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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The reliability is rated Klimish 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ciment Fondu®:
- Substance type: mineral
- Physical state: drak grey fine powder
- Analytical purity: 100%
- Composition of test material, percentage of components: Ciment Fondu® is a mixture of more than 99.5% milled clinker of Ciment Fondu® which is the same substance as the registered substance here, and a grinding agent
- Purity test date: 2010-04-20
- Lot/batch No.: 91478
- Storage condition of test material: at ambient temperature
- Other : It must be kept in mind that the test was carried on with ground product of less than 100 microns diameter. The usual average content in the commercial substance is only less than 15% in weight

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l (the concentration range was chosen after a range finding study had been performed, the results of which are not provided in the report)
- Sampling method:dilution

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by the addition of the appropriate quantity of Ciment Fondu® directly to 5, 2 or 1 litre of culture medium. The larger volumes were used to aid in the weighing out of small quantities of test substance used to make the test solutions of lower concentrations. All test solutions were stirred overnight using magnetic followers and then allowed to stand for 15 minutes. The test solutions were then filtered through a 0.45 µm membrane filter. All test solutions were clear and colourless. In all cases, the final solutions contained nutrients.

- Controls: Six replicate cultures of the culture medium control and one blank vessel (without algal inoculum)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: Pseudokirchneriella subcapitata CCAP 278/4
- Source (laboratory, culture collection): Brixham Environmental Laboratory culture from strain CCAP 278/4
- Age of inoculum (at test initiation): 4 days old
- Method of cultivation: anexic conditions

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no data

Study design

Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22 +/- 2 °C
pH:
At the start of the test the pH ranged from 7.4 to 7.9 and at the end of the test the range was from 7.5 to 7.7
pH (control) : 7.5 (0h) - 7.5 (72h)
pH (0.56 nominal loading conc of Ciment Fondu) : 7.4 (0h) - 7.6 (72h)
pH (1.0 nominal loading conc of Ciment Fondu) : 7.5 (0h) - 7.7 (72h)
pH (1.8 nominal loading conc of Ciment Fondu) : 7.6 (0h) - 7.7 (72h)
pH (3.2 nominal loading conc of Ciment Fondu) : 7.7 (0h) - 7.6 (72h)
pH (5.6 nominal loading conc of Ciment Fondu) : 7.9 (0h) - 7.6 (72h)
Nominal and measured concentrations:
Nominal concentrations : 0.56, 1, 1.8, 3.2 and 5.6 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed with foam bungs
- Material, size, headspace, fill volume: glass, 250 ml nominal capacity
- Initial cells density: 0.5 × 10+4 cells/ ml
- Control end cells density: mean 53.9 x 10+4 cells/ml
- No. of organisms per vessel: 0.5 × 10+6 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: see table below



OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: "cool-white" illumination of approximately 6000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counting, using a Coulter counter and counting at a lower threshold equivalent
spherical diameter of approximately 2.3 µm.
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study : yes
- Test concentrations: 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 5.6 mg/L
Nominal / measured:
meas. (arithm. mean)
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): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no data
- Aggregation of algal cells: no data
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Reported statistics and error estimates:
The cell particle densities were examined by one-way analysis of variance and Dunnett’s procedure was used to identify significant differences (p<0.05) from the control. The EC50 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method, using the US EPA program ICPIN (version 2, June 1993) which is available for download from the US EPA website

The growth rates 0 to 72 hours were calculated for each replicate culture, according to the formula :
Growth rate = ( logn(N2/N1) ) / t
where N1 = Cell density at start of test
N2 = Cell density at end of test
t = Time interval (days)

Any other information on results incl. tables

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass. The algal cell particle densities measured at each time period are given in Table 1. The means of these values are also shown in Table 1 and are plotted as growth curves in table 1 :

Table1          Algal cell densities

Nominal loading conc of

Ciment Fondu®

Replicate

Algal cell particle density

 

(104 cells ml-1)

(mg l-1)

 

24 hours

48 hours

72 hours

Control

A

2.22

9.88

47.6

B

2.39

11.0

53.1

C

2.43

12.4

60.4

D

2.33

10.7

49.1

E

2.35

11.4

57.9

F

2.44

11.3

55.4

Mean

2.36

11.1

53.9

0.56

A

2.19

9.61

37.0

B

2.50

11.7

59.0

C

2.61

11.6

49.5

Mean

2.43

11.0

48.5

1.0

A

2.47

10.8

58.6

B

2.59

11.8

53.7

C

2.53

11.5

46.6

Mean

2.53

11.4

53.0

1.8

A

2.45

10.3

49.4

B

2.23

9.23

49.8

C

2.78

12.2

61.3

Mean

2.49

10.6

53.5

3.2

A

1.67

6.99

33.9

B

2.18

10.1

55.6

C

2.49

10.2

52.2

Mean

2.11

9.09

47.2

 

5.6

 

A

0.216

1.25

11.8

B

0.288

1.44

9.56

C

0.592

1.01

5.50

Mean

0.365

1.23

8.95

 

Inoculum (Day 0) cell density = 0.503 × 104cells ml-1

Algal cell particle densities reported to 3 significant figures

Mean yields over the test period

Nominal loading conc of

Ciment Fondu®

 

Mean yield

(0-72hours)

 

(104 cells ml-1)

Percentage ofcontrol

(mg l-1)

Control

53

-

0.56

48

90

1

53

98

1.8

53

99

3.2

47

87

5.6

8.5*

16

 

