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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 2017 to 23 Jan 2018
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:
2013
Deviations:
no
Remarks:
see 'Deviation from the Protocol' in 'Any other information on materials and methods incl. tables'.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Version / remarks:
2016
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Prior to exposure initiation, samples from one replicate of each treatment level and the control were collected and analyzed for the test substance concentration. In addition, samples from the saturator column effluent were analyzed. Results of this analysis were used to determine whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure.
- During the definitive exposure, samples were removed on days 0 (experimental initiation), 6, 14, 20, 27, and 34 (experimental termination) from one replicate of each concentration and the control for analysis of the test substance concentration. Replicates were sequentially alternated between sampling intervals.
Vehicle:
yes
Remarks:
methanol
Details on test solutions:
For this exposure, a glass wool saturator column was used to deliver the test substance to the exposure system. The glass column was packed with glass wool, and then coated with the test substance. The column was designed to provide a constant flow of saturated aqueous solution (1.0 mg/L) of the test substance to the diluter system without the use of a carrier solvent. The saturator column was constructed entirely of chemical inert materials (glass and Teflon). To construct the column, each 60 centimeter length × 4.8 centimeter diameter column was firmly packed with approximately 15% of the total column volume with glass wool. This provided ample surface area inside the column for the exposure substance to adhere once the column preparation was complete. After the column was packed, the end fittings were placed on the column. All fittings used to enclose the column and to attach the column to the water source were composed of Teflon.
To coat each column, approximately 8.0 g of the test substance technical was diluted with 190 mL of methanol. The solution was sonicated in an ultrasonicator water bath for approximately 20 minutes until no undissolved test substance remained. This solution was then slowly poured into the glass column. After all of the solution was added, the column was attached to a vacuum pump. The vacuum pump was used to draw the solution evenly throughout the column to uniformly coat the glass wool with the test substance and draw off the remaining methanol. After it had visually appeared that all of the glass wool was coated and all the solvent was drawn off, the column was detached from the
vacuum pump and attached to a pump which delivered a flow of dilution water through the column. During non-solvent dosing trials, the saturator column effluent was measured to be delivering a stable, consistent dose of approximately 1.0 mg/L for approximately one week. The column output concentration was used to calculate the appropriate flow rate of the effluent stock solution into the diluter system. Based on the functional solubility of the test substance (1.0 mg/L), a flow rate of 30 mL/min was required to achieve the high test concentration to dose the system during the definitive exposure. Saturator columns were prepared and replaced on the exposure system as needed (approximately every week).
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Sheepshead minnow
- Source: Sheepshead minnow embryos used during this testing were obtained from brood stock maintained at the test facility.
- The brood stock used to generate embryos for this exposure were approximately 26 weeks old at exposure initiation.

ACCLIMATION
- Acclimation period: 14 days prior to exposure initiation
- Temperature: 17 to 19 °C (except during the final 24 hours, when the temperature was intentionally and gradually raised over several hours to 25 °C)
- Dissolved oxygen: 92 to 99% of saturation
- Health during acclimation: No mortality was observed among the brood stock during the seven days prior to testing.
- The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure.

FEEDING DRUING POST-HATCH
- Food type: Beginning on exposure day 7 (day 1 post-hatch), the juvenile fish were fed live brine shrimp nauplii (Artemia salina). At each feeding, juvenile fish were fed live brine shrimp such that all juvenile fish were afforded equal proportions of food (nauplii supplied ad libitum).
- Frequency: Three times daily
- Fish were not fed during the 24 hours prior to study termination.
- Food quality: Representative samples of the food source were periodically analyzed for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected in the food samples at concentrations considered toxic to the test organisms. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern in agreement with ASTM (2007).
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
34 d
Remarks on exposure duration:
28-day post-hatch
Test temperature:
25 ± 1 °C
pH:
7.3 - 7.8
Dissolved oxygen:
- 4.81 - 7.40 mg O2/L
- 66 and 100% of saturation
Salinity:
20 - 22‰
Nominal and measured concentrations:
- Nominal concentration: 0(negative control), 3.1, 7.7, 19, 48, and 120 µg/L
- Measured concentration: < LOQ (negative control), 3.7, 8.1, 18, 52, and 130 µg/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Size of vessel: 30 × 14.5 × 20 cm with a 12.5 cm high side drain
- Material: Glass and silicone sealant
- Fill volume: Approximately 5.5 L
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 53 L/aquarium/day
- Embryo incubation cups: Round glass jars
- Size of incubation cups: 5 cm O.D., 8 cm high (bottoms removed and 475 µm Nitex mesh screen bottoms applied)
- No. of organisms per incubation cup: 30
- No. of incubation cup per vessel: 1
- No. of vessels per concentration: 4
- No. of vessels per control: 4

EXPOSURE SYSTEM
- Prior to exposure initiation, a pump was calibrated to continuously deliver 30 mL/min of the 1.0 mg/L saturator column effluent (i.e., 396 mL per cycle) to the diluter system's mixing chamber. The effluent flow rate, in combination with the calibrated dilution water cell, filled the mixing chamber at each cycle to a total volume of 3.296 L. The mixing chamber was positioned over a magnetic stir plate and the solution was continuous stirred with a Teflon-coated stir bar throughout the test. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration (120 µg/L) and was proportionally diluted by a constant factor of 2.5 to produce the remaining nominal test concentrations (48, 19, 7.7, and 3.1 µg/L).
- The flow-through exposure was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 24 exposure aquaria. Flow-splitting cells were employed to equally distribute the solutions to the replicate vessels at a rate of 500 mL of control or test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within 5% of the nominal value. The exposure system was designed to provide five concentrations of the test substance and a dilution water control to four replicate aquaria. The diluter delivered the control and test solutions to the exposure aquaria (approximately 53 L/aquarium/day) at a rate sufficient to provide approximately 9.7 aquarium volumes per 24-hour period, with a 90% replacement time of approximately six hours.
- The diluter system was calibrated prior to exposure initiation and confirmed at exposure termination by measuring delivery volumes of toxicant solution and dilution water. The function of the diluter system (e.g., cycle rate and column effluent flow rate) was monitored daily and a visual check of the system’s operation was performed twice daily. In addition, analysis of the exposure solutions for the test substance concentration was also used to verify proper operation of the diluter system prior to the start of the exposure. The exposure system was properly operating for four days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure aquaria.

