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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 28, 2016 to September 27, 2016
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ASTM Standard E 1241-05: Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes
Deviations:
no
GLP compliance:
yes
Remarks:
GLP Standards as published by the US EPA (40 CFR Part 160 and Part 792); OECD principles of GLP (ENV/MC/CHEM (98)17); and Japan MAFF (11 Nousan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999).
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under ambient conditions
- Stability under test conditions: was not determined in accordance with GLP standards
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations selected for use in the study were 10, 20, 40, 80 and 160 mg/L.
- Sampling method: Water samples were collected from one test chamber of each treatment and control group two 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, 20, 27 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 glass vials and processed immediately for analysis.
- Sample storage conditions before analysis: All samples were processed immediately for analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared eleven times during the test. At each preparation, the primary stock solution was prepared in well water at a nominal concentration of 2.5 mg/mL. The primary stock solution was prepared by weighing and adding a calculated amount of test substance into a 180-L stainless steel container that contained a pre-measured volume of well water and stirred for approximately four to five hours with a top-down electric mixer to mix prior to use.
The primary stock solution was stored ambient during mixing. The stock solution was stirred continuously with a submersible mixer while in use. Newly prepared stock was placed in the fluid metering pump system every three to four days during the study. The appropriate amount of the 2.5 mg /mL stock was pumped into the diluter mixing chambers assigned to treatment groups at target rates of 0.80, 1.60, 3.20, 6.40 and 12.8 mL/minute where they were mixed with dilution water delivered at rates of 199, 198, 197, 194 and 187 mL/minute to achieve the desired test concentrations.
- Controls: The negative control received dilution water only at a rate of 200 mL/min.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle was used. Test solutions were prepared in well water.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The primary stock solution appeared slightly cloudy with a layer of fine bubbles on the surface of the solution at mixing initiation and clear and colorless with no evidence of precipitate upon mixing termination.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: The fathead minnow
- Source: 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 (1, 2, 3). 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 EAG Laboratories-Easton on spawning substrates.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Identification of the species was verified by the supplier. 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 nine individual spawns and were <24 hours old when the test was initiated and were approximately between the gastrula and neurula developmental stages. 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.

POST-HATCH FEEDING
- Start date: Embryos were < 24 hours old when the test was initiated.
- Type/source of feed: live brine shrimp nauplii (Artemia sp.)

- Amount given: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.034 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 0.35 g fish/L.

- Frequency of feeding: Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period.
Hardness:
Hardness was measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Hardness was measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4).
Test temperature:
The target test temperature during the test was 25 ± 1C. 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 digital 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 digital thermometer.
pH:
pH was 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 pH were made using a Thermo Scientific Orion Dual Star pH meter. Measurements of pH ranged from 8.1 to 8.5 during the test.
Dissolved oxygen:
Dissolved oxygen was 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 Scientific Orion Star A213 dissolved oxygen meter. Dissolved oxygen concentrations remained ≥99% of saturation (8.1 mg/L).
Salinity:
Alkalinity was measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Alkalinity was measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4).
Conductivity:
Specific conductance was measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Specific conductance was measured using a Thermo Orion Star A122 Portable Conductivity meter.
Nominal and measured concentrations:
Nominal concentrations selected for use in the study were 10, 20, 40, 80 and 160 mg/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 99.4 to 102% of nominal concentrations (Table 1). Samples of the test solutions collected during the test had measured concentrations that ranged from 96.0 to 110% of nominal concentrations (Table 2). When the measured concentrations of test solution samples collected on Days 0, 7, 14, 20, 27 and 33 of the test were averaged for each treatment group, the mean measured test concentrations were 10, 20, 41, 82 and 167 mg/L, which represented 100, 100, 103, 103 and 104% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment.
- Test vessel: The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was approximately 15.4 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.

- Type of flow-through (e.g. peristaltic or proportional diluter): A continuous-flow diluter was used to deliver each concentration of the test substance and a negative dilution water) control. Fluid metering pumps (Fluid Metering, Inc., Syosset, New York) were used to deliver volumes of a single test substance stock solution to mixing chambers indiscriminately assigned to each treatment. The fluid metering pumps used to deliver the stock solution to the mixing chambers were calibrated prior to the test and calibrated/verified approximately weekly during the test. The stock solutions were diluted with U.V. sterilized 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 buildup of bacterial/fungal growth.

