Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-27 to 2012-03-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2010-10-20

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: solid, whitish to white crystals
- Storage condition of test material: ambient temperature, dark, dry and in original container
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
SAMPLING
- Samples for determination of the test item concentration were taken after 0 and 72 hours. (Separate replicates for the test item analysis after 0 hours were prepared without algae. Samples after 72 hours were taken from pooled test replicates.)
- Sample storage conditions before analysis: The original samples were stored at 6 ± 2°C until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION (especially for difficult test substances)
- Preparation of the saturated solution acc. to OECD Series, No. 23 (2000)6:
The test item was weighed out and stored overnight before preparation of the saturated solution (8 mg/L). This solution was shaken overhead for 48 hours. Undissolved particles were removed by filtration (0.45 µm).

APPLICATION
Application was carried out by adding appropriate volumes of the saturated solution to the test replicates.

CONTROL
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, 37073 Göttingen, germany
- Age of inoculum: A three day old preculture was used as inoculum. The preculture was exposed to the same environmental conditions relative to the definitive test.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE·m-2·s-1 for 24 h per day

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
nominal hardness (medium): 0.24 mmol Ca+Mg/L
Test temperature:
min.: 21.0 °C; max.: 22.0 °C; mean value: 21.5 °C
pH:
At test start: 7.85 up to 7.90
At test end: 7.95 up to 7.97
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal loading of the test item 8.00 mg/L (Saturated solution)
Mean measured test item concentration of 0.32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (volume: 250 mL), sealed with cotton wool plugs
- Test volume: 100 mL
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: Nominal approximately 5 x 103 cells/mL; Current 5466 cells/mL
- Control end cells density: mean 1134136 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes; Nutrient medium Z according to LÜTTGE et al. (1994)

TEST MEDIUM / WATER PARAMETERS
Intervals of water quality measurement:
- pH: The pH-value at the beginning of the test was measured out of one additional replicate of the limit concentration and control. At the end it was measured from pooled replicates per limit concentration and control.
- Temperature: The room temperature was measured and recorded continuously.
- Light intensity: Light intensity was measured prior to the start of the exposure.
- Dilution water: According to the OECD guideline

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity: 60 - 120 µE · m-2 · s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm after 0, 24, 48 and 72 hours. Dilution water was used as background signal.
- Microscopic evaluation of the cells at the start and end of the incubation were carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of alga cells.


PRELIMINARY RANGE FINDING TEST
- A non GLP preliminary range finding test was conducted with the saturated solution of the test item (1:1 and 1:10) over a period of 72 h. The specific algal growth rate inhibitions were negative, -7 %. The recovery rate was 100 %.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks:
(inhibition of yield)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks:
(inhibition of yield)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks:
(inhibition of yield)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Remarks:
(inhibition of yield)
Details on results:
BIOLOGICAL DATA
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.
Exponential growth in the control: yes, 207-fold after 72 h

DETERMINATION OF THE TEST ITEM
After 72 hours the measured concentration of the test item gave a recovery rate of 99 % of the initial measured concentration.

SELF FLUORESCENCE
No self fluorescence has been found in the preliminary range finding test at the saturated solution.

PHYSICOCHEMICAL DATA
The environmental conditions (pH-value, room temperature) were determined to be within the acceptable limits.
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, batch number 1450864V, purity 100.3 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from September 06 to 09, 2011. The reference item toxicity is in the valid range following test facility SOPS.

EC50 Values of the Reference Item based on nominal concentrations in mg/L (0-72 hours):
- Rate-related inhibition:
ErC50 = 0.759 (95 % confidence interval 0.732 – 0.795)
Valid Range (average ± 3 x SD) 0.856 ± 0.420

- Yield inhibition:
EyC50 = 0.396 (95 % confidence interval 0.371 – 0.431)
Valid Range (average ± 3 x SD) 0.455 ± 0.260

(SD = Standard deviation)


Reported statistics and error estimates:
Since effects were not observed at the tested limit concentration, EC-values of the growth rate and yield inhibition were estimated with > limit concentration.

Any other information on results incl. tables

Cell Densities

 

 

Initial measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

0.319

1

5466

21282

128965

1048250

 

2

5466

20925

156671

1174714

 

3

5466

19383

120755

1052347

 

4

5466

23175

133911

1273203

 

5

5466

20314

154034

1131131

 

6

5466

19852

129881

1067098

 

Mean

5466

20822

137370

1124457

 

Control

1

5466

21416

134463

1062293

 

2

5466

22601

134046

1015395

 

3

5466

26851

149273

1238001

 

4

5466

20593

142799

1098873

 

5

5466

21897

147329

1124724

 

6

5466

25287

150726

1265529

 

Mean

5466

23108

143106

1134136

 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Initial measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield 

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

0.319

1

 

1.76

 1.42

 

1042784

  7.61

2

 

1.74

-0.72

 

1169248

 -3.60

3

 

1.81

 1.35

 

1046881

  7.25

4

 

1.77

-2.23

 

1267737

-12.3

5

 

1.78

-0.01

 

1125665

  0.27

6

 

1.82

 1.08

 

1061632

  5.94

Mean

 (-)

1.78

 0.15

(-)

1118991

  0.86

Control

1

 

1.75

 

 

1056827

 

2

 

1.79

 

 

1009929

 

3

 

1.75

 

 

1232535

 

4

 

1.82

 

 

1093407

 

5

 

1.78

 

 

1119258

 

6

 

1.76

 

 

1260063

 

Mean

 

1.78

 

 

1128670

 

negative inhibitions = increase of growth

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours) 

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.37

1.84

2.07

1.76

0.358

20.4

18.4

2

1.42

1.78

2.03

1.74

0.305

17.5

3

1.59

1.72

2.12

1.81

0.274

15.1

4

1.33

1.94

2.04

1.77

0.386

21.8

5

1.39

1.91

2.03

1.78

0.342

19.2

6

1.53

1.79

2.13

1.82

0.299

16.5

 

 

 

Mean

1.78

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.62

 

SD = Standard deviation CV = Coefficient of variation

Concentrations and Percent of the initial measured concentrationin the Definitive Test

Sampling date

2012-02-27

(Fresh media, 0 h)

2012-03-01

(Old media, 72 h)

 

Start of analysis

2012-02-27

2012-03-01

Nominal

test item

concentration

[mg/L]

test item

Measured

concentration*

[mg/L]

Measured

concentration*

[mg/L]

 

[%]

Saturated solution

0.319

0.317

99

Control

<LOQM

<LOQM

 

*                             = Mean value of 2 injections

LOQM                    = Limit of quantification of the analytical method (0.1 mg test item/L)

%                           = Percent of the initial measured test item concentration

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, an inhibition of growth of the freshwater green alga Pseudokirchneriella subcapitata was not observed after 72 hours of exposure to the test item. All effect levels are based on measured concentrations of the substance. The EC50 and the EC10 for the inhibition of growth rate and yield after 72 hours were estimated with > 0.32 mg/L, respectively.