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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:
key study
Study period:
January - April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: Dibutyl ether
Chemical name: 1-butoxybutane
CAS number: 142-96-1
Batch/Lot number: 7J05163A01
Description: Colourless liquid
Purity: 99.7%
Expiry date: 21 November 2019
Storage conditions: Controlled room temperature (15-25oC, <70% relative humidity), protected from light and humidity (tight closed container), under inert gas
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were performed from the control and at the applied test concentration levels at the beginning and at the end of the renewal periods.
Vehicle:
yes
Details on test solutions:
A saturated stock solution with a nominal loading rate of 100.0 mg/L was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) before the start of the renewal periods. This solution was stirred for about 2 hours (based on the analytical results that indicated that was sufficient) at the test temperature in a closed system with reduced headspace in order to minimize potential losses due to volatilization. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the 100 % saturated solution. The test solutions were prepared by appropriate dilution of this stock solution just before the introduction of the Daphnia (start of the treatments).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Breeding The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd. The health of the stock animals was monitored on a daily basis by visual checking. Abnormal behaviour or significant decrease of the population was recorded.
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: 20 animals per test group and control group respectively, divided into 4 replicates per group (5 animals / replicate)
Age of the animals: Less than 24 hours old at the beginning of the test
Age of the animals: Less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water (ISO medium) had a total hardness of 248 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start and at the end of the renewal periods in each test vessel. The test temperature was in the range of 20.3 – 20.8°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.9 and 21.0°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in each test vessel and was in the range of 7.06 – 7.72.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 7.0 – 8.7 mg/L.
Nominal and measured concentrations:
The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution (of 100 mg/L nominal loading rate).

Test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test item concentrations were: 0.73, 2.26, 4.62, 10.32 and 18.76 mg/L. The biological results are described based on the measured geometric mean test item concentrations.

Details on test conditions:
The test was conducted in a closed system with minimized headspace in the test vessels under semi-static conditions. The test duration was 48 hours. The frequency of the water renewal periods was 24 hours Twenty animals, divided into four replicates (in Erlenmeyer flasks with stopper) of five animals each (at least 5 mL test solution/animal) were used per test concentrations and controls. The test vessels were not aerated during the test.

A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions. The frequency of the water renewal periods was 24 hours.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 18.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 18.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 18.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
18.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of dibutyl ether were: 24 and 48h EC50 value:  > 18.76 mg/L (measured)
Executive summary:

The acute toxicity of dibutyl ether on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. Because significant immobility was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under semi-static conditions. The test concentrations were analytically determined at the start and at the end of the renewal periods. The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions (of 100 mg/L nominal loading rate). The corresponding measured geometric mean test item concentrations were: 0.73, 2.26, 4.62, 10.32 and 18.76 mg/L. The biological results are based on the measured geometric mean test item concentrations. Twenty animals per group were divided into four replicates of five animals and placed into glass beakers. Four replicates per group were used for the control and treated groups. The 24- and 48-hour EC50values of the test item could not be calculated due to the fact that only slight effects were observed. The 24- and 48-hour EC50 and the 48 hour EC100values of the test item were determined directly from the raw data..

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of dibutyl ether were the following:

The 24 and 48h EC50 value:  > 18.76 mg/L (measured)

The 48h EC100 value: > 18.76 mg/L (measured)

The 48h No-Observed Effect Concentration (NOEC):  10.32 mg/L (nominal)

The 48h Lowest Observed Effect Concentration (LOEC): 18.76 mg/L (nominal)

