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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
other information
Study period:
30 May - 01 Jun 1095
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original report not available and documentation insufficient for assessment.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION - Chemical name of vehicle: castor oil ethoxylate
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Common name: water flea- Age at study initiation: < 24h
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
20 ± 1 °C
Nominal and measured concentrations:
Nominal: 10 mg/L
Details on test conditions:
TEST SYSTEM- No. of organisms per vessel: 20 EFFECT PARAMETERS MEASURED: Immobilisation after 24 and 48 h test duration.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: EC 50 (48h)> solubility limit
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Oct - 12 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD, EU, ISO test guidelines and conducted in a GLP accredited laboratory
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment number 23, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: On the day of analysis, the samples were defrosted at room temperature. The test samples were extracted in a 3:1 (v:v) ratio with ethyl acetate. The shaking time was 30 seconds. If necessary, the samples were diluted with ethyl acetate extracted with ISO-medium to obtain concentrations within the calibration range.- Sample storage conditions before analysis: The samples were stored in the freezer (≤ -15°C).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was left to stabilize for approximately 3-3.5 hours where after the Water Soluble Fraction (WSF) was siphoned off and used as the highest test concentration for the combined limit/range-finding test. For the final test the WSF was only used as a stock solution to prepare the test concentrations. The WSF prepared for the combined limit/range-finding test was observed to be hazy, while the WSF prepared for the final test was observed to be clear. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. These lower test solutions were all clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Common name: water flea- Source: In-house laboratory culture with a known history. At least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.- Age at study initiation (mean and range, SD): <24 hours, from parental daphnids of more than two weeks old- Method of breeding:Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.Maximum age of the cultures:4 weeksRenewal of the cultures: After 7 days of cultivation half of the medium twice a week.Temperature of medium: 18-22 °CFeeding: Daily, a suspension of fresh water algae.- Feeding during test: none
Test type:
static
Water media type:
freshwater
Total exposure duration:
48 h
Hardness:
180 mg/L as CaCO3 (medium)
Test temperature:
18.9 - 20.4 °C
pH:
7.8 - 8.0
Dissolved oxygen:
9.1 - 9.2 mg O2/L
Nominal and measured concentrations:
0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10% of a WSF prepared at a loading rate of 100 mg/L and a control
Details on test conditions:
TEST SYSTEM- Test vessel: - Material, size, headspace, fill volume: 100 mL, all-glass; filled with 80 mL of test solution- Aeration: none- No. of organisms per vessel: 5- No. of vessels per concentration (replicates): 4- No. of vessels per control (replicates): 4TEST MEDIUM / WATER PARAMETERS- Culture medium different from test medium: No; M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).- Intervals of water quality measurement:OTHER TEST CONDITIONS- Photoperiod: 16 hours light, 8 hours darkEFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality) at 24 hours and at 48 hours
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: filtered test solution
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: filtered test solution
Basis for effect:
mobility
Remarks on result:
other: physical effects, daphnids were trapped in the surface of the test solutions
Results with reference substance (positive control):
The 24h-EC50 was 0.66 mg/L with a 95% confidence interval between 0.59 and 0.77 mg/L.The 48h-EC50 was 0.38 mg/L with a 95% confidence interval between 0.34 and 0.45 mg/L.

Combined limit/range-finding test:

After 48 hours of exposure all daphnids exposed to 10 and 100% of the WSF were immobilised. In the replicates of both concentrations a floating layer was observed and test substance was stuck to the daphnids, indicating that the concentrations tested were above the solubility limit in test medium. Immobility observed in the replicates containing 1.0% of the WSF should be considered with caution. After 24 hours of exposure test substance was observed to be stuck to the daphnids but no longer after 48 hours of exposure. Daphnids recorded as immobile after 48 hours of exposure were moving about but were no longer able to swim up. No immobility was observed at the lowest test concentration.

Based on these results samples taken from 0.10 and 10% of the WSF were analysed. Actual concentrations proved to be below the lowest calibration solution (i.e. 0.03 mg/L) at 0.10% of the WSF. The initial concentration in 10% of the WSF was 1.3 mg/L. This concentration decreased to 0.35 mg/L (27% of initial) at the end of the test. The expected EC50 was below an average concentration of 0.66 mg/L.

Table: Incidence of immobility in the combined limit/range-finding test

Test substance % of WSF

Prep. at 100 mg/L

Vessel number

 

Number Daphnia exposed

Response at 24 h

Response at 48 h

number

Total %

number

Total %

control

A

5

0

5

0

5

B

5

0

5

0

5

C

5

1

5

1

5

D

5

0

5

0

5

0.10 (<0.03)

A

5

0

0

0 [1]

0

B

5

0

0

0 [1]

0

1.0

A

5

0 [5]

0

3 [2]

50

B

5

0 [5]

0

2 [3]

50

10 (0.66)

A

5

5 [5]

90

5*

100

B

5

4 [5]

90

5*

100

100

A

5

5 [5]*

100

5 [5]*

100

B

5

5 [5]*

100

5 [5]*

100

C

5

5 [5]*

100

5 [5]*

100

D

5

5 [5]*

100

5*

100

( ) Average exposure concentration

[ ] Number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

* A floating layer was observed

Final test:

Analysis of the samples taken from 2.2, 4.6, 10 and 100% of the WSF at the start of the test showed measured concentrations of 0.052, 0.11, 0.25 and 2.7 mg/L. These concentrations decreased to 37 - 47% of initial at the end of the test. Given these results, the effect parameters were based on the average exposure concentrations that were calculated to be 0.034, 0.075 and 0.17 mg/L. The average exposure concentration in the stock solution (100% of WSF) was 1.7 mg/L but no daphnids were exposed to this concentration.