EXPOSURE INITIATION
- At exposure initiation, a subsample of embryos (N = 30) was collected to determine embryonic stage of development. The embryo subsample was preserved in Stockard’s solution (an 85:6:5:4 mixture of water, glycerine, formalin, and glacial acetic acid) for at least 24 hours prior to assessment. Embryonic stage was determined on each individual embryo within the subsample using guidance in the Pre-Hatching Development of the Fathead Minnow, Pimephales promelas, Rafinesque (U.S. EPA, 1996). Since there is little information on the precise developmental stages for sheepshead minnow, fathead minnow was used as a surrogate species for evaluating embryonic development. Pre - hatching development is similar between teleosts, and the time to hatching is similar between these two species at 25 °C. This document presents a classical model of teleostean embryogenesis and divides the pre-hatching development into 32 stages.
- The spawning apparatus was introduced to the brood stock aquarium at the end of the working day the evening prior to initiation (day 0). Therefore, some embryos were likely between 12 and 24 hours old. However, sheepshead minnows typically spawn in the early morning hours after dawn, or when the controlled light cycle begins, and based on the developmental stages of the embryo subsample at exposure initiation, a majority of the embryos used for this exposure were likely less than 7.5 hours post-fertilization. The exposure of sheepshead minnow embryos and juvenile fish to the test substance was initiated when the embryo incubation cups were distributed to each of the exposure aquaria. The embryos were impartially distributed to the embryo incubation cups in the following manner: twenty-four labeled embryo incubation cups were placed in a heated water bath containing dilution water maintained at 25 ºC. A container with the embryos in 25 ºC dilution water was placed in the same water bath. Prior to loading, the embryos were examined microscopically to assess embryo fertilization; all non-viable embryos were removed, leaving only fertilized embryos. The fertilized embryos were then impartially placed in the embryo incubation cups, five at a time, until each cup contained five embryos. This process was repeated until all cups contained 30 embryos. To initiate the exposure, the incubation cups were then placed in the respective exposure aquaria (one cup per replicate).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilute, natural, filtered seawater was used as dilution and control water during this study. Natural seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts. The water was collected at about 1 to 4 meters offshore and a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 6080 L polyethylene holding tank. The water was diluted with on-site laboratory well water to a salinity of 20 ± 2‰, filtered through 50 µm and 1 µm polypropylene bag filters, and heated to the required test temperature.
- Salinity: 20 to 21‰
- pH: 7.6 to 7.8
- Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2007) standard practices.
- TOC: Representative samples of the dilution water source were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the filtered seawater was 1.3 and 2.0 mg/L for the months of December 2017 and January 2018, respectively.
- Intervals of water quality measurement: At exposure initiation, dissolved oxygen concentration, pH, and temperature were measured in each replicate aquarium. Following day 0, one replicate of each concentration and the control was measured for these parameters; replicates were sequentially alternated each day thereafter.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness (Sudden transitions from light to dark, and vice versa, were avoided with a period of lower light intensity during the transition period. )
- Light intensity: 51 to 88 footcandles (550 to 950 lux)

EFFECT PARAMETERS MEASURED:
- Embryo-larval exposure: Embryos were removed from their respective incubation cup daily until hatching began. The number of dead, live, and unaccounted for embryos were counted and recorded. Dead embryos were discarded when observed at any time during the embryo incubation phase. If a loading error was suspected after the first day of embryo exposure (e.g., one or more missing embryos), then the actual number of embryos present on the first day was used as the denominator when computing percent hatch. When embryos began to hatch (test day 5), the number of live, hatched, dead, and unaccounted for embryos were counted within the incubation cup in order to avoid possible physical damage caused by removing newly hatched larvae. Dead larvae were also removed when observed during this period. Completion of hatch was considered to be exposure day 6, when all viable embryos in all control embryo incubation cups were hatched. Calculations of hatching success were based on the number of live, dead, and deformed larvae per incubation cup after hatching was completed (exposure day 6/day 0 post-hatch), compared to the number of embryos loaded in each egg cup.
- 28-day post-hatch fish exposure: The 28-day post-hatch fish exposure was initiated at completion of hatch (exposure day 6) by impartially selecting 20 surviving larvae from each incubation cup and transferring them into a Petri dish using a fire-polished glass pipette. The juvenile fish were then gently placed into each respective exposure aquarium from the Petri dish. The remaining juvenile fish were euthanized using an overdose of buffered tricaine methanesulfonate (MS-222) and then discarded. During the post-hatch exposure period, fish were observed daily; the behavior and appearance of the fish were recorded. Dead fish were removed during these observation periods and fish survival was estimated daily throughout the post-hatch period. At 28 days post-hatch, the exposure was terminated. The surviving juvenile fish in each replicate aquarium were euthanized with a buffered solution of MS-222, counted to determine juvenile fish survival, and measured individually to determine total length, wet weight, and dry weight. At exposure termination, fish that could not be accounted for were presumed to be dead at exposure termination. Following wet weight measurements, the fish were dried in an oven at approximately 60 °C for 24 hours. After a cooling period, dry weight measurements were collected. Length and weight were determined to the nearest 0.01 mm and 0.1 mg, respectively, using calibrated digital calipers and a calibrated analytical balance interfaced.

RANGE-FINDING STUDY
- Experimental design: A 20-day (14-day post-hatch) preliminary exposure was conducted from 24 October to 13 November 2017. Four replicates were maintained for each concentration and the control. The exposure was initiated with 30 embryos per replicate and larvae were thinned to 20 larvae per replicate following completion of hatch.
- Test nominal concentrations: 2.5, 7.4, 22, 67, and 200 µg/L and a control
- Results used to determine the conditions for the definitive study: Statistically significant reductions were observed in post-thinning survival, post-hatch survival, and overall survival at the 200 µg/L nominal concentration compared to the control. Statistically significant growth reductions were observed at the 67 and 200 µg/L nominal concentrations. Based on these results and consultation with the Study Sponsor, nominal concentrations of 3.1, 7.7, 19, 48, and 120 µg/L were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
8.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Mean Wet Weight and Mean Dry Weight
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
18 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
130 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Time-To-Hatch, Embryo Hatching Success, Live, Normal Larvae at Hatch, Post-thinning Survival, Post-Hatch Survival and Overall Survival
Details on results:
- At exposure initiation, a subsample of 30 embryos was collected and the embryonic stage of development was determined. The mean and median stage were both 13 (one-quarter epiboly) and the range of the developmental stages was 12 to 13 (early gastrula to one-quarter epiboly). Based on these embryonic stage characteristics, the subsample of embryos were approximately five to seven hours post-fertilization at the time of subsample preservation (i.e., exposure initiation).

- On exposure day 6, all viable embryos in the control and treatment replicates were hatched. No delays in hatch were observed at any treatment level. Time-to-hatch for all viable embryos in the control averaged 5.7 days. Time-to-hatch in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels averaged 5.8, 5.9, 5.8, 5.9, and 5.8 days, respectively. The variation in time-to-hatch was within expectations for this species. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant delay in time-to-hatch in any of the treatment levels tested compared to the control (5.7 days). Based on time-to-hatch, the No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) were determined to be 130 and > 130 µg/L, respectively.