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No information given whether the study was performed according to GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: U.S. Environmental Protection Agency: Method for acute toxicity tests with fish, macroinvertebrates, and amphibians (1975)
Deviations:
not applicable
Principles of method if other than guideline:
Method: other: U.S. Environmental Protection Agency: Method for acute toxicity tests with fish, macroinvertebrates, and amphibians (1975)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: Five to eight nominal concentrations of each chemical were tested. A negative control and, if appropriate, a solvent control were maintained concurrently with each test.
Vehicle:
not specified
Details on test solutions:
A stock solution of the chemical, in distilled water, was prepared and used to provide the desired concentrations for testing if the test material was sufficiently soluble in water. If insoluble at the desired water stock solution concentrations, a stock solution utilizing a co-solvent (triethylene glycol, ethanol, acetone or dimethylformamide, respectively, in order of preference) was prepared. If the chemical was not sufficiently soluble in either distilled water or a co-solvent for the preparation of a stock solution, then the chemical was added directly to diluent water. Using one of the methods described above (which one of these was used is unknown), the chemical was added to 500 ml of diluent water in 2 litre jars to prepare each test solution. If the chemical was soluble in the diluent water, then the 500 ml volume of test solution was divided into three 150 ml aliquots in 250 ml beakers to provide triplicate exposures. The remaining 50 ml of control, high, middle, and low test concentrations were used to measure the 0 hour dissolved oxygen concentration and pH of the solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna
- Source: from stocks cultured at EG&G Bionomics, Wareham, USA
- Age at study initiation (mean and range, SD): ≤ 24 hours old
- Weight at study initiation (mean and range, SD):
- Method of breeding: Water used to culture the organisms was deionized reconstituted well water having a total hardness of 72 ± 6 mg/l as CaCO3 and a pH of 7.0 ± 0.2. Subsequently, culture water was reconstituted according to U.S. EPA (1975) to a total hardness of 173 ± 13 mg/l as CaCO3 and a pH of 8.0 ± 2.0, to improve conditions for test organisms.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
not applicable
Hardness:
173 ± 13 mg/l as CaCO3
Test temperature:
22 ± 1 °C
pH:
7.4 - 9.4
Dissolved oxygen:
At the initiation of all tests, the dissolved oxygen concentration of diluent water was greater than 60% of saturation. Dissolved oxygen concentrations ranged from 6.5-9.1 mg/l for all tests during the 48-hour exposure period. Within any one test, the greatest range observed was 6.6-8.1 mg/l.
Salinity:
no information available
Nominal and measured concentrations:
Five to 8 nominal concentrations of each chemical were tested. A negative control and, if appropriate, a solvent control were maintained concurrently with each test
Details on test conditions:
Daphnia magna (≤ 24 hours old) from stocks cultured at EG &G Bionomics, Wareham, USA were used. Water used to culture the organisms was deionized reconstituted well water having a total hardness of 72 ± 6 mg/l as CaCO3 and a pH of 7.0 ± 0.2. Subsequently, culture water was reconstituted according to U.S. EPA (1975) to a total hardness of 173 ± 13 mg/l as CaCO3 and a pH of 8.0 ± 2.0, to improve conditions for test organisms. At the initiation of all tests, the dissolved oxygen concentration of diluent water was greater than 60% of saturation. Temperature of test solutions was maintained at 22 ± 1 °C. The chemical was added to 500 ml of diluent water in 2 litre jars to prepare each test solution. If the chemical was soluble in the diluent water, then the 500 ml volume of test solution was divided into three 150 ml aliquots in 250 ml beakers to provide triplicate exposures. The remaining 50 ml of control, high, middle, and low test concentrations were used to measure the 0-hour dissolved oxygen concentration and pH of the solutions.
Five daphnids were randomly placed in each 150 ml test solution within 30 minutes of the solution preparation. If the test material was not soluble in the diluent water, the 500 ml test mixtures were not divided into triplicate test vessels but retained in the 2 litre jars. Fifteen daphnids were placed directly into the 2 litre jars containing diluent water prior to addition of the test material. The tests were also conducted in unreplicated 500 ml solutions containing 15 daphnids if dividing the solutions into triplicate test vessels presented a risk of the loss of the test substance through volatilization or if vapors of the substance posed a high health risk to the investigators. Which one of these methods was used for testing dibutyl ether is unknown. Vessels were covered with plastic wrap secured with an elastic band
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% CL (26-36 mg/l)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% CL (21-31 mg/l)
Details on results:
The 24 hour EC50 of dibutyl ether to Dapnhia magna was 32 mg/l with 95% CL ranging 26-36 mg/l and the 48 hour EC50 of dibutyl ether to Dapnhia magna was 26 mg/l with 95% CL ranging 21-31 mg/l. The no discernable effect concentration at 48 hours was 4.6 mg/l.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Median lethal concentration (LC50) and its 95% confidence limits based on nominal concentrations were calculated using a moving average angle method, probit analysis, or binominal probability analysis. No corrections were made for control mortality, however control mortality did not exceed 10 %.

