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:
2010-02-09 to 2010-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study with GLP, no deviations

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
Details on test material:
- Name of test material (as cited in study report): Phosphorpentoxid
- Molecular formula: P4O10
- Molecular weight: 283.9 g/mol
- Physical state: Solid, white powder
- Analytical purity: 99.7 % (m/m)
- Purity test date: 2009-09-23
- Lot/batch No.: DEH2 018965
- Expiration date of the lot/batch: 2011-09-08
- Storage condition of test material: Stored in a desiccator at room temperature, protected from light.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration 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: Separate samples at start (provided without alga) were prepared. Samples at the 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 200 mg/L was freshly prepared with dilution water and agitated. The pH-value was adjusted to the pH of the dilution water ± 0.2 by adding appropriate volumes of 1 M NaOH.
- Eluate: Dilution water
- Differential loading: 12.5 - 25.0 - 50.0 - 100 - 200 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
Post exposure observation period:
No

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22.5 °C, Min: 22 °C, Max: 23 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
200 8.06 7.26
100 8.03 7.32
50.0 7.98 7.29
25.0 7.97 7.36
12.5 7.94 7.45
Control 7.92 7.56
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 1437745 cells/mL
- No. of vessels per concentration (replicates): 3
- 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 Tests (0-72 h)
Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10 - 10 - 59
1 - 11 - 69
0.1 - 11 - 70








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

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
66.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (64.0-69.7)
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: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.614 (CI 0.551 - 0.679) mg/L
Yield Inhibition: 0.333 (CI 0.318 - 0.350) mg/L
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of
statistical analyses statistical significance of growth rates and yield.
One Way Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. 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 Tests were 0.05. The a-value for ANOVA was a=0.05. Normality test failed for yield calculation. Transformation (square root) was carried out for yield values.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression (EyCx). Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried from the best fit values, the standard error and the t-distribution with the software Graph Pad Prism.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Phosphorpentoxid(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

25.0

LOEC

50.0

ErC10

39.4 (37.2 – 41.5)

ErC20

48.6 (47.3 – 50.0)

ErC50

66.5 (64.0 – 69.7)

Inhibition of Yield

NOEC

12.5

LOEC

12.5

EyC10

12.5

EyC20

16.3 ( 12.5 – 22.8)

EyC50

38.0 (34.2 – 41.2)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

200

1

5074

17202

14943

15041

 

2

5074

16952

17694

17226

 

3

5074

13524

15443

14090

 

Mean

5074

15893

16027

15452

 

100

1

5074

16046

16780

18432

 

2

5074

17800

18707

20531

 

3

5074

16517

19904

21130

 

Mean

5074

16788

18464

20031

 

   50.0

1

5074

19740

94868

429018

 

2

5074

22499

93081

376378

 

3

5074

21249

103429

440412

 

Mean

5074

21163

97126

415269

 

   25.0

1

5074

29559

197098

1055392

 

2

5074

28600

203498

1018434

 

3

5074

30199

213026

1058897

 

Mean

5074

29453

204541

1044241

 

   12.5

1

5074

34635

218225

1151348

 

2

5074

28288

171641

1047757

 

3

5074

31822

271312

1317160

 

Mean

5074

31582

220393

1172088

 

Control

1

5074

37152

269122

1413473

 

2

5074

31576

237019

1387574

 

3

5074

28223

234740

1193152

 

4

5074

35246

302422

1489901

 

5

5074

35365

303890

1542668

 

6

5074

40695

332717

1599703

 

Mean

5074

34710

279985

1437745

 

Evaluation after 72 h

                  Statistically significant differences of growth rates and yield compared to

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

               

Nominal test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

 

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

 

200

1

0.362

80.7

9967

99.3

2

0.407

78.3

12152

99.2

3

0.340

81.9

9016

99.4

Mean

 (+)

0.370

80.3

(+)

10378

99.3

100

1

0.430

77.1

13358

99.1

2

0.466

75.2

15457

98.9

3

0.476

74.7

16056

98.9

Mean

 (+)

0.457

75.7

(+)

14957

99.0

   50.0

1

1.48

21.4

423944

70.4

2

1.44

23.7

371304

74.1

3

1.49

20.9

435338

69.6

Mean

 (+)

1.47

22.0

(+)

410195

71.4

   25.0

1

1.78

  5.40

1050318

26.7

2

1.77

  6.03

1013360

29.3

3

1.78

  5.34

1053823

26.4

Mean

 (+)*

1.78

  5.59

(+)

1039167

27.5

   12.5

1

1.81

  3.86

1146274

20.0

2

1.78

  5.53

1042683

27.2

3

1.85

  1.48

1312086

  8.42

Mean

 (+)*

1.81

  3.62

(+)

1167014

18.5

Control

1

1.88

1408399

2

1.87

1382500

3

1.82

1188078

4

1.89

1484827

5

1.91

1537594

6

1.92

1594629

Mean

1.88

1432671

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

1.99

1.98

1.66

1.88

0.189

10.1

12.6

2

1.83

2.02

1.77

1.87

0.130

6.93

3

1.72

2.12

1.63

1.82

0.262

14.4

4

1.94

2.15

1.60

1.89

0.280

14.8

5

1.94

2.15

1.63

1.91

0.265

13.9

6

2.08

2.10

1.57

1.92

0.301

15.7

Mean

1.88

 

± SD

0.03

 

CV [%]

1.84

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Phosphorpentoxid was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 66.5 (64.0 – 69.7) and 38.0 (34.2 – 41.2) mg/L, respectively.
All effect levels are given based on nominal concentrations of Phosphorpentoxid.
Executive summary:

The toxicity of Phosphorpentoxid(batch number DEH2 018965) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from February 9th to July 2nd with the definitive exposure phase from June 29th to July 2nd, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

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 2, nominal: 12.5 - 25.0 - 50.0 - 100 - 200 mg/L. In order to exclude possible effects due to the pH-value all test concentrations were tested by correction of the pH-value. 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 Phosphorpentoxidwere analysed at all concentration levels after 0 and 72 h via standardized photometric test. The measured concentrations of Phosphorpentoxidat test start were in the range of 89 – 93 % of the nominal values. At the end of the test Phosphorpentoxidgave recoveries of 79 – 97 % of the nominal values. All effect values are given based on nominal concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Phosphorpentoxid(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

25.0

LOEC

50.0

ErC10

39.4 (37.2 – 41.5)

ErC20

48.6 (47.3 – 50.0)

ErC50

66.5 (64.0 – 69.7)

Inhibition of Yield

NOEC

12.5

LOEC

12.5

EyC10

12.5

EyC20

16.3 ( 12.5 – 22.8)

EyC50

38.0 (34.2 – 41.2)