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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
Study period:
2013-01-28 to 2013-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (Pentapropylensuccinimido)-capronic acid
- Physical state: Liquid, brown viscous
- Analytical purity: 95.0 % (w/w) (100 % minus by-products)
- Purity test date: 2011-11-15
- Lot/batch No.: ESD0009272
- Expiration date of the lot/batch: 2014-04-26
- 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:
- Concentrations: 7.50 - 15.0 - 30.0 - 60.0 - 120 mg/L
- Sampling method: Each test item concentration and the control were analytically verified at the beginning and at the end of the test. Analytical evaluation of (Pentapropylensuccinimido)-capronic acid was carried out via LC-MS/MS. Samples at test start were taken from additional replicate without algae. At end of the test the samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water. Dispersion treatment was 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): 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.

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:
0.24 mmol Ca+Mg/l
Test temperature:
min.: 21.0 max.: 22.5, mean value: 21.75 °C
pH:
Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
82.2 7.75 8.14
41.3 7.85 8.20
10.4 7.94 8.26
9.26 7.95 8.34
2.17 7.99 8.41
Control 8.05 8.47
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 4332 cells/mL
- Control end cells density: Mean 653967 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:
Composition of Dilution Water
according to the guideline
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 8.1  0.2




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: 67.9



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

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]

100 59 98
20.0 2 26
10.00 -1 -3
1.00 0 1
0.10 0 2















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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 33.5-42.6 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 8.4-10.9 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

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation


Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rate 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 tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
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.

Any other information on results incl. tables

Cell Densities

 

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

82.2

1

4332

8304

10150

17971

 

2

4332

8769

10263

19736

 

3

4332

7747

12078

22632

 

Mean

4332

8273

10830

20113

 

41.3

1

4332

8593

23161

48110

 

2

4332

6549

20377

47289

 

3

4332

8929

21009

46493

 

Mean

4332

8024

21516

47297

 

10.4

1

4332

13380

53142

263766

 

2

4332

14628

68729

351574

 

3

4332

14241

63970

338585

 

Mean

4332

14083

61947

317975

 

  9.26

1

4332

16707

73418

452705

 

2

4332

14993

79085

459073

 

3

4332

16597

78664

512821

 

Mean

4332

16099

77056

474866

 

  2.17

1

4332

19245

139035

769252

 

2

4332

20654

137393

805262

 

3

4332

21944

142221

887001

 

Mean

4332

20614

139550

820505

 

Control

1

4332

22072

120520

670027

 

2

4332

18789

106953

667480

 

3

4332

19339

112529

615556

 

4

4332

17333

116224

648295

 

5

4332

19198

118810

700314

 

6

4332

23758

112642

622129

 

Mean

4332

20082

114613

653967

 

Evaluation after 72 hours

                   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]

[%]

82.2

1

0.474

71.6

13639

97.9

2

0.505

69.8

15404

97.6

3

0.551

67.0

18300

97.2

Mean

(+)

0.510

69.5

(+)

15781

97.6

41.3

1

0.802

52.0

43778

93.3

2

0.797

52.4

42957

93.4

3

0.791

52.7

42161

93.5

Mean

(+)

0.797

52.4

(+)

42965

93.4

10.4

1

1.37

18.1

259434

60.1

2

1.47

12.4

347242

46.6

3

1.45

13.1

334253

48.6

Mean

(+)

1.43

14.5

(+)

313643

51.7

  9.26

1

1.55

   7.31

448373

31.0

2

1.55

   7.03

454741

30.0

3

1.59

   4.83

508489

21.7

Mean

(+)1)

1.57

   6.39

(+)

470534

27.6

  2.17

1

1.73

  -3.26

764920

-17.8

2

1.74

  -4.17

800930

-23.3

3

1.77

  -6.10

882669

-35.9

Mean

(+)2)

1.75

  -4.51

(+)2)

816173

-25.6

Control

1

1.68

665695

2

1.68

663148

3

1.65

611224

4

1.67

643963

5

1.70

695982

6

1.66

617797

Mean

1.67

649635

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

1.63

1.70

1.72

1.68

0.0461

  2.74

8.99

2

1.47

1.74

1.83

1.68

0.189

11.3

3

1.50

1.76

1.67

1.65

0.139

  8.39

4

1.39

1.90

1.72

1.67

0.262

15.7

5

1.49

1.82

1.77

1.70

0.180

10.6

6

1.70

1.56

1.71

1.66

0.0862

  5.20

Mean

1.67

 

SD ±

0.02

 

CV [%]

0.97

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study (Pentapropylensuccinimido)-capronic acid was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values (geometric mean measured test item concentrations). The NOEC-values for inhibition of growth rate and yield after 72 hours were 9.26 and 2.17 mg/L, respectively. The LOEC-values for inhibition of growth rate and yield after 72 hours were 10.4 and 9.26 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % confidence intervals after 72 hours were 38.0 (33.5 – 42.6) and 10.3 (10.0 – 10.8) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of (Pentapropylensuccinimido)-capronic acid.
Executive summary:

The toxicity of (Pentapropylensuccinimido)-capronic acid (batch number: ESD0009272)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 2013-01-28 to 2013-02-01, with the definitive exposure phase from 2013-01-29 to 2013-02-01. 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. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of 2 (nominal): 7.50 - 15.0 - 30.0 - 60.0 - 120 mg/L, corresponding to geometric mean measured test item concentrations of 2.17 - 9.26 - 10.4 - 41.3 - 82.2 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.

Various test concentrations of (Pentapropylensuccinimido)-capronic acid and the control were analytically verified by LC-MS/MS at test start and test end.

The measured concentrations of (Pentapropylensuccinimido)-capronic acid at test start were in the range of 28 to 77 % of the nominal values. After 72 hours the measured concentrations of (Pentapropylensuccinimido)-capronic acid were in the range of 30 to 66 %. Therefore, all effect values given are based on the geometric mean measured concentrations of the test item (Pentapropylensuccinimido)-capronic acid.

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

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

Growth Rate Inhibition

NOEC

9.26

LOEC

10.4

ErC10

9.59 (8.42 – 10.9)

ErC20

14.6 (13.2 – 16.3)

ErC50

38.0 (33.5 – 42.6)

Yield Inhibition

NOEC

2.17

LOEC

9.26

EyC10

8.46 (7.74 – 8.90)

EyC20

8.92 (8.34 – 9.25)

EyC50

10.3 (10.0 – 10.8)