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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:
25 Sep 1995 to 6 Dec 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
Version / remarks:
1989
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test concentrations were verified by chemical analysis at 0 and 96 hours using an HPLC analysis.
Vehicle:
no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Mean length control group at study termination: 53 mm (range: 50 – 57 mm)
- Mean weight control group at study termination: 1.35 g (range: 1.1.0 – 1.69 g)

ACCLIMATION
- Acclimation period: 20 days

FEEDING DURING TEST
No feeding during test
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
116 mg/L as CaCO3
Test temperature:
12.6 - 13.6
pH:
7.7 - 8.0
Dissolved oxygen:
74 - 81%
Conductivity:
Not reported
Nominal and measured concentrations:
- Nominal concentration: 0 (control) and 100 mg/L (based on pre-test which showed a 96h LC50 > 100 mg/L)
- Mean measured concentration: 125 mg/L. Measured test concentrations of the test substance at the beginning, at 24h, 48h, and the end of exposure were 39%, 243% (both values due to initial problems of the dosing system), 108% and 111% of the nominal concentration, respectively.
An overview of the analytical results is provided in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 L Glass vessels
- Fill volume: 15 L water
- Aeration: Gentle aeration provided
- Type of flow-through: Continuous flow of water (26 L/h) was provided by high precision positive displacement pumps.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily measurements of the test solutions were undertaken throughout the 96 hour period for pH, temperature and dissolved oxygen concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 hours fluorescent light and 8 hours dark with 30 minute dawn and dusk transition periods
- Other: The test was conducted in a temperature controlled water-bath.

EFFECT PARAMETERS MEASURED: mortality, sublethal effects, weight and length
At 2, 24, 48, 72, and 96 hours, observations of mortality and sublethal symptoms, such as abnormal behavioural activity and stress were made (swimming behaviour, loss of equilibrium, respiratory function, pigmentation and other observations). Dead fish, if any, were removed from the test solutions at least at the above mentioned intervals. At test termination the fish in the control were retained for measurements of weight and length
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
Remarks on result:
other: critical effect value assessed by the registrant as the mean measured concentration is based on unreliable analytical measurements and 100 mg/L is the regulatory limit concentration.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 125 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: critical effect value in the original report
Details on results:
- Mortality: No mortality in the control and the 100 mg/L treatment was recorded throughout the test
- Sublethal effects: Not reported
- Test substance solubility: The test substance appeared homogenously distributed in all test vessels at all times.
Reported statistics and error estimates:
Not reported
Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The acute toxicity of the test substance to rainbow trout Oncorhynchus mykiss was determined under flow-through conditions. Fish were exposed to a single nominal concentration of 100 mg a.s./L (equivalent to 125 mg a.s./L mean measured), alongside a dilution water control. Based on mean measured concentrations, the 96 hour LC50 was > 125 mg a.s./L (corrected to >100 mg/L by the registrant).
Executive summary:

The short-term toxicity to freshwater fish was determined in a GLP-compliant guideline study according to OECD TG 203. In this study groups of 7 Rainbow Trout (Oncorhynchus mykiss) were exposed to 0 (control) and a limit concentration of 100 mg/L for 96 hours under continuous flow through conditions. Test concentrations were analytical verified. The mean measured concentration of the limit dose was determined to be 125 mg/L. Observations for mortalities and symptoms of toxicity were made at 2, 24, 48, 72, and 96 hours exposure. No deaths occurred during the 96 hours test period at the limit concentration. Based on this, the 96-h LC50 value was determined at 125 mg/L (corrected to >100 mg/L by the registrant).

