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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct 2013 to 20 Nov 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Version / remarks:
1996
Qualifier:
according to guideline
Guideline:
other: ASTM Standard E 1241-05: Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes
Version / remarks:
2005
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Duplicate water samples were collected from each treatment and control group two days prior to the start of the test after conditioning the diluter for approximately three days. Duplicate water samples also were collected from test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test 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 frozen for possible future analysis.
Vehicle:
no
Details on test solutions:
Stock solutions were prepared three times during the study. A primary test substance stock solution was prepared in reverse osmosis (RO) water at a nominal concentration of 80 mg technical test substance/mL. Proportional dilutions of the primary stock were made in RO water to prepare additional stock solutions at nominal concentrations of 2.5, 5.0, 10, 20 and 40 mg technical test substance/mL. The stock solutions were mixed by inversion and ranged in appearance from clear and light brown to opaque and very dark brown, with color intensity increasing with increasing concentration. No evidence of precipitate was noted in any of the stock solutions. Stock solutions were stored under ambient conditions and fresh aliquots were placed in the syringe pumps every one or two days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 20.0 μL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
Upon receipt, the embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development.
- Common name: Fathead minnow
- Source: Chesapeake Cultures, Inc., Hayes, Virginia
- Age at study initiation: Embryos collected for use were <24 hours old between stage 8 (gastrula) and stage 9 (neurula) when the exposure was initiated.
- Feeding during test : Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
5-Day Hatch and 28-Day Post-Hatch
Hardness:
128 - 144 mg/L as CaCO3
Test temperature:
23.9 - 25.5 °C; temperature measured continuously during the in the test in the negative control replicate A test chamber ranged from approximately 24.09 to 26.02°C, measured to the nearest 0.01°C.
pH:
8.0 - 8.2
Dissolved oxygen:
7.6 - 8.2 mg O2/L
Conductivity:
308 - 413 mg/L μS/cm
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 mg technical test substance/L.
- Mean measured concentrations:
Details on test conditions:
TEST SYSTEM
- Emybro cups: To initiate the exposure, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control test chamber. Embryos were held in incubation cups constructed from glass cylinders 50 mm in diameter with 425-μm nylon screen attached to the bottom with silicone sealant. One cup was suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (2 rpm) facilitated circulation of test water around the embryos during incubation.
- Test vessel: The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was 16.1 cm.
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test substance and a negative control (dilution water). Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver the six test substance stocks into mixing chambers assigned to each treatment. The syringe pumps were calibrated prior to the exposure. The stock solutions were diluted with freshwater in the mixing chambers to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to exposure initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately ten volume additions of test water to each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.026 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.27 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. 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, 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: 158 - 184 as mg/L CaCO3
- Culture medium different from test medium: Same

WATER QUALITY PARAMETERS
- Tempetrature: The target test temperature during the test was 25 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a Fulscope ER/C Recorder and a validated environmental monitoring system (Amegaview Central Monitoring System), which were calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Model 850Aplus dissolved oxygen meter and pH was measured using a Thermo Orion Model 525Aplus pH meter.
- Hardness, alkalinity and specific conductance: Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Hardness and alkalinity were measured 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
- Adjustment of pH: No
- Photoperiod: Lighting 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 test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: 537 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: embryo mortality, hatching, larave mortality, sublethal effects, length and weight weight (wet and dry) of surviving fish
During the 24 hours of exposure, embryos were observed twice for mortality and for eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached >90% in the control groups on Day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were maintained in the egg cups until they hatched and were released into the respective test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the number of mortalities and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos, which hatched successfully.
Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day posthatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60°C for approximately. The fish were dried for 48 hours. After drying, the fish were then measured for dry weight data collection.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
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:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: pure test substance
Basis for effect:
other: growth measured as total length, wet and dry weight,
Remarks on result:
other: recalculated value, expressed as pure substance, see ''Any other information on results incl. tables' for respective calculation
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth measured as total length, wet and dry weight,
Remarks on result:
other: Original value presented in study report (technical test item)
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: pure test substance
Basis for effect:
other: growth measured as total length, wet and dry weight,
Remarks on result:
other: recalculated value, expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
0.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth measured as total length, wet and dry weight,
Remarks on result:
other: Original value presented in study (technical test item)
Details on results:
TIME TO HATCH AND SUCCESS
All viable embryos in the control, 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment replicates hatched by Days 4 or 5 of the test, and larvae were released into the test chambers on Day 5 of the test when >90% of the viable embryos in the negative control replicates hatched. Daily observations of all the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the treatment groups. Hatching success in the negative control group was 95.0%. Hatching success in the 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment groups was 97.5, 88.8, 91.3, 100, 95.0 and 98.8%, respectively. Fisher’s Exact test showed that there were no statistically significant decreases in hatching success at any of the treatment groups when compared to the negative control (p > 0.05).