- At the completion of hatch, hatching success in the control averaged 90%. Hatching success in the 3.7, 8.1, 18, 52, and 130 µg/L mean measured treatment levels averaged 89, 77, 88, 87, and 84%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in hatching success among embryos exposed to any of the treatment levels tested compared to the control (90%). Based on hatching success, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At the completion of hatch, the percent of live, normal larvae in the control averaged 100%. The mean percent of live, normal larvae in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 100, 99, 100, 100, and 100%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in percent of live, normal larvae among embryos exposed to any of the treatment levels tested compared to the control (100%). Based on percent of live, normal larvae at hatch, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively.
- At exposure termination (28 days post-hatch), post-thinning survival in the control averaged 99%. The mean post-thinning survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 99, 98, 98, 98, and 98%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in post-thinning survival among juvenile fish exposed to any of the treatment levels tested compared to the control (99%). Based on post-thinning survival, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At exposure termination, post-hatch survival in the control averaged 99%. The mean post-hatch survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 99, 98, 98, 98, and 98%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in post-hatch survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (99%). Based on post-hatch survival, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At exposure termination, overall survival in the control averaged 89%. The mean overall survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 88, 75, 85, 84, and 82%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in overall survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (89%). Based on overall survival, the NOEC and LOEC were determined to be 130 and >130 µg/L, respectively. At exposure termination, the total length of juvenile fish exposed to the control averaged 19.67 mm. The mean total length of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 19.45, 19.59, 19.37, 18.97, and 17.41 mm, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean total length of surviving juvenile fish exposed to the 52 and 130 µg/L treatment levels compared to the control (19.67 mm). Based on total length of juvenile fish, the NOEC and LOEC were determined to be 18 and 52 µg/L, respectively. At exposure termination, the wet weight of juvenile fish exposed to the control averaged 0.1084 g. The mean wet weight of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 0.1080, 0.1077, 0.1012, 0.0959, and 0.0741 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean wet weight of surviving juvenile fish exposed to the 18, 52, and 130 µg/L treatment levels compared to the control (0.1084 g). Based on the wet weight of juvenile fish, the NOEC and LOEC were determined to be 8.1 and 18 µg/L, respectively. At exposure termination, the dry weight of juvenile fish exposed to the control averaged 0.0272 g. The mean dry weight of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 0.0264, 0.0268, 0.0252, 0.0238, and 0.0175 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean dry weight of juvenile fish exposed to the 18, 52, and 130 µg/L treatment levels compared to the control (0.0272 g). Based on the dry weight of juvenile fish, the NOEC and LOEC were determined to be 8.1 and 18 µg/L, respectively.
Reported statistics and error estimates:
See Statistical Analysis in 'Any other information on materials and methods incl. tables'.

Table 3. Endpoint Summary

Mean Measured

Concentration

(µg/L)

 

 

Replicate

Time-to- Hatch

(Days)

Embryo

Hatching

Success

(%)a

Live, Normal

Larvae at

Hatch

(%)

Juvenile Fish (28 Days Post-Hatch)

Post - Thinning

Survival

(%)b

Post-Hatch

Survival

(%)c

Overall

Survival

(%)d

Mean Total

Length

(mm)

Mean Wet

Weight

(g)

Mean Dry

Weight

(g)

 

Control

A

5.9

90

100

100

100

90

19.25 (0.75)

0.1009 (0.0137)

0.0257 (0.0038)

B

5.6

87

100

100

100

87

20.10 (0.92)

0.1117 (0.0177)

0.0288 (0.0051)

C

5.8

93

100

95

95

89

19.53 (1.12)

0.1100 (0.0200)

0.0271 (0.0048)

D

5.7

90

100

100

100

90

19.79 (1.11)

0.1113 (0.0200)

0.0273 (0.0054)

Mean (SD)e

5.7 (0.13)

90 (3)

100 (0)

99 (3)

99 (3)

89 (2)

19.67 (0.36)

0.1084 (0.0051)

0.0272 (0.0013)

3.7

A

5.8

87

100

100

100

87

19.38 (0.98)

0.1096 (0.0320)

0.0265 (0.0053)

B

6.0

83

100

95

95

79

19.92 (1.23)

0.1091 (0.0198)

0.0280 (0.0049)

C

5.8

90

100

100

100

90

19.07 (1.73)

0.0993 (0.0267)

0.0262 (0.0050)

D

5.8

97

100

100

100

97

19.41 (1.12)

0.1139 (0.0326)

0.0248 (0.0070)

Mean (SD)

5.8 (0.091)

89 (6)

100 (0)

99 (3)

99 (3)

88 (7)

19.45 (0.35)

0.1080 (0.0062)

0.0264 (0.0013)

8.1

A

5.8

70

100

100

100

70

19.75 (1.13)

0.1092 (0.0282)

0.0261 (0.0053)

B

5.9

77

96

100

100

77

19.46 (1.49)

0.1043 (0.0277)

0.0256 (0.0065)

C

5.9

90

100

95

95

86

19.72 (1.08)

0.1104 (0.0191)

0.0289 (0.0052)

D

5.9

70

100

95

95

67

19.44 (1.32)

0.1069 (0.0350)

0.0266 (0.0056)

Mean (SD)

5.9 (0.069)

77 (9)

99 (2)

98 (3)

98 (3)

75 (8)

19.59 (0.16)

0.1077 (0.0027)

0.0268 (0.0014)

18

A

5.8

80

100

100

100

80

18.81 (1.28)

0.0930 (0.0206)

0.0229 (0.0059)

B

5.9

97

100

100

100

97

19.52 (1.00)

0.1002 (0.0137)

0.0251 (0.0035)

C

5.7

83

100

95

95

79

19.44 (0.83)

0.1044 (0.0157)

0.0256 (0.0045)

D

5.8

90

100

95

95

86

19.72 (0.96)

0.1073 (0.0172)

0.0271 (0.0048)

Mean (SD)

5.8 (0.078)

88 (7)

100 (0)

98 (3)

98 (3)

85 (8)

19.37 (0.39)

0.1012f (0.0062)

0.0252f(0.0017)

a Values presented represent hatching success at the completion of hatch (day 6).

b Post-thinning survival percentage of hatched larvae, based on the initial number of thinned larvae at completion of hatch.

c Proportion of post-hatch fish that survived to the end of the test equals the percentage of fish that survived to completion of hatch multiplied by the percentage of juvenile fish that survived to the

end of the test (based on the initial number of thinned larvae at completion of hatch).

d Proportion of fertilized eggs that hatch and survived to the end of the test equals the embryo hatching success percentage multiplied by the post-hatch survival percentage.

e Mean values are presented with standard deviations (SD) in parentheses.

f Significantly reduced compared to the control, based on Williams’ Multiple Comparison Test.

Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations and juvenile fish wet and dry body weight (the most sensitive indicators of toxicity), the NOEC was determined to be 8.1 µg/L for sheepshead minnow exposed to the test substance.
Executive summary:

This study was performed to determine the potential chronic toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) embryos, larvae, and juvenile fish under flow-through conditions, following OECD TG 210 and OCSPP Guideline 850.1400. The ASTM E: 1241-05 – Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes (ASTM, 2013) was also used as a supporting document in the development of this test design. The study was in compliance with GLP criteria.

