Paraquat-dichloride

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 Oct 2013 to 11 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition 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 test chamber of 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 each test chamber in each treatment and the 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 were 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

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 (unfiltered saltwater) at a concentration of 2.24 mg a.i./mL. The stock 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 target concentrations of 1.35, 0.808, 0.485 and 0.291 mg a.i./mL. The five stocks were injected into the diluter mixing chambers (at a rate of 18 mL/minute) where they mixed with dilution water (at a rate of 334 mL/minute) to achieve the desired test concentrations. After mixing, the test solutions appeared clear and colorless.

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: Eastern oyster
- Justification for species other than prescribed by test guideline: The eastern oyster is representative of an important group of aquatic organisms and was selected for use in the test based upon past history of use and ease of holding in the laboratory.
- Feeding: To supplement the diet of the oysters and to enhance their condition and growth, the oysters were provided with a suspension of marine microalgae. This suspension was provided continuously during holding at a nominal rate of approximately 2.9E+09 cells/oyster/day and during the test at a nominal rate of
approximately 5.8E+09 cells/oyster/day. Peristaltic pumps were used to deliver the supplemental algal solution to the test chambers. The pumps were calibrated prior to the test and confirmed daily during the test.
- Average length at study initiation: The average length of the oysters was 33.6 (± 2.31) mm with a range of 30.1 to 38.7 mm (n=20).

HOLDING PERIOD
- Holding conditions: The oysters were held in water from the same source as used during the test. The oysters were supplied unfiltered natural seawater during holding. During the 10 days preceding the test, water temperatures ranged from 19.7 to 20.8 °C. The pH of the water ranged from 7.7 to 8.3, salinity was 2.0%, and dissolved oxygen was ≥7.0 mg/L (≥88% of saturation).
- Holding period: Test organisms were held for 10 days prior to test initiation.
- Health during holding: During the holding period the oysters showed no signs of disease or stress.

SHELL PRETREATMENT METHOD PRIOR TO STUDY INITIATION
On Day 0 prior to exposure initiation, recently deposited shell was removed from all oysters in the test lot by grinding the periphery of the oysters with an electric grinder. The lengths of 20 impartially-selected oysters were determined by measuring the longest distance from the umbo to the distal valve edge. Measurements were made to the nearest 0.1 mm using calipers to confirm that the oysters fell within the acceptable 30 to 50 mm size criterion.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

19.7 - 20.4 °C by manual temperature measurements. Temperature monitored continuously in the negative control during the test ranged from approximately 19.70 to 20.77ºC, measured to the nearest 0.01°C.

pH:

7.8 - 8.0

Dissolved oxygen:

7.4 - 8.1 mg O2/L (A dissolved oxygen concentration of 4.8 mg/L represents 60% saturation at 20 °C in saltwater with a salinity of 2%)

Salinity:

2%

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 3.1, 6.3, 13, 25, 50 and 100 mg technical test material
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were 54-L glass aquaria. The depth of the test water in a representative chamber was 16.1 cm.
- Fill volume: 27 L, the depth of the test water in a representative test chamber was approximately 6.2 cm.
- 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 into 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 so that each test chamber received at least one liter of test solution per oyster per hour. During the test, each chamber received approximately 18 volume additions of test water every 24 hours. 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 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: 20
- No. of vessels per concentration: 1
- No. of vessels per control: 1

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 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

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 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 quality: Lighting used to illuminate the holding and test chambers during holding and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone® 50).
- Light intensity: Light intensity was 321 lux over the surface of water in the negative control. Light intensity was measured at the water surface of one representative test chamber at exposure initiation using a SPER Scientific Model 840006 light meter.

WATER QUALITY MEASUREMENTS
- Temperature: 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. Measurements of pH were taken in each test chamber at the beginning of the test, at the approximate mid-point of the test (48 hours), and
at the end of the test using a Thermo Orion Model 525Aplus pH meter.
- Dissolved oxygen and pH: Dissolved oxygen was measured in each test chamber at the beginning of the test, at approximately 24-hour intervals during the test, and at the end of the test using a Thermo Orion Model 850Aplus dissolved oxygen meter.
- Salinity: Salinity was measured in each test chamber at the beginning and end of the test using a VitalSine refractometer.

