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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 17, 2014 to May 30, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Deviation:
The concentrations of the test substance in solution were satisfactorily maintained within ± 20% of the mean measured values, with the exception of the 8.0 μg/L nominal treatment concentration where the percent coefficient of variation was slightly above 20% (it was 20.8%). Since the deviation from the guideline was very slight, the study was considered to be valid.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
Water samples were collected from one test chamber of each treatment and control group four days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 7, 14, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. All samples were collected at mid-depth in the test chambers, placed in 40 mL Teflon®-lined tubes and separatory funnels and processed immediately for analysis.
Vehicle:
yes
Details on test solutions:
Stock solutions were prepared three times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 500 μg/mL. Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations of 13, 32, 80 and 200 μg/mL. The stock solutions were mixed by inversion and appeared clear and colorless. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every two days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 20 μL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 1.3, 3.2, 8.0, 20 and 50 μg/L. The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all Reofos 35 treatment groups was 0.1 mL/L.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnows are one of the preferred fish species to test the toxicity of test substances during the early life-stages of fish. This species was selected for use in the test based upon past use and ease of handling in the laboratory. Fathead minnow embryos used in the test were supplied by Chesapeake Cultures, Inc., Hayes, Virginia and were received at Wildlife International on spawning substrates. 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. Embryos collected for use in the test were from 11 individual spawns and were <24 hours old when the test was initiated. To initiate the test, 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.
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.
Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.036 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 0.37 g fish/L.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
5-day hatching period plus a 28-day post-hatch growth period.
Post exposure observation period:
No post exposure observation period detailed in the study report.
Hardness:
Hardness (mg/L as CaCO3) 137 - 139
Test temperature:
23.72 to 25.60°C, measured to the nearest 0.01°C.
pH:
8.1 - 8.2
Dissolved oxygen:
7.7 - 8.2 mg/L
Salinity:
Not applicable - freshwater study
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations selected for use in the study were 1.3, 3.2, 8.0, 20 and 50μg/L.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 78.6 to 101% of nominal concentrations.
Details on test conditions:
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 37,800-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 nd was passed through an ultraviolet (UV) sterilizer.
The well water is characterized as moderately-hard water.

Test Apparatus
A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver the five test substance stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into mixing chambers assigned to each treatment and the solvent control. The syringe pumps were calibrated prior to the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test 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 at approximately weekly intervals 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 10 volume additions of test water in 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 end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the build-up of bacterial/fungal growth.
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 approximately 15.7 cm. Test chambers were labeled with the project number, test concentration and replicate. Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant. The cups were 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.

Environmental Conditions
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were 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 was measured at the water surface of one representative test chamber at test initiation using a SPER Scientific Model 840006C light meter.
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 or digital thermometer.
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 were measured in alternating replicates of the negative control (dilution water), solvent control and each 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.

Biological Observations and Measurements
During the first day of exposure, embryos were observed twice for mortality and 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, the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the 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 numbers of mortalities and the numbers 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 that hatched successfully.
Post-hatch growth of the fathead 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 42 hours to obtain dry weight data.