Four replicate exposure aquaria, each containing 30 embryos, were established for each exposure concentration and the control. Based on preliminary testing and in consultation with the Study Sponsor, the following nominal concentrations were chosen for the nominal exposure: 3.1, 7.7, 19, 48, and 120 µg/L (3.7, 8.1, 18, 52, and 130 µg/L mean measured). The test was conducted using an exposure system consisting of a glass wool saturator column, an intermittent-flow proportional diluter, temperature-controlled water bath, and a set of 24 exposure aquaria. The exposure system was designed to provide five concentrations of the test substance and a control to four replicate exposure aquaria. The exposure system maintained a temperature range of 24 to 26 °C, with the exception of test day 32, where minimum-maximum readings were inadvertently not recorded, and a photoperiod of 16 hours of light with a light intensity range of 51 to 88 footcandles (550 to 950 lux) and 8 hours of dark. Exposure solution concentrations were analytically confirmed on exposure days 0 (experimental initiation), 6, 14, 20, 27, and 34 (experimental termination). Results of this study are based on mean measured concentrations.

On exposure day 6, all viable embryos in the control and treatment replicates were hatched. No delays in hatch were observed at any treatment level. Time-to-hatch for all viable embryos in the control averaged 5.7 days. Time-to-hatch in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels averaged 5.8, 5.9, 5.8, 5.9, and 5.8 days, respectively. The variation in time-to-hatch was within expectations for this species. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant delay in time-to-hatch in any of the treatment levels tested compared to the control (5.7 days). Based on time-to-hatch, the No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) were determined to be 130 and > 130 µg/L, respectively.

At the completion of hatch, hatching success in the control averaged 90%. Hatching success in the 3.7, 8.1, 18, 52, and 130 µg/L mean measured treatment levels averaged 89, 77, 88, 87, and 84%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in hatching success among embryos exposed to any of the treatment levels tested compared to the control (90%). Based on hatching success, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At the completion of hatch, the percent of live, normal larvae in the control averaged 100%. The mean percent of live, normal larvae in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 100, 99, 100, 100, and 100%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in percent of live, normal larvae among embryos exposed to any of the treatment levels tested compared to the control (100%). Based on percent of live, normal larvae at hatch, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively.

At exposure termination (28 days post-hatch), post-thinning survival in the control averaged 99%. The mean post-thinning survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 99, 98, 98, 98, and 98%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in post-thinning survival among juvenile fish exposed to any of the treatment levels tested compared to the control (99%). Based on post-thinning survival, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At exposure termination, post-hatch survival in the control averaged 99%. The mean post-hatch survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 99, 98, 98, 98, and 98%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in post-hatch survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (99%). Based on post-hatch survival, the NOEC and LOEC were determined to be 130 and > 130 µg/L, respectively. At exposure termination, overall survival in the control averaged 89%. The mean overall survival in the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 88, 75, 85, 84, and 82%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in overall survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (89%). Based on overall survival, the NOEC and LOEC were determined to be 130 and >130 µg/L, respectively. At exposure termination, the total length of juvenile fish exposed to the control averaged 19.67 mm. The mean total length of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 19.45, 19.59, 19.37, 18.97, and 17.41 mm, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean total length of surviving juvenile fish exposed to the 52 and 130 µg/L treatment levels compared to the control (19.67 mm). Based on total length of juvenile fish, the NOEC and LOEC were determined to be 18 and 52 µg/L, respectively. At exposure termination, the wet weight of juvenile fish exposed to the control averaged 0.1084 g. The mean wet weight of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 0.1080, 0.1077, 0.1012, 0.0959, and 0.0741 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean wet weight of surviving juvenile fish exposed to the 18, 52, and 130 µg/L treatment levels compared to the control (0.1084 g). Based on the wet weight of juvenile fish, the NOEC and LOEC were determined to be 8.1 and 18 µg/L, respectively. At exposure termination, the dry weight of juvenile fish exposed to the control averaged 0.0272 g. The mean dry weight of juvenile fish exposed to the 3.7, 8.1, 18, 52, and 130 µg/L treatment levels was 0.0264, 0.0268, 0.0252, 0.0238, and 0.0175 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean dry weight of juvenile fish exposed to the 18, 52, and 130 µg/L treatment levels compared to the control (0.0272 g). Based on the dry weight of juvenile fish, the NOEC and LOEC were determined to be 8.1 and 18 µg/L, respectively.

Based on mean measured concentrations and juvenile fish wet and dry body weight (the most sensitive indicators of toxicity), the NOEC was determined to be 8.1 µg/L, and the LOEC was determined to be 18 µg/L for sheepshead minnow exposed to the test substance.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Oct 2017 to 21 Nov 2017
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:
2013
Deviations:
no
Remarks:
see Deviation from the Protocol in "Any other information on materials and methods incl. tables"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Version / remarks:
2016
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Prior to exposure initiation, samples from one replicate of each treatment level and the control were collected and analyzed for the test substance concentration. In addition, triplicate samples from the saturator column effluent were analyzed. Results of this analysis were used to determine whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure.
During the definitive exposure, samples were removed on days 0 (experimental initiation), 3, 10, 17, 24, and 32 (experimental termination) from one replicate of each concentration and the control for analysis of the test substance concentration. Replicates were sequentially alternated between sampling intervals. One sample of the column effluent was also sampled and analyzed at each interval.
Vehicle:
yes
Remarks:
methanol
Details on test solutions:
For this exposure, a glass wool saturator column was used to deliver the test substance to the exposure system. The glass column was packed with glass wool, and then coated with the test substance. The column was designed to provide a constant flow of saturated aqueous solution (1.3 mg/L) of the test substance to the diluter system without the use of a carrier solvent. The saturator column was constructed entirely of chemical inert materials (glass and Teflon). To construct the column, each 60 centimeter length × 4.8 centimeter diameter column was firmly packed with approximately 15% of the total column volume with glass wool. This provided ample surface area inside the column for the exposure substance to adhere once the column preparation was complete. After the column was packed, the end fittings were placed on the column. All fittings used to enclose the column and to attach the column to the water source were composed of Teflon.
To coat each column, approximately 8.0 g of the test substance was diluted with 190 mL of methanol. The solution was sonicated in an ultrasonicator water bath for approximately 10 minutes until no undissolved test substance remained. This solution was then slowly poured into the glass column. After all of the solution was added, the column was attached to a vacuum pump. The vacuum pump was used to draw the solution evenly throughout the column to uniformly coat the glass wool with the test substance and draw off the remaining methanol. After it had visually appeared that all of the glass wool was coated and all the solvent was drawn off, the column was detached from the vacuum pump and attached to a pump which delivered a flow of dilution water through the column. During non-solvent dosing trials, the saturator column effluent was measured to be delivering a stable, consistent dose of approximately 1.3 mg/L for approximately one week. The column output concentration was used to calculate the appropriate flow rate of the effluent stock solution into the diluter system. Based on the functional solubility of the test substance (1.3 mg/L), a flow rate of 41 mL/min was required to achieve the high test concentration to dose the system during the definitive exposure. Saturator columns were prepared and replaced on the exposure system as needed (approximately every week). A set of control aquaria were also established which contained the same dilution water and were maintained under the same conditions as the treatment aquaria, but contained no test substance.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Age at study initiation: between 12 - 24 hours old
- Source: Fathead minnow embryos were obtained from a brood stock maintained at the test facility. The brood stock used to generate embryos for this exposure was approximately five months old.

