Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 December 2018 to 17 January 2019

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1241-05 (Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test solution samples were collected from one test chamber of each treatment and control group four and two days prior to the start of exposure to confirm concentrations of the test material after conditioning the diluter system for three and five days, respectively. Samples of the one-day old stock solutions were also collected from the syringes delivering to the test system four days prior to the start of exposure to confirm that the test system was being dosed appropriately. Test solution samples were collected from one replicate test chamber in each treatment and control group at the beginning of exposure, approximately weekly during the test, and at the end of the test to measure concentrations of the test substance.
Additional samples were collected from the 0.10 mg/L treatment group on Day 19 and 20 of the test due to a syringe leak. Test solution samples were collected at the time this issue was discovered on Day 19 and again the next morning on Day 20 after approximately 6.8 test solution turnovers, in order to demonstrate that the measured concentration had returned to normal. All test solution samples were collected at mid-depth in the test chambers, placed in glass vials containing 2.00 mL of 0.5 % formic acid in methanol, and processed immediately for analysis. Stock solution samples were collected directly from the syringes delivering to the test system, placed in glass vials, and processed immediately for analysis.

Vehicle:

yes

Remarks:

HPLC-grade dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solutions were prepared daily during the test. At each preparation, individual stocks were prepared in HPLC-grade DMF at nominal concentrations of 0.063, 0.13, 0.25, 0.5, and 1.0 mg/mL. The individual stock solutions were sonicated for approximately 15 minutes and inverted to mix, and appeared dark purple and opaque. The opacity of the solutions precluded any observations of a colour gradient or observations of precipitation. However, staining of the glassware appeared to increase in intensity with increasing concentration.
Stock solutions were used immediately and were placed on the syringe pumps daily during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 20 µL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 0.0063, 0.013, 0.025, 0.050 and 0.10 mg/L. The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all test material treatment groups was 0.10 mL/L.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Obtained from brood fish that had been cultured at the testing facility using water from the same source as used in the test and were deposited upon spawning substrates.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: 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 (approximately blastula to neurula). Embryos collected for use in the test were from ten individual spawns and were < 24 hours old when the test was initiated. Identification of the species was verified by the original supplier.

POST-HATCH FEEDING
- Type/source of feed and 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 and holidays. Fish were not fed for approximately 24 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:

32 d

Remarks on exposure duration:

32 Days (4-Day Hatch and 28-Day Post-Hatch)

Hardness:

136 - 144 mg/L (as CaCO3)

Test temperature:

24.3 - 25.5 °C

pH:

8.1 - 8.2

Dissolved oxygen:

7.0 - 8.2 mg/L
Results confirmed dissolved oxygen concentrations remained ≥ 85 % of saturation (≥ 7.0 mg/L).

Salinity:

not applicable

Conductivity:

307 - 365 µS/cm

Nominal and measured concentrations:

0.0063, 0.013, 0.025, 0.05 and 0.1 mg

Details on test conditions:

TEST SYSTEM
- Emybro cups: 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 (approximately 4 rpm) facilitated circulation of test water around the embryos during incubation.
- Test vessel: The test chambers were 9 L glass aquaria.
- Material, size, headspace, fill volume: 9 L glass aquaria filled with 7 L of test solution. The depth of the test water in a representative test chamber was approximately 16 cm.
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test material. A continuous-flow diluter was used to deliver each concentration of the test material, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus, Holliston, Massachusetts) were used to deliver the five test material stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into mixing chambers assigned to each treatment and the solvent control. The syringe pumps were calibrated prior to the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at approximately weekly intervals during the test and near the end of the test to ensure that flow rates varied by no more than ± 10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately ten volume additions of test water 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.
- Biomass loading rate: Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.026 g of fish per litre of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per litre of water in the tank) at the end of the test was 0.27 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the Eurofins – Easton site. The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37 800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterised as moderately-hard water.
- Intervals of water quality measurement: Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, approximately weekly during the test and at the end of the test. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using a Thermo Scientific Orion Star A122 Portable Conductivity meter.
The target test temperature during the test was 25 ± 1 °C. Temperature was measured in each test chamber at the beginning of the test, approximately weekly during the test, and at the end of the test using a digital thermometer. Water temperature was also monitored continuously during the test in one negative control test chamber using a validated environmental monitoring system (Amegaview Central Monitoring System). The system measurements were calibrated prior to exposure initiation and verified or recalibrated approximately weekly during the test with a digital thermometer.
pH was measured in alternating replicates of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test.
Dissolved oxygen was measured in alternating replicates of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Star A213 Benchtop RDO/DO meter and pH was measured using a Thermo Scientific Orion DUAL STAR pH/ISE meter.