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Feb 1997 to 10 Feb 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. EPA 540/9-82-024.
Version / remarks:
1982
Deviations:
no
Qualifier:
according to
Guideline:
other: Standard Evaluation Procedure, Acute Toxicity Test/or Estuarine and Marine Organisms (Estuarine Fish 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office of Pesticide Programs. EPA 540/9-85-009. Washington, D.C.
Version / remarks:
1985
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard E729-88a. Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. Philadelphia, PA.
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Water samples were collected from one replicate test chamber of the low and high treatment groups prior to the test (pre-test) to determine if the test substance had reached equilibrium in the diluter system. Samples were also collected from each replicate test chamber of each treatment and the control group at the beginning and end of the test to measure concentrations of the test substance. Samples were collected from mid-depth of the test chambers and placed in glass scintillation vials. Samples from the A replicates at test initiation and the B replicates at test termination were analyzed immediately without storage. Samples not analyzed were held as backups.
Vehicle:
no
Details on test solutions:
One stock solution was prepared for each of the five concentrations tested. The first stock was prepared by dissolving the test substance in dilution water (filtered saltwater) at a concentration of 2.28 mg a.i./mL. The stock solution was stirred with an electric mixer to aid solubilization of the test substance. Aliquots of the stock solution were proportionally diluted with dilution water to prepare four additional stock solutions at concentrations of 1.37, 0.821, 0.492 and 0.295 mg a.i./mL. The five stocks were injected into the diluter mixing chambers (at a rate of 5 mL/minute) where they mixed with dilution water (at a rate of 95 mL/minute) to achieve the desired test concentrations. After mixing, the test solutions appeared clear and colourless.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISMS
- Common name: Sheepshead minnow
- Source: Sheepshead minnows used in the test were hatched from maintained cultures
- Mean length of control at end of study: 23 mm (18 - 29 mm)
- Mean wet weight (blotted dry) at end of study: 0.35 g (0.13 - 0.58g)

CULTURING AND HOLDING
- Culture medium: The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity of approximately 2% with well water. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 2Sμm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered to remove microorganisms and particles.
- Holding period: The sheepshead minnows were held in water from the same source and at approximately the same temperature as used during the test. The fish were held for 107 to 121 days prior to testing. During the holding and acclimation periods the fish showed no signs of disease or stress.
- Holding conditions: During the 14-day holding period preceding the test, water temperatures ranged from 23.6 to 24.0 °C. The pH of the water ranged from 7.7 to 7.8, salinity ranged from 20 to 23%0 (parts per thousand) and dissolved oxygen ranged from 6.4 to 6.8 mg/L.
- Feeding: During the holding period, the sheepshead minnows were fed a commercially-prepared diet; live brine shrimp nauplii (Artemia sp.); and frozen brine shrimp (Artemia sp.).

ACCLIMATION
The fish were acclimated to test conditions for approximately 49 hours prior to test incubation. At test initiation, the sheepshead minnows were collected from the acclimation tank and transferred to the test chambers.
- Feeding: The fish were not fed during the acclimation period (at least 48 hours prior to the test) or during the test.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21.9 - 22.1 °C
pH:
8.2 - 8.3
Dissolved oxygen:
6.0 - 6.9 mg O2/L
Salinity:
2%
Nominal and measured concentrations:
- Nominal test concentrations: 0 (control), 16, 26, 43, 72 and 120 mg a.i./L.
- Mean measured concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers were 8-L Teflon®-lined polyethylene aquaria. The depth of the test water in a representative chamber was approximately 17 cm.
- Type (delete if not applicable): Closed, the water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross-contamination.
- Fill volume: Approximately 6.5 L of test water
- Aeration: Yes, with spray nozzles.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- Biomass loading rate: 0.051 g/fish/L/day
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test substance and a negative (dilution water) control. A peristaltic pump (Cole-Parmer Instrument Company) was used to deliver the five test substance stock solutions into mixing chambers assigned to each treatment. The stock solutions were mixed with dilution water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters. The flow of test water from each mixing chamber was split and allowed to flow into replicate test chambers. The proportion of test water that was split into each replicate was checked prior to the test to ensure that flow rates varied by no more than ± 10% of the mean for the two replicates. The diluter was adjusted so that each test chamber received approximately 10.5 volume additions of test water every 24 hours. The stock solution delivery pump and the dilution water rotameters were calibrated before the test, and the general operation of the diluter was checked visually two times per day during the test, and once on the last day of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity of approximately 2% with well water.
- Culture medium different from test medium: Same

OTHER TEST CONDITIONS
- Photoperiod: A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity: Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone® 50). Light intensity at test initiation was approximately 346 lux at the surface of the water.