LARVAL SURVIVAL AND CLINICAL OBSERVATIONS
- Larval survival: Larval survival in the negative control group at test termination was 89.5%. Larval survival in the 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment groups was 92.3, 90.1, 93.2, 86.3, 78.9 and 30.4%, respectively. Fisher’s Exact test determined that there was a statistically significant decrease in larval survival noted in the 9.1 mg technical test substance/L treatment group when compared to the negative control data (p ≤0.05).
- Sublethal effects: The majority of the fish in the negative control and the 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment groups appeared normal throughout the test, with occasional observations of fish that were small, weak, lying on the bottom of the test chamber, exhibited erratic swimming or loss of equilibrium and/or were deformed (curled). These observations were made in the negative control as well as in the test substance technical treatment groups and are considered within the normal biological variability for this species. Therefore, none of the sublethal effects noted for the fish in the 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment groups were considered to be treatment-related.

GROWTH
Statistically significant reductions in mean wet weight were noted among fish in the 4.5 mg technical test substance/L treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). Statistically significant reductions in mean dry weight were noted among fish in the 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test substance/L treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). No statistically significant reductions in mean total length were noted in any of the treatment groups in comparison to the negative control (Dunnett’s one-tailed test, p > 0.05).
Reported statistics and error estimates:
A description of the reported statistics and error estimates are presented in 'Any other information on materials and methods incl. tables'.

Table: Summary of hatching success, larval survival and growth of fathead minnows exposed to the test substance

Technical test substance mean measured concentration (mg/L)

Cation equivalent concentration (mg/L)

Number exposed

Total number hatched

Hatching success (%)

Number surviving to termination

Post-hatch survival (%)

Mean total length ± Std. Dev. (mm)

Mean wet weight ± Std. Dev. (mg)

Mean dry weight ± Std. Dev. (mg)

Negative Control

Negative Control

80

76

95

68

89.5

24.0 ± 0.00

93.8 ± 6.25

19.3 ± 1.12

0.28

0.094

80

78

97.5

72

92.3

23.5 ± 0.58

86.7 ± 2.14

17.6 ± 0.854

0.54

0.18

80

71

88.8

64

90.1

23.8 ± 0.50

89.5 ± 4.02

17.2 ± 0.538*

1.1

0.37

80

73

91.3

68

93.2

24.0 ± 0.00

85.5 ± 1.32

16.9 ± 1.09*

2.2

0.74

80

80

100

69

86.3

24.0 ± 0.00

88.3 ± 3.12

17.3 ± 0.676*

4.5

1.5

80

76

95

60

78.9

23.5 ± 0.58

81.5 ± 1.11*

15.7 ± 0.359*

9.1

3.0

80

79

98.8

24

30.4*

23.8 ± 1.89

87.7 ± 13.3

16.3 ± 2.31*

* Indicates a significant difference (p≤0.05) from the control.

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:

NOEC: 0.463 x 0.28 mg technical test material/L = 0.13 mg pure test substance/L.

LOEC: 0.463 x 0.54 mg paraquat dichloride technical material/L = 0.25 mg pure test substance/L.

