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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At test initiation (new solutions), at the 24-hour renewal interval (new solutions) and test termination (aged solutions), one sample was removed from each test solution and the controls for analysis of methyl isoamyl ketone concentration. Samples removed at test initiation and new solutions at the 24-hour test interval were taken from the intermediate vessels prior to division into replicate test vessels. The aged samples at test termination were a composite of all replicates within each concentration. Three quality control (QC) samples were also prepared at each sampling interval at nominal methyl isoamyl ketone concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.

The pH, dissolved oxygen concentration and temperature were measured at 0 (new solutions), 24 (new and aged solutions) and 48 (aged solutions) hours. Measurements at 0 hour and 24 hours (new) were taken from the intermediate mixing vessels, measurements at 24 hours (aged) were taken from a composite of replicates A through D and measurements at 48 hours (aged) were taken from replicate A of the treatment levels and the control. Continuous temperature monitoring was performed in the waterbath containing the test vessels throughout the exposure period.
Vehicle:
no
Details on test solutions:
A 100 mg/L stock solution was prepared prior to test initiation and at the 24-hour renewal period by placing approximately 0.3000 g (range of 0.3003 to 0.3004 g) of test substance in a 3.0-L intermediate vessel and bringing it to volume with dilution water. The resultant solution was mixed with a glass rod for one minute. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. The 100 mg/L primary stock solution was used to prepare the exposure solutions. The test solutions were mixed with a glass rod for approximately one minute prior to division into replicate exposure vessels. Following mixing, all test solutions were observed to be clear and colorless with no visible undissolved test substance. Each test solution was divided into four replicate vessels, each containing approximately 290 mL of solution. A set of control solutions was also prepared containing dilution water only. A similar procedure was used to prepare test solutions at the 24-hour solution renewal.
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers Laboratories. The culture water was prepared by fortifying well water based on the formula for hard water and filtering it through an Amberlite XAD 7 resin column to remove any potential organic contaminants. The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light at an intensity of 58 to 77 footcandles (620 to 830 lux) at the surface of the culture solutions was provided by fluorescent bulbs. Daphnids were fed a unicellular green algae, Ankistrodesmus falcatus (4 x 107 cells/mL), in addition to a suspension of YCT (yeast, cereal leaves and flaked fish food) daily. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. Daphnids, < 24 hours old, were impartially selected and distributed to intermediate vessels by adding no more than two daphnids to each vessel until all vessels contained two daphnids. This process was repeated until each vessel contained five daphnids. The test was initiated when the daphnids from each intermediate vessel were added to each exposure vessel. Daphnids were carefully transferred at the renewal (24 hours) period to new test solutions using a wide bore pipet. Daphnids were not fed during the exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L as CaCO3
Test temperature:
Continuous temperature monitoring in the waterbath containing the test vessels established a temperature range of 18 to 20 ºC during the exposure period.
pH:
8.0 -8.2
Dissolved oxygen:
7.9-9.4 mg/L
Salinity:
specific conductivity of 550 µmhos/cm
Nominal and measured concentrations:
Nominal Concentrations: 6.3, 13, 25, 50 and 100 mg/L
Mean measured concentrations ranged from 79 to 95% of nominal and defined the treatment levels as 5.0, 12, 23, 45 and 91 mg/L.
Details on test conditions:
The toxicity test was conducted in 250-mL glass screw-top flasks, each containing approximately 290 mL of test solution. These flasks were filled to capacity and capped to reduce the amount of headspace in the vessels. Four replicate test vessels were established for each concentration and the control. Each test vessel was labeled with the study number, concentration and replicate designation. The test area was illuminated with fluorescent bulbs at an intensity range of 79 to 88 footcandles (850 to 950 lux) at the solutions’ surface. Light intensity was measured with a VWR Traceable light meter. The test vessels were placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 20 ± 1 ºC. Test concentrations were selected based on preliminary testing conducted at Springborn Smithers). Based on these results and consultation with the Study Sponsor, the nominal concentrations selected for the definitive test were 6.3, 13, 25, 50 and 100 mg/L. The number of immobilized daphnids in each replicate test vessel was recorded at 24 and 48 hours of exposure. Immobilization was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 91 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Following 48 hours of exposure, 5% immobilization was observed among daphnids exposed to the 12 mg/L nominal treatment level. No immobilization or adverse effects were observed among daphnids exposed to the control or any of the remaining treatment levels tested (5.0, 23, 45 and 91 mg/L). Since a clear dose-response was not established, the immobilization observed among daphnids exposed to the 12 mg/L treatment level was not considered to be toxicant-related.
Reported statistics and error estimates:
The 48-hour EC50 value for for Daphnia magna and methyl isoamyl ketone was extrapolated to be >100 mg a.i./L (mean measured concentration) using linear regression. As no effects were observed in the highest test concentration the NOEC was determined emperically.
Validity criteria fulfilled:
yes
Executive summary:

