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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
Study period:
20 Mar 2017 to 24 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
2016
Principles of method if other than guideline:
- This study was conducted according to the test facility protocol entitled “Acute Toxicity to Rainbow Trout (Oncorhynchus mykiss) Under Flow-Through Conditions”. The methods described in this protocol meet the testing requirements specified in the OECD TG 203 Guideline (OECD, 1992) and the Ecological Effects Test Guidelines OCSPP 850.1075 Fish Acute Toxicity Test, Freshwater and Marine (U.S. EPA, 2016).
- For deviations, see Deviations from the Protocol in "Any other information on materials and methods incl. tables".
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour), the 48-hour interval, and exposure termination (96 hours), two samples from each treatment level and control were collected. One sample was analyzed for the test substance concentration while the duplicate was stored frozen as an archive backup sample. Each sample was collected from the approximate midpoint of the test vessel using a pipet.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
Prior to exposure initiation, a 10 mg/mL primary stock solution was prepared by placing 4.9905 g of the test substance in a glass volumetric flask and bringing it to a full volume of 500 mL with DMF. This stock solution was used to prepare a series of secondary solutions. The preparation scheme is provided in Table 1 in "Any other information on materials and methods incl. tables".
The resulting stock solutions were mixed by inversion of the flasks. The 10, 5.0, and 2.5 mg/mL solutions were observed to be clear with a slight yellow tint and no visible undissolved test substance. The yellow tint was observed to decrease with decreasing concentrations. The 1.3 and 0.63 mg/mL solutions were observed to be clear and colorless with no visible undissolved test substance.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Commercial supplier
- Mean length at study initiation: 43 mm (range: 40 to 49 mm)
- Mean wet weight at study initiation: 0.78 g (range: 0.52 to 1.0 g)

ACCLIMATION
- Acclimation period: 14 days
- Acclimation contrainer: 500 L fiberglass tank
- Photoperiod: 16 hours light and 8 hours darkness
- Water source: From an on-site 100 meter bedrock well supplemented with dechlorinated well water
- Water hardness: 60 to 62 mg/L as CaCO3
- Total alkalinity: 22 to 24 mg/L as CaCO3
- Conductivity: 450 µS/cm
- pH: 7.3 - 7.5
- Temperature: 12 to 14 °C
- Dissolved Oxygen: 96 to 102% saturation
- Type and amount of food during acclimation: commercially prepared fish food
- Feeding frequency during acclimation: At least once daily (Fish were not fed during the 48 hours prior to exposure initiation nor during the 96-hour exposure period)
- Food quality: Food source were analyzed periodically for the presence of toxic metals, pesticides, and PCBs. None of these compounds have been detected at concentrations that are considered toxic in the food samples analyzed, in agreement with ASTM (ASTM, 2007) standard practice. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern.
- Health during acclimation (any mortality observed): No mortality was observed among the test fish population during the 7 days prior to testing.