- Renewal rate of test solution (frequency/flow rate): The appropriate amount of the 2.5 mg /mL stock was pumped into the diluter mixing chambers assigned to treatment groups at target rates of 0.80, 1.60, 3.20, 6.40 and 12.8 mL/minute where they were mixed with dilution water delivered at rates of 199, 198, 197, 194 and 187 mL/minute to achieve the desired test concentrations. The negative control received dilution water only at a rate of 200 mL/min.

- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.034 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 0.35 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for testing was freshwater obtained from a well approximately 40 meters deep located on the EAG Laboratories-Easton site.
- Total organic carbon (mg C/L): < 1
- Particulate matter: 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 and was passed through an ultraviolet (UV) sterilizer.
- Specific Conductance (μS/cm): 332 (N=4); range: 325 – 338
- Hardness (mg/L as CaCO3): 140 (N=4); range: 140 – 140
- Alkalinity (mg/L as CaCO3) : 176 (N=4); range 174-178
- pH: 8.0; range: 8.0 – 8.1
- Metals: see table below;
- Pesticides: see table below;
- Chlorine: see table below;
- Culture medium different from test medium: no
- Intervals of water quality measurement: the parameters of well water were measured during the four-week period immediately preceding the test.

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 16 hours of light and 8 hours of darkness
- Light intensity: 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 840006 light meter. Light intensity at test initiation was 310 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 group on Day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were 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 48 hours to obtain dry weight data.

VEHICLE CONTROL PERFORMED: no; untreated medium served as control

RANGE-FINDING STUDY
- Test concentrations: The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data.

POST-HATCH DETAILS
- Begin of post-hatch period: from day 5
- No. of hatched eggs (alevins)/treatment released to the test chamber:
- Release of alevins from incubation cups to test chamber on day no.: 5
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
82 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
167 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: length and wet weight
Remarks:
Fathead minnows exposed to the test material at a concentration of 167 mg/L had statistically significant reductions in total length and wet weight in comparison to the negative control.
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: Survival of the fathead minnow larvae through Day 28 post-hatch is summarized in Table 4 (please see below), and individual replicate data are presented in Appendix 11. Larval survival in the negative control groups was 87%. Larval survival in the 10, 20, 41, 82 and 167 mg/L treatment groups was 89, 92, 86, 90 and 88%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any of treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for larval survival was 167 mg/L and the LOEC was >167 mg/L.
Biological/clinical observations of sublethal effects during the 28-day post-hatch period are presented in Appendix 12. In general, the majority of the fish in the control groups and in the treatment groups appeared normal throughout the test. There were a few observations of organisms that appeared small, weak, pale or had a crooked spine. However, these observations were infrequent and were comparable in the control.

- Days to hatch or time to release of young: Hatching success of the fathead minnow embryos is summarized in Table 4. Time to hatch data were not evaluated by formal statistical tests due to lack of variation in the endpoint across the treatment and control groups. Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the test material 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. A few embryos remained in the incubation chambers until they hatched on the Day 6 or 7 of the test.
Hatching success in the negative control group was 88%. Hatching success in the 10, 20, 41, 82 and 167 mg/L treatment groups was 91, 91, 99, 79 and 90%, respectively. Fisher’s Exact test indicated that there were no statistically significant differences in any of the treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for hatching success was 167 mg/L and the LOEC was >167 mg/L.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: Growth measurements at the end of the 28-day post-hatch period are summarized in Table 4, and the individual replicate data for total length, wet weight and dry weight are presented in Appendices 13, 14 and 15, respectively. Dunnett’s test indicated there was a statistically significant reduction in total length and wet weight among fish in the 167 mg/L treatment groups in comparison to the negative control (p ≤ 0.05). Consequently, the NOEC for growth was 82 mg/L and the LOEC was 167 mg/L.

- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: no
Results with reference substance (positive control):
No positive control substance was used.
Reported statistics and error estimates:
Please see "Any other information on materials and methods incl. tables".

Weekly measurements of specific conductance, hardness and alkalinity in the negative control water and in the highest test concentration are summarized in Table 3.

