2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 February 2002 to 18 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPP 72–3 (Acute Toxicity Test for Estuarine and Marine)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

During the 96-hour definitive study, one sample was removed from each treatment level and the controls at each sampling interval. Each sample was collected by pipette from the approximate midpoint of the test vessel. 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:

yes

Details on test solutions:

A 200 mg a.i./mL primary stock solution was prepared by placing 10.5817g of test material (10.000 g as active ingredient) in a 50-mL volumetric flask and diluting to volume with dimethylformamide (DMF). The primary stock solution was observed to be clear and amber in colour. Secondary stock solutions were prepared from dilutions of the 200 mg a.i./mL primary stock solution. Exposure solutions were prepared by diluting the appropriate volume of the appropriate stock solution with natural filtered seawater.
The two lowest concentration solutions, 13 and 22 mg a.i./L, were observed to be clear and colourless. All remaining exposure solutions were observed to be clear with a slight yellow tint, increasing in intensity with increasing concentration.
The control solution was prepared with natural filtered seawater without the addition of test material.
A 0.50 mL/L solvent control solution was prepared by diluting 7.5 mL of DMF with 15.0 L of natural seawater.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Length at study initiation (mean and range for representative sample of 30 fish): 28 mm (23 - 32 mm)
- Weight at study initiation (mean and range for representative sample of 30 fish): 0.38 g (0.20 - 0.58 g)
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: Prior to testing, the fish were held in a 500-L fiberglass tank with a closed-loop recirculating filtration system under a photoperiod of 16 hours of light and 8 hours of darkness. The seawater which flowed into this tank had a salinity range of 33 to 35 ‰. Other parameters monitored in the holding tank were pH with a range of 7.6 to 7.8 and dissolved oxygen concentration with a range of 82 to 92 % of saturation. The temperature in the holding tank was 22 to 23 °C during this period. The fish were fed a dry commercial flaked food, ad libitum, daily except during the 48 hours prior to testing.
- Health during acclimation (any mortality observed): No mortality was observed among the test fish population during the 48-hour period prior to test initiation.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21 - 23 °C

pH:

6.8 - 7.9

Dissolved oxygen:

5.0 - 8.2 mg/L

Salinity:

33 - 35 ‰

Nominal and measured concentrations:

0, 13, 22, 36, 60, 100 mg a.i./L (nominal)
0, 12, 21, 34, 64, 120 mg a.i./L (mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: 19.5-L glass aquaria (39 x 20 x 25 cm)
- Fill volume: 15 L of test solution
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was prepared by filtering natural seawater collected from the Cape Cod Canal, Bourne, Massachusetts. The seawater was transferred with a pump (fibreglass reinforced thermoplastic housing) and a polyvinyl chloride (PVC) pipe and was then transported to the laboratory in a 3400-L fibreglass tank. In the laboratory, the seawater was passed through a series of polypropylene core filters (20- and 5-micron) and then recirculated within an epoxy-lined concrete reservoir prior to use. The seawater was pumped to the laboratory under constant pressure through PVC pipe and a polypropylene heat exchanger system. The seawater used during this study had a salinity range of 31 to 32 ‰ and a pH range of 7.9 to 8.0.
- Total organic carbon: < 2.0 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Representative samples of the dilution water were analysed 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 analysed.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 50 to 70 footcandles at the solutions’ surface

EFFECT PARAMETERS MEASURED
All aquaria were examined at 0, 6, 24, 48, 72 and 96 hours of exposure as follows: mortalities were recorded and removed, biological observations, including sublethal effects (e.g., loss of equilibrium), of the exposed fish 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. Dead fish were removed and the findings 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).
During the definitive exposure, the pH, dissolved oxygen concentration, temperature and salinity were measured daily in each test vessel. The pH was measured with a Jenco Model 60 pH meter and combination electrode; dissolved oxygen concentration was measured with a YSI Model #57 dissolved oxygen meter and probe; the daily temperature was measured with a VWR alcohol thermometer; and salinity concentrations were measured with a Vista refractometer. In addition, temperature was monitored continuously in the solvent control using a Fisher Scientific Min-Max Thermometer.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (dilution water control), 0.10, 1.0, 10, 100 mg a.i./L. Juvenile sheepshead minnow (10 fish per treatment level and the control).
- Results used to determine the conditions for the definitive study: Yes. At test termination (96 hours), no mortality or sublethal effects (e.g., lethargy) were observed among fish exposed to the treatment levels tested or the control. Based on these results, the nominal test material concentrations selected for the definitive study were 13, 22, 36, 60 and 100 mg a.i./L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

At test termination (96 hours), no mortality or sublethal effects were observed among fish exposed to the treatment levels or the controls.

