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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:
2013-03-11 to 2013-03-15
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
- Sampling method: 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 the end of the test samples were taken from pooled replicate with algae.
- Sample storage conditions before analysis:All original samples were stored at room temperature until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
100 mg/L of the test item were weighed out and transferred with an appropriate amount of dilution water into a brown glass flask. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
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:
0.24 mmol Ca+Mg/l
Test temperature:
min.: 22.0 max.: 23.0, mean value: 22.5 °C
pH:
Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 8.10 8.13
Control 8.14 8.29
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 631901 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 Loading
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 1 3
10 1 3
1.0 -4 -22















Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 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

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:
NOEL/LOEL were 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 NOEL/LOEl 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 -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.
The Normality Test failed for the growth rate, therefore the data were transformed (y = 10y).

Cell Densities

 

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

100

1

4332

18987

101569

505431

 

2

4332

16644

87831

540623

 

3

4332

18151

83375

499958

 

4

4332

18456

96769

501908

 

5

4332

16254

88662

500304

 

6

4332

17890

90756

587924

 

Mean

4332

17730

91494

522691

 

Control

1

4332

21516

128924

846474

 

2

4332

20355

140510

737311

 

3

4332

25205

155071

723662

 

4

4332

24576

144812

698597

 

5

4332

26051

143007

811565

 

6

4332

22714

143171

693502

 

Mean

4332

23403

142583

751852

 

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 loading

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

1.59

8

501099

33

2

1.61

6

536291

28

3

1.58

8

495626

34

4

1.58

8

497576

33

5

1.58

9

495972

34

6

1.64

5

583592

22

Mean

(+)*

1.60

7

(+)

518359

31

Control

1

1.76

842142

2

1.71

732979

3

1.71

719330

4

1.69

694265

5

1.74

807233

6

1.69

689170

Mean

1.72

747520

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

1.60

1.79

1.88

1.76

0.142

8.09

7.52

2

1.55

1.93

1.66

1.71

0.198

11.6

3

1.76

1.82

1.54

1.71

0.146

8.57

4

1.74

1.77

1.57

1.69

0.106

6.27

5

1.79

1.70

1.74

1.74

0.0461

2.65

6

1.66

1.84

1.58

1.69

0.135

7.99

Mean

1.72

 

SD ±

0.03

 

CV [%]

1.59

 

Validity criteria fulfilled:
yes
Conclusions:
In this study caused slightly inhibition on the growth rate and yield of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Fatty acids, C16-18-, reaction products with diethanolamine. All effect values given are based on the nominal loading level of the test item Fatty acids, C16-18-, reaction products with diethanolamine.
Executive summary:

The toxicity of Fatty acids, C16-18-, reaction products with diethanolamine to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-03-11 to 2013-03-15, with the definitive exposure phase from 2013-03-12 to 2013-03-15. 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. A water accommodated fraction (WAF)* with a nominal loading of 100 mg/L was tested. Six replicates were tested for the limit loading and the control. Environmental conditions were determined to be within the acceptable limits.

The test item Fatty acids, C16-18-, reaction products with diethanolaminewas analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (72 h) of the WAF with a nominal loading of 100 mg/L and the control. Details for the analytical method are presented in part 14.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.660 mg/L. The measured concentration in the WAF at the end of the exposure (72 h) was 0.370 mg/L. All effect values are given based on the nominal loading of the test item.

NOEL, LOEL, EL-values of Fatty acids, C16-18-, reaction products with diethanolamine (0-72 hours)

                   based on the nominal loading with 100 mg test item/L

                               

Growth Rate Inhibition

NOEL

  100

LOEL

> 100

ErL10

> 100

ErL20

> 100

ErL50

> 100

Yield Inhibition

NOEL

100

LOEL

   100

EyL10

100

EyL20

100

EyL50

> 100

Description of key information

In this study caused slightly inhibition on the growth rate and yield of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Fatty acids, C16-18-, reaction products with diethanolamine. All effect values given are based on the nominal loading level of the test item Fatty acids, C16-18-, reaction products with diethanolamine.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L
EC10 or NOEC for marine water algae:
100 mg/L

Additional information

The toxicity ofFatty acids,C16-18-, reaction products with diethanolamine to the unicellular freshwater green algaDesmodesmus subspicatuswas determined according to the principles of OECD 201 (2006)atDr.U.Noack-Laboratorienin 31157 Sarstedt, Germanyfrom2013-03-11 to 2013-03-15, with the definitive exposure phase from2013-03-12 to 2013-03-15.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.A water accommodated fraction (WAF)*with a nominal loading of 100 mg/Lwas tested.Sixreplicates were tested for the limit loading and the control.Environmental conditionswere determined to be within the acceptable limits.

The test itemFattyacids, C16-18-, reactionproducts with diethanolaminewas analytically verified by LC-MS/MSat the start of the exposure(0 h) and at the end of the exposure (72 h) of the WAF with a nominal loading of 100 mg/L and the control. Details for the analytical method are presented inpart14.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.660 mg/L. The measured concentration in the WAF at the end of the exposure (72 h) was 0.370 mg/L. The analytical results are presented inTable 8.All effect values are given based on the nominal loading of the test item.

 

Table1:  NOEL, LOEL, EL-values ofFatty acids, C16-18-, reaction products with diethanolamine(0-72 hours)

                  based on the nominal loading with 100 mg test item/L

                               

 

Growth Rate Inhibition

NOEL

 100

LOEL

> 100

ErL10

> 100

ErL20

> 100

ErL50

> 100

 

Yield Inhibition

NOEL

< 100

LOEL

  100

EyL10

< 100

EyL20

< 100

EyL50

> 100