day)

Ir (%)

r (1/day)

Ir (%)

r (1/day)

Ir (%)

Control

-

1,56

0,0

1,27

0,0

1,34

0,0

1:32

3,1

1,55

0,8

1,27

0,1

1,33

0,8

1:16

6,2

1,52

2,6

1,28

-0,3

1,32

1,9

1:8

12

1,42

9,3

1,26

0,7

1,30 (*)

3,2

1:4

25

1,59

-1,6

1,29

-1,1

1,33

1,1

1:2

50

1,49

4,8

1,27

-0,1

1,28 *

4,3

Undiluted

100

1,65

-5,5

1,28

-0,5

1,03 *

23,0

#: loading rate corrected for the water content of the test item

- % inhibition: increase in growth relative to that of control

*: mean value significantly lower than in control (according to Dunnett-test, one sided smaller, α = 0,05)

(*): statistically significantly lower than control. However, no concentration-effect relationship was determined. Therefore, the significant result was considered to be caused by the very low variation of the biological results in this test and not by a toxic effect caused by the test item.

Table 4: Yield (y) and percentage inhibition of y (Iy) during the test period

Treatment / Dilution

Loading rate #

(mg/L)

Yield y and inhibition of y

0-24 h

0-48 h

0-72 h

yield

Iy (%)

yield

Iy (%)

yield

Iy (%)

Control

-

1.89

0.0

5.88

0.0

27.51

0.0

1:32

3.1

1.87

1.3

5.85

0.4

26.65

3.1

1:16

6.2

1.80

4.8

5.95

-1.3

25.40

7.7

1:8

12

1.57

17.2

5.75

2.1

24.22(*)

12.0

1:4

25

1.95

-3.1

6.05

-3.0

26.35

4.2

1:2

50

1.72

9.3

5.88

-0.1

23.05*

16.2

Undiluted filtrate

100

2.10

-11.0

5.95

-1.3

10.60*

61.5

 

#: loading rate corrected for the water content of the test item

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in the control (according to Dunnett’s tests, one-sided, alpha = 0.05)

(*): statistically significantly lower than in the control. However, no concentration-effect relationship was determined. Therefore, the significant result was considered to be caused by the very low variation of the biological results in this test and not by a toxic effect caused by the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control, the cell density increased by a factor of 56 during the test period of 72 hours (> 16), the coeff. of variation of the daily growth rates was 24% (<35%), and the coeff. of variation of the average specific growth rates was 1.1% (< 7%).
Conclusions:
The test item had a statistically significant inhibitory effects on the growth of Scenedesmus subspicatus after the test period of 72 h first at the loading rate of 50 mg/L. Thus, this loading rate was determined as the 72h-LOELR. The 72h-NOELR was determined to be the loading rate of 25 mg/L.
Executive summary:

The influence of dicerium tricarbonate on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72 -hour static test according to guidelines EU C.3 (1992) and OCDE 201 (2006). The GLP were stated.

Algal suspensions were exposed to the test item with a loading rate of 100 mg/L (corrected for water content) and to dilutions 1:2, 1:4, 1:8, 1:16, 1:32. Additionally, a control was tested in parallel.

The algal cell densities were counted daily, and inhibition of algal growth was assessed. Inhibition of growth rate is summarized in the table below:

Based on the loading rates of the test item

(corrected from its water content):

Based on initially measured concentrations of

cerium carbonate in the test media 

72-h LL50 > 100 mg/L

72-h LL10 = 67 mg/L

72-h LL90 > 100 mg/L

72-h NOELR = 25 mg/L

72-h LOELR = 50 mg/L

72-h LL50 > 6.6 µg/L

72-h LL10 = 4.4 µg/L

72-h LL90 > 6.6 µg/L

72-h NOELR = 1.6 µg/L

72-h LOELR = 3.3 µg/L

The inhibition of algal growth was presumably caused by a secondary effect, the complexation of the essential algal nutrient phosphate by the test item. A statistically significant decrease of the phosphate concentration was determined at the loading rates of 50 and 100 mg/L. Thus the growth inhibition determined at these loading rates may have been caused by depletion of phosphate in the test medium.