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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:
2018-06-18 to 2018-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZMG-195668
- Expiration date of the lot/batch: 2019-03-07


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep containers tightly closed in a dry, cool and well-ventilated place. Keep cool and protect from sunlight.
- Stability under test conditions: yes


OTHER SPECIFICS:
- Water solubility: 90.3 g/L (20°C, pH 6.4)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.00, 3.16, 10.0, 31.6, 100 mg/L
- Sampling method: At the start of the exposure (0 hours), samples from the freshly prepared media with algae were taken after preparation of each concentration and the control. At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analyses.
- Sample storage conditions before analysis: Extracts of the samples were stored at room temperature and until the start of the analysis on an autosampler, if necessary.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green Algae - Chlorophytes
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2590 - 5180 lux corresponding to 35 - 70 µE m-2 s-1 for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.0 - 24.0°C
pH:
start: 7.56 - 7.95;
end: 8.42 - 9.01
Nominal and measured concentrations:
nominal: 1.00, 3.16, 10.0, 31.6 and 100 mg/L
measured (geometric mean): 0.839, 2.71, 8.33, 26.4 and 82.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used.
- Type (delete if not applicable): open (Due to the analytical recoveries of the headspace replicate, an open study design with headspace is proposed for the definitive study)
- Fill volume: 100 mL
- Aeration: No
- Initial cells density: 5 x 10^3 - 10^4 cells/mL (6442 cells/mL)
- Control end cells density: 5.437 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guidelines


OTHER TEST CONDITIONS
- 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) :
- Determination of cell density: The cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.


- Determination of cell concentrations: Coulter Counter Z2, Beckman Coulter, Krefeld
- Chlorophyll measurement: 96-well micro-plates, No. 655 101, Greiner bio-one, Frickenhausen; Fluorescence Microplate Reader, Tecan infinite F200, Tecan Group Ltd. Männedorf, Switzerland



TEST CONCENTRATIONS
- Spacing factor for test concentrations: app. 3
- Range finding study: yes, a non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility.
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 d
Dose descriptor:
NOEC
Effect conc.:
26.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 d
Dose descriptor:
LOEC
Effect conc.:
82.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 d
Dose descriptor:
EC10
Effect conc.:
80 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % C.l.: 58.1 - > 82.4 mg/L
Duration:
72 d
Dose descriptor:
EC20
Effect conc.:
> 82.4 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.:
> 82.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes (281-fold; specific growth rate 1.88 day-1
- Observation of abnormalities (for algal test): no abnormalities observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 1.09 mg/L (CL 95%: 0.843 - 1.31 mg/L)
- EyC50: 0.548 mg/L (CL 95%: 0.439 - 0.660 mg/L)

Table 1: Cell Densities

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

82.4

1

6442

19376

136684

1045388

2

6442

19403

101661

857264

3

6442

23452

162552

1127360

Mean

6442

20744

133632

1010004

26.4

1

6442

27367

207286

1500331

2

6442

22516

189084

1474414

3

6442

24872

198627

1549385

Mean

6442

24918

198332

1508043

 8.33

1

6442

31495

237046

1972155

2

6442

28891

166146

1467314

3

6442

27702

204714

1256103

Mean

6442

29363

202635

1565191

 2.71

1

6442

27913

192898

1798777

2

6442

29375

185937

1621427

3

6442

26071

206680

1377000

Mean

6442

27786

195172

1599068

 0.839

1

6442

28951

180048

1498742

2

6442

26448

208210

1396016

3

6442

26034

242697

1782045

Mean

6442

27144

210318

1558934

Control

1

6442

32965

252229

2246868

2

6442

31595

197962

1725990

3

6442

33159

217862

1738651

4

6442

24641

230820

1570536

5

6442

28767

202272

1776584

6

6442

32104

240740

1815906

Mean

6442

30539

223648

1812423

Table 2: Evaluation after 72 hours

Geometric mean measured test item concentration

Replicate

Growth rate

Inhibition of growth rate

[mg/L]

No.

[d-1]

[%]

82.4

1

 

1.70

10

2

 

1.63

13

3

 

1.72

8

Mean

(+)

1.68

10

26.4

1

 

1.82

3

2

 

1.81

4

3

 

1.83

3

Mean

(-)

1.82

3

 8.33

1

 

1.91

-2

2

 

1.81

4

3

 

1.76

6

Mean

(-)

1.83

3

 2.71

1

 

1.88

0

2

 

1.84

2

3

 

1.79

5

Mean

(-)

1.84

2

 0.839

1

 

1.82

3

2

 

1.79

5

3

 

1.87

0

Mean

(-)

1.83

3

Control

1

 

1.95

 

2

 

1.86

 

3

 

1.87

 

4

 

1.83

 

5

 

1.87

 

6

 

1.88

 

Mean

 

1.88

 

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Validity criteria fulfilled:
yes
Conclusions:
Tert-butyl glycidyl ether was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentration): The EC50-values with 95% confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 82.4 mg/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 80.0 (58.1 – > 82.4) mg/L and < 0.839 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 26.4 mg/L, respectively.

Executive summary:

In a 72 hour algal growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain CCAP 278/4) were exposed to tert-butyl glycidyl ether at nominal concentrations of 0 (control), 1.00, 3.16, 10.0, 31.6 and 100 mg/L, corresponding to geometric mean measured concentrations of 0, 0.839, 2.71, 8.33, 26.4 and 82.4 mg/L under static conditions in accordance with the OECD Guideline 201 (23 March 2006), Council Regulation (EC) No. 266/2016 Method C.3 and under GLP.

The % growth inhibition in the treated algal culture as compared to the control ranged from 3 % at a concentration of 0.839 mg/L to 10% at a concentration of 82.4 mg/L.

There were no compound related phytotoxic effects.  

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata (Strain No. CCAP 278/4)

Test Type: Static

As the measured test item concentrations are not within ± 20 % of the nominal concentrations, according to OECD 201 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.

Specific Growth Rate:

72 hr EC50: > 82.4 mg/L;

72 hr EC20: > 82.4 mg/L;

72 hr EC10: 80.0 mg/L (95 % C.l. 58.1 - > 82.4);

72 hr LOEC: 82.4 mg/L

72 hr NOEC: 26.4 mg/L.  

 

Endpoint(s) Effected:  Specific growth rate

Description of key information

In a 72 hour algal growth inhibition test reported by Klix (2018), cultures of Pseudokirchneriella subcapitata were exposed to tert-butyl glycidyl ether under static conditions in accordance with the OECD Guideline 201, EU Method C.3 and under GLP. Tert-butyl glycidyl ether was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentration): The EC50-value with 95% confidence intervals for inhibition of growth rate (ErC50) after 72 hours was > 82.4 mg/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) after 72 hours was 80.0 (58.1 – > 82.4) mg/L. The NOEC-values was 26.4 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
35 mg/L
EC10 or NOEC for freshwater algae:
80 mg/L

Additional information