EFFECT PARAMETERS MEASURED: mortality, sub-lethal effects and shell growth
All organisms were observed daily throughout the exposure period to determine the number of mortalities in each treatment group. Oysters having open shells and not responding to gentle prodding were considered dead. The numbers of individuals exhibiting signs of toxicity or abnormal behavior compared to the control organisms (e.g., reduced feeding or reduced fecal production) also were evaluated. Observations were made approximately 5, 24, 48, 72 and 96 hours after exposure initiation. At the end of the test, the longest finger of new shell growth on each oyster was measured to the nearest 0.1 mm using calipers.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

33.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: pure test substance

Basis for effect:

other: oyster shell growth

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:

EC50

Effect conc.:

73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: oyster shell growth

Remarks on result:

other: 95% C.I.:50 - 87 mg/L

Remarks:

Original value as presented in the study report

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: pure test substance

Basis for effect:

other: oyster shell growth

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:

NOEC

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: oyster shell growth

Remarks on result:

other: Original value as presented in the study report

Details on results:

- Mortality: All organisms survived the test period.
- Appearance: All oysters appeared normal throughout the 96-hour exposure period.
- Shell growth: After 96 hours, the mean shell deposition in the negative control group was 5.5 mm. Mean shell deposition in the 3.7, 7.5, 15, 27, 54 and 106 mg technical test material/L treatment groups was 4.5, 4.7, 4.1, 3.7, 3.4 and 1.6 mm, respectively. Dunnett’s test indicated that there was a statistically significant decrease in shell deposition in the 15, 27, 54 and 106 mg technical test material/L treatment groups in comparison to the negative control (p ≤ 0.05). Percent inhibition of shell growth was calculated relative to the negative control data. Inhibition of shell growth in the 3.7, 7.5, 15, 27, 54 and 106 mg technical test material/L treatment groups was 19, 15, 26, 32, 38 and 72%, respectively. Mean shell deposition and shell growth inhibition are summarised in 'Any other information on materials and methods incl. tables'.

Reported statistics and error estimates:

The EC50 value, the concentration of test substance that would inhibit shell deposition by 50% relative to the negative control, was calculated using linear interpolation. The shell deposition data were evaluated for normality and homogeneity of variance using the Shapiro-Wilks test and Levene’s test, respectively. Since the data passed the assumptions of normality and homogeneity, the data in the treatment groups were compared to the negative control data using Dunnett’s test to identify any significant differences. The no-observed effect concentration (NOEC) was determined from the statistical analysis of the data and an assessment of the concentration-response pattern. All statistical tests were performed using a personal computer with SAS and TOXSTAT® computer software.

Table: Mean shell deposition and shell growth inhibition at 96 hours

Technical Test material Mean Measured Concentration (mg/L)	Test substance Cation Equivalent Concentration (mg/L)	Mean Shell Deposition ± Standard Deviation (mm)	Shell Growth Inhibition (1,2) (%)
Negative Control	Negative Control	5.5 ± 1.91	---
3.7	1.2	4.5 ± 1.86	19
7.5	2.5	4.7 ± 1.49	15
15	5.0	4.1 ± 1.25	26*
27	9.0	3.7 ± 1.37	32*
54	18	3.4 ± 1.38	38*
106	36	1.6 ± 1.05	72*

(1) Shell growth inhibition was calculated relative to the negative control.

(2) 96-hour EC50 (95% confidence interval) = 73 mg technical test material/L (equivalent to 24 mg test substance cation/L) (50 – 87 mg technical test material/L (equivalent to 17 - 29 mg test substance cation/L)).

* Statistically significant difference from the negative control using Dunnett’s test.

 

Calculation of key result

The doses of the test substance were expressed in mg technical test material/L, 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 73 mg technical test material/L = 33.8 mg pure test substance/L. The recalculated NOEC value is 3.74 mg pure test substance/L

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 96-hour EC50 value for eastern oysters was determined to be 73 mg technical test material/L. This value corresponds to a recalculated value of 33.8 mg pure substance/L.

