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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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: 04620/MA
- Expiration date of the lot/batch: September 2018 (Retest date)
- Purity test date: September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under Nitrogen at Room Temperature (15-30ºC)
- Stability under test conditions: Not stable, therefore semi-static test design


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: WAF including filtration
- Preliminary purification step (if any): None

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all
- Sampling method: 10 mL samples freshly prepared test media (at test start and 24h) takenfrom preparation flasks; 10 mL samples taken from the pooled old test media test vessel replicates (at 24h and test end).
- Sample storage conditions before analysis: frozen storage at nominally -20°C - -10°C (In prior storage experiments, Elendt M4 medium fortified with dihexyl ether showed acceptable stability, 80 to 120% mean recovery, after 8 days of frozen storage).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF preparation with filtration step
- Controls: filtered and non-filtered Elendt M4 medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Justification for species other than prescribed by test guideline: not applicable
- Source: MicroBioTests Inc., Belgium
- Feeding during test: no


CULTURE
The Daphnia magna were cultured in 1 litre glass beakers containing 800 mL of Elendt M4 medium. New beakers were initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 10 - 15 daphnids per litre. The cultures were fed daily with a concentrated suspension of Chlorella vulgaris.
Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before the test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.


Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No
Test temperature:
18°C - 22°C
pH:
7.3 - 7.9
Dissolved oxygen:
>7.87 mg/l
Nominal and measured concentrations:
Nominal: 50%, 25%, 12.5% 6.25%, 3.125% of filtered WAF (10 mg/l stock solution)
Measured: 0.371, 0.0993, 0.0325, 0.00940, 0.00246 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: screw top glass jars
- Type: closed
- Material, size, headspace, fill volume: glass, 50ml, none, ~50 ml
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency): after 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 (non-filtered)
- No. of vessels per additional control (replicates): 4 (filtered)

TEST MEDIUM / WATER PARAMETERS
- Standard test medium: yes (Elendt M4 medium)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light - 8h dark
- Light intensity: 1000-1500lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility (0, 24 and 48 h)

VEHICLE CONTROL PERFORMED: no, filtered and non-filtered controls were performed

RANGE-FINDING STUDY
- Test concentrations: nominal test substance concentrations of 1, 10 and 100% of filtered water accommodated fractions (WAF) and 100% unfiltered WAF were testedt. A filtered and unfiltered control group was also included in the test.
Four replicate test vessels were used for the controls and for the non-filtered and filtered 100% WAF concentrations. Two replicate test vessels were used for the remaining treatments (1 and 10% filtered WAF concentrations).
- Results used to determine the conditions for the definitive study: Chemical analysis at 0, 24 and 48 hours showed that the test substance was not stable in test media over a 24 hour test period and therefore this confirmed the requirement for a semi-static test design with daily renewals. The results of the range-finding test suggested that the 48-hour EC50 value would be between 0.0333 and 0.432 mg/L, based on time-weighted mean measured concentrations.
Reference substance (positive control):
yes
Remarks:
Regular tests are conducted using a reference toxicant.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.194 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI (0.194 – 0.194)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.344 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.099 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.371 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none
- Observations on body length and weight: not observed
- Other biological observations: 2 daphnids mobile and at surface after 48h (one in filtered control and one in 12.5%WAF)
- Immobility of control: none
- Other adverse effects control: no
- Abnormal responses: not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no (WAF test setup)
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Reference substance: Potassium dichromate
- Test period: 07.November 2016 - 09. November 2016
- Relevant effect levels: Control, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- Results: The 48-hour EC50 value and NOEC were determined to be 0.626 and 1.0 mg/L, respectively. This result is within the expected range.
Reported statistics and error estimates:
Statistical analysis was performed using the CETIS program v 1.8.6.8.
A non-parametric control vs treatment comparison test (Steel Many-One Rank Sum Test) was performed in order to estimate 24 and 48 hour NOEC values.
Linear interpolation analysis was performed in order to estimate 24 and 48 hour EC50 values. Where possible, 95% confidence limits were calculated for the EC50 values.

Immobilisation of Daphnia magna after 48 hours exposure during the Definitive test

 Nominal Concentration (% Filtered WAF)    Time-Weighted Mean Measured Concentration (mg/L)      Number of Daphnia magna exposed      Mobile Daphnia magna      Immobile Daphnia magna    
 Submerged Surface   Submerged Surface 
 Control (Not Filtered) - 20 20 0 0

0

 Control (Filtered)

-

20

19

1

0

0

3.125

0.00246

20

20

0

0

0

6.25

0.00940

20

20

0

0

0

12.5

0.0325

20

19

1

0

0

25

0.0993

20

17

0

2

1

50

0.371

20

0

0

20*

0

*Includes the 11 immobile Daphnia magna recorded at 24 hours

Validity criteria fulfilled:
yes
Remarks:
The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied.
Conclusions:
The 48-hour acute toxicity of dihexyl ether to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004). Based on time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be 0.194 mg/L. The test isconsidered valid.
Executive summary:

The objective of the study was to determine the 48-hour acute toxicity of the test substance against the mobility of the freshwater planktonic crustacean, Daphnia magna.

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004).

Based on the results of the range finding/limit test, a definitive test was conducted at 3.125, 6.25, 12.5, 25 and 50% filtered water accommodated fractions (WAF) nominal concentrations and 0.00246, 0.00940 0.0325, 0.0993, 0.371 mg/l measured based on time-weighted average (TWA), respectively

At the start of the test, five juvenile Daphnia magna were added to each test vessel, resulting in 20 daphnids per control or test treatment. The Daphnia magna in each test vessel were observed at 24 and 48 hours and were not fed during the test.

After 24 hours there was 55% immobilisation at 0.371 mg/L. No immobilisation was observed at 24 hours at any other treatment.

After 48 hours there were 15 % and 100% immobilisation at 0.0993 and 0.371 mg/L, respectively. No immobilisation was observed at 48 hours at any other treatment.

Analysis of the test media samples was conducted on fresh media at 0 and 24 hours and on corresponding old media at 24 and 48 hours The results showed a decline in concentration over each 24-hour exposure period. Based on time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be 0.194 mg/L. The EC50  values, the corresponding NOEC  and LOEC values were statistically derived from the data and are presented in the table below:

 

 

 

 Time-Weighted Mean Measured Concentrations (mg/L)

 24-hours

 48-hours

 EC50

 0.344

 0.194

 LOEC

 0.371

 0.371

 NOEC

 0.0993

 0.0993

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test isconsidered valid.

Description of key information

The 48-hour acute toxicity of dihexyl ether to the freshwater planktonic crustacean, Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004). Based on time-weighted mean measured concentrations, the 48-hour EC50 value was determined to be 0.194 mg/L.

The validity criteria for control immobility (≤10%) and dissolved oxygen (≥3 mg/L) were both satisfied. The test is therefore considered valid.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.194 mg/L

Additional information

In addition, a reliable QSAR model (Ecological Structure-Activity Relationship (ECOSAR) version 1.11) was used to predict the acute toxicity of dihexyl ether to Daphnia magna. The 48 hour LC50 was 0.242 mg/L for the neutral organic class. The calculated value based on the calculated logKow value reflects the experimentally determined EC50 and therefore the calculated results for the acute toxicity towards fish and algae, as well as the calculated results for chronic toxicity towards fish, daphnids and alge are considered reliable.