ACCLIMATION
- Acclimation period: 14 days
- Temperature: 23 to 25 °C
- Dissolved oxygen: 76 to 100% of saturation
- Health during acclimation: The fish appeared to be in good health at the time of embryo collection and no mortality was observed in the brood stock 48 hours prior to testing.
- The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure.

FEEDING DURING TEST
- Food type: Beginning on exposure day 5 (day 1 post-hatch), the juvenile fish were fed live brine shrimp nauplii (Artemia salina) At each feeding, juvenile fish were fed live brine shrimp such that all juvenile fish were afforded equal proportions of food (nauplii supplied ad libitum).
- Frequency: Three times daily
- Fish were not fed during the 24 hours prior to study termination.
- Food quality: Representative samples of the food source were periodically analyzed for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected in the food samples at concentrations considered toxic to the test organisms. Based on these analyses, food sources were considered to be of acceptable quality since all analyte concentrations were below levels of concern in agreement with ASTM (2007).
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Remarks on exposure duration:
28-day post hatch
Hardness:
60 - 84 mg/L as CaCO3
Test temperature:
25 ± 1 °C
pH:
7.0 - 8.0
Dissolved oxygen:
- 7.28 - 8.32 mg O2/L
- 87.8 to 99.9% of saturation
Conductivity:
520 - 610 µS/cm
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 11, 23, 45, 90, and 180 µg/L
- Measured concentration: < MDL (negative control), 13, 27, 48, 110 and 220 µg/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Size of vessel: 30 × 14.5 × 20 cm with a 12.5 cm high side drain
- Material of vessel: Glass and silicone sealant
- Filled volume: Approximately 5.5 L
- Embryo incubation cups: Round glass jars
- Size of incubation cups: 5 cm outer diameter, 8 cm high (the bottoms removed and 475 µm Nitex mesh screen bottoms applied)
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 54 L/aquarium/day
- No. of embryos per incubation cup: 30
- No. of incubation cup per vessels: 1
- No. of vessels per concentration: 4
- No. of vessels per control: 4

EXPOSURE SYSTEM
- Prior to exposure initiation, a pump was calibrated to continuously deliver 41 mL/min of the 1.3 mg/L saturator column effluent (i.e., 537 mL per cycle) to the diluter system's mixing chamber. The effluent flow rate, in combination with the calibrated dilution water cell, filled the mixing chamber at each cycle to a total volume of 3.872 L. The mixing chamber was positioned over a magnetic stir plate and the solution was continuously stirred with a Teflon-coated stir bar throughout the exposure. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration (180 µg/L) and was proportionally diluted by a constant factor of 2.0 to produce the remaining nominal test concentrations (90, 45, 23, and 11 µg/L).
- Flow-splitting cells were employed to equally distribute the solutions to the replicate vessels at a rate of 500 mL of control or test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within 5% of the nominal value. The exposure system was designed to provide five concentrations of the test substance and a dilution water control to four replicate aquaria. The diluter delivered the control and test solutions to the exposure aquaria (approximately 54 L/aquarium/day) at a rate sufficient to provide approximately 9.8 aquarium volumes per 24-hour period, with a 90% replacement time of approximately six hours.
- The diluter system was calibrated prior to exposure initiation and confirmed at exposure termination by measuring delivery volumes of toxicant solution and dilution water. The function of the diluter system (e.g., cycle rate) was monitored daily and a visual check of the system's operation was performed twice daily. In addition, analysis of the exposure solutions for the test substance concentration was also used to verify proper operation of the diluter system prior to the start of the exposure. The exposure system was properly operating for nine days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure aquaria.
- The embryos were impartially distributed to the embryo incubation cups in the following manner: twenty-four labeled embryo incubation cups were placed in a heated water bath containing dilution water maintained at 25 ºC. A container with the embryos in 25 ºC dilution water was placed in the same water bath. Prior to loading, the embryos were examined microscopically to assess embryo fertilization; all non-viable embryos were removed, leaving only fertilized embryos. The fertilized embryos were then impartially placed in the embryo incubation cups, five at a time, until each cup contained five embryos. This process was repeated until all cups contained 30 embryos.

EXPOSURE INITIATION
- At exposure initiation, a subsample of embryos (N = 30) was collected to determine embryonic stage of development. The embryo subsample was preserved in Stockard’s solution (an 85:6:5:4 mixture of deionized water, glycerine, formaldehyde, and glacial acetic acid) for at least 24 hours prior to assessment. Embryonic stage was determined on each individual embryo within the subsample using guidance in the Pre-Hatching Development of the Fathead Minnow Pimephales promelas Rafinesque.
- Spawning substrates were introduced to the brood stock aquaria at the end of the working day the evening prior to initiation (day 0). Therefore, some embryos were likely between 12 and 24 hours old. However, fathead minnows typically spawn in the early morning hours after dawn, or when the controlled light cycle begins, and based on the developmental stages of the embryo subsample collected at exposure initiation, a majority of the embryos used for this exposure were likely less than or equal to six hours post-fertilization.
- The exposure of fathead minnow embryos and larvae to the test substance was initiated when the embryo incubation cups were distributed to each of the exposure aquaria.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was a mixture of unadulterated water from a 100 meter bedrock well and de-chlorinated Town of Wareham well water. This mixture was pumped into a 5700 liter polyethylene tank that was continuously circulated through de-gassing chambers to ensure proper mixing and equilibration of dissolved gases with the laboratory atmosphere. The water was then pumped through aged PVC piping to a heat exchanger that heated the dilution water to test temperature prior to use.
- Total organic carbon: Representative samples of the dilution water source were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 0.96 and 0.76 mg/L for October and November 2017, respectively.
- pH: 6.6 to 7.2
- Hardness: 60 to 88 mg/L as CaCO3
- Alkalinity: 16 to 21 mg/L as CaCO3
- Conductivity: 430 to 610 µS/cm
- Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected in any of the water samples analyzed at concentrations that are considered toxic, in agreement with ASTM (2007) standard practice.
- Intervals of water quality measurement: At exposure initiation, dissolved oxygen concentration, pH, and temperature were measured in each replicate aquarium. Following day 0, one replicate of each concentration and the control was measured for these parameters; replicates were sequentially alternated each day thereafter.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness
- Light intensity: 59 to 99 footcandles (630 to 1100 lux); Sudden transitions from light to dark, and vice versa, were avoided with a period of lower light intensity during the transition period.

