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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2013-01-04 to 2013-01-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1 mg/L, freshly prepared with dilution water. The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL).

- Eluate: Dilution water
- Differential loading: Saturated solution* corresponding to a nominal test item concentration of 1.00 mg/L.

- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None

Test conditions

Hardness:
Not measured; the synthetic medium used as dilution water had a nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
Mean: 22.5 °C, Min: 22.0 °C, Max: 23.0 °C
pH:
Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.67 8.29
Control 7.91 8.47


Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Nominal limit concentration of test item used to prepare saturated solutions: 1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4332 cells/mL
- Control end cells density: Mean 944265 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)

Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
Saturated Solution 1% 8%
















Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.631 (CI 0.583 - 0.687) mg/L
Yield Inhibition: 0.256 (CI 0.247 - 0.265) mg/L
Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

Detection of statistically significant differences was considered negligible when inhibition values were ≤ 10 %. These findings were marked as “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are biologically significant.


Any other information on results incl. tables

NOEC, LOEC, EC - values of test item (0-72 hours)

                   based on nominal concentration [mg/L]

Growth Rate Inhibition

NOEC

1.00

LOEC

> 1.00

ErC10

> 1.00

ErC20

> 1.00

ErC50

> 1.00

Inhibition of Yield

NOEC

1.00

LOEC

> 1.00

EyC10

> 1.00

EyC20

> 1.00

EyC50

> 1.00

Cell Densities

 

Nominal concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

1.00

1

4332

20789

146674

1030721

 

2

4332

22044

152279

1037420

 

3

4332

19751

155084

1141677

 

4

4332

22082

164079

1099880

 

5

4332

20887

148630

1047767

 

6

4332

20251

167016

1099597

 

Mean

4332

20967

155627

1076177

 

Control

1

4332

23780

173092

980307

 

2

4332

23774

162544

938132

 

3

4332

23107

156763

936434

 

4

4332

23104

157116

988798

 

5

4332

19956

151159

998768

 

6

4332

20428

160701

823151

 

Mean

4332

22358

160229

944265

 

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal concentration

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1.00

1

1.82

-1.67

1026389

   -9.20

2

1.83

-1.79

1033088

   -9.91

3

1.86

-3.57

1137345

-21.0

4

1.85

-2.87

1095548

-16.6

5

1.83

-1.97

1043435

-11.0

6

1.85

-2.87

1095265

-16.5

Mean

(+)1

1.84

-2.46

(+)1

1071845

-14.0

Control

1

1.81

975975

2

1.79

933800

3

1.79

932102

4

1.81

984466

5

1.81

994436

6

1.75

818819

Mean

1.79

939933

negative inhibitions = increase of growth - 1) Due to stimulation 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

1.70

1.99

1.73

1.81

0.155

8.56

9.74

2

1.70

1.92

1.75

1.79

0.115

6.42

3

1.67

1.92

1.79

1.79

0.120

6.71

4

1.67

1.92

1.84

1.81

0.124

6.86

5

1.53

2.03

1.89

1.81

0.257

14. 2

6

1.55

2.06

1.63

1.75

0.275

15.7

Mean

1.80

 

SD ±

0.02

 

CV [%]

1.33

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study no inhibition of growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L.
The test item caused a slight stimulation of the algae growth. All effect levels are based on the nominal concentration of the test item.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of
OECD 201 compliant to GLP (reliability category 1)
. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4332 cells/mL. A saturated solution* with a nominal concentration of 1 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for determination of the test item could be established: The test item is highly insoluble in water. All effect values of the test item are given based on the nominal concentration.

*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.

NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on nominal concentration [mg/L]

Growth Rate Inhibition

NOEC

1.00

LOEC

> 1.00

ErC10

> 1.00

ErC20

> 1.00

ErC50

> 1.00

Inhibition of Yield

NOEC

1.00

LOEC

> 1.00

EyC10

> 1.00

EyC20

> 1.00

EyC50

> 1.00