Statistical Analyses
Data on time to hatch was evaluated by visual interpretation of the data. Test endpoints analysed statistically for the juvenile fish were hatching success, larval survival and growth (total length, wet weight and dry weight). The statistical analyses used to evaluate the data were based on the procedures provided in the OECD 210 guideline. The following conditions were used to judge whether the ECx values (e.g., EC10 or EC20) would be determined and reported:
1.) The test concentrations must bracket the ECx so that the ECx comes from interpolation rather than extrapolation.
2.) The ECx will be estimated so that (i) the 95% confidence interval reported for ECx does not contain zero and is not overly wide, (ii) the 95% confidence interval for the predicted mean at ECx does not contain the control mean, and (iii) there is no significant lack-of-fit of regression model to the data.
Because the data of the survival and growth endpoints met the above criteria for ECx, the ECx values based on survival and growth endpoints were calculated and reported. Since the EC 10 and EC20 based on hatching success and EC20 based on dry weight were outside of the data used for the calculation, the EC10 and EC20 for hatching success and EC20 based on dry weight were not reported. The 95% confidence interval values for the EC10 based on wet weight were overly wide, the values were not reported. 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.
Data from the negative and solvent control groups for each parameter were compared using an appropriate statistical tests (using Chi-square and Fisher’s Exact test, α = 0.05 for percent hatching success and survival and t-test, α = 0.05 for growth parameters). No statistically significant differences were detected in the hatching success, survival, wet weight or dry weight endpoints among the control groups (p > 0.05). Therefore, the negative and solvent control data were pooled for comparison with the treatment data for these endpoints. There was a statistically significant difference in total length endpoint between the negative and solvent control groups (p ≤ 0.05). However, the differences were very slight (~2.2%) and not considered to be biologically meaningful. Therefore, the control data were pooled for comparison with the treatment total length data.
Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch 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 (α = 0.05) from the pooled controls. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test, and for homogeneity of variance using Levene’s test (α = 0.01). Since the growth data passed the assumptions of normality and homogeneity of variances, those treatments that were significantly different from the pooled control means were identified using Dunnett’s one-tailed test (α = 0.05). All statistical tests were performed using a personal computer using TOXSTAT or SAS software.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
3.1 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
8.2 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
33 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration
Effect conc.:
5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Maximum Acceptable Toxicant Concentration
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 1.3, 3.2, 8.0, 20 and 50μg/L. During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. 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.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 78.6 to 101% of nominal concentrations. Samples of the test solutions collected during the test had measured concentrations that ranged from 72.3 to 128% of nominal concentrations. When the measured concentrations of test solution samples collected on Days 0, 7, 14, 21, 28 and 33 of the test were averaged for each treatment group, the mean measured test concentrations were 1.3, 3.1, 8.2, 19 and 51 μg/L representing 100, 97, 103, 95 and 102% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Physical and Chemical Measurements of Water
Water temperatures were within the 25 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥88% of saturation (7.2 mg/L). Measurements of pH ranged from 8.0 to 8.2 during the test.
Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment groups and did not appear to be influenced by Reofos 35 concentration. Light intensity at test initiation was 272 lux at the surface of the water of one representative test chamber.

Time to Hatch and Hatching Success
Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control groups and any of the Reofos 35 treatment groups. The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 4 and 5 of the test. Hatching reached >90% in the control groups on Day 5 of the test, at which time the larvae were released to their respective test chambers.
Hatching success in the negative and solvent control groups was 99 and 100%, respectively. There were no statistically significant differences in hatching success between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Hatching success in the pooled control, 1.3, 3.1, 8.2, 19 and 51 μg/L treatment groups was 99, 100, 100, 100, 100 and 99%, respectively. No statistically significant differences in hatching success were found in any of the Reofos 35 treatment groups in comparison to the pooled controls (Fisher’s Exact, p > 0.05). Consequently, the NOEC for hatching success was 51 μg/L and the LOEC was greater than 51 μg/L. Both the EC10 and EC20 values were determined to be greater than 51 μg/L.

Larval Survival and Clinical Observations
One fish in replicate D of the 1.3 μg/L treatment group was inadvertently injured during transfer (i.e. hemorrhaging around the gills) and was observed swimming erratically. A dead fish found in this replicate the following day was presumed to be the injured fish, and was excluded from the analysis of survival at test end as its mortality was not treatment related. Larval survival in the negative and solvent control groups was 91 and 96%, respectively. There were no statistically significant differences in larval survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Larval survival in the pooled control, 1.3, 3.1, 8.2, 19 and 51 μg/L treatment groups was 94, 96, 99, 94, 74 and 9%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 19 and 51 μg/L treatment groups in comparison to the pooled controls (p ≤ 0.05). Consequently, the NOEC for larval survival was 8.2 μg/L and the LOEC was 19 μg/L. The EC10 value was 12 μg/L, with the 95% confidence interval of 5.7 to 14 μg/L. The EC20 value was 17 μg/L, with the 95% confidence interval of 13 to 21 μg/L.
In general, the majority of the fish in the control groups and in the 1.3 and 3.1 μg/L treatment groups appeared normal throughout the test. There were some observations of organisms in the 1.3 and 3.1 μg/L treatment groups that appeared small, were weak, had a curled or crooked spine, were discoloured (pale) and/or were lying on the bottom of the test chamber. However, these observations were infrequent and comparable to observations of fish in the control group. In the 8.2, 19 and 51 μg/L treatment groups, the numbers of small fish increased with increasing concentration, with all surviving fish in the 19 and 51 μg/L treatments noted as small and/or weak by Day 3 of the post-hatch period. In addition, some surviving fish in the 19 and 51 μg/L treatment groups did not have clearly developed air bladders and were unable to swim to the surface, while some of the larvae were noted with distended air bladders and were noted to remain on the surface of the test solutions. These sublethal effects in the 8.2, 19 and 51 μg/L treatment groups were considered to be treatment related effects.