EFFECT PARAMETERS MEASURED:
- Embryo-larval exposure: Embryos were removed from their respective incubation cup daily until hatching began. The number of dead, live, and unaccounted for embryos were counted and recorded. Dead embryos were discarded when observed at any time during the embryo incubation phase. If a loading error was suspected after the first day of embryo exposure (e.g., one or more missing embryos), then the actual number of embryos present on the first day was used as the denominator when computing percent hatch. When embryos began to hatch (test day 3), the number of live, hatched, dead, and unaccounted for embryos were counted within the incubation cup in order to avoid possible physical damage caused by removing newly hatched larvae. Dead larvae were also removed when observed during this period. Completion of hatch was considered to be exposure day 4, when all viable embryos in all control embryo incubation cups were hatched. Any unhatched embryos on day 4 in remaining replicates were allowed to continue incubating in their respective cups. For example, one embryo in the 11 µg/L treatment level replicates C and D, and the 45 µg/L treatment level replicate D, hatched on day 5. The last remaining embryo hatched on day 6 in the 11 µg/L treatment level replicate C. Slight variation in time to hatch completion in a small percentage of embryos is typical for this species; greater than 90% of viable embryos were hatched in every control and treatment replicate on test day 4. Calculations of hatching success were based on the number of live, dead and deformed larvae per incubation cup after hatching was completed (exposure day 4/day 0 post-hatch), compared to the number of embryos loaded in each egg cup.
- Post hatch fish exposure: The 28-day post-hatch fish exposure was initiated at completion of hatch (exposure day 4) by impartially selecting 20 surviving larvae from each incubation cup and transferring them into a Petri dish using a fire-polished glass pipette. The juvenile fish were then gently placed into each respective exposure aquarium from the Petri dish. The remaining juvenile fish were euthanized using an overdose of buffered tricaine methanesulfonate (MS-222) and then discarded. During the post-hatch exposure period, larval fish were observed daily; the behavior and appearance of the larval fish were recorded. In addition, dead larvae were removed during these observation periods and larval survival was estimated daily throughout the post-hatch period. At 28 days post-hatch, the exposure was terminated. The surviving juvenile fish in each replicate aquarium were euthanized with a buffered solution of MS-222, counted to determine juvenile fish survival, and measured individually to determine total length, wet weight, and dry weight. At exposure termination, fish that could not be accounted for were presumed to be dead. Following wet weight measurements, the fish were dried in an oven at approximately 60 °C for 24 hours. After a cooling period, dry weight measurements were collected. Length and weight were determined to the nearest 0.01 mm and 0.1 mg, respectively, using calibrated digital calipers and a calibrated analytical balance interfaced. The mean and standard deviation were calculated using individual measurements (total length, wet weight, and dry weight) for each replicate aquarium.

RANGE-FINDING STUDY
- Study design: A 19-day (15-day post-hatch) preliminary exposure was conducted from 27 July to 15 August 2017. Four replicates were maintained for each concentration and the control. The exposure was initiated with 30 embryos per replicate and larvae were thinned to 20 larvae per replicate following completion of hatch.
- Test nominal concentrations: 0.86, 2.6, 7.8, 23, and 70 µg/L and a control
- Results used to determine the conditions for the definitive study: No statistically significant effects were observed for any endpoint. Based on these results and consultation with the Study Sponsor, nominal concentrations of 11, 23, 45, 90, and 180 µg/L were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
110 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Mean Total Length and Mean Wet Weight
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
220 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Time-To-Hatch, Embryo Hatching Success, Live, Normal Larvae at Hatch, Post-Thinning Survival, Post-Hatch Survival, Overall Survival and Mean Dry Weight
Details on results:
An overview of the results is provided in Table 3 in 'Any other information on results incl. tables'.

- At exposure initiation, a subsample of 30 embryos was collected and the embryonic stage of development was determined. The mean and median were both 11 (flat blastula stage) and the range of the developmental stages was 10 to 12 (high blastula to early gastrula). Based on these embryonic stage characteristics, the subsample of embryos were approximately four and a half to six hours post-fertilization at exposure initiation (i.e., the time of subsample preservation).

- On exposure day 4, all viable embryos in the control and the majority of treatment replicates were hatched. Greater than 90% of viable embryos had hatched for all remaining replicates on this day. No delays in hatch were observed at any treatment level. The variation in time to completion of hatch was within expectations for this species. Time-to-hatch for all viable embryos in the control averaged 3.6 days. Time-to-hatch in the 13, 27, 48, 110, and 220 µg/L treatment levels averaged 3.9, 3.7, 3.8, 3.8, and 3.9 days, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant delay in time-to-hatch in any of the treatment levels tested compared to the control (3.6 days). Based on time-to-hatch, the No-Observed-Effect Concentration (NOEC) and Lowest−Observed−Effect Concentration (LOEC) were determined to be 220 and > 220 µg/L, respectively.

- At the completion of hatch, hatching success in the control averaged 96%. Hatching success in the 13, 27, 48, 110, and 220 µg/L mean measured treatment levels averaged 100, 100, 100, 99, and 100%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in hatching success among embryos exposed to any of the treatment levels tested compared to the control (96%). Based on hatching success, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At the completion of hatch, the percent of live, normal larvae in the control averaged 96%. The mean percent of live, normal larvae in the 13, 27, 48, 110, and 220 µg/L treatment levels was 92, 96, 92, 96, and 96%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in percent of live, normal larvae among embryos exposed to any of the treatment levels tested compared to the control (96%). Based on percent of live, normal larvae at hatch, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively.

- At exposure termination (28 days post-hatch), post-thinning survival in the control averaged 94%. The mean post-thinning survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 96, 95, 95, 93, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in post-thinning survival among juvenile fish exposed to any of the treatment levels tested compared to the control (94%). Based on post-thinning survival, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At exposure termination, post-hatch survival in the control averaged 90%. The mean larval survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 94, 93, 92, 92, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in post-hatch survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (90%). Based on post-hatch survival, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At exposure termination, overall survival in the control averaged 86%. The mean overall survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 94, 93, 92, 91, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in overall survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (86%). Based on overall survival, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At exposure termination, the total length of juvenile fish exposed to the control averaged 24.04 mm. The mean total length of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 23.72, 24.28, 23.66, 23.34, and 22.88 mm, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean total length of surviving juvenile fish exposed to the 220 µg/L treatment level compared to the control (24.04 mm). Based on total length of juvenile fish, the NOEC and LOEC were determined to be 110 and 220 µg/L, respectively. At exposure termination, the wet weight of juvenile fish exposed to the control averaged 0.1139 g. The mean wet weight of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 0.1138, 0.1219, 0.1124, 0.1072, 0.0970 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean wet weight of surviving juvenile fish exposed to the 220 µg/L treatment level compared to the control (0.1139 g). Based on the wet weight of juvenile fish, the NOEC and LOEC were determined to be 110 and 220 µg/L, respectively. At exposure termination, the dry weight of juvenile fish exposed to the control averaged 0.0221 g. The mean dry weight of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 0.0226, 0.0243, 0.0224, 0.0218, and 0.0202 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined no significant reduction in mean dry weight of juvenile fish exposed to any of the treatment levels tested compared to the control (0.0221 g). Based on the dry weight of juvenile fish, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively.
Reported statistics and error estimates:
See statistical analysis in 'Any other information on materials and methods incl. tables'.