Sublethal observations / clinical signs:

Analytical Chemistry

Results of the analyses established measured concentrations which were consistent between sampling intervals (i.e., 0 and 96 hours). Mean measured concentrations defined the exposure concentrations as 12, 21, 34, 64 and 120 mg a.i./L. Analysis of the quality control samples resulted in measured concentrations which were consistent with the predetermined recovery range and ranged from 93.7 to 103 % (N = 6) of the nominal fortified levels (10.0 to 100 mg a.i./L). These results established that the appropriate quality control was maintained during the analysis of the exposure solutions.

Water Quality Parameters

The pH of the test solutions decreased with increasing test material concentration throughout the 96-hour exposure. The remaining water quality parameters measured were unaffected by the concentrations of test material tested and remained within acceptable levels for the survival of sheepshead minnow. Continuous temperature monitoring of the solvent control solution established that the test solution temperature ranged from 21 to 24 °C during the exposure period. Daily monitoring of the test solutions established a salinity range of 33 to 35 ‰ during the exposure period.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96 hour LC50 was determined to be in excess of 120 mg/L, based on mean measured concentration of active ingredient.

Executive summary:

The acute toxicity of the test material to the saltwater fish, sheepshead minnow, was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1075.

The study was conducted under static conditions with dose solutions prepared at a target concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L. The control solution was prepared with natural filtered seawater without the addition of test material. A 0.50 mL/L solvent control solution was prepared by diluting 7.5 mL of DMF with 15.0 L of natural seawater.

The definitive study was initiated when fish were added to each test vessel (10 per treatment level and controls). Following exposure solution preparation fish were impartially added to the test aquaria, two at a time until each aquarium contained ten fish. All aquaria were examined at 0, 6, 24, 48, 72 and 96 hours of exposure for mortality and sublethal effects.

At test termination (96 hours), no mortality or sublethal effects were observed among fish exposed to the treatment levels or the controls. Therefore, under the conditions of the study, the 96 hour LC50 was determined to be in excess of 120 mg/L (active ingredient), based on mean measured concentrations; the NOEC was 120 mg a.i./L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 December 2001 to 17 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

At test initiation (0 hour) and test termination (96 hours), one sample from each replicate exposure and control aquarium was analysed for test material residue. Each sample was collected by pipette from the approximate midpoint of the test vessel. Three quality control (QC) samples were also prepared at each sampling interval at nominal test material concentrations of 80.0 to 110 mg a.i./L and remained with the exposure solution samples throughout the analytical process. This range was selected because of the solubility limit of the test material. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.

Vehicle:

yes

Details on test solutions:

Prior to test initiation, a 100 mg a.i./L exposure solution was prepared by placing 5.291 g of test material (5.000 g as active ingredient) in a 100 mL glass beaker and wetting the substance with 5.0 mL of dimethylformamide (DMF). This produced a yellow slurry which was quantitatively transferred to a 100 L aquarium by repeated washing with dilution water, and brought to a final volume of 50 L. The solution initially contained some undissolved test material and was stirred vigorously for approximately 15 minutes with a Tamco® laboratory stirrer. The solution was then stirred gently for approximately 2.5 hours with two stir bars and magnetic stir plates. Following mixing, the stirred solution was allowed to settle for approximately 30 minutes, and was observed to be clear with a very slight yellow colour.
Three individual 15-L aliquots of this solution were added to separate aquaria. Each replicate solution was stirred for 30 seconds with a glass rod.
Three solvent control aquaria were prepared by dissolving 1.5 mL of DMF in 15 L of dilution water (final solvent concentration of 0.1 mL/L). In addition, three control aquaria were also established and maintained under the same conditions as the treatment level solutions. Each aquarium contained dilution water and no test material.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Length at study initiation (mean and range for representative sample of 30 fish): 36 mm (23 - 44 mm)
- Weight at study initiation (mean and range for representative sample of 30 fish): 0.54 g (0.18 - 0.92 g)
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days minimum
- Acclimation conditions: Prior to testing, the fish were held in a 500-L fiberglass tank under a photoperiod of 16 hours of light and 8 hours of darkness. The culture water was drawn from a 100-metre deep bedrock well into an epoxy-coated concrete reservoir where it was aerated and supplemented with well water supplied by the Town of Wareham, Massachusetts. The water which flowed into this holding tank was characterised as having total hardness and total alkalinity ranges as calcium carbonate (CaCO₃) of 49 to 56 mg/L and 30 to 36 mg/L, respectively, and a specific conductance range of 130 to 150 micromhos per centimetre (μmhos/cm). Other parameters monitored in the holding tank were pH with a range of 7.4 to 7.7 and dissolved oxygen concentration with a range of 97 to 101 % of saturation. The temperature in the holding tank was 21 °C during the 48 hours prior to test initiation.
- Type and amount of food: The fish were fed a dry commercial flaked food, ad libitum, daily. Fish were not fed during the 48-hour period prior to test initiation.
- Health during acclimation (any mortality observed): No mortality was observed among the test fish population during the 48-hour period prior to testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

22 ± 1 °C

pH:

5.6 - 7.0

Dissolved oxygen:

6.7 - 9.6 mg/L 75 - 109 % saturation)

Nominal and measured concentrations:

0, 100 mg/L (nominal and mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 19.5-L glass aquaria
- Fill volume: 15 L
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The mean organism loading concentration was 0.36 g of biomass per litre of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was from the same source as the water which flowed into the fish holding tank and was characterised as having total hardness and alkalinity ranges as CaCO₃ of 52 to 54 mg/L and 30 to 34 mg/L, respectively, a pH range of 7.3 to 7.4, and a specific conductivity range of 130 to 140 μmhos/cm.
- Total organic carbon: 0.63 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Representative samples of the dilution water source were analysed 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 analysed.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness
- Light intensity: 60 to 80 footcandles at the solutions’ surface

EFFECT PARAMETERS MEASURED
All aquaria were examined at 0, 24, 48, 72 and 96 hours of exposure as follows: mortalities were recorded and removed, biological observations, including sublethal effects (e.g., loss of equilibrium), of the exposed bluegill sunfish 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. Dead fish were removed and the findings 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).
The pH was measured with a Jenco Model 601A pH meter and combination electrode. Dissolved oxygen concentration was measured with a Yellow Springs Instrument (YSI) Model #57 dissolved oxygen meter and probe. Daily temperature was measured with a VWR Brand alcohol thermometer. Temperature was continuously monitored throughout this study using a Fisher Scientific Min-Max thermometer.

TEST CONCENTRATIONS
- Range finding study: no

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

Following 96 hours of exposure, no mortalities or sublethal effects were observed among fish exposed to the treatment level tested or the controls.

Sublethal observations / clinical signs:

Chemical Analysis

Throughout the test period, replicate measurements ranged from 97 to 100 % of nominal and defined the treatment level as 100 mg a.i./L. Analysis of the quality control samples resulted in measured concentrations which were consistent with the pre-determined recovery range and ranged from 93.6 to 102 % of the nominal fortified levels (80.0 to 110 mg a.i./L). Based on the results of these analyses, it was established that the appropriate precision and quality control was maintained during the analyses of the exposure solutions.

Water Quality Parameters

Throughout the exposure period, the pH of the 100 mg a.i./L test solutions was lower than the controls. The remaining water quality parameters measured were unaffected by the concentrations of test material tested and remained within acceptable ranges for the survival of bluegill sunfish. Daily measurement of the temperature in the exposure solutions and continuous temperature monitoring established that the temperature ranged from 21 to 23 °C throughout the definitive study.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96 hour LC50 was determined to be in excess of 100 mg/L.