Growth
Due to statistically significant effects on survival, the growth data from the 19 and 51 μg/L treatment groups were excluded from the statistical analysis of growth endpoints. There were no statistically significant differences in the wet and dry weight parameters between the negative and solvent control groups (p > 0.05). Therefore, the control data for wet and dry weight were pooled for comparisons with the treatment groups. Dunnett’s one-tailed test indicated there was a statistically significant reduction in wet weight and dry weight among fish in the 1.3 and 8.2 μg/L treatment groups in comparison to the pooled controls (p ≤ 0.05).
There was a statistically significant difference in total length endpoint between the negative and solvent control groups (p ≤ 0.05). However, the differences were very slight (~2.2%) and not considered to be biologically meaningful. Therefore, the control data were pooled for comparison with the treatment total length data. Dunnett’s one-tailed test indicated statistically significant reductions in mean total length in the 1.3 and 8.2 μg/L treatment groups from the pooled control (p≤0.05). Since the reductions noted in the 1.3 μg/L treatment groups did not follow a dose response pattern, the statistically significant difference noted in this treatment level was not considered to be biologically meaningful. In addition, the reduction in total length in the 1.3 μg/L treatment group from the pooled control was very slight (~2.7%). Consequently, the NOEC for growth was 3.1 μg/L and the LOEC was 8.2 μg/L.
Results with reference substance (positive control):
Reference substance not used in this study.
Reported statistics and error estimates:
There were no statistically significant differences in hatching success between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups.
There were no statistically significant differences in larval survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 19 and 51 μg/L treatment groups in comparison to the pooled controls (p ≤ 0.05).
There were no statistically significant differences in the wet and dry weight parameters between the negative and solvent control groups (p > 0.05). Therefore, the control data for wet and dry weight were pooled for comparisons with the treatment groups. Dunnett’s one-tailed test indicated there was a statistically significant reduction in wet weight and dry weight among fish in the 1.3 and 8.2 μg/L treatment groups in comparison to the pooled controls (p ≤ 0.05).

Measured Concentrations of Reofos 35 in Test Solution Samples

Nominal Test Concentration (μg/L)

Sample Number (616A-118-)

Sampling Time (Days)

Measured Concentration (μg/L)1,2

Percent of Nominal2

Mean Measures Concentration (μg/L)

Mean Measured Percent of Nominal

Negative Control

1

0

<LOQ

--

<LOQ

--

8

7

<LOQ

--

15

14

<LOQ

--

22

21

<LOQ

--

29

28

<LOQ

--

36

33

<LOQ

--

Solvent Control

2

0

<LOQ

--

<LOQ

--

9

7

<LOQ

--

16

14

<LOQ

--

23

21

<LOQ

--

30

28

<LOQ

--

37

33

<LOQ

--

1.3

3

0

1.06

81.5

1.3 ± 0.161 CV = 12.4%

100

10

7

1.31

101

17

14

1.53

118

24

21

1.15

88.5

31

28

1.30

100

28

33

1.29

99.2

3.2

4

0

2.84

88.8

3.1 ± 0.459 CV = 14.8%

97

11

7

2.95

92.2

18

14

3.06

95.6

25

21

2.78

86.9

32

28

4.00

125

39

33

2.88

90.0

8.0

5

0

5.78

72.3

8.2 ± 1.71

CV = 20.8%

103

12

7

10.2

128

19

14

9.00

113

26

21

6.66

83.3

33

28

7.99

99.9

40

33

9.49

119

20

6

0

16.6

83.0

19 ± 2.34

CV = 12.3%

95

13

7

21.9

110

20

14

19.9

99.5

27

21

16.5

82.5

34

28

20.4

102

41

33

21.3

107

50

7

0

43.6

87.2

51 ± 4.90

CV = 9.61%

102

14

7

56.5

113

21

14

55.2

110

28

21

48.2

96.4

35

28

54.3

109

42

33

51.0

102

1The limit of quantification (LOQ) was 0.300 μg/L, calculated as the product of the concentration of the lowest calibration standard (3.00 μg/L) and the dilution factor of the matrix blank samples (0.100).

2Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

 

Means and Ranges of Water Quality Measurements Taken During the 33-Day Exposure to Reofos 35

Mean Measured Concentration (μg/L)

Mean ± SD and Range of Measured Parameters

Temperature1(°C)

DO2(mg/L)

pH

Hardness (mg/L as Ca CO3)

Alkalinity (mg/L as CaCO3)

Conductivity (μS/cm)

Negative Control

25.0 ± 0.32 (24.4 – 25.5)

8.2 ± 0.00 (8.2 – 8.2)

8.2 ± 0.05 (8.1 – 8.2)

137 ± 7

(128 – 148)

176 ± 2

(172 – 178)

370 ± 8

(361 – 380)

Solvent Control

25.2 ± 0.23 (24.9 – 25.6)

7.9 ± 0.33 (7.3 – 8.2)

8.1 ± 0.05 (8.0 – 8.1)

139 ± 7

(128 – 148)

178 ± 2

 (176 – 180)

367 ± 4

 (360 – 372)

1.3

25.2 ± 0.24 (24.8 – 25.5)

8.1 ± 0.22 (7.7 – 8.2)

8.1 ± 0.04 (8.1 – 8.2)

137 ± 5

 (128 – 140)

179 ± 1

 (178 – 180)

364 ± 4

 (358 – 371)

3.1

25.2 ± 0.23 (24.8 – 25.5)

8.0 ± 0.24 (7.7 – 8.2)

8.1 ± 0.08 (8.0 – 8.2)

138 ± 6

(128 – 144)

180 ± 2

 (176 – 182)

366 ± 4

 (360 – 371)

8.2

25.1 ± 0.27 (24.7 – 25.5)

7.7 ± 0.41 (7.2 – 8.2)

8.1 ± 0.05 (8.0 – 8.1)

139 ± 7

 (128 – 148)

179 ± 3

 (176 – 182)

367 ± 3

 (362 – 370)

19

25.2 ± 0.24 (24.8 – 25.5)

7.9 ± 0.22 (7.7 – 8.2)

8.1 ± 0.05 (8.1 – 8.2)

139 ± 5

(132 – 144)

179 ± 1

 (178 – 180)

365 ± 3

 (361 – 370)

51

25.1 ± 0.21 (24.8 – 25.4)

7.9 ± 0.33 (7.4 – 8.2)

8.1 ± 0.08 (8.0 – 8.2)

139 ± 6

 (132 – 148)

180 ± 2

(178 – 182)

366 ± 4

 (361 – 371)

1Temperature measures continuously during the test ranged from approximately 23.72 to 25.60°C, measured to the nearest 0.01°C.

2A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation at 25°C in freshwater.

 

Summary of Hatching Success, Larval Survival and Growth at Fathead Minnows Exposed to Reofos 35

Mean Measured Concentration (μg/L)

Percent Hatching Success1

Percent Survival to Day 28 Post-Hatch

Growth Parameters at Day 28 Post-Hatch2

Mean Total Length ± SD (mm)2

Mean Wet Weight ± SD (mg)2

Mean Dry Weight ± SD (mg)2

Negative Control

99

91

26.7 ± 0.42

129.7 ± 8.0

26.2 ± 2.3

Solvent Control

100

96

26.1 ± 0.22

119.5 ± 4.1

23.7 ± 1.0

Pooled Control

99

94

26.4 ± 0.483

124.6 ± 8.0

24.9 ± 2.1

1.3

100

96

25.7 ± 0.30Δ

115.3 ± 3.5*4

22.5 ± 0.97*4

3.1

100

99

26.2 ± 0.16

124.5 ± 3.0

25.0 ± 0.55

8.2

100

94

24.8 ± 0.32Δ,

108.4 ± 4.1*

21.9 ± 0.71*

19

100

74*

17.7 ± 1.15

51.7 ± 13.1

10.6 ± 2.86

51

99

9*

11.1 ± 1.66

10.4 ± 4.9

2.1 ± 0.96

EC10 (95% Confidence Interval) (μg/L):

NA

12 (5.7 – 14)

8.3 (8.24 – 8.4)

0.69 (NA)

9.8 (0.62 – 154)

EC20 (95% Confidence Interval) (μg/L):

NA

17 (13 – 21)

9.0 (8.8 – 9.2)

8.6 (8.3 – 8.9)

NA

*Indicates a significant difference in survival (Fisher’s Exact test, p≤0.05) and in mean wet and dry weight (Dunnett’s one-tailed test, p≤0.05).