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 33-d early-life stage toxicity NOEC of the test item was calculated to be 0.28 mg technical test material/L. This value corresponds to a recalculated value of 0.13 mg/L pure substance.
Executive summary:

The early-life stage toxicity to freshwater fish was determined in a study according to OECD TG 210 and in compliance with GLP criteria. In this study, groups of 80 embryo fathead minnows (Pimephales promelas) were exposed for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period) under flow-through conditions to nominal concentrations concentrations of 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 mg technical test material/L. Test concentrations were analytically verified and determined to be <LOQ (control), 0.28, 0.54, 1.1, 2.2, 4.5 and 9.1 mg technical test material/L (arithmic mean), respectively. There were no statistically significant treatment-related effects on hatching success, survival or growth at concentrations of 0.28 mg/L. Growth (measured as total length, wet and dry weight) was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to concentrations ≥0.54 mg/L exhibited statistically significant reductions in dry weight in comparison to the negative control. Consequently, the NOEC, based on growth, was 0.28 mg technical test material/L (equivalent to 0.094 mg cation/L). The LOEC was 0.54 mg technical test material/L (equivalent to 0.18 mg cation/L) and the MATC was calculated to be 0.39 mg technical test material/L (equivalent to 0.13 mg cation/L).

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Nov 2013 to 10 Dec 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ASTM Standard E 1241-05: Standard Guide for Conducting Early Life- Stage Toxicity Tests with Fishes
Version / remarks:
2005
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Duplicate water samples were collected from each treatment and control group one day prior to the start of the test after conditioning the diluter for approximately two days. Duplicate water samples also were collected from test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test 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 refrigerated for possible future analysis.
Vehicle:
no
Details on test solutions:
Stock solutions were prepared four times during the study. A primary test substance stock solution was prepared in reverse osmosis (RO) water at a nominal concentration of 100 mg technical test material/mL. Proportional dilutions of the primary stock were made in RO water to prepare additional stock solutions at nominal concentrations of 6.3, 13, 25 and 50 mg technical test material/mL. The stock solutions were mixed by inversion and ranged in appearance from clear and light brown to opaque and very dark brown, with color intensity increasing with increasing concentration. No evidence of precipitate was noted in any of the stock solutions. Stock solutions were stored under ambient conditions and fresh aliquots were placed in the syringe pumps every one or two days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 20.0 μL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Sheepshead minnow
- Age at study initiation: Embryos collected for use in the test were 24 to 27 hours old at exposure initiation. Based on a subset evaluated, the eggs were at early stages of differentiation of the embryo and had passed the stage of differentiation of the embryonic shield and embryonic axis. The undifferentiated embryos also did not extend beyond half way around the circumference of the yolk indicating they were approximately 24 hours but less than 28 hours of age when the exposure was initiated.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Egg evaluation: Upon receipt, the embryos were examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development.

POST-HATCH FEEDING
Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from INVE Aquaculture, Salt Lake City, Utah. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
5-Day Hatch and 28-Day Post-Hatch
Test temperature:
24.6 - 26.1; Temperature measured continuously during the test in the negative control replicate A test chamber ranged from approximately 22.39 to 27.50°C, measured to the nearest 0.01°C.
pH:
7.9 - 8.0
Dissolved oxygen:
7.1 - 7.4 mg O2/L
Salinity:
2%
Nominal and measured concentrations:
- Nominal test concentrations: 0 (control), 0.63, 1.3, 2.5, 5.0 and 10 mg technical test material/L
- Mean measured concentrations:
Details on test conditions:
TEST SYSTEM
- Emybro cups: Embryos were held in incubation cups constructed from glass cylinders 50 mm in diameter with 425-μm nylon screen attached to the bottom with silicone sealant. One cup was suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (2 rpm) facilitated circulation of test water around the embryos during incubation.
- Introduction of embryo: To initiate the exposure, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control test chamber.
- Test vessel: The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria. The depth of the test water in a representative test chamber was 16.2 cm.
- Fill volume: 7 L
- Aeration: not reported
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test substance and a negative control (dilution water). Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver the five test substance stocks into mixing chambers assigned to each treatment. The syringe pumps were calibrated prior to the exposure. The stock solutions were diluted with saltwater in the mixing chambers to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by
rotameters, which were calibrated prior to exposure initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately ten volume additions of test water to each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.021 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.22 g fish/L.

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 Wildlife International 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.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 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 quality: ambient laboratory light from fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: 462 at study initiation; 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 PARAMETERS
- Tempetrature: The target test temperature during the test was 25 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a Fulscope ER/C Recorder and a validated environmental monitoring system (Amegaview Central Monitoring System), which were calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer.
- Dissolved oxygen, pH and salinity: Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. Salinity was measured in one alternating replicate of the negative control and highest test concentration at the beginning of the test, weekly during the test and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Model 850Aplus dissolved oxygen meter and pH was measured using a Thermo Orion Model 525Aplus pH meter.