All measurements are quoted to 2 significant figures and percentages to the nearest integer

*Significant difference (p <0.05) from the control

The results obtained from these statistical analyses, based on nominal loading concentrations, were as follows:

 

No Observed Effect (p <0.05) Concentration                                        = 3.2 mg l-1

Lowest Observed Effect (p <0.05) Concentration                                  = 5.6 mg l-1

Median effect concentration, yield (EyC50)                                            = 4.5 mg l-1

95% confidence limits                                                                             = 3.7 - 4.7 mg l-1

The mean growth rates wer as followed :

Mean growth rates over the test period

Nominal loading conc of

Ciment Fondu®

 

(mg l-1)

Mean growth rate

(0-72hours)

 

(104 cells ml-1day-1)

Percentage ofcontrol

Control

1.6

-

0.56

1.5

98

1

1.6

100

1.8

1.6

100

3.2

1.5

97

5.6

0.96*

62

 

All measurements are quoted to 2 significant figures and percentages to the nearest integer

*Significant difference (p <0.05) from the control

The results obtained from these growth rate analyses, based onnominalloading concentrations, were as follows:

 

No observed effect (p <0.05) concentration                                  = 3.2 mg l-1

Lowest observed effect (p <0.05) concentration                           = 5.6 mg l-1

Median effective concentration, growth rate (ErC50)                                >5.6 mg l-1

The data suggests that the growth rate of the 5.6 mg l-1 test concentration was negative in the

first 24 hours of the study but becomes comparable to the control in the 24 to 72 hour period.

In order to investigate this further, the growth rates of each time period (0 - 24,

24 - 48 and 48 - 72 hours) of each test concentration were examined by one-way analysis of

variance and Dunnett’s procedure to identify significant differences (p<0.05) from the

control. The results are given below :

 Nominal loading conc. of Ciment fondu® (mg/l)  Mean growth rate (0 -24 hours) (10000 cells/ml/day) Mean growth rate (0 -24 hours) (10000 cells/ml/day) Mean growth rate (0 -24 hours) (10000 cells/ml/day) 
 control 1.5  1.5  1.6 
 0.56  1.6  1.5  1.5
 1  1.6 1.5  1.5 
 1.8 1.6  1.4  1.6 
3.2  1.4  1.5  1.6 
5.6  -0.41* 1.3  1.9*

All measurement are quoted to 2 significant figures

* Significant difference (p<0.05) from the control

The results obtained show that the growth rate in the 5.6 mg/l test concentration was

significantly lower than in the control during the 0 – 24 hour period. In the 24 – 48 hour

period there was no significant difference from the control in any test concentration and in 48 – 72

hour period the 5.6 mg/l test concentration had a significantly higher growth rate than the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on growth rate over the test period, the result obtained, based on nominal loading concentration of Ciment fondu® is :
EC50 > 5.6 mg/l
Based on cell density over the test period, the result obtained, based on nominal loading concentration of Ciment fondu® is :
EC50 = 4.5 mg/l
Executive summary:

The toxicity of Ciment Fondu® to the green alga Pseudokirchneriella subcapitata was determined according to OECD guideline 201.

Six replicate cultures of the culture medium control and triplicate cultures of each

concentration of Ciment Fondu® were employed. One blank vessel (without algal

inoculum) was incubated concurrently for each test concentration and the control.

The algal cell densities of the inoculum and test cultures were determined by electronic

particle counting. Each replicate test vessel was inoculated with

0.505 ml of the inoculum culture to give a nominal cell density of 0.5 × 104 cells ml-1. One

100 ml volume of Coulter electrolyte, inoculated in the same manner, had a cell density of

0.503 × 104 cells ml-1. This value was used for growth calculations.

After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The

appropriate blank particle count was subtracted from that of the test culture to obtain the cell

density.

At the end of the test microscopic observations were made on samples taken from a single

replicate of the control and each test substance concentration.

The test solutions were prepared by the addition of the appropriate quantity of Ciment Fondu®

directly to 5, 2 or 1 litre of culture medium. The larger volumes were used to aid in the

weighing out of small quantities of test substance used to make the test solutions of lower

concentrations. All test solutions were stirred overnight using magnetic followers and then

allowed to stand for 15 minutes. The test solutions were then filtered through a 0.45 μm

membrane filter. All test solutions were clear and colourless.

The cell particle densities were examined

The growth rates 0 to 72 hours were calculated for each replicate culture.

Conclusion :

Cell particle densities : The results obtained from these statistical analyses, based on nominal loading concentrations,

were as follows:

No Observed Effect (p <0.05) Concentration = 3.2 mg l-1

Lowest Observed Effect (p <0.05) Concentration = 5.6 mg l-1

Median effect concentration, yield (EyC50) = 4.5 mg l-1

95% confidence limits = 3.7 - 4.7 mg l-1

Growth rate : The results obtained from these growth rate analyses, based on nominal loading

concentrations, were as follows:

No Observed Effect (p <0.05) Concentration = 3.2 mg l-1

Lowest Observed Effect (p <0.05) concentration = 5.6 mg l-1

Median effective concentration, growth rate (ErC50) > 5.6 mg l-1

The results obtained show that the growth rate in the 5.6 mg l-1 test concentration was

significantly lower than in the control during the 0 – 24 hour period. In the 24 – 48 hour

period, there was no significant difference from the control in any test concentration and in the 48 – 72 hour period the 5.6 mg l-1 test concentration had a significantly higher growth rate than the control