OTHER TEST CONDITIONS
- Photoperiod: 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: 590 lux. Light intensity was measured at the water surface of one representative test chamber at test initiation using a SPER Scientific Model 840006 light meter.

EFFECT PARAMETERS MEASURED:
- During the first day of exposure, embryos were observed twice for mortality and eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached > 90 % in the control groups on Day 4 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any un-hatched 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 and overall survival, and growth at test termination 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. Overall survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos exposed at the beginning of the test.
- 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 46 hours to obtain dry weight data.

VEHICLE CONTROL PERFORMED: Yes. A solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control were conducted.

RANGE-FINDING STUDY
- Test concentrations: Nominal concentrations of 0.25, 0.70, 2.0 and 4.0 mg.
- Results used to determine the conditions for the definitive study: The concentrations were selected in consultation with the Sponsor, and were based on the results of an exploratory non-GLP range-finding toxicity test and non-GLP diluter trial. Less than 24-hour old fathead minnow embryos were exposed from the embryonic stage through 14-days post-hatch. Treatment-related adverse effects compared to the solvent control, based on visual interpretation of the data, were observed at 2.0 and 4.0 mg/L for post-hatch survival, and at 0.70, 2.0 and 4.0 mg/L for mean wet weight. Hatching success was not affected.

Reference substance (positive control):

no

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.072 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: No effects observed

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 0.072 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: No effects observed

Details on results:

- Days to hatch or time to release of young: The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 3 and 4 of the test. Hatching reached > 90 % in the control groups on Day 4 of the test, at which time the larvae were released to their respective test chambers.
A few embryos in the negative and solvent control and the 0.015 mg/L treatment group remained in the incubation chambers until they hatched or died on Day 7 of the test. The mean time to hatch in the negative control and solvent control groups was day 3.9 and 3.8, respectively. Since there were no statistically significant differences in mean time to hatch between the negative and solvent control (p > 0.05), the control data were pooled for comparisons among the treatment groups.
The mean time to hatch in the pooled control and the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was day 3.9, 3.4, 3.8, 4.2, 3.7 and 3.8, respectively. According to both Dunnett’s one-tailed test and the one-sided Jonckheere-Terpstra step-down trend test, there were no statistically significant increases in mean time to hatch in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for mean time to hatch were determined to be 0.072 mg/L and > 0.072 mg/L, respectively. The IC10 and IC20 values for mean time to hatch could not be estimated, since the calculated ICx values were extrapolated beyond the data range used in the calculation and/or the 95% confidence intervals were overly wide.

- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Hatching success in the negative and solvent control groups was 100 and 100 %, respectively. Since percent hatching success in the negative control and solvent control was the same, the control data were pooled for comparisons among the treatment groups. Hatching success in the pooled control and the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 100, 100, 100, 97.5, 100, and 97.5 %, respectively. According to Fisher’s Exact test, there were no statistically significant decreases in hatching success for any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for hatching success were determined to be 0.072 mg/L and >0.072 mg/L, respectively. Since there was less than 10% inhibition in hatching success in any treatment group in comparison to the pooled control, the IC10 and IC20 values were empirically estimated to be greater than the highest test concentration.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: The mean total length, wet weight and dry weight of surviving fish in the negative control at test termination was 23.8 mm, 93.6 mg and 20.3 mg, respectively. The mean total length, wet weight and dry weight of surviving fish in the solvent control at test termination was 22.8 mm, 79.2 mg and 16.3 mg, respectively. The negative control and solvent control were found to be statistically different for all growth endpoints (p ≤ 0.05). Therefore, the treatment group data were compared to the solvent control data only, per guidance in the OECD 210 Fish, Early-life Stage Toxicity Test and OECD Current Approaches in the Statistical Analysis of Ecotoxicity Data guidelines.
Mean total length at test termination of surviving fish in the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 22.9, 22.7, 22.8, 23.1 and 22.7 mm, respectively. Mean wet weight at test termination of surviving fish in the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 84.0, 81.6, 87.6, 88.0 and 85.0 mg, respectively. Mean dry weight at test termination of surviving fish in the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 16.9, 16.0, 18.1, 17.5 and 16.9 mg, respectively. According to both Dunnett’s one-tailed test and the one-sided Jonckheere-Terpstra step-down trend test, there were no statistically significant decreases evident in total length, wet weight or dry weight in any treatment group, when the data was compared to the solvent control (p > 0.05). Consequently, the NOEC and LOEC for the growth endpoints were determined to be 0.072 mg/L and >0.072 mg/L, respectively. Since there was a less than 10% inhibition in total length, wet weight and dry weight in any treatment group in comparison to the solvent control, the IC10 and IC20 values for the growth endpoints were empirically estimated to be greater than the highest concentration tested.

- Number of healthy fish at end of test: Post-Hatch and Overall Survival: Both post-hatch larval survival and overall survival in the negative and solvent control groups was 95.0 and 95.0% at test termination, respectively. Since there were no statistically significant differences in post-hatch larval survival or overall survival between the negative and solvent control (p > 0.05), the control data were pooled for comparisons among the treatment groups. At test termination, post-hatch larval survival in the pooled control and the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 95.0, 98.8, 93.8, 97.4, 98.8 and 96.2 %, respectively. Overall survival in the pooled control and the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 95.0, 98.8, 93.8, 95.0, 98.8 and 93.8 %, respectively. According to Fisher’s Exact test, there were no statistically significant decreases in either post-hatch larval survival or overall survival in any treatment group, in comparison to the pooled control (p > 0.05). Consequently, the NOEC and LOEC for both post-hatch larval survival and overall survival were determined to be 0.072 mg/L and > 0.072 mg/L, respectively. Since there was less than 10% inhibition in both post-hatch larval survival and overall survival in any treatment group in comparison to the pooled control, the LC10 and LC20 values were empirically estimated to be greater than the highest test concentration.

- Type of and number with morphological abnormalities: In general, the majority of the fish in the control groups and in all treatment groups appeared normal throughout the test. Observations included unusual behaviour or appearance such as appearing small, weak, discoloured (pale), erratic swimming, loss of equilibrium, morphologically deformed (e.g. curled, crooked spine, deformed jaw, deformed/protruding operculum), and lying on the bottom of the tank with little motion other than minor gill movement. However, these observations were not prevalent in the majority of the population, did not follow a dose-responsive pattern, and were comparable in the negative and solvent control groups and therefore, were not considered to be treatment-related.

LC/ICx Values 

	LC10/IC10 (95 % CI) (mg/L)	LC20/IC20 (95 % CI) (mg/L)
Hatching Success	> 0.072*	> 0.072*
Time to Hatch	NC	NC
Post-Hatch Larval Survival	> 0.072*	> 0.072*
Overall Larval Survival	> 0.072*	> 0.072*
Total Length	> 0.072*	> 0.072*
Wet Weight	> 0.072*	> 0.072*
Dry Weight	> 0.072*	> 0.072*

* Empirically estimated to be greater than the highest test concentration, since there was less than 10 % inhibition in any treatment group in comparison to the pooled control (or solvent control when the control groups cannot be pooled).

NC Not calculable, since the estimated ICx value was extrapolated beyond the data range used in the calculation and/or the 95 % confidence intervals were overly wide.

Conditions for the Validity of the Test

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

- The dissolved oxygen concentration should be > 60% of the air-saturation value throughout the test. Dissolved oxygen concentrations remained ≥ 85% of air-saturation (≥ 7.0 mg/L) throughout the test.