Executive summary:

The acute toxicity of the test material to the freshwater fish, bluegill sunfish, was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72 -1 and OECD 203.

The study was conducted under static conditions with dose solutions prepared at a target concentration of test material of 100 mg/L.

Three test material treatments and three solvent (DMF) controls were included. In addition, three control aquaria were also established and maintained under the same conditions as the treatment level solutions. Each control aquarium contained dilution water and no test material.

The test was initiated when bluegill sunfish (10 fish per treatment level and the controls) were impartially selected and distributed to each aquarium. Fish were added two at a time to each aquarium until each aquarium contained 10 fish. The mean organism loading concentration was 0.36 g of biomass per litre of test solution. All aquaria were examined at 0, 24, 48, 72 and 96 hours of exposure for mortality and sublethal effects.

Following 96 hours of exposure, no mortalities or sublethal effects were observed among fish exposed to the treatment level tested or the controls. Therefore, under the conditions of the study, the 96 hour LC50 was determined to be in excess of 100 mg/L; the NOEC was determined to be 100 mg/L.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2 July 2001 to 6 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

Samples were obtained from each water control and test solution test vessel on day 0 and day 4.
On days 0 and 4 of the study, the concentration of test material in the test solutions was confirmed by collecting 3.2 mL aliquots from each test solution, as well as the laboratory dilution water (LDW) control vessels. The collected aliquots from each solution and control vessels were then transferred to a 4 mL autosampler vial containing 0.8 mL methanol acidified with 1 % acetic acid. This yielded a final sample at 80 % of the initial concentration (dilution correction factor = 1.25) in a matrix of 20/80 acidified methanol/LDW. The resulting solutions were capped and vortexed prior to analysis.
To define method precision, three additional samples were taken on day 0 from a representative test vessel at a target concentration of 100 mg test material/L LDW. These additional samples were collected, diluted, and analysed along with the other day 0 samples.

Vehicle:

no

Details on test solutions:

Six replicate test solutions with a nominal concentration of 100-mg test material/L-LDW were prepared by adding approximately 1 gram of test material to each test vessel filled with 10 L of LDW. The solutions were stirred vigorously and then sonicated for approximately 10 minutes to allow for complete dissolution of test material. Six replicate LDW (water control) vessels were also prepared.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Age at study initiation: juvenile
- Length (mean, post exposure): 49 mm; the length of the largest fish was no more than twice that o the shortest fish.
- Weight (mean, post exposure): 1137 mg
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions: The fish were placed in a 230-L holding tank. All fish were held on a 16-hour light/8-hour dark transitional photoperiod and observed for at least 14 days before testing. During holding, the fish received a standard diet at least once daily; in the 48 hours prior to the test fish were held without food. The test lot used for the definitive study was acclimated to 12.0 ± 1 °C. Temperature changes did not exceed ±3 °C in the 72-hours pre-test.
- Health during acclimation (any mortality observed): Mortality did not exceed three percent of the population in the 48 hours before testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

11.9 - 12.7 °C

pH:

5.6 - 7.1

Dissolved oxygen:

8.5 - 10.3 mg/L

Nominal and measured concentrations:

0, 100 mg/L (nominal and mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 12 L glass vessels
- Type: closed (all vessels were loosely covered)
- Fill volume: 10 L
- Aeration: Aeration was provided via a glass capillary at a rate of approximately 100 bubbles-per-minute
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Biomass loading rate: Loading of the test vessels did not exceed 0.8-g fish per litre of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The laboratory dilution water (LDW) was Lake Huron water supplied by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron near Whitestone Point, and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to treatment for municipal use. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO₂, carbon-filtered and UV-irradiated.
- Total organic carbon: < 1000 ng/mL
- Hardness: 58 mg/L CaCO₃
- Alkalinity: 36 mg/L CaCO₃
- Conductivity: 53.3 μmho/cm
- pH: 8.1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Alkalinity, conductivity, pH, and hardness are monitored weekly. Total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds are monitored biannually.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light / 8 hours of darkness