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
60 mg/L as CaCO3
Test temperature:
12 ± 1 °C
pH:
6.9
Dissolved oxygen:
- 7.9 to 10 mg O2/L
- 73 to 95% of saturation
Conductivity:
450 µS/cm
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 0 (solvent control), 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
- Measured concentration: < MDL (negative contirl), < MDL (solvent control), 0.066, 0.12, 0.23, 0.42, and 0.94 mg/L, respectively. See Table 2 in 'Any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Material of vessel: Constructed entirely of glass and silicone sealant
- Size of vessel: 30 × 15 × 20 cm
- Filled volume: Water depth was maintained at a constant level by an overflow drain 15 cm from the bottom of each aquarium. The total test
solution volume was therefore maintained at 6.8 L.
- Type of flow-through: Proportional diluter
- Flow rate: Based on the flow of water into each aquarium, approximately six volume replacements occurred per day in order to provide a
90% test solution replacement rate of approximately 9 hours.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Exposure initiation: The definitive exposure was initiated when seven rainbow trout were impartially selected from the holding tank and placed, no more than two at a time, into each test aquarium. This procedure was repeated until each aquarium contained seven fish.
- Biomass loading rate: 0.1 g of biomass per liter of solution per aquarium per day
- Exposure system for trestment: Prior to exposure initiation, a pump in conjunction with six 25 mL gas-tight syringes, was calibrated to deliver 0.0402 mL/cycle of the appropriate stock solution or solvent (DMF) into individual diluter chemical mixing chambers, which received approximately 0.402 L of dilution water per cycle. The mixing chambers were positioned over magnetic stir plates and were stirred continuously throughout the exposure. The continuous stirring aided the solubilization of the test substance with the dilution water. All mixing chamber solutions, as well as all splitter and aquaria solutions, were observed to be clear and colorless with no visible undissolved material.
- Exposure system for solvent control: The solvent control solution was prepared by using a pump to deliver 0.0402 mL/cycle of DMF to 0.402 L of dilution water per cycle, which was subsequently delivered to the solvent control vessel. This delivery method ensured the DMF concentration in the solvent control was 0.10 mL/L, which was equivalent to each treatment level (0.10 mL/L).
- Exposure system for negative control: A negative control vessel was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no test substance or DMF.
- Calibration of diluter system: The diluter system was calibrated prior to exposure initiation and the calibration was confirmed at exposure termination by measuring each dilution water cell delivery volume into each mixing chamber (i.e., test substance), the dilution water, and the solvent control. 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 seven days prior to initiation of the definitive exposure to allow equilibration of the test substance in the diluter apparatus and exposure vessels. A flow-splitting box was used between the mixing chambers and exposure vessels to promote further mixing of the toxicant solution and the diluent water. One glass delivery tube exited each box, which transferred the test solution to the exposure vessel.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water (laboratory well water) used during this study was from the same source
as the water which flowed into the fish holding tanks.
- Total organic carbon: Representative samples of the dilution water were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 0.66 mg/L for March 2017. The TOCconcentration falls within the recommendation of the guideline (i.e., < 2.0 mg/L).
- Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement
with ASTM (ASTM, 2007) standard practice.
- Intervals of water quality measurement: Temperature was continuously monitored in the 0.25 mg/L treatment level on test days 0, 1, and 2 and in the 0.13 mg/L treatment level on test days 3 and 4 during the exposure. The minimum/maximum thermometer was moved to a new test vessel due to 100% mortality observed in the 0.25 mg/L treatment level at test day 2.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light : 8 hours dark (electronic timers were used to provide a 30 minute transition period).
- Light intensity and location: 55 to 73 footcandles (590 to 790 lux) at the solutions' surface

EFFECT PARAMETERS MEASURED:
- All aquaria were examined at 0, 6, 24, 48, 72, and 96 hours of exposure. Dead fish were recorded and removed, if observed. Biological observations of adverse effects (e.g., loss of equilibrium) of the rainbow trout were made and recorded 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, if applicable. Effects for this study were based on mortality, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

RANGE FINDING STUDY
- Study design: Rainbow trout were exposed under flow-through conditions to the test substance. One test vessel containing seven fish was established for each treatment level, the negative control, and solvent control.
- Nominal test concentrations: 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg/L, a negative control and solvent (DMF) control
- Results used to determine the conditions for the definitive study: Following 96 hours of exposure, 100% mortality was observed among fish exposed to the 1.0 mg/L treatment level. No mortality or adverse effects were observed in fish exposed to the remaining treatment levels tested (0.00010, 0.0010, 0.010, 0.10 mg/L), negative control, or solvent control. Based on these results and consultation with the Study Sponsor, nominal concentration of 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L, a negative control, and a solvent (DMF) control were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other:
Remarks:
95% C.L.: 0.10 - 0.16 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
An overview of the results is provided in Table 4 and Table 5 in 'Any other information on results incl. tables'
Following 24 hours of exposure, 100% mortality was observed in the 0.94 mg/L mean measured concentration. At test termination, mortality of 43, 100, and 100% was observed in the 0.12, 0.23, and 0.42 mg/L mean measured concentrations, respectively. One fish in the 0.12 mg/L mean measured concentration was observed to be on the bottom of the test vessel. No mortality or adverse effects were observed among fish exposed to the remaining treatment level tested (0.066 mg/L), the negative control, or solvent control during the exposure.
Sublethal observations / clinical signs:

Table 4. Mean Measured Concentrations Tested, Corresponding Cumulative Percent Mortality, Number of Mortalities, and Observations Made during the 96-Hour Flow-Through Acute Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test substance

Mean Measured Concentration (mg/L)

Cumulative Mortality and Sub-Lethal Effects

6-Hour

24-Hour

48-Hour

72-Hour

96-Hour

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

 

Negative Control

 

0 (0)

 

-

 

0 (0)

 

-

 

0 (0)

 

-

 

0 (0)

 

-

 

0 (0)

 

-

Solvent Control

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0.066

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0.12

0 (0)

-

0 (0)

-

0 (0)

-

29 (2)

1ERR

43 (3)

1B

0.23

0 (0)

-

0 (0)

-

100 (7)

NA

100 (7)

NA

100 (7)

NA

0.42

0 (0)

-

43 (3)

CL

100 (7)

NA

100 (7)

NA

100 (7)

NA

0.94

0 (0)

2PL

100 (7)

NA

100 (7)

NA

100 (7)

NA

100 (7)

NA

PL = partial loss of equilibrium; CL = complete loss of equilibrium; B = at bottom of exposure aquarium; ERR = Erratic Swimming; - = None; NA = Not Applicable

NOTE: The actual number of mortalities is presented in parentheses

Table 5. The LC50 Values, Corresponding 95% Confidence Intervals, and No-Observed-Effect Concentration (NOEC) Established During the 96-hour Flow-Through Acute Exposure of Rainbow Trout (Oncorhynchus mykiss) to the test substance.

Based on Mean Measured Concentrations

 

Time Interval

 

LC50

(mg/L)

95% Confidence Intervals (mg/L)

Lower

Upper

 

24-Houra

 

0.46

 

0.36

 

0.60

48-Hourb

0.17

0.12

0.23

72-Houra

0.14

0.11

0.17

96-Houra

0.13

0.10

0.16

96-Hour NOEC = 0.066 mg/L

a LC50 value and corresponding 95% confidence intervals were determined using Spearman-Kärber Estimates.

b LC50 value and corresponding 95% confidence intervals were determined using Binomial/Graphical Estimates.

Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations of the test substance, the 96-hour LC50 value was determined to be 0.13 mg/L, with 95% confidence intervals at 0.10 and 0.16 mg/L.
Executive summary:

To determine the 96-hour acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss), the exposure was conducted under flow-through conditions following OECD TG 203 and U.S. EPA Guideline 850.1075. The test was in compliance with GLP criteria. One aquarium, containing seven fish, was established for each treatment level, (i.e., 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L nominal and 0.066, 0.12, 0.23, 0.42, and 0.94 mg/L mean measured) negative control and solvent control (dimethylformamide, DMF). These vessels were maintained in a temperature-controlled water bath at a temperature of 11 to 13°C with a 16 hour light and 8 hour dark photoperiod at a light intensity of 55 to 73 footcandles (590 to 790 lux). Aquaria were examined at 0, 6, 24, 48, 72, and 96 hours of exposure as follows: dead fish were recorded and removed, biological observations including adverse effects (i.e., loss of equilibrium) of the exposed rainbow trout and observations of the physical characteristics of the test solutions (i.e., presence of precipitate, film on the solution's surface) were made and recorded, if applicable. Effects for this study were based on mortality, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

No sub-lethal effects or mortalities were observed among fish exposed to the negative control or solvent control during the exposure. Following 24 hours of exposure, 100% mortality was observed in the 0.94 mg/L mean measured concentration. By the 96-hour interval, mortality of 43, 100, 100, and 100% was observed in the 0.12, 0.23, 0.42, and 0.94 mg/L mean measured concentrations, respectively. One fish in the 0.12 mg/L mean measured concentration was observed to be on the bottom of the test vessel. No mortalities or sub-lethal effects were observed among fish exposed to the 0.066 mg/L mean measured concentration. Based on mean measured concentrations, the 96-hour LC50 was determined using Spearman-Kärber Estimates to be 0.13 mg/L, with 95% confidence intervals of 0.10 to 0.16 mg/L. The NOEC, based on lethal effects was empirically estimated to be 0.066 mg/L. The highest concentration producing no mortality was 0.066 mg/L and the lowest concentration producing 100% mortality was 0.23 mg/L.