Table 3. Endpoint Summary

Mean Measured

Concentration

(µg/L)

 

 

Replicate

Time-toHatch

(Days)

Embryo

Hatching

Success

(%)a

Live, Normal

Larvae at

Hatch

(%)

Juvenile Fish (28 Days Post-Hatch)

Post - Thinning

Survival

(%)b

Post-Hatch

Survival

(%)c

Overall

Survival

(%)d

Mean Total

Length

(mm)

Mean Wet

Weight

(g)

Mean Dry

Weight

(g)

Control

A

3.7

87

96

100

96

83

23.53 (2.06)

0.1043 (0.0322)

0.0200 (0.0061)

B

3.8

97

97

100

97

93

24.05 (1.59)

0.1077 (0.0216)

0.0213 (0.0041)

C

3.3

100

93

85

79

79

24.62 (2.21)

0.1271 (0.0334)

0.0249 (0.0067)

D

3.6

100

100

90

90

90

23.97 (1.90)

0.1166 (0.0309)

0.0222 (0.0055)

Mean (SD)e

3.6 (0.22)

96 (6)

96 (3)

94 (8)

90 (8)

86 (6)

24.04 (0.45)

0.1139 (0.0102)

0.0221 (0.0021)

13

A

3.9

100

93

100

93

93

23.71 (1.90)

0.1059 (0.0278)

0.0207 (0.0051)

B

3.9

100

97

90

87

87

23.36 (5.38)

0.1187 (0.0653)

0.0247 (0.0135)

C

4.0

100

90

100

100

100

23.57 (2.85)

0.1104 (0.0400)

0.0220 (0.0081)

D

3.7

100

90

95

95

95

24.22 (2.61)

0.1200 (0.0357)

0.0231 (0.0068)

Mean (SD)

3.9 (0.13)

100 (0)

92 (3)

96 (5)

94 (5)

94 (5)

23.72 (0.37)

0.1138 (0.0068)

0.0226 (0.0017)

27

A

3.7

100

97

100

97

97

23.67 (2.32)

0.1116 (0.0299)

0.0221 (0.0059)

B

3.9

100

93

100

93

93

22.97 (3.11)

0.1039 (0.0418)

0.0208 (0.0084)

C

3.3

100

97

90

90

90

25.30 (1.59)

0.1344 (0.0287)

0.0264 (0.0057)

D

3.9

100

97

90

90

90

25.17 (3.51)

0.1380 (0.0548)

0.0279 (0.0111)

Mean (SD)

3.7 (0.27)

100 (0)

96 (2)

95 (6)

93 (3)

93 (3)

24.28 (1.14)

0.1219 (0.0168)

0.0243 (0.0034)

48

A

4.0

100

97

100

100

100

22.98 (4.44)

0.1035 (0.0457)

0.0211 (0.0092)

B

3.7

100

83

95

89

89

23.21 (2.56)

0.1077 (0.0373)

0.0215 (0.0073)

C

3.8

100

97

100

97

97

23.84 (2.22)

0.1127 (0.0313)

0.0222 (0.0064)

D

3.9

100

90

85

82

82

24.61 (1.57)

0.1256 (0.0279)

0.0250 (0.0055)

Mean (SD)

3.8 (0.11)

100 (0)

92 (6)

95 (7)

92 (8)

92 (8)

23.66 (0.73)

0.1124 (0.0096)

0.0224 (0.0017)

a Values presented represent hatching success at the completion of hatch (day 4).

b Post-thinning survival percentage of hatched larvae, based on the initial number of thinned larvae at completion of hatch.

c Proportion of post-hatch fish that survived to the end of the test equals the percentage of fish that survived to completion of hatch multiplied by the percentage of juvenile that survived to the end of the test (based on the initial number of thinned larvae at completion of hatch).

d Proportion of fertilized eggs that hatched and survived to the end of the test equals the embryo hatching success percentage multiplied by the post-hatch survival percentage).

e Mean values are presented with standard deviations (SD) in parentheses.

Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations and total larval length and wet weight (the most sensitive indicators of toxicity), the NOEC was determined to be 110 µg/L for fathead minnow exposed to the test substance.
Executive summary:

This study was performed to determine the potential chronic toxicity of the test substance to fathead minnow (Pimephales promelas) embryos, larvae, and juvenile fish under flow-through conditions, following OECD TG 210 and OCSPP Guideline 850.1400. The ASTM E: 1241-05 – Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes (ASTM, 2013) was also used as a supporting document in the development of this test design. The study was in compliance with GLP criteria.

Four replicate exposure aquaria, each containing 30 embryos, were established for each exposure concentration and the control. Based on preliminary testing and in consultation with the Study Sponsor, the following nominal concentrations were chosen for the definitive exposure: 11, 23, 45, 90, and 180 µg/L (13, 27, 48, 110, and 220 µg/L mean measured). The test was conducted using an exposure system consisting of a glass wool saturator column, an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 24 exposure aquaria. The exposure system was designed to provide five concentrations of the test substance and a control to four replicate exposure aquaria. The exposure system maintained a temperature range of 24 to 26 °C (a measurement of 27.9 °C was recorded on test day 3 that was attributed to a typographical error), and a photoperiod of 16 hours of light with a light intensity range of 59 to 99 footcandles (630 to 1100 lux) and 8 hours of dark. Exposure solution concentrations were analytically confirmed on exposure days 0 (experimental initiation), 3, 10, 17, 24, and 32 (experimental termination). Results of this study are based on mean measured concentrations. Time-to-hatch, embryo hatching success, percentage of embryos that produce live, normal larvae at hatch, juvenile fish survival, post-hatch survival, overall survival, and juvenile fish growth (total length, wet weight, and dry weight) values were used to determine the No-Observed-Effect Concentration (NOEC) and the Lowest-Observed-Effect Concentration (LOEC).

On exposure day 4, all viable embryos in the control and the majority of treatment replicates were hatched. Greater than 90% of viable embryos had hatched for all remaining replicates on this day. No delays in hatch were observed at any treatment level. The variation in time to completion of hatch was within expectations for this species. Time-to-hatch for all viable embryos in the control averaged 3.6 days. Time-to-hatch in the 13, 27, 48, 110, and 220 µg/L treatment levels averaged 3.9, 3.7, 3.8, 3.8, and 3.9 days, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant delay in time-to-hatch in any of the treatment levels tested compared to the control (3.6 days). Based on time-to-hatch, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At the completion of hatch, hatching success in the control averaged 96%. Hatching success in the 13, 27, 48, 110, and 220 µg/L mean measured treatment levels averaged 100, 100, 100, 99, and 100%, respectively. Statistical analysis (Dunn’s Test with Bonferroni-Holm’s Adjustment) determined no significant reduction in hatching success among embryos exposed to any of the treatment levels tested compared to the control (96%). Based on hatching success, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively.