- The water temperature will not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the 25 ± 1 °C range specified for the test species. Water temperatures manually measured in the test chambers throughout the test ranged from 24.3 to 25.5 °C, while temperature continuously monitored in one replicate of the negative control ranged from 23.86 to 25.38 °C.

- Evidence will be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ± 20 % of the mean measured value. The coefficient of variation calculated based on the time-weighted mean measured concentrations and time-weighted standard deviations for the 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L treatment groups was 4.1, 4.7, 3.0, 3.3 and 9.8%, respectively.

- The percent hatching success of fertilized eggs in the control group and, where relevant, in the solvent-only control group will be > 70%, and the minimum percent post-hatch larval survival will be 75%. Hatching success in the negative control and solvent control was 100 and 100%, respectively. Post-hatch larval survival at test termination in the negative control and solvent control was 95.0 and 95.0%, respectively.

Measurement of Test Concentrations

Nominal concentrations selected for use in the study were 0.0063, 0.013, 0.025, 0.050, and 0.10 mg/L. The test solutions appeared clear and colourless, with no evidence of precipitation observed during the test, in the negative control and solvent control test chambers, and in the diluter mixing chambers that delivered test solution for these groups. Test solutions in the 0.0044, 0.0074, and 0.015 mg/L test chambers appeared clear and colourless with no precipitates observed at test initiation and termination. The solution in the diluter mixing chamber that delivered test solution for the 0.0044, 0.0074, and 0.015 mg/L test concentration also appeared clear and colourless, however, purple staining, increasing in intensity with increasing concentration was noted on the side of the mixing chamber. The solution for the 0.037 mg/L appeared clear and colourless with staining of the side in the mixing chamber, while the solution in the tanks appeared clear and very slight purple at test initiation and termination. Additionally at termination, slight staining at the water line of the tanks was observed. The solutions in the mixing chamber of the 0.072 mg/L appeared clear and slight purple with heavy purple staining on the side of the mixing cup and siphon cap at test initiation and termination. The solutions in the test chambers were clear and purple with staining at the water line on the tanks at initiation and termination. Since there were no visible precipitates observed in the test solutions, test solution samples were analysed without centrifugation only throughout the test.

Measured concentrations of the stock solution samples ranged from 87.3 to 112% of the nominal stock concentrations, indicating the test system was being dosed appropriately. Measured concentrations of the samples collected during the pre-test period ranged from 59.9 to 90.0% of the nominal concentrations. With the exception of the sample collected from the highest treatment group on Day 19, measured concentrations of the samples collected during the exposure phase of the test ranged from 41.9 to 96.0 % of the nominal concentrations.

At the second check of the diluter system on Day 19, the syringe delivering stock solution for the highest treatment group (0.10 mg/L) was found to be leaking. The syringe had been observed to be delivering correctly earlier that day when the syringes were replaced. Test solution samples were collected at the time this issue was discovered. In order to demonstrate that the measured concentration had returned to normal, test solution samples were also collected the next morning after approximately 6.7 test solution turnovers. Analysis of the sample collected from the highest treatment group at the time the issue was discovered yielded a measured recovery that was 27.2% of the nominal test concentration, while analysis of the sample collected the following morning yielded a measured recovery that was 75.5% of the nominal test concentration. Since the syringe leak was discovered and corrected approximately three hours after it was observed to be functioning correctly and the test solution inside each test chamber is completely replaced every 2.4 hours, the measured concentration in the test chambers for the highest treatment group was believed to have been impacted for less than six hours. This amount of time that the measured concentration was impacted equates to less than 1% of the overall exposure duration. Consequently, there was no impact on the interpretation of the study results.

Due to the disruption in stock solution delivery for the highest treatment group, the mean measured concentrations for all treatment groups were calculated using the time-weighted averages. This allows for the brevity of the disruption in stock solution delivery to be factored in so that it does not artificially inflate the coefficient of variation for the affected treatment group. When the measured concentrations of test solution samples collected on Days 0, 7, 13, 19, 20, 21, 28 and 32 of the test were averaged over time for each treatment group, the time-weighted mean measured test concentrations were calculated to be 0.0044, 0.0074, 0.015, 0.037, and 0.072 mg/L, representing 70.3, 57.1, 61.0, 74.8 and 72.2 % of nominal concentrations, respectively. The results of the study were based on the time-weighted mean measured concentrations.