 


Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Mar 2017 to 31 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Principles of method if other than guideline:
- This study was conducted according to the test facility protocol entitled 'Acute Toxicity to Sheepshead Minnow (Cyprinodon variegatus) Under Flow-Through Conditions'. The methods described in this protocol meet the testing requirements specified in the OECD TG 203 Guideline (OECD, 1992) and the Ecological Effects Test Guidelines OCSPP 850.1075 Fish Acute Toxicity Test, Freshwater and Marine (U.S. EPA, 2016).
- For deviations, see Deviations from the Protocol in "Any other information on materials and methods incl. tables".
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour), the 48-hour interval, and exposure termination (96 hours), two samples from each treatment level and control were collected. One sample was analyzed for the test substance concentration while the duplicate was stored frozen as an archive backup sample. Each sample was collected from the approximate midpoint of the test vessel using a pipet.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
Prior to exposure initiation, a 10 mg/mL primary stock solution was prepared by placing 4.9905 g of the test substance in a glass volumetric flask and bringing it to a full volume of 500 mL with DMF. This stock solution was used to prepare a series of secondary solutions. The preparation scheme is provided in Table 1 in 'Any other information on materials and methods incl. tables'.
The resulting stock solutions were mixed by inversion of the flasks. The 10, 5.0, and 2.5 mg/mL solutions were observed to be clear with a slight yellow tint and no visible undissolved test substance. The yellow tint was observed to decrease with decreasing concentrations. The 1.3 and 0.63 mg/mL solutions were observed to be clear and colorless with no visible undissolved test substance.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Sheepshead minnow
- Source: A commercial supplier
- Mean length at study initiation: 19 mm (range: 16 to 23 mm).
- Mean wet weight at study initiation: 0.16 g (range: 0.080 to 0.32 g)

ACCLIMATION
- Acclimation period: 14 days
- Acclimation containter: 30 L glass tank
- Photoperiod: 16 hours light and 8 hours darkness
- Salinity: 19 to 21‰
- Temperature: 17 to 23°C during the 14 days prior to exposure initiation and was 22°C during the seven days prior to exposure initiation
- Type of food during acclimation: Commercially prepared fish food
- Feeding frequency during acclimation: At least once daily. Fish were not fed during the 48 hours prior to exposure initiation nor during the 96-hour exposure period.
- Food quality: Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in the food samples analyzed, in agreement with ASTM (2007) standard practice. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern.

Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22 ± 1 ºC
pH:
7.4 - 7.8
Dissolved oxygen:
4.0 - 8.2 mg O2/L
Salinity:
19 - 21‰
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 0 (solvent control), 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
- Measured concentration: < MDL (negative control), < MDL (solvent control), 0.064, 0.14, 0.27, 0.54, and 0.94 mg/L, respectively. See Table 2 in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Material of vessel: Constructed entirely of glass and silicone sealant
- Size of vessel: 30 × 15 × 20 cm
- Filled volume: Water depth was maintained at a constant level by an overflow drain 15 cm from the bottom of each aquarium. The total test
solution volume was therefore maintained at 6.8 L.
- Type of flow-through: Proportional diluter
- Flow rate: Based on the flow of water into each aquarium, approximately six volume replacements occurred per day in order to provide a 90% test solution replacement rate of approximately 9 hours. Due to dropping dissolved oxygen levels at test day 1, the flow rate was increased to provide 10 volume replacements per vessel per day which provided a 90% test solution replacement rate of approximately five hours.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Exposure initiation: The definitive exposure was initiated when seven sheepshead minnow were impartially selected from the holding tank and placed, no more than two at a time, into each test aquarium. This procedure was repeated until each test aquarium contained seven fish.
- Exposure system for trestment: Prior to exposure initiation, a pump in conjunction with six 20 mL gas-tight syringes, was calibrated to deliver 0.0401 mL/cycle of the appropriate stock solution or solvent (DMF) into individual diluter chemical mixing chambers, which received approximately 0.401 L of dilution water per cycle. The mixing chambers were positioned over magnetic stir plates and were stirred continuously throughout the exposure. The continuous stirring aided the solubilization of the test substance with the dilution water. All mixing chamber solutions, as well as all splitter and aquaria solutions, were observed to be clear and colorless with no visible undissolved material.
- Exposure system for solvent control: The solvent control solution was prepared by using the pump to deliver 0.0401 mL/cycle of DMF to 0.401 L of dilution water per cycle, which was subsequently delivered to the solvent control vessels. This delivery method ensured the DMF concentration in the solvent control was 0.10 mL/L, which was equivalent to each treatment level (0.10 mL/L).
- Exposure system for negative control: A negative control vessel was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no the test substance or DMF.
- Caliberation of diluter system: The diluter system was calibrated prior to exposure initiation and the calibration was confirmed at exposure termination by measuring each dilution water cell delivery volume into each mixing chamber (i.e., test substance), the dilution water, and the solvent control. 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 seven days prior to initiation of the definitive exposure to allow equilibration of the test substance in the diluter apparatus and exposure vessels. A flow-splitting box was used between the mixing chambers and exposure vessels to promote further mixing of the toxicant solution and the diluent water. One glass delivery tube exited each box, which transferred the test solution to the exposure vessel.
- Biomass loading rate: 0.027 g of biomass per liter of solution per aquarium per day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water (natural, filtered seawater) used during this study was prepared by filtering natural seawater collected from the Cape Cod Canal, Bourne, Massachusetts. The water was collected at about 1 to 4 meters offshore and a depth of approximately 0.5 meters. The seawater was transferred with a pump (fiberglass reinforced thermoplastic housing) and a polyvinyl chloride (PVC) pipe and was then transported to the laboratory in a 6080 L polyethylene holding tank. In the laboratory, the seawater was diluted to a salinity of 20 ± 3‰ with laboratory well water and filtered through 20, 5, and 1 µm polypropylene core filters prior to use. The diluted seawater was mixed thoroughly prior to characterization and use. The seawater was pumped to the laboratory under constant pressure through
PVC pipe.
- Total organic carbon: Representative samples of the dilution water were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 1.3 mg/L for March 2017. The TOC concentration falls within the recommendation of the guideline (i.e., ≤ 2.0 mg/L).
- Salinity: 19 to 20‰
- pH: 7.5 to 7.9
- Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2007) standard practice.
- Intervals of water quality measurement: Temperature was continuously monitored throughout this study in the 0.25 mg/L nominal treatment level.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light : 8 hours dark. Electronic timers were used to provide a 30 minute transition period.
- Light intensity and location: 48 to 71 footcandles (520 to 760 lux) at the solutions’ surface

EFFECT PARAMETERS MEASURED:
All aquaria were examined at 0, 6, 24, 48, 72, and 96 hours of exposure. Dead fish were recorded and removed, biological observations of adverse effects (e.g., loss of equilibrium) of the sheepshead minnow were made and recorded, 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, if applicable. Effects for this study were based on mortality, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

RANGE FINDING STUDY
- Study design: Sheepshead minnow were exposed under flow-through conditions to the test substance. One test vessel containing three fish was established for each treatment level, negative control, and solvent control.
- Test nominal concentrations: 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg/L, a negative control, and a solvent control (DMF)
- Results used to determine the conditions for the definitive study: Following 96 hours of exposure, 100% mortality was observed among fish exposed to the 1.0 mg/L treatment level. All of the fish exposed to the 0.10 mg/L treatment level were observed to be lethargic. No mortality or adverse effects were observed in fish exposed to the remaining treatment levels tested (0.00010, 0.0010, 0.010 mg/L), negative control, or solvent control. Based on these results and consultation with the Study Sponsor, nominal concentrations of 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other:
Remarks:
95% C.L.: 0.22 - 0.37 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
An overvirew of the results is provided in Table 4 and Table 5 in 'Any other information on results incl. tables'. Following 72 hours of exposure, 100% mortality was observed among fish exposed to the 0.54 and 0.94 mg/L mean measured concentration. By the 96-hour interval, mortality of 43% was observed in the 0.27 mg/L mean measured concentration. No mortalities or sub-lethal effects were observed among fish exposed to the remaining treatment levels tested (0.14 or 0.064 mg/L), the negative control, or the solvent control during the exposure.
Sublethal observations / clinical signs:

Table 4. Mean Measured Concentrations Tested, Corresponding Cumulative Percent Mortality, Number of Mortalities, and Observations Made during the 96-Hour Flow-Through Acute Exposure of Sheepshead Minnow (Cyprinodon variegatus) to the test substance

Mean measured concentration (mg/L)

Cumulative mortality and sub-lethal effects

6-Hour

24-Hour

48-Hour

72-Hour

96-Hour

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

Mortality

Sub-lethal

 Negative Control

 0 (0)

 -

 0 (0)

 -

 0 (0)

 -

 0 (0)

 -

 0 (0)

 -

Solvent Control

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0.064

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0.14

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0 (0)

-

0.27

0 (0)

-

0 (0)

-

0 (0)

-

14 (1)

1CL, 5B

43 (3)

1PL, 1CL, 2B

0.54

0 (0)

-

0 (0)

2PL, 1B

71 (5)

1PL, 1B

100 (7)

NA

100 (7)

NA

0.94

0 (0)

-

14 (1)

4PL, 2CL

86 (6)

B

100 (7)

NA

100 (7)

NA

PL = partial loss of equilibrium; CL = complete loss of equilibrium; B = at bottom of exposure aquarium; - = None; NA = Not Applicable

NOTE: The actual number of mortalities is presented in parentheses.

Table 5. The LC50 Values, Corresponding 95% Confidence Intervals, and No-Observed-Effect Concentration (NOEC) Established during the 96-Hour Flow-Through Acute Exposure of Sheepshead Minnow (Cyprinodon variegatus) to the test substance

Based on Mean Measured Concentrations

 

Time Interval

 

LC50

(mg/L)

95% Confidence Intervals (mg/L)

Lower

Upper

24-Houra

 > 0.94

 NAb

 NA

48-Hourb

0.46

0.33

0.63

72-Houra

0.35

0.29

0.41

96-Houra

0.29

0.22

0.37

96-Hour NOEC = 0.14 mg/L

a LC50 value was empirically estimated; therefore, 95% confidence intervals could not be determined.

b NA = Not Applicable.

c LC50 value and corresponding 95% confidence intervals were determined using Trimmed Spearman-Kärber Estimates.

d LC50 value and corresponding 95% confidence intervals were determined using Spearman-Kärber Estimate


Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations, the 96-hour LC50 was 0.29 mg/L, with 95% confidence intervals of 0.22 to 0.37 mg/L.
Executive summary:

To determine the 96-hour acute toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus), the exposure was conducted under flow-through conditions following OECD TG 203 and U.S. EPA Guideline 850.1075. The test was in compliance with GLP criteria. One aquarium, containing seven fish, was established for each treatment level, (i.e., 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L nominal and 0.064, 0.14, 0.27, 0.54, and 0.94 mg/L mean measured) negative control and solvent control (dimethylformamide, DMF). These vessels were maintained in a temperature-controlled water bath at a temperature range of 22 to 23°C with a 16-hour light and 8-hour dark photoperiod at a light intensity range of 30 to 100 footcandles (320 to 1100 lux). Aquaria were examined at 0, 6, 24, 48, 72, and 96 hours of exposure as follows: dead fish were recorded and removed, biological observations including adverse effects (i.e., loss of equilibrium) of the exposed sheepshead minnow and observations of the physical characteristics of the test solutions (i.e., presence of precipitate, film on the solution's surface) were made and recorded, if applicable. Effects for this study were based on mortality, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

No sub-lethel effects or mortalities were observed among fish exposed to the negative control or solvent control during the exposure. Following 72 hours of exposure, 100% mortality was observed among fish exposed to the 0.54 and 0.94 mg/L mean measured concentrations. By the 96-hour interval, mortality of 43% was observed in the 0.27 mg/L mean measured concentration. No mortalities or sub-lethal effects were observed among fish exposed to the remaining treatment levels tested (0.14 or 0.064 mg/L), the negative control or solvent control during the exposure.