Table 3. Means and Ranges of Water Quality Measurements Taken During the 33-Day Exposure to the test material

Mean Measured Concentration (mg/L)   Mean ± SD and Range of Measured Parameters  
Temperature1(°C) DO2(mg/L) pH

Hardness3

(mg/L as CaCO3)

Alkalinity3

(mg/L as CaCO3)

Conductivity3(µS/cm)
Negative Control 25.5 ± 0.15 8.2 ± 0.05 8.3 ± 0.19 141 ± 8 172 ± 4 348 ± 20
  (25.3 - 25.7) (8.1 - 8.2) (8.1 - 8.5) (128 - 148) (168 - 178) (329 - 379)
10 25.6 ± 0.12 8.2 ± 0.04 8.2 ± 0.11 -- -- --
  (25.4 - 25.8) (8.1 - 8.2) (8.1 - 8.3) -- -- --
20 25.5 ± 0.14 8.2 ± 0.04 8.2 ± 0.10 -- -- --
  (25.3 - 25.7) (8.1 - 8.2) (8.1 - 8.3) -- -- --
41 25.5 ± 0.13 8.2 ± 0.0 8.2 ± 0.08 -- -- --
  (25.3 - 25.7) (8.2 - 8.2) (8.1 - 8.3) -- -- --
82 25.5 ± 0.14 8.2 ± 0.04 8.2 ± 0.08 -- -- --
  (25.3 - 25.8) (8.2 - 8.2) (8.1 - 8.3) -- -- --
167 25.5 ± 0.15 8.2 ± 0.04 8.2 ± 0.08 145 ± 2 171 ± 4 347 ± 18
  (25.3 - 25.8) (8.2 - 8.2) (8.1 - 8.3) (144 - 148) (164 - 176) (324 - 368)

1 Temperature measured continuously during the test ranged from approximately 24.69 to 25.44°C, measured to the nearest 0.01°C.

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

3 -- = no measurements scheduled.

Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by the test material concentration.

Conditions for the Validity of the Test

The following criteria were used to judge the validity of the test and were met in this study:

1) the dissolved oxygen concentration was >60% of the air saturation value (>99% of saturation; 8.1 mg/L) throughout the test;

2) the water temperature measurements did not differ by more than ± 1.5ºC between test chambers or between successive days at any time during the test, and were within 25 ± 1°C;

3) the measured concentrations of the test substance in solution were satisfactorily maintained within C.V. ± 20% (1.72 to 3.30% in test) of the mean measured values throughout the test;

4) the percentage of embryos in the negative control that hatched successfully was 88% and the post-hatch survival in the negative control was 87%. The criteria for validity was >70% control hatch ability and >75% control larval survival; and

5) a solubilizing agent was not used, therefore, a solvent-only control group was not needed for this test.

Table 4. Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed to test material

Mean Measured Concentration (mg/L) Number Exposed Total Number Hatched Hatching Success (%)1 Number Surviving to Termination Post-Hatch Survival (%)1

Mean Total Length ± Std. Dev.

(mm)

Mean Wet Weight ± Std. Dev. (mg)

Mean Dry Weight ± Std. Dev.

(mg)1

Negative Control

80

70

88

61

87

24.6 ± 0.44

122 ± 7.1

23.9 ± 2.0

10

80

73

91

65

89

24.6 ± 0.21

126 ± 4.2

24.1 ± 1.2

20

80

73

91

66

92

24.5 ± 0.37

124 ± 4.1

23.8 ± 1.0

41

80

79

99

68

86

24.4 ± 0.42

118 ± 3.9

22.3 ± 1.1

82

80

63

79

57

90

24.7 ± 0.62

130 ± 8.6

25.6 ± 1.5

167

80

72

90

63

88

23.4 ± 0.68*

110 ± 6.5*

22.0 ± 0.94

EC10& 95% confidence interval (mg/L) 

 

>167 

 

>167

>167

>167 

>167

EC20 & 95% confidence interval (mg/L)

 

 

>167

 

>167

>167

>167

>167

1 There were no statistically significant differences in percent hatching success, survival or mean dry weight from the control (Fisher’s Exact test, p>0.05 for hatching success and survival and Dunnett’s one-tailed test p>0.05 for mean dry weight).

* Indicates a statistically significant difference in mean total length and wet weight from the control (Dunnett’s one-tailed test, p≤0.05).

Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to the test material at mean measured concentrations of 10 to 167 mg/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, survival or dry weight in any of the treatment groups. Growth, measured as total length and wet weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to the test material at a concentration of 167 mg/L had statistically significant reductions in total length and wet weight in comparison to the negative control. Consequently, the NOEC, based on growth, was 82 mg/L and the LOEC was 167 mg/L.
Executive summary:

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

Fathead minnow embryos were exposed to a geometric series of test concentrations and a negative (dilution water) control under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 10, 20, 40, 80 and 160 mg/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.