EFFECT PARAMETERS MEASURED: embryo mortality, hatching, larave mortality, sublethal effects, length and weight weight (wet and dry) of surviving fish
During the first 24 hours of exposure, embryos were observed twice for mortality and for eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached >90% in the control group on Day 6 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were maintained in the egg cups until they hatched and were released into the respective test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the number of mortalities and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch survival and growth were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos, which hatched successfully. Post-hatch growth of the sheepshead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60°C for approximately. The fish were dried for approximately 49 hours. After drying, the fish were then measured for dry weight data collection.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
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:
33 d
Dose descriptor:
NOEC
Effect conc.:
2.55 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: pure test substance
Basis for effect:
other: growth (total length and wet weight)
Remarks on result:
other: recalculated value, expressed as pure substance, see ''Any other information on results incl. tables' for respective calculation
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
5.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth (total length and wet weight)
Remarks on result:
other: Original value as presented in study report
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
5.09 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: pure test substance
Basis for effect:
other: growth (total length and wet weight)
Remarks on result:
other: recalculated value, expressed as pure substance, see ''Any other information on results incl. tables' for respective calculation
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth (total length and wet weight)
Remarks on result:
other: Orignial value as presented in study report
Details on results:
TIME TO HATCH AND HATCHING SUCCESS
Results on hatching success of the sheepshead minnow embryos is presented in 'Any other information on results incl. tables'. All viable embryos in the control, 0.70, 1.5, 2.8, 5.5 and 11 mg technical test material/L treatment replicates hatched by Days 5,6 or 7 of the test, and larvae were released into the test chambers on Day 6 of the test when >90% of the viable embryos in the negative control replicates hatched. Daily observations of all the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the test substance treatment groups. Hatching success in the negative control group was 98.8%. Hatching success in the 0.70, 1.5, 2.8, 5.5 and 11 mg technical test material/L treatment groups was 95.0, 97.5, 96.3, 96.3 and 98.8%, respectively. Fisher’s Exact test showed that there were no statistically significant decreases in hatching success at any of the treatment groups when compared to the negative control (p > 0.05).

LARVAL SURVIVAL AND CLINICAL OBSERVATIONS
Results on survival of the sheepshead minnow larvae through Day 28 post-hatch is presented in 'Any other information on results incl. tables'. Larval survival in the negative control group at test termination was 98.7%. Larval survival in the 0.70, 1.5, 2.8, 5.5 and 11 mg technical test material/L treatment groups 97.4, 100, 100, 97.4 and 100%, respectively. Fisher’s Exact test showed that there were no statistically significant decreases in larval survival at any of the test substance treatment groups when compared to the negative control (p > 0.05). The majority of the fish in the negative control and the 0.70, 1.5, 2.8, 5.5 and 11 mg technical test material/L treatment groups appeared normal throughout the test, with occasional observations of fish that were small, lethargic, weak and/or discolored (pale). These observations were made in the negative control as well as in the test substance treatment groups and are considered within the normal biological variability for this species. Therefore, none of the sublethal effects noted for the fish in the 0.70, 1.5, 2.8, 5.5 and 11 mg/L treatment groups were considered to be treatment-related.

GROWTH
Results on growth measurements at the end of the 28-day post-hatch period is presented in 'Any other information on results incl. tables'. Growth was evaluated at the end of the test by measuring the total length, wet weight and dry weight of each surviving fish. There were no statistically significant reductions in mean dry weight in any treatment group in comparison to the negative control (Dunnett’s one-tailed test, p ≤ 0.05). There was a statistically significant reduction in mean total length in the 11 mg/L treatment group. There was a statistically significant reduction in mean wet weight in the 0.70 and 11 mg/L treatment group. While there was a statistically significant decrease in the 0.70 mg/L treatment group in comparison to the negative control, it was not considered to be treatment related since the decrease was slight and nondose-responsive.
Reported statistics and error estimates:
Data on time to hatch was evaluated by visual interpretation of the data. Test endpoints analyzed statistically for the juvenile fish were hatching success, larval survival and growth (total length, wet weight and dry weight). The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and MATC. However, scientific judgment was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. The NOEC was defined as the highest test concentration that produced no significant treatment-related effects on hatching success, survival or growth. The LOEC was defined as the lowest test concentration that produced a significant treatment-related effect on hatching success, survival or growth. The MATC is calculated as the geometric mean of the NOEC and LOEC. Hatching success was calculated as the percentage of embryos that hatched successfully. Posthatch survival was calculated from the number of larvae that survived to test termination as a percentage of the number of embryos that hatched successfully. Hatching success and survival data were considered to be discrete-variable data, while growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (p ≤ 0.05) from the negative control.

