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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published pre-GLP study conducted by reliable methodology and with sufficent documentation
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Analytical monitoring:
All test exposure chambers were sampled at approximately mid-depth at 0 and 96 hours, and one of each duplicate exposure chamber at 24, 48, and 72 hours. All samples were analyzed immediately or adequately preserved for later analysis.

Water Quality Monitoring:
Temperature, dissolved oxygen, total hardness, total alkalinity, and pH were routinely measured in the tests. Water temperature was determined using a partial immersion mercury thermometer. Measurements were made in each exposure chamber daily. Dissolved oxygen was determined in the high, medium, lowand control exposure chambers at least three times (0, 24, 96 hours) during a test. Determinations were made with an oxygen sensitive electrodode (Yellow Springs Instrument Model 54) which was calibrated weekly using the azide modification of the Winkler method. Total hardness was determined at least once for each test using the compleximetric or EDTA method. Total alkalinity was determined at least once for each test by a titrametric method. The pH was measured at least once during each test in the high, medium, low and control exposure chambers. Measurments were made with a pH meter which had been calibrated shortly prior to each test.
Vehicle:
no
Details on test solutions:
The majority of the tests reported in this publication were conducted at the U.S. Environmental Protection Agency (USEPA) Environmental Research Laboratory-Duluth , Duluth, MN using Lkae Superior water. A few tests were conducted at the University of Wisconsin-Superior campus using dechlorinated City of Superior, WI water. The two waters were similar in all measured chemical parameters. The test was conducted with a flow-through cycling proportional diluter (Mount and Brungs, 1967) with duplicate exposures for every tested concentration. The test substance was introduced by direct injection of the full strength sample to provide nominal exposure concentrations of 0, 125, 208, 447, 579, and 965 mg/L. The diluter provided 4.5 tank volumes of exposure solutions per day.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The fish utilized in the study were cultured from brood stock provided by the USEPA Environmental Research Laboratory-Duluth. Adults were held at 25 °C in flowing water with a 16 hour light controlled photperiod and fed frozen adult brine shrimp. They were provided spawing substrate and eggs were spawned and fertilized on the undersides of these substrates. The embryos were tended by the male parent until eye-up at which time the substrates were removed to another 25 °C bath where hatching occured. Fry of similar age (within 24 hours) were transferred to rearing tanks and were fed freshly hatched brine shrimp to excess three times daily until 24 hours before a test. From these fish 30 day old fish were utilized in this test. Two replcates of 25 fish were used for each exposure concentration.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
43 mg/L as CaCO3
Test temperature:
25.1 °C (Standard Deviation = 0.34)
pH:
7.6 (Standard Deviation = 0.00)
Dissolved oxygen:
6.5 (Standard Deviation = 0.58)
Nominal and measured concentrations:
Nominal: 0, 125, 208, 447, 579, and 965 mg/L.
Measured average corrected for percent recovery: (replicate 1/replicate 2): (0/0), ( 27/23), (49/41), (83/80), (136/133), (186/189)
Details on test conditions:
During exposures to toxicants the fish were routinely observed for behavioral reponses and death. Death was defined as the cessation of opercular movements and the inability to respond when prodded. Dead fish were removed and recorded at 3, 6, 12, 24, 48, 72, and 96 hours from initial exposure. Observations of affected and dead fish exposed to toxicants in duplicate exposures were combined, resulting in a single LC50 and EC50 estimate.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
159 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
159 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
behaviour
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 135 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
For the mortality LC50 endpoint complete mortality was observed in the highest exposure concentration and no mortalities observed in the control and 4 lowest exposure concentrations. For the behavior EC50 endpoint all fish in the highest concentration and 1 fish in the next to the highest concentration were observed to lose equilibrium and they did not school prior to death.
Reported statistics and error estimates:
Estimation of the LC50 and EC50 was made using the Trimmed Spearman-Karber method. Confidence limits (95%) were calculated when possible.
Validity criteria fulfilled:
yes
Executive summary:

A 96-hour acute toxicity test of 5-methyl-2 -hexanone (CAS 110 -12 -3) was conducted using fathead minnows (Pimephales promelas) in a flow-through diluter system. The test was conducted with 5 exposure concentrations and a control with two replicates of 25 juvenile fish in each exposure replicate. LC50 (mortlity) and EC50 (behavior) values were calculated based upon mean measured exposure concentrations. The LC50 and EC50 values were both estimated to be 159 mg/L. A NOEC value for mortality was not calculated in the study, but as there was no mortality observed in any concentration up to and including the 135 mg/L concentration, the NOEC for mortality can be emperically estimated to be ≥ 135 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Water Quality Measurements
Total hardness and alkalinity of the test solutions were determined according to APHA et al. Specific conductivity was monitored with a YSI Model No. 33 salinity-conductivity-temperature (SCT) meter. Total hardness, alkalinity and specific conductivity were measured at 0 hour in each treatment level and the control. Dissolved oxygen concentration, temperature and pH were measured once daily in each treatment level aquaria and the control throughout the exposure period. The pH was measured using a Yellow Springs Instrument (YSI) Model pH100 pH meter and combination electrode; the dissolved oxygen concentration and daily temperature were measured with either a YSI Pro 20 or YSI Model No. 550A dissolved oxygen meter/temperature probe. Test solution temperature was continuously monitored during the definitive test in the 6.3 mg a.i./L (nominal) solution using a VWR Minimum/Maximum thermometer.