EFFECT PARAMETERS MEASURED
The fish were observed and mortality and sublethal effects recorded at 24, 48, 72, and 96 hours. Mortality was defined as a lack of response to prodding of the caudal peduncle accompanied by an absence of opercular movement. Behavioural effects were noted and reported (e.g., swimming at the surface, complete or partial loss of body equilibrium, lethargy, or hyperactivity). The presence of gross pathological conditions was also noted and reported (e.g., exophthalmia (bulging eyes), ascites (fluid accumulation in the abdomen), haemorrhaging (discharge of blood), excess mucus, sloughing of epidermis, and melanosis (increased amount of dark pigmentation)). Dead fish were removed when observed.
Dissolved oxygen, pH, and temperature data were recorded in each test vessel at 0, 24, 48, 72 and 96 hours. Water temperature was continuously monitored from one test vessel throughout the study.

TEST CONCENTRATIONS
- Range finding study
Two probe studies were conducted between 05 March 2001 and 06 April 2001 to determine the concentrations to be used for the definitive study. With the dose levels of both studies combined, five fish (five fish/replicate; one replicate/dose level) were exposed to nominal concentrations of 0 (water control), 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg test material/L, over a 96-hour exposure period. No morality or sublethal effects were observed at any of the dose levels following 96 hours of exposure. Therefore, the definitive study was conducted as a limit test at a nominal concentration of 100 mg test material/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The mean post-exposure length and weight of all surviving fish used in this study were measured and recorded. The fish used in control treatments were 48 ± 4 mm and 1076 ± 248 mg. Mean post-exposure length and weights for the test material treatment fish were 50 ± 3 mm and 1199 ± 198 mg, respectively, indicating that fish used in this study were of a similar size class. Pooled length and weight means for all fish were 49 ± 3 mm and 1137 ± 231 mg. The resulting biological loading rate was 569-mg fish/L.
The fish were observed and mortality and sublethal effects recorded at 24, 48, 72, and 96 hours of exposure. Partial loss of equilibrium was observed in 7 % (2/30) of fish at the 100 mg/L dose level following 96-hours of exposure; no other sublethal effects were observed during the conduct of this study. No mortality was observed in the water control or the nominal 100-mg test material/L test vessels during this study.

Reported statistics and error estimates:

Based on the design of this study (i.e., limit test), no statistical analysis programs were used for the data analysis and the statistical determination of a NOEC value was not attempted.

Sublethal observations / clinical signs:

Chemical Analysis

Daily residual values were calculated along with the daily percent of target values (daily value divided by the target value). Averaging the day 0 and day 4 values calculated an overall study average, which was 100 % of target. None of the analyses of the laboratory dilution water controls exhibited a peak eluting at the retention time of the test material at a concentration exceeding the lowest level quantified (LLQ) of 5.9 mg test material/L laboratory dilution water (concentration of the lowest standard analysed times the dilution factor of 1.25).

Water Quality Parameters

Dissolved oxygen levels averaged 89 % of saturation and remained greater than or equal to 81 % over the 96-hour exposure period. Temperature in the vessels ranged from 11.9 – 12.7 °C (12.4 ± 0.2 °C), and the pH ranged from 5.6 – 7.1 (6.6 ± 0.4).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 96 hour LC50 was determined to be in excess of 100 mg/L.

Executive summary:

The acute toxicity of the test material to the freshwater fish, rainbow trout, was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72 -1, OECD 203 and EU Method C.1.

The study was conducted under static conditions with dose solutions prepared on day 0 at a target concentration of test material of 100 mg/L. Thirty fish were exposed per dose level in six replicates per concentration with five fish per replicates. Test and control solutions were analysed to determine test material concentrations on days 0 and 4 of the study. Collected samples were analysed by high-performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV).

Mean measured concentrations were less than LLQ of 5.9 mg/L for the water controls and 100 mg/L for the test material dose solutions. The fish were observed and mortality and sublethal effects recorded at 24, 48, 72, and 96 hours of exposure. Partial loss of equilibrium was observed in 7 % (2/30) of fish at the 100 mg/L dose level following 96-hours of exposure; no other sublethal effects were observed during the conduct of this study. No mortality was observed in the water control or the nominal 100 mg/L test vessels during this study.