Executive summary:

The acute toxicity to saltwater invertebrates was determined in a study according to EPA OPPTS 850.1025 and in compliance with GLP criteria. In this study Eastern oysters (C. virginica, 20 per concentration) were exposed to nominal concentrations of 0 (control), 3.1, 6.3, 13, 25, 50 and 100 mg technical test material for 96 hours under flow-through conditions. Analytical confirmation of nominal test concentrations showed that all test concentrations remained within ± 20% of nominal concentrations throughout the test. The mean measured concentrations were <LOQ (control), 3.7, 7.5, 15, 27, 54 and 106 mg technical test material/L. Oysters were observed daily during the test for mortality and clinical signs of toxicity. At the end of the test, the longest finger of new shell growth was measured to the nearest 0.05 mm. After 96 hours, the mean shell deposition in the negative control group was 5.5 mm. Mean shell deposition in the 3.7, 7.5, 15, 27, 54 and 106 mg technical test material/L treatment groups was 4.5, 4.7, 4.1, 3.7, 3.4 and 1.6 mm, respectively. Dunnett’s test indicated that there was a statistically significant decrease in shell deposition in the 15, 27, 54 and 106 mg technical test material/L treatment groups in comparison to the negative control (p ≤ 0.05). Percent inhibition of shell growth was calculated relative to the negative control data. Inhibition of shell growth in the 3.7, 7.5, 15, 27, 54 and 106 mg technical test material/L treatment groups was 19, 15, 26, 32, 38 and 72%, respectively. Based on these findings, The 96-hour EC50 value was determined to be 73 mg technical test material/L. This value corresponds to a recalculated value of 33.8 mg pure substance/L . The 96-h NOEC value was determined to be 7.5 mg technical test material/L, corresponding to a recalculated value of 3.74 mg pure substance/L .

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Oct 2013 to 13 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1035 (Mysid 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 one test chamber of 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 alternating replicate 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 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

Details on test solutions:

Individual stock solutions were prepared for each of the five concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into reverse osmosis water at a nominal concentration of 20 mg technical test substance/mL. Four secondary stock solutions were prepared in R.O. water at nominal concentrations of 1.3, 2.5, 5.0 and 10 mg technical test substance/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and ranged in appearance from clear and very light brown to clear and dark brown. Stock solutions were stored ambient in glass amber bottles, and aliquots of each stock were placed in the syringe pump every three days during the study. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 12.50 μL/minute where they were mixed with dilution water delivered at a rate of 125 mL/minute to achieve the desired test concentrations. The negative control received dilution water only.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Saltwater mysid
- Source: Cultures maintained by the test facility
- Age: Juveniles (<24 hours old)

HOLDING
- Acclimation period: 2 weeks
- Acclimation conditions: Similar to test; during the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 25.6 to 26.1ºC, measured with a liquid-in-glass thermometer. The pH of the water ranged from 8.0 to 8.3, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥7.0 mg/L (≥95% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. Salinity of the filtered saltwater ranged from 20 to 21 parts per thousand (‰), measured with a VitalSine refractometer.
- Feeding: Mysids in the cultures were fed live brine shrimp nauplii (Artemia sp.) daily. The brine shrimp periodically were enriched with a nutrient enrichment (A1 DHA Selco). During the test, the mysids were fed live approximately 0.1 mL of brine shrimp nauplii (Artemia sp.) one to two times a day. Excess food and waste were siphoned out daily during observations.
- Health during acclimation: During the holding period the adults showed no signs of disease or stress.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23.2 - 24.9 °C

pH:

7.9 - 8.0

Dissolved oxygen:

7.4 mg/L

Salinity:

20‰

Nominal and measured concentrations:

- Nominal concentrations: 0.13, 0.25, 0.50, 1.0 and 2.0 mg technical test substance/L.
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period. Test chambers were 25-L Teflon-lined stainless steel aquaria filled with 15 L of test water. The depth of the test water in a representative chamber was 18.0 cm. Each test chamber contained one test compartment constructed from a glass container approximately 12 cm in diameter and 19 cm in height, with nylon screen attached to two holes (approximately 5 cm in diameter) on the sides of the container. The surface of the water in the test chamber was located approximately halfway up the holes on the side of the test compartment. The split delivering fresh solution to each replicate was positioned towards one of the holes on the side of the test compartment. The stand-pipe drain for each test chamber was located in the center of each aquaria. The depth of the test water in a representative compartment was 10.0 cm.
- 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 6 volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers. 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. The proportion of the test water that was split into each replicate test chamber was checked prior to the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. 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 twice per day during the test.
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- Introduction of individuals: To initiate the exposure, the juvenile mysids were collected by first removing the adults from a culture tank using a mesh screen and then placing these adult mysids into a clean tank. The time was recorded when this transfer was conducted. The following day, the adults were again removed using the mesh screen and any young remaining were enumerated for use in the study. The young were then indiscriminately transferred one and two at a time into transfer chambers until each chamber contained 10 mysids. Each group of mysids then was transferred to the test compartment in an indiscriminately assigned test chamber to initiate the exposure. All transfers were performed beneath the water surface using widebore pipettes.

