Paraquat-dichloride

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:

key study

Study period:

02 Oct 2013 to 08 Oct 2013

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate water samples were collected from each treatment and the control group one day prior to the start of the exposure after conditioning the diluter for approximately one day. Duplicate water samples also were collected from test chambers in each treatment and control group at the beginning of the test and at 48 and 96 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected from mid-depth, placed in plastic vials, and two to three drops of 10% H3PO4 was added to each vial. One set of samples was processed immediately for analysis and the other set was stored under refrigeration for possible future analysis.

Vehicle:

no

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest.
- Common name: Fathead minnow
- Length at study termination: The average total length of 7 negative control fish measured at the end of the test was 2.8 cm, with a range of 2.6 to 3.0 cm.
- Weight at study termination: The average wet weight (blotted dry) of 7 negative control fish measured at the end of the test was 0.17 grams, with a range of 0.15 to 0.20 grams.

ACCLIMATION
- Acclimation period: The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test.
- Acclimation conditions: During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 21.2 to 22.4°C, measured with a liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥8.0 mg/L (≥92% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.
- Feeding: Daily during the holding period, except during periods of fasting prior to testing, the fish were fed a brine shrimp nauplii (Artemia sp.). The fish were not fed for at least two days prior to the test or during the test.
- Health during acclimation: During the 2-week period prior to the test the fish showed no signs of disease or stress. At exposure initiation, fathead minnows were collected from the culture tank and impartially distributed one and two at a time to the test chambers until each contained 7 fish.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

132 mg/L as CaCO3

Test temperature:

21.5 - 22.1 °C. Temperature monitored continuously in the negative control during the test ranged from approximately 21.72 to 22.66°C, measured to the nearest 0.01°C.

pH:

8.0 - 8.2

Dissolved oxygen:

8.0 - 8.1 mg O2/L

Conductivity:

364 μS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.3, 2.5, 5.0, 10 and 20 mg/L.
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were 25-L Teflon-lined stainless steel aquaria filled with approximately 15 L of test water. The depth of the test water in a representative chamber was 17.0 cm. The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
- Type of flow-through (e.g. peristaltic or proportional diluter): The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver test substance stock solutions to impartially assigned
mixing chambers where the stocks were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately 7 volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber delivered test water to one replicate test chamber. The syringe pumps used to deliver stock solutions to the mixing chambers, and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test. Delivery of test solutions to the test chambers was initiated two days prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at
least once on the first and last days of the test and at least two times per day during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was 0.0112 g fish/L/day. Instantaneous loading was 0.081 g fish/L of test water present in the test chambers at any given time.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep. The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37800-L storage tank where the water was aerated with spray nozzles. Prior to use in the test, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Alkalinity: 174 - 178 mg/L as CaCO3
- Culture medium different from test medium: Same

WATER QUALITY PARAMETERS
- Tempetrature: Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in the negative control test chamber using a validated environmental monitoring system (Amegaview Central Monitoring System), which was calibrated prior to exposure initiation with a digital thermometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter.
- Hardness, alkalinity and specific conductance: Hardness, alkalinity and specific conductance in the dilution water were measured at the beginning of the test. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using an Acorn Series Model CON6 conductivity-temperature meter.

OTHER TEST CONDITIONS
- Photoperiod: Light was controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light type: Ambient laboratory light was used to illuminate the exposure systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: 755 lux; light intensity was measured at the water surface of one representative test chamber at exposure initiation using a SPER Scientific Model 840006 light meter.

EFFECT PARAMETERS MEASURED: mortality and sub-lethal effects
All organisms were observed daily throughout the exposure period to determine the number of mortalities in each treatment group. Lethality was defined as the lack of visible movement (e.g., lack of fin or opercular movement) in the fish. The numbers of individuals exhibiting signs of toxicity or abnormal behavior compared to the control organisms (e.g., lethargy) also were evaluated. Observations were made approximately 6, 24, 48, 72 and 96 hours after exposure initiation.

TEST CONCENTRATIONS
Nominal test concentrations were selected based on the results of exploratory range finding toxicity data (not further reported).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.48 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

mortality (fish)

Remarks on result:

other: recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks on result:

other: 95% C. I.: 9.1 - 25 mg/L

Remarks:

Original value presented in study

Details on results:

- Biological observations: All fish in the negative control group appeared normal throughout the test. All fathead minnows in the 1.5, 2.7 and 5.8 mg/L treatment groups also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortality in the 12 and 23 mg/L treatment groups at test termination was 57 and 71%, respectively.
- Test substance solution: The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.

