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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2017 - July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for analysis (10 mL) were taken at 0, 24, 48 and 72 hours for all test concentrations. At 0 hours the samples were taken from freshly prepared control and test media flasks. Samples at 24 and 48 hours (old media) were taken from additional inoculated analytical vessels, which were disposed of after sampling. Samples of inoculated and non-inoculated vessels were sampled at 72 hours (old media) from either the pooled inoculated control and test vessels used for the biology cell counts or from additional non-inoculated vessels. All samples taken were immediately frozen upon sampling. On the day, one set of samples were defrosted and analysed. The samples were stored for a maximum of 9 days before analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A 10 mg/L stock solution was prepared, sealed and then stirred in the dark for 1 hour. After stirring, the test solution was settled for 1 hour and then the test solution was vacuum filtered using 0.45 µm cellulose nitrate membrane filters and combined to produce the 100 % saturated solution. The 90, 80, 64, 51, 41 and 33 % saturated solutions were prepared by diluting appropriate quantities of the prepared 100 % saturated solution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: na (Green algae)
- Strain: Strain 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK.
- Age of inoculum (at test initiation): min. 72h
- Method of cultivation: Semi-axenic liquid culture

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.1 – 23.3°C
pH:
7.22 – 10.24 (As this test was conducted in sealed vessels an increased pH was expected. This change in pH was not considered to be detrimental to this study since increases in pH of greater than 1.5 units are commonly observed and this change had no impact on the growth of the control, which met the established validity criteria for the Precautions were taken to try and limit pH increase, e.g. reduced light and inoculum, however, this did not prevent the pH increase in the control vessels.)
Nominal and measured concentrations:
The geometric mean measured concentrations were 0.00832, 0.0174, 0.00991, 0.0246, 0.0526, 0.0565 and 0.0670 mg/L for the 33, 41, 51, 64, 80, 90 and 100 % saturated solutions, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer (conical) flasks
- Type:closed
- Material, size, headspace: glass, 250 mL, no headspace
- Initial cells density: 1 x 10^4 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: EC medium according to Annex 3 of the OECD test guideline 201
- Culture medium different from test medium: no
- Intervals of water quality measurement: At test initiation the pH of freshly prepared test media, before and after alga cell inoculation, was determined. The pH in each test vessel was also determined at the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: no
- Photoperiod: Continous light
- Light intensity: maintained within 4680 - 5680 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter (Z2 Coulter Counter®) during the test and microscope observations were conducted on the algae culture and on pooled samples from all treatments at the end of the test.


TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test (0.1, 1.0, 10 and 100 % saturated solution) and an initial definitive test (1.0, 3.2, 10, 32 and 100 % saturated solutions), the reported test was conducted at nominal saturated concentrations of 33, 41, 51, 64, 80, 90 and 100 %. A filtered control group (0.45 µm cellulose nitrate membrane filtered) was also included. No unfiltered control was included in this test to allow for extra test treatments. No difference was seen between the unfiltered control and filtered control in the initial definitive test.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.067 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.:
0.038 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
- Observation of abnormalities (for algal test): none
- Unusual cell shape: At 80% saturated test solution and higher, algae cells appear straighthened and smaller.
- Colour differences: At 80% saturated test solution and higher, algae cells appear light green up to yellowish with dark patches at the 100% saturated test solution.
- Flocculation: At 80% saturated test solution and higher, flocculations were observed.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: Not reported.
- Other: None
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No observations made, but substance and/or breakdown products known to be volatile.
- Effect concentrations exceeding solubility of substance in test medium: No (WAF based test method).
Results with reference substance (positive control):
- Results with reference substance valid?
The 72h-ErC50 for growth rate was 1.16 mg/l which was within the expected toxicity range (72h-ErC50: 0.38-2.60 mg/l) for Potassium dichromate and all validity criteria were met.The test was conducted in March 2018.

Growth of Pseudokirchneriella subcapitata cultures exposed to Dihexyl Ether during the Definitive Test

Nominal Concentration

[% Saturated Solution]

Geometric Mean Measured Concentration  

[mg/L]

Cell Concentration [cells/mL]

Mean Cell Concentration [cells x 10^4/ mL]

 24h

48h

72h

24h 

48h

72h

Filtered control

 

 

 

 

 

 

 

 

 

 

Filtered control

 

 

 

 

 

 

 

 

 

 

 29585

 124100

 260300

2.387

9.645

 21.773 

 23560

85680 

 228820

 22480

85430 

194900 

 28475

114280 

228280 

19645 

 87630

203020 

19450

81550

191080

 33      

0.00832

23675 

90970 

205600 

 2.430  10.019

 22.104

24765 

104730 

235700 

24450

104880

221820 

41      

0.0174

20250 

106020 

217940 

2.334

10.138 

 21.873 

23115 

93720 

207560 

26640

104390

230680

51

0.00991

 12970

88970 

214220 

1.793 

 9.201 

 19.771

22135 

98530 

192700 

18695

91220

186200

 64      

0.0246

20800 

90000 

197460 

2.364 

 9.272

 20.429 

28235 

105600 

229980 

21885

82550

185440

80       

0.0526

8820

45530 

128280 

 0.952

 4.250

11.190

8170 

35080 

85020 

11580

46890

122400

90

0.0565

9235 

27690 

32640 

0.960*

4.039* 

2.054*

9965 

53080 

8440 

 18000

90310

228780

100

0.0670

12440 

 22300

 132660

0.922

3.722

8.510

8225 

37190 

82320 

7000

52170

40320

* The third replicate at the 90 % saturated solution treatment was identified as a statistical outlier at 72 hours and therefore excluded from the calculations.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour ErC50 and ErC10 values based on inoculated geometric mean measured concentrations was estimated to be > 0.067 mg/L and 0.0385 mg/l, respectively. The corresponding NOEC value for specific growth rate after 72 hours based on geometric mean measured concentrations was estimated to be 0.0246 mg/L. All validity criteria were met therefore the test was considered valid.
Executive summary:

The effects of dihexyl ether on the growth of the unicellular green alga, Pseudokirchneriella subcapitata were determined during a 72-hour growth inhibition test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

The 72-hour ErC50 and ErC10 values based on inoculated geometric mean measured concentrations was estimated to be > 0.067 mg/L and 0.0385 mg/l, respectively. The corresponding NOEC value for specific growth rate after 72 hours based on geometric mean measured concentrations was estimated to be 0.0246 mg/L. All validity criteria were met therefore the test was considered valid.

Description of key information

The 72-hour ErC50 and ErC10 values based on inoculated geometric mean measured concentrations was estimated to be > 0.067 mg/L and 0.0385 mg/l, respectively. The corresponding NOEC value for specific growth rate after 72 hours based on geometric mean measured concentrations was estimated to be 0.0246 mg/L. All validity criteria were met therefore the test was considered valid.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.038 mg/L

Additional information

A reliable QSAR model (Ecological Structure-Activity Relationship (ECOSAR) version 1.11) was used to predict the toxicity of dihexyl ether to algae. The 96 hour EC50 was 0.562 mg/L for the neutral organic class. In addition, the algae 96 hour ChV was 0.272 mg/L for the neutral organic class. The ChV was divided by2to estimate a NOEC equivalent value of 0.192 mg/l. The NOEC is almost an factor of 10 higher than the experimentally determined ErC10 value. Due to the low solubility and volatility of dihexyl ether it is highly likely that the experimental value is considerably lower as the ECx calculations are based on the measured geo. mean values to account for the decrease of test substance concentration over the course of the test.