Based on the biological findings, the 96 -hour LC50 was found to be greater than 100 mg/L, NOEC ≥ 100 mg/L.

Description of key information

Freshwater fish
96 hour LC50 > 100 mg/L (rainbow trout), EPA OPP 72 -1, OECD 203, EU Method C.1, Marino et al. (2001)
96 hour LC50 > 100 mg/L (bluegill sunfish), EPA OPP 72 -1, OECD 203, Machado (2003)
Saltwater fish
96 hour LC50 > 120 mg/L (sheepshead minnow), EPA OPP 72-3, EPA OPPTS 850.1075, Machado (2002)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Marine water fish

Marine water fish

Effect concentration:

120 mg/L

Additional information

Three studies, investigating the short-term toxicity of the substance to fish are available. All three studies were conducted under GLP conditions and in accordance with standardised guidelines. All three studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

In the study reported by Marino et al. (2001) the acute toxicity of the test material to the freshwater fish rainbow trout was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-1, OECD 203 and EU Method C.1.

The study was conducted under static conditions with dose solutions prepared on day 0 at a target concentration of test material of 100 mg/L. Thirty fish were exposed per dose level in six replicates per concentration with five fish per replicates. Test and control solutions were analysed to determine test material concentrations on days 0 and 4 of the study. Collected samples were analysed by high-performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV).

Mean measured concentrations were less than LLQ of 5.9 mg/L for the water controls and 100 mg/L for the test material dose solutions. The fish were observed and mortality and sublethal effects recorded at 24, 48, 72, and 96 hours of exposure. Partial loss of equilibrium was observed in 7 % (2/30) of fish at the 100 mg/L dose level following 96-hours of exposure; no other sublethal effects were observed during the conduct of this study. No mortality was observed in the water control or the nominal 100 mg/L test vessels during this study.

Based on the biological findings, the 96-hour LC50 was found to be greater than 100 mg/L, NOEC ≥ 100 mg/L.

 

In the study reported by Machado (2003) the acute toxicity of the test material to the freshwater fish, bluegill sunfish, was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-1 and OECD 203.

The study was conducted under static conditions with dose solutions prepared at a target concentration of test material of 100 mg/L.

Three test material treatments and three solvent (DMF) controls were included. In addition, three control aquaria were also established and maintained under the same conditions as the treatment level solutions. Each control aquarium contained dilution water and no test material.

The test was initiated when bluegill sunfish (10 fish per treatment level and the controls) were impartially selected and distributed to each aquarium. Fish were added two at a time to each aquarium until each aquarium contained 10 fish. The mean organism loading concentration was 0.36 g of biomass per litre of test solution. All aquaria were examined at 0, 24, 48, 72 and 96 hours of exposure for mortality and sublethal effects.

Following 96 hours of exposure, no mortalities or sublethal effects were observed among fish exposed to the treatment level tested or the controls. Therefore, under the conditions of the study, the 96 hour LC50 was determined to be in excess of 100 mg/L; the NOEC was determined to be 100 mg/L.

 

In the study reported by Machado (2002) the acute toxicity of the test material to the saltwater fish, sheepshead minnow, was assessed in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 72-3 and EPA OPPTS 850.1075.

The study was conducted under static conditions with dose solutions prepared at a target concentrations of test material of 13, 22, 36, 60 and 100 mg a.i./L. The control solution was prepared with natural filtered seawater without the addition of test material. A 0.50 mL/L solvent control solution was prepared by diluting 7.5 mL of DMF with 15.0 L of natural seawater.

The definitive study was initiated when fish were added to each test vessel (10 per treatment level and controls). Following exposure solution preparation fish were impartially added to the test aquaria, two at a time until each aquarium contained ten fish. All aquaria were examined at 0, 6, 24, 48, 72 and 96 hours of exposure for mortality and sublethal effects.

At test termination (96 hours), no mortality or sublethal effects were observed among fish exposed to the treatment levels or the controls. Therefore, under the conditions of the study, the 96 hour LC50 was determined to be in excess of 120 mg/L (active ingredient), based on mean measured concentrations; the NOEC was 120 mg a.i./L.