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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-21 to 2012-09-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was 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: None, saturated solution as limit concentration
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- 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): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 21.5 max.: 22.5, mean value: 22
pH:
Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.97 8.01
Control 8.24 8.28
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Due to the test item being highly insoluble in water, a saturated solution prepared at a nominal concentration of 1 mg/L was tested (limit test).
No analytical evaluation of the limit test concentration and the control was carried out since no suitable method for determination of the test item could be established.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, 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: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 957221 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol/L Ca+Mg
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


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, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



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

Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Saturated Solution -1 -6





Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations [mg/L], (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.540 0.686 ± 0.470
95 % confidence interval 0.528 - 0.551
Yield Inhibition
EyC50 0.324 0.319 ± 0.140
95 % confidence interval 0.304 - 0.347



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.

Cell Densities

 

Nominal concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

1.00

1

4332

27938

257303

979766

 

2

4332

25771

199246

994044

 

3

4332

25875

215852

908657

 

4

4332

24947

205498

942120

 

5

4332

25743

225324

1025714

 

6

4332

29526

222318

1022286

 

Mean

4332

26633

220924

978765

 

Control

1

4332

19544

179722

951272

 

2

4332

22422

181999

1072103

 

3

4332

15842

157216

954700

 

4

4332

22220

170551

941994

 

5

4332

23541

168148

889756

 

6

4332

18893

171683

933503

 

Mean

4332

20410

171553

957221

 

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

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1.00

1

1.807

-0.46

975434

-2.37

2

1.812

-0.73

989712

-3.86

3

1.782

  0.93

904325

  5.10

4

1.794

  0.26

937788

  1.58

5

1.822

-1.31

1021382

-7.19

6

1.821

-1.25

1017954

-6.83

Mean

(-)

1.806

-0.43

(-)

974433

-2.26

Control

1

1.797

946940

2

1.837

1067771

3

1.798

950368

4

1.794

937662

5

1.775

885424

6

1.791

929171

Mean

1.799

952889

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

1.51

2.22

1.67

1.80

0.374

20.8

17.3

2

1.64

2.09

1.77

1.84

0.232

12.6

3

1.30

2.30

1.80

1.80

0.499

27.8

4

1.64

2.04

1.71

1.79

0.215

12.0

5

1.69

1.97

1.67

1.78

0.166

   9.36

6

1.47

2.21

1.69

1.79

0.377

21.0

Mean

1.80

 

SD ±

0.02

 

CV [%]

1.15

 

Validity criteria fulfilled:
yes
Conclusions:
In this study no inhibition on 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 on the algae growth. All effect levels are given based on the nominal test item concentration.
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 and 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. Due to the test item being highly insoluble in water 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. All effect values of the test item are given based on the nominal concentration.

Table 1:   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

Description of key information

One toxicity studie to algae of reliability 1 for Pigment Orange 34 on Desmodesmus subspicatus is available.
Under the conditions of this test the NOEC and the EC10/50 of the submission substance after 72 hours was 1 mg/L and > 1 mg/L, respectively (based an nominal concentration) and is above the solubility limit of the test substance in the test water (3.5 μg/L).


For all three pigments of the Diazopyrazolone group consistently no aquatic toxicity has been found up to nominal concentrations well above the solubility limit. Therefore it is justified to use the data for all members of the group.

Key value for chemical safety assessment

Additional information

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