None

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 24 hour EC50 of dibutyl ether to Dapnhia magna was 32 mg/l with 95% CL ranging 26-36 mg/l and the 48 hour EC50 of dibutyl ether to Dapnhia magna was 26 mg/l with 95% CL ranging 21-31 mg/l. The no discernable effect concentration at 48 hours was 4.6 mg/l.
Executive summary:

Daphnia magna (≤ 24 hours old) from stocks cultured at EG &G Bionomics, Wareham, USA were used. Water used to culture the organisms was deionized reconstituted well water having a total hardness of 72 ± 6 mg/l as CaCO3 and a pH of 7.0 ± 0.2. Subsequently, culture water was reconstituted according to U.S. EPA (1975) to a total hardness of 173 ± 13 mg/l as CaCO3 and a pH of 8.0 ± 2.0, to improve conditions for test organisms. At the initiation of all tests, the dissolved oxygen concentration of diluent water was greater than 60% of saturation. Temperature of test solutions was maintained at 22 ± 1 °C. The chemical was added to 500 ml of diluent water in 2 litre jars to prepare each test solution. If the chemical was soluble in the diluent water, then the 500 ml volume of test solution was divided into three 150 ml aliquots in 250 ml beakers to provide triplicate exposures. The remaining 50 ml of control, high, middle, and low test concentrations were used to measure the 0-hour dissolved oxygen concentration and pH of the solutions.

Five daphnids were randomly placed in each 150 ml test solution within 30 minutes of the solution preparation. If the test material was not soluble in the diluent water, the 500 ml test mixtures were not divided into triplicate test vessels but retained in the 2 litre jars. Fifteen daphnids were placed directly into the 2 litre jars containing diluent water prior to addition of the test material. The tests were also conducted in unreplicated 500 ml solutions containing 15 daphnids if dividing the solutions into triplicate test vessels presented a risk of the loss of the test substance through volatilization or if vapors of the substance posed a high health risk to the investigators. Which one of these methods was used for testing dibutyl ether is unknown. Vessels were covered with plastic wrap secured with an elastic band.

During the tests, the dissolved oxygen concentration, pH, and temperature of test solutions were measured at the initiation and the termination of the toxicity tests in the high, middle, and low concentrations and the controls. Dissolved oxygen concentrations ranged from 6.5-9.1 mg/l for all tests during the 48-hour exposure period. Within any one test, the greatest range observed was 6.6-8.1 mg/l. The range of pH values measured for tests conducted in water with a mean hardness of 173 mg/l as CaCO3 (like in the test with dibutyl ether) was 7.4-9.4. Test animals were observed at 24 and 48 hours of exposure and any mortality was recorded. Median lethal concentration (LC50) and its 95% confidence limits based on nominal concentrations were calculated using a moving average angle method, probit analysis, or binominal probability analysis. No corrections were made for control mortality, however control mortality did not exceed 10 %.

Under the conditions of the study, the 24 hour EC50 of dibutyl ether to Dapnhia magna was 32 mg/l with 95% CL ranging 26-36 mg/l and the 48 hour EC50 of dibutyl ether to Dapnhia magna was 26 mg/l with 95% CL ranging 21-31 mg/l. The no discernable effect concentration at 48 hours was 4.6 mg/l.

Description of key information

At the request of ECHA a new study was conducted to examine the acute toxicity of dibutyl ether on Daphnia magna in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. The 24h and 48h EC50 value in this study was > 18.76 mg/L (measured).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
18.76 mg/L

Additional information