WATER QUALITY MEASUREMENTS
- Temperature: Temperature was measured in each test chamber at the beginning and end of the test using a hand-held thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ERIC Recorder. The target test temperature during the study was 22 ± 1 °C.
- Dissolved oxygen and pH: Dissolved oxygen and pH measurements were made on water samples collected from alternate replicate test chambers at test initiation and at approximately 24-hour intervals thereafter. Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter.
- Salinity: Salinity was measured in the dilution water at test initiation. Salinity was measured using an Aquafauna Bio-Marine, Inc. refractometer.

EFFECT PARAMETERS MEASURED: mortality, sub-lethal effects
Observations were made to determine the number of mortalities. The number of individuals exhibiting clinical signs of toxicity or abnormal behaviour also were evaluated. Observations were made approximately 1, 24, 48, 72, and 96 hours after test initiation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 111 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
Sheepshead minnows in the negative control group appeared healthy and normal throughout the test. Sheepshead minnows in all test substance treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity.
Reported statistics and error estimates:
Not required
Validity criteria fulfilled:
not specified
Conclusions:
The 96-hour LC50 value for sheepshead minnows exposed to the test substance was >111 mg a.i./L. Due to the lack of a concentration-response, the 95% confidence limits could not be calculated. Based on the mortality and observation data, the 96-hour no mortality concentration and the NOEC were 111 mg a.i./L, which was the highest concentration tested.
Executive summary:

The short-term toxicity to saltwater fish was determined in a study according to FIFRA Guideline 72 -3 a and in compliance with GLP criteria. In this study, groups of 20 (10 per replicate) sheepshead minnow (Cyprinodon variegatus) were exposed for 96 hours to nominal concentrations of 0 (control), 16, 26, 43, 72 and 120 mg a.i./L for 96 hours under flow-through conditions. Water samples of the nominal test concentrations were taken at t = 0h and 96h for test substance analysis. Throughout the test, the geometric mean measured concentrations remained within ±20% of the nominal. Incidences of mortality and sub-lethal effects were recorded after 1, 24, 48, 72 and 96 hours exposure. Sheepshead minnows in the negative control group appeared healthy and normal throughout the test. Sheepshead minnows in all test substance treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity. Based on these findings, the 96-h LC50 was determined to be >111 mg a.i./L, the NOEC was set at 111 mg a.i./L

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

No quantitative acute effect value can be determined as no ≥50% effect was reached in any of the available studies.

The 96-h LC50 value is >100 mg/L in freshwater fish (Oncorhynchus mykiss), OECD 203, Rufli 1996

The 96-h LC50 value is >111 mg/L in saltwater fish (Cyprinodon variegatus), FIFRA 72-3-a, Drottar 1997

Key value for chemical safety assessment

Additional information

Table: Overview of available data on the toxicity to fish

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Oncorhynchus mykiss

(freshwater)

OECD 203/ GLP

96-h LC50

>100 mg a.i./L

Flow-through regime. Measured concentrations did not remain within 20% of nominal (125 mg/L). Still the nominal concentration is used by the registrant as the analytical measurements were unreliable. Furthermore, the 100 mg/L concentration is the regulatory cut-off value and is therefore valid for the risk characterization.

Rufli, 1996

Cyprinodon variegatus

(saltwater)

FIFRA 72-3 a / GLP

96-LC50

>111 mg a.i./L

Flow-through regime. Measured concentrations remained within 20% of nominal.

Drottar, 1997