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Diss Factsheets

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:
2017-10-24 to 2018-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide
EC Number:
855-895-8
Cas Number:
212908-67-3
Molecular formula:
C27H47NO5
IUPAC Name:
2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide
Test material form:
solid: particulate/powder
Details on test material:
N-Acyl Phytosphingosine 95.8%, of which N-Salicyloyl 99.6%
Batch: E817500879

Sampling and analysis

Analytical monitoring:
no
Remarks:
No analytical monitoring was carried out due to the low solubility of the test item.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi-component test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture).
Exposure are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
Five water accommodated fraction (WAF) was prepared with a nominal loading of the test item in the range of 1.00 to 100 mg/L set up in geometric series with a factor of square of 10: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. For each loading level an appropriate amount of the test item was weighed. The test item was applied onto a glass slide. The glass slide with the test item was inserted in abrown glass flask with an appropriate amount of dilution water. These disruptions was shaken for 24 hours with 20 rpm at room temprature, the aqueous phase or WAF's were removed by siphoning (from the approximate centre of the glass flask). The resulting water accomodated fractions (WAF) were used in the test.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulture (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): a four days old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567–5130 lux for 24 h per day
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

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

Test conditions

Hardness:
river water total hardness [mg CO3/l]: 80.1
Test temperature:
Nominal range: 21 - 24°C, controlled at ± 2°C
pH:
start: 8.24 (test 100 mg/l) 8.28 (control); end: 8.48 (test 100 mg/l) 8.59 (control)
Dissolved oxygen:
8.81 mg O2/L (river water)
Conductivity:
266 µS/m (river water)
Nominal and measured concentrations:
1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L loading rate water accomodated fraction
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, provided with cotton wool plugs.
- Fill volume: 100 mL
- Initial cells density: nominal: approximately 5 x 10 E03 E04 cells/mL; current: 6839 cells/mL
- Control end cells density: 1232642 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, natural river water, for details see section "any other information on materials and methods"

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: The natural river water was stored at 6 ± 2 °C and at - 18± 2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: square root of 10
Range finding study
- Test concentrations: 1.00 - 10.0 - 100 mg/L nominal
- Results used to determine the conditions for the definitive study:
Growth Rate Inhibition: 100: 31%, 10.0: 20%, 1.00: 3% each concentration mg/l nominal (WAF)
Yield Inhibition: 100: 76%, 10.0: 61%, 1.00: 14% each concentration mg/l nominal
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal test item loadings
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal test item leadings
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.
Results with reference substance (positive control):
- Results with reference substance valid?
- ErC50: 0.535 (95 % c.l. 0.523 - 0.547 mg/L); valid range: 0.745 ± 0.524
- EyC50: 0.320 (95 % c.l. 0.249 - 0.339), valid range: 0.398 ± 0.291
- Other: tested from 2017-10-10 to 2017-10-13
Reported statistics and error estimates:
Statistical Significance (NOEL/LOEL) – Growth Rate

Normality Test (Shapiro-Wilk): Passed (P = 0.968)
Equal Variance Test: Failed (P < 0.050)

A NOEC of 31.6 mg/L is suggested by the program. No transform was fould to pass the equal variance test.

Any other information on results incl. tables

Table4:    Cell Density

Nominal test item loading

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6839

32146

253711

1074092

2

 6839

92152

300130

663574

3

6839

32398

372741

530382

Mean

6839

52232

308861

756016

31.6

1

6839

31113

274006

1620756

2

 6839

36155

268096

1920554

3

 6839

28017

238789

1426324

Mean

 6839

31762

260297

1655878

10.0

1

 6839

26186

258721

1428606

2

 6839

37969

235804

1274874

3

 6839

29355

244220

1475012

Mean

 6839

31170

246248

1392831

3.16

1

 6839

32304

298784

1549830

2

 6839

30010

251347

1642262

3

 6839

21577

158523

1663885

Mean

 6839

27964

236218

1618659

1.00

1

 6839

31730

226453

961763

2

 6839

30443

153385

1250998

3

 6839

34016

217429

1078276

Mean

 6839

32063

199089

1097012

Control

1

 6839

27938

161624

972940

2

 6839

23880

123891

964016

3

 6839

27941

195098

1043398

4

 6839

35171

163216

1430830

5

 6839

32146

143015

1447508

6

 6839

27739

166049

1537160

Mean

 6839

29136

155816

1232642

Table 5: 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

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.69

2

 

1067253

13

2

 

1.53

12

 

656735

46

3

 

1.45

16

 

523543

57

Mean

(+)

1.55

10

(+)

749177

39

31.6

1

 

1.82

-6

 

1613917

-32

2

 

1.88

-9

 

1913715

-56

3

 

1.78

-3

 

1419485

-16

Mean

(-)

1.83

-6

(-)

1649039

-35

10.0

1

 

1.78

-3

 

1421767

-16

2

 

1.74

-1

 

1268035

-3

3

 

1.79

-4

 

1468173

-20

Mean

(-)

1.77

-3

(-)

1385992

-13

3.16

1

 

1.81

-5

 

1542991

-26

2

 

1.83

-6

 

1635423

-33

3

 

1.83

-6

 

1657046

-35

Mean

(-)

1.82

-6

(-)

1611820

-31

1.00

1

 

1.65

4

 

954924

22

2

 

1.74

-1

 

1244159

-2

3

 

1.83

2

 

1071437

13

Mean

(-)

1.69

2

(-)

1090173

11

Control

1

 

1.65

 

 

966101

 

2

 

1.65

 

 

957177

 

3

 

1.68

 

 

1036559

 

4

 

1.78

 

 

1423991

 

5

 

1.79

 

 

1440669

 

6

 

1.81

 

 

1530321

 

Mean

 

1.73

 

 

1225803

 

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 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.41

1.76

1.80

1.65

0.213

12.9

20.2

2

1.25

1.65

2.05

1.65

0.401

24.3

3

1.41

1.94

1.68

1.68

0.268

16.0

4

1.64

1.54

2.17

1.78

0.342

19.2

5

1.55

1.49

2.32

1.79

0.460

25.7

6

1.40

1.79

2.23

1.81

0.413

22.9

 

 

 

Mean

1.73

 

 

 

 

 

SD ±

0.07

 

 

 

 

CV [%]

4.22

 

SD: Standard deviation, CV: Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
72 h NOEL of Phytosphingosine SLC to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 31.6 mg/l water accomodated fraction (nominal).
72h LOEL based on growth rate and yield inhibition are 100 mg/l WAF (nominal).
72 h ErL10, ErL20, ErL50 and EyL50 based on growth rate are > 100 mg/l WAF (nominal).
72 h EyL10 based on yield inhibition is 90.4 mg/l WAF (nominal).
72 h EyL20 based on yield inhibition is 94.2 mg/l WAF (nominal).
Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201, the cultures of Pseudokirchneriella subcapitata were exposed to Phytosphingosine SLC with a nominal loading of the test item in the range of 1.00 to 100 mg/L (1.00 - 3.16 - 10.0 - 31.6 - 100) mg/L water accommodated fraction in natural river water under static conditions. The concentration was not measured due to low water solubility.

NOEL based on growth rate and NOEL based on yield inhibition were 31.6mg/L WAF (nominal).

ErL10, ErL20, ErL50 and EyL50 based on growth rate are > 100 mg/l WAF (nominal).

EyL10 based on yield inhibition is 90.4 mg/l WAF (nominal).

EyL20 based on yield inhibition is 94.2 mg/l WAF (nominal).

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Pseudokirchneriella subcapitata.