Reported statistics and error estimates:

The mortality data were analyzed using the computer program of C. E. Stephan. The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. In this study, nonlinear interpolation was used to calculate 48-hour LC50 value and binominal probability was used to calculate the 95% confidence intervals. Probit analysis was used to calculate the 72 and 96-hour LC50 values. For the 96-hour LC50 value, the slope of the dose response curve was 4.0. Since there was <50% mortality at 24 hours, the 24-hour LC50 value, as well as the no-mortality concentration and NOEC, were determined by visual interpretation of the mortality and observation data.

Sublethal observations / clinical signs:

Table: Cumulative Mortality and Observations

Mean measured concentration	Mean measured concentration (cation equivalent)	No. Exposed	6 Hours		24 Hours		48 Hours		72 Hours		96 Hours		Cumulative Percent Mortality
			No Dead	Effects	No Dead(1)	Effects(2)	No Dead(1)	Effects(2)	No Dead(1)	Effects(2)	No Dead(1)	Effects(2)
Negative Control	Negative Control	7	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0
1.5	0.50	7	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0
2.7	0.90	7	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0
5.8	1.9	7	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0	7 AN	0
12	4.0	7	0	7 AN	0	7 AN	0	7 AN	2	4 AN; 1 R	4	3 AN	57.1
23	7.7	7	0	7 AN	0	6 AN; 1 C	4	3 AN	5	2 AN	5	2 AN	71.4

(1) Cumulative number of dead fish.

(2) Observations: AN = appear normal; C = lethargic; R = fish lying on bottom with little motion other than gill movement

Calculation of key result

The doses of the test substance were expressed in technical test material, which relates to an aqueous solution of the registered substance. The key effect levels are calculated by correction for the amount of water: 0.463 x 14 mg technical test material/L = 6.48 mg pure test substance/L.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

The 96-hour LC50 of the test item was calculated to be 14 mg technical test material/L. This value corresponds to a recalculated value of 6.48 mg pure substance/L.

Executive summary:

The short-term toxicity to freshwater fish was determined in a study according to OECD TG 203 and in compliance with GLP criteria. In this study, groups of 7 fathead minnows (Pimephales promelas) were exposed for 96 hours under flow-through conditions to nominal concentrations of 0 (control), 1.3, 2.5, 5.0, 10 and 20 mg/L. Test concentrations were analytically verified and determined to be <LOQ (control), 1.5, 2.7, 5.8, 12 and 23 mg/L (arithmetic mean). All fish in the negative control group appeared normal throughout the test. All fathead minnows in the 1.5, 2.7 and 5.8 mg/L treatment groups also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortalities in the 12 and 23 mg/L treatment groups at test termination were 57 and 71%, respectively. Based on these findings, the 96-hour LC50 value was 14 mg technical test material/L (equivalent to 4.7 mg cation/L), with a 95% confidence interval of 9.1 to 25 mg technical test material/L (equivalent to 3.0 to 8.4 mg cation/L). The no-mortality concentration and the NOEC were both 5.8 mg technical test material/L (equivalent to 1.9 mg cation/L). The 96-h LC50 value recalculated for the pure substance is 6.48 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct 2013 to 22 Oct 2013

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:

1996

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

Version / remarks:

2007

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate water samples were collected from each treatment and the control group one day prior to the start of the exposure after conditioning the diluter for approximately one day. Duplicate water samples also were collected from test chambers in each treatment and control group at the beginning of the test and at 48 and 96 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected from mid-depth, placed in plastic vials, and two to three drops of 10% H3PO4 was added to each vial. One set of samples was processed immediately for analysis and the other set was stored under refrigeration for possible future analysis.

Vehicle:

no

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest.
- Common name: Sheepshead minnow
- Length at study termination: The average total length of 7 negative control fish measured at the end of the test was 2.0 cm, with a range of 1.9 to 2.3 cm.
- Weight at study termination: The average wet weight (blotted dry) of 7 negative control fish measured at the end of the test was 0.13 grams, with a range of 0.099 to 0.17 grams.

ACCLIMATION
- Acclimation period: The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test.
- Acclimation conditions: During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 21.4 to 23.1°C, measured with a liquid-in-glass thermometer. The pH of the water ranged from 7.9 - 8.1, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥7.5 mg/L (≥96% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. Salinity of the water ranged from 20 to 21 parts per thousand (‰), measured with a VitalSine refractometer
- Feeding: Daily during the holding period, except during periods of fasting prior to testing, the fish were fed a brine shrimp nauplii (Artemia sp.). The fish were not fed for at least two days prior to the test or during the test.
- Health during acclimation: During the 2-week period prior to the test the fish showed no signs of disease or stress. At exposure initiation, sheepshead minnows were collected from the culture tank and impartially distributed one and two at a time to the test chambers until each contained 7 fish.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21.8 - 21.9 °C. Temperature monitored continuously in the negative control during the test ranged from approximately 20.30 to 22.11°C, measured to the nearest 0.01°C.

pH:

7.8 - 8.0

Dissolved oxygen:

7.4 - 7.9 mg O2/L

Salinity:

2%

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 7.5, 13, 30, 60 and 120 mg technical test material/L.
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were 25-L Teflon-lined stainless steel aquaria filled with approximately 15 L of test water. The depth of the test water in a representative chamber was 18.5 cm. The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
- Type of flow-through: The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance and a negative control (dilution water) to test chambers. Syringe pumps were used to deliver test substance stock solutions to impartially assigned
mixing chambers where the stocks were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately 7 volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber delivered test water to one replicate test chamber. The syringe pumps used to deliver stock solutions to the mixing chambers, and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test. Delivery of test solutions to the test chambers was initiated two days prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was 0.0083 g fish/L/day. Instantaneous loading was 0.060 g fish/L of test water present in the test chambers at any given time.

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. The freshly-collected seawater was pumped into a 5000-gallon holding tank and ozonated, before being filtered through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the test facility site and was aerated with spray nozzles. Prior to use in the test, the 20‰ water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Culture medium different from test medium: Same

WATER QUALITY PARAMETERS
- Tempetrature: Temperature was measured in each test chamber at the beginning and end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in the negative control test chamber using a validated environmental monitoring system, which was calibrated prior to exposure initiation with a digital thermometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter.
- Salinity: Salinity was measured in the dilution water at the beginning and end of the test using a VitalSine refractometer.

OTHER TEST CONDITIONS
- Photoperiod: Light was controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light type: Ambient laboratory light was used to illuminate the exposure systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: 329 lux; light intensity was measured at the water surface of one representative test chamber at exposure initiation using a SPER Scientific Model 840006 light meter.

EFFECT PARAMETERS MEASURED: mortality and sub-lethal effects
All organisms were observed daily throughout the exposure period to determine the number of mortalities in each treatment group. Lethality was defined as the lack of visible movement (e.g., lack of fin or opercular movement) in the fish. The numbers of individuals exhibiting signs of toxicity or abnormal behavior compared to the control organisms (e.g., lethargy) also were evaluated. Observations were made approximately 6, 24, 48, 72 and 96 hours after exposure initiation.

RANGE-FINDING STUDIES
Nominal test concentrations were selected based on the results of exploratory range finding toxicity data (not further reported).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 57 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

mortality (fish)

Remarks on result:

other: recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 123 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Original value presented in study

Details on results:

- Biological observations: All fish in the negative control alltreatment groups appeared normal throughout the test with no mortalities.
- Test substance solution: The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.

Reported statistics and error estimates:

Not required based on the findings

Sublethal observations / clinical signs:

Calculation of key result

The doses of the test substance were expressed in technical test material, which relates to an aqueous solution of the registered substance. The key effect levels are calculated by correction for the amount of water: 0.463 x >123 mg technical test material/L = >57 mg pure test substance/L.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'.

Conclusions:

The 96-hour LC50 of the test item was calculated to be >123 mg technical test material/L. This value corresponds to a recalculated value of >57 mg/L pure substance.

Executive summary:

The short-term toxicity to saltwater fish was determined in a study according to OECD TG 203 and in compliance with GLP criteria. In this study, groups of 7 sheepshead minnows (Cyprinodon variegatus) were exposed for 96 hours under flow-through conditions to nominal concentrations of 0 (control), 7.5, 13, 30, 60 and 120 mg/L. Test concentrations were analytically verified and determined to be <LOQ (control), 6.9, 14, 31, 65 and 123 mg/L (arithmetic mean). All fish in the negative control all treatment groups appeared normal throughout the test with no mortalities. Based on these findings, the 96-hour LC50 value was determined to be >123 mg technical test material/L. The recalculated 96-h LC50 value for the pure substance is >57 mg/L.

Description of key information

The 96-h LC50 value is 6.48 mg pure test substance/L (recalculated) for fathead minnow (Pimephales promelas), OECD 203, flow-through, Claude 2014

The 96-h LC50 value is >57 mg pure test substance/L (recalculated) for sheepshead minnow (Cyprinodon variegatus), OECD 203, flow-through, Claude 2014

The studies representing the worst-case effects were included as key studies. Other studies are included as supporting information.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

6.48 mg/L

Additional information

Table: Overview of available data on the acute toxicity to fish

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Pimephales promelas (freshwater)	OECD 203 / GLP	96-h LC50	6.48 mg/L (recalculated)	Flow-through regime. Measured concentrations remained within 80 - 120% of nominal concentrations. The mean measured concentrations were used for the effect values	Claude, 2014
Cyprinodon variegatus (marine)	OECD 203/ GLP	96-h LC50	>57 mg/L (recalculated)	Flow-through regime. Measured concentrations remained within 80 - 120% of nominal concentrations. The mean measured concentrations were used for the effect values	Claude, 2014