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:
2012-04-30 to 2012-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP and no defiencies

Data source

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

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): Diethyleneglycoldibutylether
- Physical state: Colourless liquid
- Water solubility: 2.8 g/L (20°C)
- Density: 0.883 g/cm3
- pH: 6.5 - 7.5 (20°C, 100 g/L in isopropanol : water 4 : 1)
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
All 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:
APPLICATION
- Method: Stock solution 100 mg/L, freshly prepared with dilution water.
Dispersion treatment Agitation
- Eluate: Dilution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 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): 3 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.

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:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 22.0 max.: 23.0, mean value: 22.5
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.78 7.84
31.6 7.82 8.00
10.0 7.80 8.20
3.16 7.78 8.22
1.00 7.82 7.89
Control 7.80 7.84


Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
See window "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, 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 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 1713179 cells/mL
- No. of vessels per concentration (replicates): 3
- 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 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, mean value: 68.3



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)
Nominal Test Item Concentration
[mg/L] Specific Growth Rate Inhibition Yield Inhibition
[%] [%]
2800 100 100
1400 100 100
700 100 100
350 100 100
100 82 99
10.0 4 20
1.00 0 1






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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
41.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 39.5-43.6 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 14.4-17.8 mg/L
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.

Any other information on results incl. tables

 Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

100

1

4910

9713

9151

7902

 

2

4910

11317

8762

7959

 

3

4910

11534

9357

7262

 

Mean

4910

10855

9090

7708

 

  31.6

1

4910

17156

48881

208842

 

2

4910

18636

53664

218449

 

3

4910

20304

66906

242658

 

Mean

4910

18699

56484

223316

 

  10.0

1

4910

35628

237073

1272260

 

2

4910

34109

209472

1409082

 

3

4910

32589

229579

1576030

 

Mean

4910

34109

225375

1419124

 

    3.16

1

4910

29207

244276

1687803

 

2

4910

27213

245806

1810001

 

3

4910

27230

272501

1897402

 

Mean

4910

27883

254194

1798402

 

    1.00

1

4910

31150

260755

1719013

 

2

4910

29684

239605

1845408

 

3

4910

32734

244960

1750493

 

Mean

4910

31189

248440

1771638

 

Control

1

4910

37456

274159

1705743

 

2

4910

32883

247052

1561610

 

3

4910

34714

239646

1877634

 

4

4910

37233

324542

1812709

 

5

4910

30930

250200

1693557

 

6

4910

32352

241839

1627821

 

Mean

4910

34261

262906

1713179

 

Evaluation after 72 hours

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]

[%]

100

1

0.159

91.9

2992

99.8

2

0.161

91.8

3049

99.8

3

0.130

93.3

2352

99.9

Mean

(+)

0.150

92.3

(+)

2798

99.8

  31.6

1

1.25

35.9

203932

88.1

2

1.27

35.2

213539

87.5

3

1.30

33.4

237748

86.1

Mean

(+)

1.27

34.8

(+)

218406

87.2

  10.0

1

1.85

    5.05

1267350

25.8

2

1.89

    3.31

1404172

17.8

3

1.92

    1.39

1571120

     8.03

Mean

(+)1

1.89

    3.25

(+)

1414214

17.2

    3.16

1

1.95

    0.22

1682893

     1.49

2

1.97

   -0.97

1805091

    -5.67

3

1.99

   -1.78

1892492

-10.8

Mean

(-)

1.97

   -0.84

(-)

1793492

     -4.99

    1.00

1

1.95

   -0.09

1714103

    -0.34

2

1.98

   -1.3

1840498

    -7.74

3

1.96

   -0.4

1745583

    -2.18

Mean

(-)

1.96

   -0.60

(-)

1766728

    -3.42

Control

1

1.95

1700833

2

1.92

1556700

3

1.98

1872724

4

1.97

1807799

5

1.95

1688647

6

1.94

1622911

Mean

1.95

1708269

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

2.03

1.99

1.83

1.95

0.108

    5.53

5.86

2

1.90

2.02

1.84

1.92

0.0879

    4.58

3

1.956

1.93

2.06

1.98

0.0673

    3.39

4

2.03

2.17

1.72

1.97

0.228

11.6

5

1.84

2.09

1.91

1.95

0.129

    6.61

6

1.89

2.01

1.91

1.94

0.0675

    3.49

Mean

1.95

 

SD ±

0.02

 

CV [%]

1.16

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Diethyleneglycoldibutylether was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 3.16 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 31.6 mg/L and 10.0 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 41.3 (39.5 – 43.6) and 16.4 (14.6 – 18.8) mg/L, respectively.
All effect levels are given based on nominal concentrations of Diethyleneglycoldibutylether.
Executive summary:

The toxicity of Diethyleneglycoldibutylether (batch number: DEG4136740) 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 2012-04-30 to 2012-05-03. 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 4910 cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a nominal dilution factor of : 1.00 - 3.16 - 10.0 - 31.6 - 100 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 Diethyleneglycoldibutyletherwere analytically verified by GC-MS at test start and test end of all test concentrations and the control. The measured concentrations of Diethyleneglycoldibutyletherat test start were in the range of 102 to 115 % and after 72 hours in the range of 89 to 112 % of the nominal values. Therefore, all effect values given are based on the nominal test item concentrations of the test item Diethyleneglycoldibutylether.

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

               based on nominal test item concentrations [mg/L]

                               

Growth Rate Inhibition

NOEC

10.0

LOEC

31.6

ErC10

16.1 (14.4 – 17.8)

ErC20

22.8 (21.4 – 23.9)

ErC50

41.3 (39.5 – 43.6)

Inhibition of Yield

NOEC

3.16

LOEC

10.0

EyC10

8.48 (7.25 – 9.83)

EyC20

10.6 (9.37 – 12.0)

EyC50

16.4 (14.6 – 18.8)