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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:
2015-07-27 to 2016-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified in replicates prepared after 0 hours (start of exposure), 24 hours, 48 hours and 72 hours (end of exposure).
- Sample storage conditions before analysis: All samples were stored in a refrigerator at 6 ± 2 °C until sample preparation.Prepared samples were stored at ambient temperature in an auto sampler rack prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Considering the density of the test substance (0.8705) appropriate volumes of the test item were pipetted directly into measuring flasks, containing the respective volume of the dilution water. The test solutions were stirred for at least 15 minutes at approximately 1100 rpm at room temperature. Thereafter, the readily prepared test solutions were transferred to the test vessels which were tightly closed, without headspace. The test concentrations were selected based on the results of a non- GLP preliminary range finding test.
- Controls: negative control (Six replicates (without test item) were exposed under the same conditions as the test concentrations); positive control: Potassium dichromate
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green freshwater algae
- Strain: Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar and cultivated in nutrient medium Z according to Lüttge et al. (1994) under a light intensity of 2567 - 5130 lux, corresponding to 35 - 70 µE • m-2 * s-1 for 24 hours per day at a temperature of 21 - 24 °C.

ACCLIMATION
- Acclimation period: 4 days in AAP medium.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.5 - 22°C
pH:
at 0 hours: 7.47 - 7.57
at 72 hours: 7.63 - 8.39
Nominal and measured concentrations:
Nominal: 0 (control), 2.65, 4.76, 8.57, 15.4, 27.8, 50 mg test substance/L
Measured (mean): < LOQ (control), 0.250, 0.977, 2.32, 5.23, 10.1, 33.8 mg test substance/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks (volume 59 mL) with aluminum tops with PTFE seals.
- Type (delete if not applicable): Closed, without headspace.
- Material, size, headspace, fill volume: 59 mL
- Aeration: No.
- Renewal rate of test solution (frequency/flow rate): No.
- Initial cells density: Approximately 5 x 10^3 - 10^4 cells/mL, Actual: 6305 cells/mL.
- Control end cells density: 494842 cells/mL.
- No. of vessels per concentration (replicates): 3 (Separate replicates for each measuring time were prepared.)
- No. of vessels per control (replicates): 6 (Separate replicates for each measuring time were prepared.)

GROWTH MEDIUM
- Standard medium used: Yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 3 x AAP medium with compounds additionally added to enable sufficient growth under conditions without headspace.
- Ca/Mg ratio: 1/3
- Culture medium different from test medium: Yes.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes.
- Adjustment of pH: No data.
- Photoperiod: 24 hours light
- Light intensity and quality: 5475 (range: 4751 - 6107) µE*m-2*s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: The cells were checked by microscopic evaluation for unusual cell shapes, color differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: None.
- Range finding study: Yes.
- Test concentrations: 1.00, 10.0 and 100 mg/L of the test item were used in the range-finding study.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield inhibition
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield inhibition
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- EC50: 0.796 ± 0.509 mg/L (growth rate inhibition), 0.419 + 0.275 mg/L (yield inhibition)

Table 1: Cell Densities

Time weighted average mean measured test item concentration
(mg/L)

Cell density [cells/mL]

0 hours

24 hours

48 hours

72 hours

33.8

6305

16055

17677

n.a.

10.1

6305

23885

44063

213618

5.23

6305

28170

112540

603844

2.32

6305

33371

168414

667022

0.977

6305

36767

184836

575123

0.250

6305

14858

191568

545185

Control

6305

38799

192542

494842

 

Table 2: Evaluation after 72 hours

Time weighted average mean measured test item concentration
(mg/L)

Growth rate
[1/d]

Inhibition of growth rate [%]

Yield [cells/mL]

Inhibition of yield [%]

33.8

(+) n.a.

100

(+) n.a.

100

10.1

(+) 1.17

19

(+) 207313

58

5.23

(-) 1.52

-4

(-) 597539

-22

2.32

(-) 1.55

-7

(-) 660717

-35

0.977

(-) 1.50

-3

(-) 568818

-16

0.250

(-) 1.49

-2

(-) 538880

-10

Control

1.45

488537

 

Table 3: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) and Old Medium (72 hours)

Sampling

date

0 hours without algae

24 hours with algae

48 hours with algae

72 hours with algae

Geometric

Mean

(Time

weighed

average)

Nominal loading level of the test item [mg/L]

Meas.

cone.

[mg/L]

%

Meas.

cone.

[mg/L]

%

Meas.

cone.

[mg/L]

%

Meas.

cone.

[mg/L]

%

mg/L

50

44.7

89

40.7

81

45.7

91

9.59

19

33.8

27.8

22.6

81

23.0

83

18.9

68

< LOQ

-

10.1

15.4

12.5

81

12.7

82

6.36

74

< LOQ

-

5.23

8.57

7.07

82

7.38

86

1.28

8

< LOQ

-

2.32

4.76

4.26

89

3.61

76

< LOQ

-

< LOQ

-

0.977

2.65

0.185

7

0.170

6

0.180

7

< LOQ

-

0.250

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. Conc. = Measured concentration of the test item (enrichment and dilution factor taken into account)

% = Percent of the nominal concentration of the test item

LOQ= Limit of quantification of the analytical method (0.5 mg/L of the test item)

Validity criteria fulfilled:
yes
Conclusions:
An Algae test with Pseudokirchneriella subcapitata according to OECD 201 and in compliance with GLP was conducted with Ethyl hexanoate. The following values were estimated:
ErC50 (72 h): 11.8 mg/L based on growth rate.
ErC10 (72h) = 9.41 mg/L based on growth rate.
EyC50 (72 h): 9.97 mg/L based on yield inhibition.
EyC10 (72h) = 9.29 mg/L based on yield inhibition.
NOEC (72h) = 5.23 mg/L based on growth rate and yield inhibition.
(based on geometric mean measured concentrations)

Description of key information

ErC50 (72 h): 11.8 mg/L

ErC10 (72h) = 9.41 mg/L

EyC50 (72 h): 9.97 mg/L

EyC10 (72h) = 9.29 mg/L

NOEC (72h) = 5.23 mg/L based on growth rate and yield inhibition

(based on geometric mean measured concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:
11.8 mg/L
EC10 or NOEC for freshwater algae:
9.41 mg/L

Additional information

The toxicity of the test item to algae and cyanobacteria was determined in a static test performed according to OECD 201 and GLP. Cultures of Pseudokirchneriella subcapitata were exposed to nominal test concentrations of 2.65, 4.76, 8.57, 15.4, 27.8 and 50.0 mg/L, equivalent to geometric mean measured concentrations of 0.250, 0.977, 2.32, 5.23, 10.1 and 33.8 mg/L. The test concentrations were analysed by GC-FID determinations daily. After 72 h 10% inhibition of growth rate was observed at 9.41 mg/L, 10% inhibition of yield was recorded at 9.29 mg/L. The ErC50 (72 h) was calculated at 11.8 mg/L (measured), the EyC50 (72 h) at 9.97 mg/L (measured). The NOEC values for both endpoints was estimated as 5.23 mg/L.