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 37800-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, the 20‰ water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Salinity: 20‰
- Culture medium different from test medium: Same

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Ambient laboratory light was used to illuminate the exposure systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight
- Light quality: 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 intensity: 536 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.

WATER QUALITY MEASUREMENTS
- Temperature: 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 a validated environmental monitoring system, which was calibrated prior to exposure initiation with a digital thermometer.
- Dissolved oxyge nad pH: Dissolved oxygen and pH were measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24-hour intervals during the test, and at the end of the test, with measurements typically alternating between replicates in each group at each measurement interval. 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 meter. If 100% mortality occurred in a test chamber, measurements of temperature, dissolved oxygen and pH were taken in that test chamber and then discontinued.
- Salinity: Salinity was measured in the dilution water at exposure initiation and termination using a VitalSine refractometer.


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. The criteria for death include lack of movement, absence of respiratory movement and lack of reaction to gentle prodding. 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 3.5, 24, 48, 72 and 96 hours after exposure initiation.

VEHICLE CONTROL PERFORMED: no

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

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

mortality

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.:

0.68 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: original value presented in the study report (technical test substance)

Details on results:

Daily observations of mortality and signs of toxicity observed during the test are presented in 'Any other information on results incl. tables'. All mysids in the negative control group appeared normal throughout the test, with one mortality noted at 96-hours. All saltwater mysids in the 0.15 mg technical test substance/L treatment group also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortality in the 0.30, 0.56, 1.1 and 2.2 mg technical test substance/L treatment groups at test termination was 10, 20, 90 and 100%, respectively.

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 and 72-hour LC50 values and binominal probability was used to calculate the 95% confidence intervals. Probit analysis was used to calculate the 96-hour LC50 value. For the 96-hour LC50 value, the slope of the dose response curve was 5.0. 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.

Table: Cumulative Mortality and Observations

Technical Test Substance Mean Measured Concentration (mg/L)	Cation Equivalent	Rep.	No. Exposed	3.5 Hours		24 Hours		48 Hours		72 Hours		96 Hours		Cumulative Percent Mortality
				No. Dead (1)	Obs. (2)	No. Dead (1)	Obs. (2)	No. Dead (1)	Obs. (2)	No. Dead (1)	Obs. (2)	No. Dead (1)	Obs. (2)
Negative control	Negative control	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	1	9 AN; 1 M	5
		B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
0.15	0.05	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0
		B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
0.3	0.1	A	10	0	10 AN	0	10 AN	0	10 AN	1	9 AN	2	8 AN; 1 M	10
		B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
0.56	0.19	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	2	8 AN; 1 M	20
		B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	2	8 AN
1.1	0.37	A	10	0	10 AN	0	10 AN	5	5 AN	9	1 AN; 1 M	9	1 AN	90
		B	10	0	10 AN	0	10 AN	7	3 AN	9	1 AN	9	1 AN
2.2	0.74	A	10	0	10 AN	0	3 A; 7 C	10	--	10	--	10	--	100
		B	10	0	10 AN	3	4 A; 3 C	10	--	10	--	10	--

(1) Cumulative number of dead mysids.

(2) Observations: AN = appear normal; A = surfacing; C = lethargy; M = missing and assumed dead.

Calculation of key result

The doses of the test substance were expressed in mg technical test material/L, 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 0.68 mg technical test substance/L = 0.31 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 0.68 mg technical test substance/L. This value corresponds to a recalculated value of 0.31 mg/L pure substance.