 Based on mean measured concentrations, the 96-hour LC50 was determined using Spearman-Kärber Estimates to be 0.29 mg/L, with 95% confidence intervals of 0.22 to 0.37 mg/L. The NOEC, based on lethal effects, was empirically estimated to be 0.14 mg/L. The highest concentration producing no mortality was 0.14 mg/L and the lowest concentration producing 100% mortality was 0.54 mg/L.

Description of key information

Freshwater, 96-h LC50 = 0.13 mg/L (corresponding 95% confidence intervals of 0.10 to 0.16 mg/L), Oncorhynchus mykiss, OECD TG 203, Shaw 2018

Marine water, 96-h LC50 = 0.29 mg/L (corresponding 95% confidence intervals of 0.22 to 0.37 mg/L), Cyprinodon variegatus, OECD TG 203, Shaw 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
0.13 mg/L

Marine water fish

Marine water fish
Dose descriptor:
LC50
Effect concentration:
0.29 mg/L

Additional information

Freshwater


The 96-hour acute toxicity of the substance to rainbow trout (Oncorhynchus mykiss) was studied under flow-through conditions to OECD TG 203 and GLP. The substance was dissolved in dimethylformamide, DMF, before dilution in test water. Seven fish per aquarium were exposed to nominal concentrations of 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L (0.066, 0.12, 0.23, 0.42, and 0.94 mg/L mean measured). All vessels were maintained at a temperature of 11 to 13 °C with a 16 hour light and 8 hour darkness photoperiod at a light intensity of 590 to 790 lux. No sub-lethal effects or mortalities were observed among fish exposed to the negative control or solvent control during the exposure. Following 24 hours of exposure, 100% mortality was observed in the 0.94 mg/L mean measured concentration. By the 96-hour interval, mortality of 43, 100, 100, and 100% was observed in the 0.12, 0.23, 0.42, and 0.94 mg/L mean measured concentrations, respectively. One fish in the 0.12 mg/L mean measured concentration was observed to be on the bottom of the test vessel. No mortalities or sub-lethal effects were observed among fish exposed to the 0.066 mg/L mean measured concentration. The 96-hour LC50 was 0.13 mg/L, with 95% confidence intervals of 0.10 to 0.16 mg/L. Similar studies were conducted with common carp and fathead minnow showing that rainbow trout was the most sensitive test species.


Marine water


The 96-hour acute toxicity of the substance to sheepshead minnow (Cyprinodon variegatus) was studied under flow-through conditions to OECD TG 203 and GLP. The substance was dissolved in dimethylformamide, DMF, before dilution in test water. Seven fish per aquarium were exposed to nominal concentrations of 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L (0.064, 0.14, 0.27, 0.54, and 0.94 mg/L mean measured). All vessels were maintained at a temperature range of 22 to 23 °C with a 16-hour light and 8-hour darkness photoperiod at a light intensity range of 320 to 1100 lux. No sub-lethal effects or mortalities were observed among fish exposed to the negative control or solvent control during the exposure. Following 72 hours of exposure, 100% mortality was observed among fish exposed to the 0.54 and 0.94 mg/L mean measured concentrations. By the 96-hour interval, mortality of 43% was observed in the 0.27 mg/L mean measured concentration. No mortalities or sub-lethal effects were observed among fish exposed to the remaining treatment levels tested (0.14 or 0.064 mg/L), the negative control or solvent control during the exposure. The 96-hour LC50 was 0.29 mg/L, with 95% confidence intervals of 0.22 to 0.37 mg/L.