ΔStatistically significant reductions in mean total length in the 1.3 and 8.2 μg/L treatment groups from the pooled control (Dunnett’s one-tailed test, p≤0.05). However, the reductions noted in the 1.3 μg/L treatment group did not follow a dose response pattern and was very slight (~2.7%), the statistically significant reduction in mean total length in the 1.3 μg/L treatment group was not considered to be biologically meaningful.

1There was no statistically significant reduction in percent hatching success from the pooled control (Fisher’s Exact test, p≥0.05).

2Growth data from the 19 and 51 μg/L treatment groups were excluded from analyses of growth end points due to significant effects on survival.

3Since the differences in mean total length between negative control and solvent control was slight (~2.2%) and not considered to be biologically meaningful, the treatment total length data were compared to the pooled control Dunnett’s one-tailed test.

4The statistically significant reductions in mean wet and dry weight in the 1.3 μg/L treatment groups from the pooled control (Dunnett’s one-tailed test, p≤0.05) did not follow a dose response pattern and was not considered to be biologically meaningful.

NA = not applicable; since the calculated ECx value was extrapolated beyond the data range used in the calculation or the 95% confidence interval contains zero or was overly wide.

Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to Reofos 35 at mean measured concentrations of 1.3 to 51 μg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success at concentrations ≤51 μg/L. Statistically significant treatment-related effects on survival were found in fish exposed to Reofos 35 at concentrations ≥19 μg/L. Growth, measured as total length,
wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to Reofos 35 at concentrations ≥8.2 μg/L had statistically significant reductions in total length, wet weight and dry weight in comparison to the controls. Consequently, the NOEC, based on growth, was 3.1 μg/L. The LOEC was 8.2 μg/L and the MATC was calculated to be 5.0 μg/L.
Executive summary:

The objective of this study was to determine the effects of Reofos 35 on the time to hatch, hatching success, survival, and growth of fathead minnows, Pimephales promelas, during early life-stage development.

The study was conducted according to the protocol, “Reofos 35: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephales promelas)”. The protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 210: Fish, Early-life Stage Toxicity Test; U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1400: Fish, Early-Life Stage Toxicity Test; and ASTM Standard E 1241-05: Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes.

Fathead minnows (Pimephales promelas) were exposed to Reofos 35 at mean measured concentrations of 1.3 to 51 μg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success at concentrations ≤51 μg/L. Statistically significant treatment-related effects on survival were found in fish exposed to Reofos 35 at concentrations ≥19 μg/L. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to Reofos 35 at concentrations ≥8.2 μg/L had statistically significant reductions in total length, wet weight and dry weight in comparison to the controls. Consequently, the NOEC, based on growth, was 3.1 μg/L. The LOEC was 8.2 μg/L and the MATC was calculated to be 5.0 μg/L.

Description of key information

The recent 33-d FEL study is taken as the value for hazard assessment.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
3.1 µg/L

Additional information

The data set available for the phosphates as a group indicates that these do pose hazardous effects to f ish. However as detailed within the registration dossier, such effects are considered to be attributable to the content of the impurity, triphenyl phosphate, CAS 115-86-6, (EC No. 204-112-2). The content of this impurity is considered to adversely affect the toxicity of the substance within aquatic organisms. The registration dossier details that two separate classifications applicable to the substance for this endpoint as follows:

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate >5%] - H410: Very toxic to aquatic life with long lasting effects.

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate < 5%] - H413: May cause long lasting harmful effects to aquatic life.

It is considered that given the age of this data, the Triphenyl phosphate is considered to be present in these products at and above the 5% threshold proposed. As such, this data as presented is considered to represent the classification where the Triphenyl phosphate content is greater than 5%.

For this data where Triphenyl phosphate >5%, the 33 – day NOEC from the recent Fish, Early-Life Stage Toxicity Test (OECD 210) is taken as the value for hazard assessment. This gives a value of 3.1 µg/L.