Executive summary:

The short-term toxicity to aquatic invertebrates was determined in a GLP-compliant study in accordance with EPA OPPTS 850.1035. In this study, saltwater mysids (Americamysis bahia) were exposed for 96 hours under flow-through conditions to five mean measured concentrations of the test substance ranging from 0.15 to 2.2 mg technical test substance/L. The 96-hour LC50 value was 0.68 mg technical test substance/L, with a 95% confidence interval of 0.56 to 0.82 mg/L. The recalculated 96-h LC50 value for the pure substance is 0.31 mg/L.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1981 adopted as Daphnia sp.14-day Reproduction Test including an Acute Immobilisation Test

Qualifier:

according to guideline

Guideline:

other: EPA-660/3-75-009: Methods for Acute Toxicity with Fish, Macro-Invertebrates and Amphibians.

Version / remarks:

1975

Qualifier:

according to guideline

Guideline:

other: ASTM E729-80: Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macro-Invertebrates and Amphibians.

Version / remarks:

1980

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of each test concentration were analysed for the test substance at 0 and 48 hours.

Vehicle:

no

Details on test solutions:

PREPARATION OF AND APPLICATION OF TEST SOLUTION
- The reconstituted water (hard) used to prepare the test concentrations was made by dissolving the following quantities of reagents in deionised water NaHCO3 (192 mg/L), CaSO4.2H20 (120 mg/L), MgSO4.7H20 (245 mg/L) and KCl (8 mg/L).
- The top concentration (10 mg a.i./L) was prepared by adding 52.1 μL of the test material directly to 2L of reconstituted water. Lower concentrations were prepared from this by serial dilution.
- Controls: reconstituted water only.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age: Less than 24 hours
- Food type: a diet of yeast and Chlorella vulgaris
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

192 mg/L as NaHCO3

Test temperature:

20 to 21°C

pH:

8.1 to 8.3

Dissolved oxygen:

Dissolved oxygen levels remained >8.9 mg/ L (97% saturation) throughout the test

Nominal and measured concentrations:

- Nominal concentrations (based on range finder): 0 (control), 0.778, 1.3, 2.16, 3.60, 6.00 and 10.00 mg a.i./L
- Measured concentrations (0 hours): <0.06 (LOD), 0.874, 1.47, 2.41, 4.91, 6.52 and 10.7 mg a.i./L
- Measured concentrations (48 hours): <0.06 (LOD), 0.842, 1.41, 2.39, 3.91, 6.20 and 10.5 mg a.i./L
- The mean measured concentrations were in the range 106 to 123% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type: Closed; test vessels were individually covered to reduce evaporation
- Fill volume: 200 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration: 3
- No. of vessels per control: 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water (hard) used to prepare the test concentrations was made by dissolving the following quantities of reagents in de-ionised water NaHCO3 (192 mg/L), CaSO4.2H20 (120 mg/L), MgSO4.7H20 (245 mg/L) and KCl (8 mg/L).
- Culture medium different from test medium: Same

OTHER TEST CONDITIONS
- Photoperiod: 16 hour daylight / 8 hours darkness
- Light: Fluorescent lighting provided by one 30 watt "warm white" tubes
- Light intensity: approximately 800 lux at the water surface

WATER QUALITY MEASUREMENTS
- Dissolved oxygen and pH: At the beginning and end of the test, dissolved oxygen levels and pH were measured in representative test solutions. The zero-time measurements were taken in the absence of Daphnia from separate aliquots of test solutions, to avoid cross-contamination. The final measurements at 48 hours, were taken in test solutions containing Daphnia. The dissolved oxygen concentration and pH were measured by YSI model 57 and Philips PW 9421 meters, respectively.
- Temperature: The temperature of the water bath, containing the Daphnia test vessels, was recorded at each assessment time using a maximum-minimum thermometer which was re-set after each reading.

EFFECT PARAMETERS MEASURED: immobility
The toxicity of the test chemical to the organisms was assessed after 3, 9, 24 and 48 hours. Daphnia were recorded as affected if they were immobilised or showing only minor movements of appendages after gentle agitation of the vessel.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
An initial range-finding test was performed in duplicate at nominal concentrations of 0 (control), 0.11, 0.33, 1.0, 3.0, 9.0 and 27.0 mg a.i./L. Immobility was recorded after 24 and 48 hours. After 48 hours, the percentage affected daphnids was 0 (control), 0, 0, 0, 35, 100 and 100%.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

mobility

Remarks on result:

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

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mobility

Remarks on result:

other: Original value presented in study

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mobility

Remarks on result:

other: Original value presented in study

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

11.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mobility

Remarks on result:

other: Original value presented in study

Details on results:

- The 24 and 48 hour EC50 values, based on nominal concentrations, were 11.8 and 4.4 mg test substance cation/ L, respectively. The 48 hour no observed effect level was 2.16 mg/L, based on nominal concentration. Details are provided in the 'any other information on results incl. tables' section.
- The results of the dose-range finding study are summarized in the 'any other information on results incl. tables' section.