While the mean measured concentrations for the study were below the 80 to 120 % guideline recommended range, every effort was employed to maximize the measured concentrations of the test solutions throughout the test. Prior to conducting the definitive test, a non-GLP diluter trial was conducted to investigate the solubility of the test material in both DMF and dilution water. Quantitation of the test material in samples collected during the non-GLP diluter trial was based on the average of three precursor ions (437, 530, and 541). However, quality control samples prepared during the non-GLP diluter trial returned inconsistent recoveries when quantified in this manner. Consequently, the test material was quantified based on the analysis of only precursor ion 437 during the definitive test.

The definitive study was conducted under flow-through test conditions, with ten test solution turnovers in each test chamber per day. This is well over the guideline requirement five test solution turnovers per test chamber per day. During the definitive test, the DMF stock solutions were prepared individually and daily to ensure the test system was being appropriately dosed with the test material. Measured concentrations of the stock solutions during the pre-test period confirmed that the test system was being dosed at the appropriate nominal concentrations. The EPA OCSPP 850.1000 Background and Special Considerations guideline states: “The most important consideration is maintenance of a constant exposure, regardless of the percentage of the nominal concentration that is attained. Whenever possible, the exposure scenario should be selected so that the measured concentration of test material at each treatment level remains plus or minus (±) 20% of the time-weighted average concentration for the duration of the test”. The coefficient of variations calculated based on the time-weighted mean measured concentrations and standard deviations for each treatment group ranged from 3.0 to 9.8%, indicating that organisms were exposed to consistent measured concentrations of the test material throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the NOEC of the test material was 0.072 mg/L, the highest dose tested. The LOEC was > 0.072 mg/L.

Executive summary:

The effects of exposure to early life stage fish was assessed in accordance with the standardised guidelines OECD 210, EPA OCSPP 850.1400 and ASTM Standard E 1241-05, under GLP conditions.

During the study, Fathead minnows (Pimephales promelas) were exposed to the test material at mean measured concentrations of 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on mean time to hatch, hatching success, survival or growth at time-weighted mean measured concentrations of ≤ 0.072 mg/L, in comparison to the pooled control.

Since the negative control and solvent control data were found to be statistically different (p ≤ 0.05) for the growth endpoints, the treatment group data were compared to the solvent control alone, per OECD guidance. In comparison to the solvent control, there were no statistically significant decreases in growth, measured as total length, wet weight and dry weight, at time-weighted mean measured concentrations of ≤ 0.072 mg/L.

Under the conditions of the study, the NOEC of the test material was 0.072 mg/L. The LOEC was > 0.072 mg/L.

Description of key information

Under the conditions of the study, the NOEC of the test material was 0.072 mg/L, the highest dose tested. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.072 mg/L

Additional information

The effects of exposure to early life stage fish was assessed in accordance with the standardised guidelines OECD 210,EPA OCSPP 850.1400and ASTMStandard E 1241-05, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, Fathead minnows (Pimephales promelas) were exposed to the test material at mean measured concentrations of 0.0044, 0.0074, 0.015, 0.037 and 0.072 mg/L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on mean time to hatch, hatching success, survival or growth at time-weighted mean measured concentrations of ≤ 0.072 mg/L, in comparison to the pooled control.

Since the negative control and solvent control data were found to be statistically different (p ≤ 0.05) for the growth endpoints, the treatment group data were compared to the solvent control alone, per OECD guidance. In comparison to the solvent control, there were no statistically significant decreases in growth, measured as total length, wet weight and dry weight, at time-weighted mean measured concentrations of ≤ 0.072 mg/L.

Under the conditions of the study, the NOEC of the test material was 0.072 mg/L. The LOEC was > 0.072 mg/L.