Delivery of the test solutions to the test chambers was initiated four days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of N-butylpyrrolidone on time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC).

Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the test material 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. A few embryos remained in the incubation chambers until they hatched on the Day 6 or 7 of the test.

Hatching success in the negative control group was 88%. Hatching success in the 10, 20, 41, 82 and 167 mg/L treatment groups was 91, 91, 99, 79 and 90%, respectively. Fisher’s Exact test indicated that there were no statistically significant differences in any of the treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for hatching success was 167 mg/L and the LOEC was >167 mg/L.

Larval survival in the negative control groups was 87%. Larval survival in the 10, 20, 41, 82 and 167 mg/L treatment groups was 89, 92, 86, 90 and 88%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any of treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for larval survival was 167 mg/L and the LOEC was >167 mg/L.

In general, the majority of the fish in the control groups and in the treatment groups appeared normal throughout the test. There were a few observations of organisms that appeared small, weak, pale or had a crooked spine. However, these observations were infrequent and were comparable in the control.

Dunnett’s test indicated there was a statistically significant reduction in total length and wet weight among fish in the 167 mg/L treatment groups in comparison to the negative control (p ≤ 0.05). Consequently, the NOEC for growth was 82 mg/L and the LOEC was 167 mg/L.

Description of key information

The objective of the key study was to determine the effects of N-Butylpyrrolidone on the time to hatch, hatching success, survival, and growth of fathead minnows, Pimephales promelas, during early life-stage development. (OECD 210, EAG (Wildlife International), 2016).

Fathead minnow embryos were exposed to a geometric series of test concentrations and a negative (dilution water) control under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 10, 20, 40, 80 and 160 mg/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.

Delivery of the test solutions to the test chambers was initiated four days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of N-butylpyrrolidone on time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC).

Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control group and any of the test material 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. A few embryos remained in the incubation chambers until they hatched on the Day 6 or 7 of the test.

Hatching success in the negative control group was 88%. Hatching success in the 10, 20, 41, 82 and 167 mg/L treatment groups was 91, 91, 99, 79 and 90%, respectively. Fisher’s Exact test indicated that there were no statistically significant differences in any of the treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for hatching success was 167 mg/L and the LOEC was >167 mg/L.

Larval survival in the negative control groups was 87%. Larval survival in the 10, 20, 41, 82 and 167 mg/L treatment groups was 89, 92, 86, 90 and 88%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any of treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOEC for larval survival was 167 mg/L and the LOEC was >167 mg/L.

In general, the majority of the fish in the control groups and in the treatment groups appeared normal throughout the test. There were a few observations of organisms that appeared small, weak, pale or had a crooked spine. However, these observations were infrequent and were comparable in the control.

Dunnett’s test indicated there was a statistically significant reduction in total length and wet weight among fish in the 167 mg/L treatment groups in comparison to the negative control (p ≤ 0.05). Consequently, the NOEC for growth was 82 mg/L and the LOEC was 167 mg/L.

In a supporting study the chronic toxicity of test substance to Rare Minnow was investigated in a definitive test under semi-static conditions (24 h intervals) according to OECD Guideline 215. Juvenile fish in exponential growth phase were placed, after being weighed, in test chambers and exposed to a range of sublethal concentrations of the test substance dissolved in water (dechlorinated tap water). The test duration was 28 days. Effects on growth rates as well as abnormalities in morphology and behaviour were analyzed. All measured concentrations can be maintained within ± 20% of nominal concentrations, therefore, test results was described based on nominal concentrations. At the end of the test, no abnormal behaviors and mortalities were observed in blank control and the test groups (1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L). At the end of the test, "pseudo" specific growth rate (mean ± standard deviation) of blank control and 1.0 mg/L,3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L test groups were 1.66±0.59 (d-1), 1.60±0.30 (d-1), 1.67±0.30 (d-1), 1.63±0.40 (d-1), 1.54±0.40 (d-1) and 1.66±0.55 (d-1), respectively. The mean "pseudo" specific growth rate for each test concentration group were compared with blank control's using statistics method, there was not a significant difference (p>0.05). Hence, NOEC (28 d) on the growth of juvenile with Rare Minnow was 100 mg/L, and LOEC (28 d) was greater than 100 mg/L based on nominal concentration. No toxicological effects were observed at the highest test concentration (100 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
82 mg/L

Additional information