Analytical Measurements
The high, middle and low treatment levels and the dilution water control were sampled once prior to the start of the definitive exposure and analyzed for the test substance concentration present in each vessel. Results of these pretest analyses were used to judge whether sufficient quantities of methyl isoamyl ketone (MIAK) were being delivered and maintained in the exposure in order to initiate the definitive exposure. During the 96-hour definitive study, samples were removed from each treatment level and the controls at each sampling interval (i.e., 0 and 96 hours). Samples were collected from the approximate midpoint of the test vessel by volumetric pipet. Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples through the analytical process. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
no
Details on test solutions:
The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter. During the definitive study, methyl isoamyl ketone (MIAK) (density 0.888 g/mL, 888 mg/mL) was delivered directly into the diluter’s mixing chamber via syringe. The solution was observed to be clear and colorless. A Glenco® 100-mL gas-tight syringe in conjunction with a Harvard Apparatus syringe pump was calibrated to deliver 0.0861 mL/cycle of the 888 mg a.i./mL methyl isoamyl ketone (MIAK) solution into the diluter system's chemical mixing chamber which also received 0.765 L of dilution water per cycle. The mixing chamber was positioned over a magnetic stir plate and partially submerged within an ultrasonic water bath which aided in mixing the stock solution with the dilution water. The concentration of methyl isoamyl ketone (MIAK) in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (100 mg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (50, 25, 13 and 6.3 mg a.i./L). The diluter system was calibrated prior to test initiation and at test termination by measuring delivery volumes of test substance and dilution water. The function of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check was performed twice each day. The exposure system was in operation for five days prior to initiation of the definitive exposure to allow equilibration of the test substance in the diluter apparatus and exposure vessels. Each glass exposure aquarium measured 30 x 15 x 20 centimeters (cm). Water depth was maintained at a constant level by an overflow drain 14.5 cm from the bottom of each aquarium. The total test solution volume was therefore maintained at 6.8 L. The flow of exposure solution provided approximately ten solution volume replacements per day in order to provide a 90% test solution replacement rate of approximately six hours.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The juvenile rainbow trout used during this study (SSL Lot No. 10A27) were obtained from TroutLodge, Inc., Sumner, Washington. Prior to testing, these fish were held in a 500-L fiberglass tank under a photoperiod of 16 hours light and 8 hours darkness. The fish used during the definitive exposure were maintained under these conditions for at least 14 days prior to testing. The fish were fed a dry commercial flaked fish food (Ziegler Brothers Prime Tropical Flaked Fish Food), ad libitum, daily. Fish were not fed during the 24-hour period prior to test initiation or during the exposure period. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analyzed. Based on the analysis for pesticides, PCBs and toxic metals, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern. No mortality was observed among the test fish population during the 48-hour period prior to testing. A representative sample (N = 30) of the fish from the test population had a mean wet weight of 0.90 g (range 0.56 to 1.2 g) and a mean total length of 47 mm (range 39 to 55 mm).
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
72-76 mg/L as Ca CO3
Test temperature:
13-15 °C
pH:
6.6 - 7.2
Dissolved oxygen:
8.8-10 mg/L
Nominal and measured concentrations:
Nominal: 0, 6.3, 13, 25, 50, 100 mg a.i./L
Measured: 0, 4.8, 11, 20, 37, 74 mg a.i./L
Details on test conditions:
The dilution water (well water) used during this study was from the same source as the water used to culture rainbow trout and was characterized as having a total hardness and total alkalinity range (as CaCO3) of 68 mg/L and 18 to 20 mg/L, respectively, a pH range of 6.9 to 7.0 and a specific conductance range of 430 to 450 μmhos/cm. The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brung), a temperature-controlled water bath and a set of seven exposure vessels. The exposure system, constructed entirely of glass and silicone sealant, was designed to provide five concentrations of the test substance and a dilution water control. Test vessels were impartially positioned in a water bath containing circulating chilled water to maintain the test solution temperatures at 14 ± 1 ºC. Test solutions were not aerated. Exposure vessels were maintained throughout the 96-hour test in an area illuminated by Sylvania Oktron fluorescent bulbs at an intensity of 34 to 61 footcandles at the surface of the test solutions. The photoperiod was 16 hours light, 8 hours dark. Sudden transitions from light to dark and vice versa were avoided. Light intensity was measured with a VWR Traceable light meter. The definitive study was initiated when rainbow trout were selected impartially from the holding tank. Fish were added two at a time until all tanks contained two fish. This procedure was repeated until each aquarium contained ten fish for each treatment level and the control. The resulting test organism loading concentration was 0.13 g/L grams of biomass per liter of solution per day. All aquaria were examined after 0, 24, 48, 72 and 96 hours of exposure as follows: mortalities were recorded and dead fish removed, biological observations, including sub-lethal effects (e.g., lethargy, loss of equilibrium) of the exposed rainbow trout, and observations of the physical characteristics of the test solutions (e.g., presence of precipitate, film on the solution's surface) were made and recorded. Effects for this study were based on death, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).
Reference substance (positive control):
not required
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Details on results:
No mortality was observed in the test. Since no concentration tested resulted in ≥ 50% mortality, the 96-hour LC50 value for methyl isoamyl ketone (MIAK) and Oncorhynchus mykiss was empirically estimated to be > 74 mg a.i./L, the highest mean measured concentration tested. The No Observed Effect Concentration (NOEC) was determined to be ≥ 74 mg a.i./L.
Reported statistics and error estimates:
As no mortality was observed int he study the LC50 and NOEC were empirically estimated.
Validity criteria fulfilled:
yes
Executive summary:

The acute toxicity of methyl isoamyl ketone (MIAK) to rainbow trout (Oncorhynchus mykiss) was determined under flow-through conditions following OECD Guideline #203. This exposure was conducted at the maximum nominal concentration required by the OECD guideline (100 mg/L). However, due to the behavior of the compound under test conditions, recoveries did not closely approximate nominal exposure concentrations. No mortality was observed in the 96 hour study. Since no concentration tested resulted in mortality, the 96-hour LC50 value for methyl isoamyl ketone (MIAK) and Oncorhynchus mykiss was empirically estimated to be > 74 mg a.i./L, the highest mean measured concentration tested. The No-Observed-Effect Concentration (NOEC) was determined to be ≥ 74 mg a.i./L. Since no mortality was observed during this exposure, there was no possibility of extrapolation of the LC50 value. Based on the highest no-effect level observed and the fact that an extraordinarily steep dose-response curve would be required to generate an LC50 value between 74 and 100 mg a.i./L, it is reasonable to assert that the LC50 value is likely to be > 100 mg a.i./L. Additional testing to define a calculated LC50 value would require the use of additional vertebrates and other resources and would likely only confirm that the LC50 value is greater than 100 mg a.i./L.

Endpoint:
short-term toxicity to fish
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.1 (Acute Toxicity for Fish)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The exposure solution was prepared by the addition of 100 µL/L of the test substance to a glass vessel containing 20 L of dilution water. Acetone (0.5 mL/L final test volume) was mixed with the test substance to enhance dispersion.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Not reported
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
100 µL/L: 4.8-8.6
Salinity:
not reported
Nominal and measured concentrations:
100 uL/L nominal only
Details on test conditions:
The water used in this test was pumped from Lake Ontario by the Kodak Park Lake Station Water Treatment Facility into 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. The acute aquatic effects test was a static exposure and was performed in seamless pyrex glass 30.5-cm cuboidal chromatography jars. Each test vessel contained approximately 20 L of exposure solution. After the exposure solutions were prepared, fathead minnows, as uniform in size as possible, were removed from the holding tanks and transferred to the test vessels. The light/dark cycle of the photoperiod was 16 hours on/8 hours off with a 20-minute transition period. Observations for mortality and signs of stress were made during this test at 0, 6, 24, 48, 72, and 96 hours. The temperature, dissolved oxygen concentration, and pH of each exposure solution were measured at test start and 24-hour intervals from test start to test end.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: µL/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
The 96-hour LC50 value for Pimephales promelas was determined to be >100 µL/L. The highest tested concentration causing no mortality within the period of the test was determined to be 100 µL/L.
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 the fathead minnow (Pimephales promelas) was detennined in a 96-hour, static, aquatic effects 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 LC50 values for Pimephales promelas were estimated to be >100 µL/L. The highest tested concentration causing no mortality within the period of the test was 100 µL/L. The minnows in the diluent water control and the vessel containing the test substance exhibited nonnal behavior and appearance throughout the test.

Description of key information

Three reliable studies using two species of fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
159 mg/L

Additional information

Three short-term 96 -hour acute fish studies have been conducted with methyl isoamyl ketone (5 -methly-2 -hexanone). One study with fathead minnows (Pimephales promelas) was conducted as a limit test at a nominal concentration of 100 µL/L and no mortalities were observed. Two additional studies were conducted as flow-through studies with measured exposure concentrations. Rainbow trout were exposed to 5 nominal concentrations up to 100 mg/L in a flow-through system. In that study no mortality was observed up through the highest test concentration which had a mean measured concentration of 74 mg/L. The key study for this endpoint was a second flow-through study with fathead minnows that resulted in a LC50 of 159 mg/Land an NOEC for mortality of 135 mg/L (both endpoints based upon mean measured concentrations).