At the completion of hatch, the percent of live, normal larvae in the control averaged 96%. The mean percent of live, normal larvae in the 13, 27, 48, 110, and 220 µg/L treatment levels was 92, 96, 92, 96, and 96%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in percent of live, normal larvae among embryos exposed to any of the treatment levels tested compared to the control (96%). Based on percent of live, normal larvae at hatch, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively.

At exposure termination (28 days post-hatch), post-thinning survival in the control averaged 94%. The mean post-thinning survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 96, 95, 95, 93, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in post-thinning survival among juvenile fish exposed to any of the treatment levels tested compared to the control (94%). Based on post-thinning survival, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At exposure termination, post-hatch survival in the control averaged 90%. The mean post-hatch survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 94, 93, 92, 92, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in juvenile fish survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (90%). Based on post-hatch survival, the NOEC and LOEC were determined to be 220 and >220 µg/L, respectively. At exposure termination, overall survival in the control averaged 86%. The mean overall survival in the 13, 27, 48, 110, and 220 µg/L treatment levels was 94, 93, 92, 91, and 83%, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant reduction in overall survival among embryos and juvenile fish exposed to any of the treatment levels tested compared to the control (86%). Based on overall survival, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively. At exposure termination, the total length of juvenile fish exposed to the control averaged 24.04 mm. The mean total length of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 23.72, 24.28, 23.66, 23.34, and 22.88 mm, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean total length of surviving juvenile fish exposed to the 220 µg/L treatment level compared to the control (24.04 mm). Based on total length of larvae, the NOEC and LOEC were determined to be 110 and 220 µg/L, respectively. At exposure termination, the wet weight of juvenile fish exposed to the control averaged 0.1139 g. The mean wet weight of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 0.1138, 0.1219, 0.1124, 0.1072, 0.0970 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined a significant reduction in mean wet weight of surviving juvenile fish exposed to the 220 µg/L treatment level compared to the control (0.1139 g). Based on the wet weight of juvenile fish, the NOEC and LOEC were determined to be 110 and 220 µg/L, respectively. At exposure termination, the dry weight of juvenile fish exposed to the control averaged 0.0221 g. The mean dry weight of juvenile fish exposed to the 13, 27, 48, 110, and 220 µg/L treatment levels was 0.0226, 0.0243, 0.0224, 0.0218, and 0.0202 g, respectively. Statistical analysis (Williams’ Multiple Comparison Test) determined no significant reduction in mean dry weight of juvenile fish exposed to any of the treatment levels tested compared to the control (0.0211 g). Based on the dry weight of juvenile fish, the NOEC and LOEC were determined to be 220 and > 220 µg/L, respectively.

Based on mean measured concentrations and juvenile fish total length and wet weight (the most sensitive indicators of toxicity), the NOEC was determined to be 110 µg/L, and the LOEC was determined to be 220 µg/L for fathead minnow exposed to the test substance.

Description of key information

Freshwater, 32-d NOEC = 110 µg/L, Pimephales promelas, juvenile fish total length and wet weight, OECD TG 210, Marini 2018

Marine water, 34-d NOEC = 8.1 µg/L, Cyprinodon variegatus, juvenile fish mean wet and dry weight, OECD TG 210, Marini 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
110 µg/L

Marine water fish

Marine water fish
Dose descriptor:
NOEC
Effect concentration:
8.1 µg/L

Additional information

Freshwater


The chronic toxicity of the substance to fathead minnow (Pimephales promelas) embryos, larvae, and juvenile fish under flow-through conditions was studied under GLP to OECD TG 210 over a period of 32 days. Four replicate exposure aquaria, each containing 30 embryos, were established for each exposure concentration and the control. The nominal concentrations for the definitive exposure were 11, 23, 45, 90, and 180 µg/L (13, 27, 48, 110, and 220 µg/L mean measured). The exposure system maintained a temperature range of 24 to 26 °C (a measurement of 27.9 °C was recorded on test day 3 that was attributed to a typographical error), and a photoperiod of 16 hours of light with a light intensity range of 630 to 1100 lux and 8 hours of dark. Concentrations were analytically confirmed on exposure days 0 (experimental initiation), 3, 10, 17, 24, and 32 (experimental termination). Results of this study were based on mean measured concentrations. Time-to-hatch, embryo hatching success, percentage of embryos that produce live, normal larvae at hatch, juvenile fish survival, post-hatch survival, overall survival, and juvenile fish growth (total length, wet weight, and dry weight) values were used to determine the No-Observed-Effect Concentration (NOEC) and the Lowest-Observed-Effect Concentration (LOEC). No significant effects on the majority of surveyed endpoints were seen, including time to hatch, hatching success, percent of live, normal larvae among embryos, post-thinning survival among juvenile fish, reduction in juvenile fish survival, overall survival among embryos and juvenile fish, and mean dry weight of juvenile fish. However, a significant reduction in mean total length and mean wet weight of surviving juvenile fish exposed to the 220 µg/L treatment level compared to the control (24.04 mm, 0.1139 g) was found. Based on total length and wet weight of juvenile fish, the NOEC and LOEC were determined to be 110 and 220 µg/L, respectively.


Marine water


The chronic toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) embryos, larvae, and juvenile fish under flow-through conditions was investigated under GLP to OECD TG 210 over a period of 34 days. Four replicate exposure aquaria, each containing 30 embryos, were established for each exposure concentration and the control. The nominal concentrations for the definitive test were 3.1, 7.7, 19, 48, and 120 µg/L (3.7, 8.1, 18, 52, and 130 µg/L mean measured). The exposure system maintained a temperature range of 24 to 26 °C, with the exception of test day 32, where minimum-maximum readings were inadvertently not recorded, and a photoperiod of 16 hours of light with a light intensity range of 550 to 950 lux and 8 hours of dark. Concentrations were analytically confirmed on exposure days 0 (experimental initiation), 6, 14, 20, 27, and 34 (experimental termination). Results of this study were based on mean measured concentrations. No significant effects on the majority of surveyed endpoints were observed, including the time to hatch, hatching success, percent of live, normal larvae, post-thinning survival among juvenile fish, post-hatch survival and overall survival of embryos. However, a significant reduction in mean total length, wet weight and dry weight of surviving juvenile fish was found compared to the control (19.67 mm, 0.1084 g, 0.0272 g). Based on these effects the NOEC and LOEC were determined to be 18 and 52 µg/L for total length and 8.1 and 18 µg/L for wet and dry weight.


It occurred that the tested marine fish species sheepshead minnow was slightly more susceptible to the substance than the freshwater species fathead minnow.