Immobility at 10% of the WSF was significantly lower (60%) than during the combined limit range-finding test. This can be explained by the lower average exposure concentration during the final test. After 24 hours all daphnids were trapped at the surface of the test solutions starting at 4.6% of the WSF onwards. However, no floating layer was observed and no test substance was sticking to the daphnids after 48 hours of exposure. Nevertheless, even if no floating layer or sticking of test substance was observed after 48 hours, the daphnids were very likely already adversely affected by the contact with the surface layer after 24 hours. Thus the observed effects are considered to be physical effects and not substance specific toxicological effects.

Table: Acute immobilisation of daphnids after 24 and 48 hours in the final test

Test substance % of WSF

Prep. at 100 mg/L

Vessel number

Number Daphnia exposed

Response at 24 h

Response at 48 h

number

Total %

number

Total %

control

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0

0

0

0

0.10

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0

0

0

0

0.22

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0

0

0

0

0.46

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0

0

0

0

1.0

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0 [2]

0

0

0

2.2 (0.034)

A

5

0

0

0

0

B

5

0

0

0

0

C

5

0

0

0

0

D

5

0

0

0

0

4.6 (0.075)

A

5

0 [5]

0

0 [1]

5

B

5

0 [5]

0

0 [2]

5

C

5

0 [5]

0

01 [1]

5

D

5

0 [5]

0

0

5

10 (0.17)

A

5

2 [5]

10

5 [1]

60

B

5

0 [5]

10

2 [2]

60

C

5

0 [5]

10

2 [3]

60

D

5

0 [5]

10

3 [2]

60

( ) Average exposure concentration

[ ] Number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate did not induce acute immobilisation of Daphnia magna at an average exposure concentration of 0.034 mg/l after 48 hours of exposure (NOEC). The 48h-EC50 was 0.16 mg/l (95% confidence interval between 0.12 and 0.24 mg/l).
Executive summary:

 Acute Toxicity Study inDaphnia magnawith 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate.

 

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008, the ISO International Standard 6341, 1996 and the OECD series on testing and assessment number 23, 2000.

 

The batch of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate tested was a clear colourless liquid with a purity of >98% (based on GC-FID area-%) and not completely soluble in test medium at the loading rate initially prepared.

 

A final test was performed based on the results of a preceding range-finding test. Preparation of test solutions started with a loading rate of 100 mg/l applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was left to stabilize for approximately 3.5 hours where after the clear Water Soluble Fraction (WSF) was siphoned off and used as a stock solution. The test concentrations were prepared by subsequent dilutions of the WSF in test medium. These test solutions were all clear and colourless.

 

Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10% of the WSF prepared at a loading rate of 100 mg/l and a control. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test.

 

Analysis of the samples taken from 2.2, 4.6, 10 and 100% of the WSF at the start of the test showed measured concentrations of 0.052, 0.11, 0.25 and 2.7 mg/l. These concentrations decreased to 37-47% of initial at the end of the test. Given these results, the effect parameters were based on the average exposure concentrations that were calculated to be 0.034, 0.075 and 0.17 mg/l. The average exposure concentration in the stock solution (100% of WSF) was 1.7 mg/l but no daphnids were exposed to this concentration.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate did not induce acute immobilisation ofDaphnia magnaat an average exposure concentration of 0.034 mg/l after 48 hours of exposure (NOEC).

 

The 48h-EC50was 0.16 mg/l (95% confidence interval between 0.12 and 0.24 mg/l).

 

 

Description of key information

No toxic effects up to the limit of water solubility (< 0.01 mg/L) for Daphnia magna (OECD 202) 

Key value for chemical safety assessment

Additional information

One study investigating the short-term toxicity of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS No. 28510-23-8) to aquatic invertebrates is available. The GLP study was conducted under static conditions according to OECD guideline 202 using Daphnia magna as test organism (Bouwman, 2012b). Preparation of test solutions started with a loading rate of 100 mg/l applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was left to stabilize for approximately 3.5 hours where after the clear Water Soluble Fraction (WSF) was siphoned off and used as a stock solution. The test concentrations were prepared by subsequent dilutions of the WSF in test medium. These test solutions were all clear and colourless.

Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10% of the WSF prepared at a loading rate of 100 mg/l and a control. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test.

Analysis of the samples taken from 2.2, 4.6, 10 and 100% of the WSF at the start of the test showed measured concentrations of 0.052, 0.11, 0.25 and 2.7 mg/l. These concentrations decreased to 37-47% of initial at the end of the test. Given these results, the effect parameters were based on the average exposure concentrations that were calculated to be 0.034, 0.075 and 0.17 mg/l. The average exposure concentration in the stock solution (100% of WSF) was 1.7 mg/l but no daphnids were exposed to this concentration.

After 24 hours all daphnids were trapped at the surface of the test solutions starting at the 4.6% solution. However, no floating layer was observed and no test substance was sticking to the daphnids after 48 hours of exposure. It is well established that daphnids are very sensitive towards physical effects. Once they are trapped in an oily film or surface, their filtration apparatus is adversely affected. Thus, even if no floating layer or sticking of test substance was observed after 48 hours, the daphnids were very likely already adversely affected by the contact with the surface layer after 24 hours. The observed effects (EC50 = 0.16 mg/L) are therefore attributed to physical effects and not substance specific toxicological effects.