Reported statistics and error estimates:

- Estimates of slope, log EC50 and EC5Q and 95% confidence intervals (for log EC50 and EC50) were obtained by the technique of iteratively reweighted least squares of probit response on log10 (concentration).
- An estimate of the no observed effect level (NOEL) was obtained by contrasting the effect at each concentration with the effect in the control group using a pooled estimate of error variance - from a one-way analysis of variance. The NOEL is the test concentration below the lowest concentration to have an effect significantly greater than the control (p=0.05).

Table: Toxicity of the test substance to first Instar Daphnia

Nominal Concentration (mg a.i./L)	3 hour			9 hour			24 hour			48 hour
	a	b	c	a	b	c	a	b	c	a	b	c
10.0	0	0	0	0	0	0	4	5	3	10	10	10
6.00	0	0	0	0	0	0	4	0	1	10	7	9
3.60	0	0	0	0	0	0	2	0	0	3	0	2
2.16	0	0	0	0	0	0	0	0	0	1	1	0
1.30	0	0	0	0	0	0	0	0	0	0	0	0
0.778	0	0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0	0	0

a, b and c are replicate chambers

Table: Toxicity observed in the dose range finding study

Nominal Concentration (mg a.i./L)	Nos Daphnia Affected
	24 hour		48 hour
	a	b	a	b
10.0	10	9	10	10
6.00	1	1	10	10
3.60	0	0	6	1
2.16	0	0	0	0
1.30	0	0	0	0
0.778	0	0	0	0
Control	0	0	0	0

a and b are replicate chambers

Calculation of key result

The doses of the test substance were expressed in test substbace cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 4.4 mg test substance cation /L = 6.1 mg pure test substance/L

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 of the test item was calculated to be 4.4 mg test substance cation/L. This value corresponds to a recalculated value of 6.1 mg/L pure substance.

Executive summary:

The acute toxicity to aquatic invertebrates was determined in a study according to OECD 202 pt 1 (1981) and in compliance with GLP criteria. In this study daphnids (D. magna, 10 per concentration) were exposed in triplicate to nominal concentrations of 0 (control), 0.778, 1.3, 2.16, 3.60, 6.00 and 10.00 mg test substance cation/L for 48 hours under static conditions. These concentrations were selected based on an initial dose range finding study. Analytical confirmation of nominal test concentrations showed that all test concentrations were between 106 and 123 % of nominal concentrations. Immobilization was recorded after 3, 9, 24 and 48 hours exposure. The EC50 values for 24 and 48 hours based on nominal concentrations were 11.8 and 4.4 mg test substance cation/L, respectively. The 48 hour NOEL was 2.2 mg test substance cation/L, based on nominal concentrations. The recalculated 48-h EC50 value for the pure substance is 6.1 mg/L.

Description of key information

The 48-h EC50 value is 6.1 mg pure substance/L (recalculated) for freshwater invertebrates (Daphnia magna), OECD 202, Allison 1990

The 96-h LC50 value is 0.31 mg pure substance/L (recalculated) for the saltwater mysid (A. bahia), OPPTS 850.1035, Claude 2013

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 invertebrates

Fresh water invertebrates

Effect concentration:

6.1 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.31 mg/L

Additional information

Table: Overview of aquatic invertebrate acute toxicity data available for the substance:

Species	Guideline/ GLP	Endpoint	Effect value	Comment	Reference
Daphnia magna (freshwater)	OECD 202/ GLP	48-hour EC50	6.1 mg/L (recalculated)	Static regime. The mean measured concentrations were used for the effect values	Allison 1990
Americamysis bahia (saltwater)	OPPTS 850.1035 / GLP	96-h LC50	0.31 mg/L (recalculated)	Flow-through regime. The mean measured concentrations were used for the effect values	Claude, 2013
Crassostrea virginica (saltwater)	OPPTS 850.1025 / GLP	96-h EC50	33.8 mg/L (recalculated)	Flow-through regime. Shell growth inhibition is the relevant effect parameter.	Claude, 2104