All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test or Chi-Square test, and for homogeneity of variance using Levene’s tests (p = 0.01). Since the data passed the assumptions of normality and homogeneity of variances, those treatments that were significantly different from the control means were identified using Dunnett’s one-tailed test (p ≤ 0.05). All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

Table: Summary of hatching success, larval survival and growth of sheepshead minnows exposed to the test substance

Technical test substance mean measured concentration (mg/L)

Cation equivalent concentration (mg/L)

Number exposed

Total number hatched

Hatching success (%)

Number surviving to termination

Post-hatch survival (%)

Mean total length ± Std. Dev. (mm)

Mean wet weight ± Std. Dev. (mg)

Mean dry weight ± Std. Dev. (mg)

Negative Control

Negative Control

80

79

98.8

78

98.7

19 ± 0.50

76.0 ± 2.97

17.8 ± 1.51

0.7

0.23

80

76

95

74

97.4

19 ± 0.50

69.3 ± 3.75*(1)

17.0 ± 0.61

1.5

0.5

80

78

97.5

78

100

19 ± 0.00

84.4 ± 3.78

19.3 ± 1.09

2.8

0.94

80

77

96.3

77

100

19 ± 0.00

72.5 ± 1.81

16.4 ± 0.48

5.5

1.8

80

77

96.3

75

97.4

19 ± 0.50

73.4 ± 2.89

17.9 ± 0.68

11

3.7

80

79

98.8

79

100

18 ± 0.00*

67.1 ± 1.86*

16.9 ± 0.53

* Indicates a significant difference (p≤0.05) from the control.

1) While there was a statistically significant decrease in comparison to the negative control, it was not considered to be treatment related since the decrease was slight and

non-dose-responsive.

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:

NOEC: 0.463 x 5.5 mg technical test material/L = 2.55 mg pure test substance/L.

LOEC: 0.463 x 11 mg paraquat dichloride technical material/L = 5.09 mg pure test substance/L.

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 33-d early-life stage toxicity NOEC of the test item was calculated to be 5.5 mg technical test material/L. This value corresponds to a recalculated value of 2.55 mg/L pure substance.
Executive summary:

The early-life stage toxicity to freshwater fish was determined in a study according to OECD TG 210 and in compliance with GLP criteria. In this study, groups of 80 embryos sheepshead minnows (Cyprinodon variegatus) were exposed for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period) under flow-through conditions to nominal concentrations concentrations of 0 (control), 0.63, 1.3, 2.5, 5.0 and 10 mg technical test substance/L. Test concentrations were analytically verified and determined to be <LOQ (control), 0.7, 1.5, 2.8, 5.5 and 11 mg technical test substance/L (arithmic mean). There were no statistically significant treatment-related effects on hatching success, or survival at any of the concentrations tested. There was a statistically significant treatment related effect on growth (as mean total length and wet weight) only in the highest treatment group. Consequently, the NOEC, based on growth, was 5.5 mg technical test material/L. The LOEC was 11 mg technical test material/L. These values correspond to recalculated values of 2.55 and 5.09 mg/L pure substance, respectively.

Description of key information

The 33-d early-life stage toxicity NOEC is 0.13 mg pure test substance/L (recalculated) for fathead minnow (Pimephales promelas), OECD TG 210, Claude 2014

The 33-d early-life stage toxicity NOEC is 2.55 mg pure test substance/L (recalculated) for sheepshead minnow (Cyprinodon variegatus), OECD TG 210, Claude 2014

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.13 mg/L

Marine water fish

Marine water fish
Effect concentration:
2.55 mg/L

Additional information

Table: Overview of available data on the long-term toxicity to fish

Species

Guideline/ GLP

Endpoint

Effect value

Comment

Reference

Pimephales promelas (freshwater)

OECD 210/ GLP

33-d NOEC

0.13 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 210/ GLP

33-d NOEC

2.55 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