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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 201 and GLP

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at start and end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 500 mg/L was frehsly prepared with dilution water. Dispersion treatment was ultrasound for 15 min at room temperature.
- Eluate: Dilution water
- Differential loading: 5 - 15.8 - 50 - 158 - 500 mg/L (factor: √10) corresponding to
geometric mean measured concentrations of 0.855 - 1.55 - 2.78 -
155 - 501 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

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 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): Same as test
- Any deformed or abnormal cells observed: No

Study design

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

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22 °C, Min: 21 °C, Max: 23 °C
pH:
Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
501 3.04 2.95
155 6.45 6.17
2.78 8.17 7.70
1.55 8.18 8.45
0.855 7.98 8.73
Control 8.17 8.84
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 5074 cells/mL
- Control end cells density: Mean 1073776 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: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- 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



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] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 19 61
10 1 5
1 - 1 - 2

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

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (21.9-42.2)
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: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05. Normality test failed for yield values. No useful transformation of yield values was possible.

Any other information on results incl. tables

NOEC, LOEC and EC - values of pyrazolic acid 4 (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

2.78

LOEC

155

ErC10

12.2 (9.21 – 20.4)

ErC20

17.3 (13.1 – 26.5)

ErC50

30.6 (21.9 – 42.2)

Inhibition of Yield

NOEC

2.78

LOEC

155

EyC10

4.67 (2.79 – 17.4)

EyC20

7.53 (4.46 – 19.5)

EyC50

13.9 (7.45 – 26.5)

Cell Densities

 

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

501

1

5074

2823

2229

2680

 

2

5074

3737

2717

2897

 

3

5074

2897

3069

2155

 

Mean

5074

3152

2672

2577

 

155

1

5074

7460

5275

5828

 

2

5074

7415

5820

4578

 

3

5074

9473

6484

5779

 

Mean

5074

8116

5860

5395

 

    2.78

1

5074

28891

238348

989402

 

2

5074

32032

240537

1016208

 

3

5074

37259

269028

1012083

 

Mean

5074

32727

249304

1005898

 

    1.55

1

5074

25467

207671

937439

 

2

5074

22749

208787

1036946

 

3

5074

32163

271144

1055039

 

Mean

5074

26793

229201

1009808

 

    0.855

1

5074

27009

219619

986065

 

2

5074

15779

149919

1028803

 

3

5074

35410

256457

1053034

 

Mean

5074

26066

208665

1022634

 

Control

1

5074

36521

249496

1073973

 

2

5074

27407

212817

1027196

 

3

5074

29645

260918

1208276

 

4

5074

25082

197938

945270

 

5

5074

32593

236486

1080693

 

6

5074

40707

273460

1107248

 

Mean

5074

31993

238519

1073776

 

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

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

                              

Geometric mean measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

501

1

-0.213

100

-2394

100

2

-0.187

100

-2177

100

3

-0.285

100

-2919

100

Mean

(+)

-0.228

100

(+)

-2497

100

155

1

0.046

  97.4

754

  99.9

2

-0.034

100

-496

100

3

0.043

  97.6

705

   99.9

Mean

(+)

0.018

  98.3

(+)

321

100

    2.78

1

1.76

    1.48

984328

    7.89

2

1.77

    0.98

1011134

    5.39

3

1.77

    1.05

1007009

    5.77

Mean

(-)

1.76

    1.17

(-)

1000824

    6.35

    1.55

1

1.74

    2.49

932365

  12.8

2

1.77

    0.60

1031872

   3.45

3

1.78

    0.28

1049965

   1.75

Mean

(-)

1.76

    1.12

(-)

1004734

   5.99

    0.855

1

1.76

    1.54

980991

   8.21

2

1.77

    0.75

1023729

   4.21

3

1.78

    0.31

1047960

   1.94

Mean

(-)

1.77

    0.87

(-)

1017560

   4.79

Control

1

1.79

1068899

2

1.77

1022122

3

1.82

1203202

4

1.74

940196

5

1.79

1075619

6

1.80

1102174

Mean

1.78

1068702

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

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.97

1.92

1.46

1.79

0.283

15.9

16.2

2

1.69

2.05

1.57

1.77

0.249

14.0

3

1.77

2.18

1.53

1.82

0.325

17.8

4

1.60

2.07

1.56

1.74

0.281

16.1

5

1.86

1.98

1.52

1.79

0.240

13.4

6

2.08

1.91

1.40

1.80

0.355

19.8

Mean

1.78

 

SD ±

0.03

 

CV [%]

1.52

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study the test substance was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 30.6 (21.9 – 42.2) and 13.9 (7.45 – 26.5) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of the test substance.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 103cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of 10, nominal: 5.0 - 15.8 - 50.0 - 158 - 500 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test substance were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations at test start were in the range of 97 – 103 % of the nominal values. At the end of the test the test substance gave recoveries of 93 and 98 % at 158 and 500 mg/L and less than the LOQ of 0.300 mg/L at the concentrations of 5 to 50 mg/L of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.

Table 1:    NOEC, LOEC and EC - values of pyrazolic acid 4 (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

2.78

LOEC

155

ErC10

12.2 (9.21 – 20.4)

ErC20

17.3 (13.1 – 26.5)

ErC50

30.6 (21.9 – 42.2)

Inhibition of Yield

NOEC

2.78

LOEC

155

EyC10

4.67 (2.79 – 17.4)

EyC20

7.53 (4.46 – 19.5)

EyC50

13.9 (7.45 – 26.5)