The acute toxicity to Daphnia magna was evaluated under semi-static renewal conditions following OECD Guideline #202. The tests was conducted using a closed sytem with minimal headspace to avoid losses due to volatility. The test was conducted using mean measured concentrations of 5.0, 12, 23, 45 and 91 mg/L. Based on mean measured concentrations, the 48‑hour EC50 value for Daphnia magna and methyl isoamyl ketone was empirically estimated to be > 91 mg/L, the highest nominal concentration tested. The 48-hour EC50 value for Daphnia magna and methyl isoamyl ketone was extrapolated to be > 100 mg a.i./L (mean measured concentration) using linear regression. The No-Observed-Effect Concentration (NOEC) was determined to be≥ 91 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-GLP study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The exposure solution was prepared by the addition of the appropriate amount of the test substance to a glass vessel containing 20 L of dilution water. A nominal exposure concentration of 100 µL/L was prepared for this test. Acetone at 0.5 mL/L final test volume was used to enhance dispersion of the test substance.
Test organisms (species):
Daphnia magna
Details on test organisms:
Adult Daphnia magna were reared within 100-L culturing tanks located within the Testing Facility. The gravid daphnids used to produce the test animals for the study were obtained from rearing tanks that had been established for at least two weeks. Prior to the study, approximately 100 gravid daphnids were transferred by net into two glass bowls containing diluent water and food. After approximately 18 hours in these bowls, all adult daphnids were removed by using nets and pipettes. From this source daphnid neonates were transferred from culturing bowls into stainless steel mesh baskets suspended in the exposure solution vessels.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Not reported
Test temperature:
19 °C (measured)
pH:
Control: 7.5-7.9
100 µL/L: 7.4-7.9
Dissolved oxygen:
Control: 5.9-8.7 mg/L
100 µL/L: 4.8-8.6 mg/L
Salinity:
Not reported
Nominal and measured concentrations:
100 µL/L nominal only
Details on test conditions:
The water used in the test was pumped from a large underground storage reservoir located near the Testing Facility. This water subsequently was pumped into the laboratory where it passed through polypropylene filter tubes, then through a series of activated carbon filter tubes, and finally through another set of polypropylene filter tubes. The filtered water stream was then treated with sodium thiosulfate via a chemical injection system to further reduce trace levels of residual chlorine. The filtered-treated water was then tempered to 20 ± 2 °C by passage through a heat-exchange unit and distributed throughout the testing facility through stainless steel piping. Upon reaching the laboratory, the filtered-treated-tempered water cascaded into an open aeration basin for seasoning prior to use. All test organisms were acclimated to the diluent water prior to the test since the same filteredtreated-tempered water and filtered, compressed air used for all laboratory water/aeration processes during the test were supplied continuously to the rearing tanks. All aquatic organism populations used in the laboratory were maintained in this water for at least two weeks before being used in the test. The acute test was perfonned by placing the organisms in stainless steel mesh baskets suspended in glass aquaria containing 20 L of exposure solution. Each basket was suspended from a small motor that slowly raised and lowered the position of the basket within the test solution. The light/dark cycle of the photoperiod during the test was 16 hours on/8 hours off, with a 20-minute transition period. At test start (time 0), the exposure solutions were prepared, their physical parameters were measured, and then ten test organisms were introduced into each test vessel. Observations for immobility and signs of stress were made during this test at 0, 6, 24, 48, 72, and 96 hours. At 24 hour intervals from test start, physical parameters of the solutions were measured.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The 24-, 48-, 72-, and 96-hour EC50 values for Daphnia magna were estimated to be>100 µL/L. The highest tested concentration causing no immobility (NOEC) within the period of the test was determined to be 100 µL/L. The Daphnia magna in the dilution water controls exhibited normal behavior and appearance throughout the test.
Validity criteria fulfilled:
yes
Conclusions:
This is a pre-GLP study conducted in 1978 with preparation of a final report from lab records in 2000. While the study was conducted as a limit test and with nominal concentrations, the procedures were adequate for that time period.
Executive summary:

The acute toxicity of Methyl Isoamyl Ketone to Daphnia magna was determined in a 96-hour, static, aquatic effects limit test. The exposure solution was prepared by the addition of the appropriate amount of the test substance to a glass vessel containing 20 L of dilution water. A nominal exposure concentration of 100 µL/L was prepared for this test. The 24-, 48-, 72-, and 96-hour EC50 values for Daphnia magna were estimated to be>100 µL/L. The highest tested concentration causing no immobility (NOEC) within the period of the test was determined to be 100 µL/L.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

The acute toxicity to Daphnia magna was evaluated under semi-static renewal conditions following OECD Guideline #202. The test was conducted using a closed system with minimal headspace to avoid losses due to volatility. The test was conducted using mean measured concentrations of 5.0, 12, 23, 45 and 91 mg/L. Based on mean measured concentrations, the 48-hour EC50 value for Daphnia magna and methyl isoamyl ketone was empirically estimated to be > 91 mg/L, the highest nominal concentration tested. The 48-hour EC50 value for Daphnia magna and methyl isoamyl ketone was extrapolated to be > 100 mg a.i./L (mean measured concentration) using linear regression. The No-Observed-Effect Concentration (NOEC) was determined to be ≥ 91 mg/L.