2,2-dimethylpropan-1-ol, tribromo derivative; 3-bromo-2,2-bis(bromomethyl)propan-1-ol

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:

Oct 2019-March 2020

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Samples of the test solution samples collected during the test had measured concentrations that ranged from 87.9 to 113% of nominal concentrations. When the measured concentrations of test solution samples collected during the test were averaged, the mean measured test concentrations were 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L, which represented 107, 95, 99, 101 and 105% of nominal concentrations, respectively.
- Sampling method:
Test solution samples were collected from one replicate test chamber of each treatment and control group three days prior to the start of exposure to confirm concentrations after conditioning the diluter system for three days, respectively. Duplicate samples of the stock solutions being delivered to the test system were collected for analysis on Day -3. One stock sample was centrifuged prior to analysis. Test solution samples also were collected from one replicate test chamber in each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test to measure concentrations of the test substance. Samples (8.0 mL) were collected from mid-depth, placed in glass vials containing 2.00 mL of acetonitrile and processed immediately for analysis.

Vehicle:

no

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Strain: fathead minnow, Pimephales promelas
- Source: Fathead minnow embryos used in the test were obtained from cultures maintained by Eurofins-Easton.
- Age at study initiation (mean and range, SD): Embryos < 24 hours old
- Length at study initiation (length definition, mean, range and SD): . Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler,
- Weight at study initiation (mean and range, SD): wet and dry weights were measured to the nearest 0.1 mg using an analytical balance.
- Method of breeding:
- Feeding during test : during the first seven days of post-hatch.
- Food type: live brine shrimp nauplii (Artemia sp.)
- Amount:
- Frequency: three times per day. Fish were not fed for at least 24 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made.

ACCLIMATION
Not required as the source is in the laboratory.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

4-Day Hatch and 28-Day Post-Hatch

Hardness:

135-141

Test temperature:

24.1-25

pH:

7.8-8.3

Dissolved oxygen:

7.7-8.2

Conductivity:

270-353

Nominal and measured concentrations:

Nominal Mean Measured
Negative Control 0.63 mg a.i./L 0.68 mg a.i./L
1.3 mg a.i./L 1.2 mg a.i./L
2.5 mg a.i./L 2.5 mg a.i./L
5.0 mg a.i./L 5.0 mg a.i./L
10 mg a.i./L 11 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): glass cylinders approximately 50 mm in diameter with 425 µm nylon screen mesh attached to the bottom with silicone sealant.
- Test vessel: 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 15.2 cm.
- Material, size, headspace, fill volume: 9-L glass aquaria , 7L
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): Newly prepared stock was placed in the fluid metering pump system every two days during the study. The diluter flow rate was adjusted to provide approximately 10 volume additions of test water in each test chamber per day.
- No. of fertilized eggs/embryos per vessel: Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): no
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:The water used for culturing 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 m 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 micron to remove fine
particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Total organic carbon:<2 (mg C/L)
- Particulate matter:
- Metals: Appendix 5 in the report
- Pesticides: Appendix 5 in the report
- Chlorine: 3.9 mg/L
- Alkalinity:170 mg/L as CaCO3
- Ca/mg ratio: 125 (mg/L as CaCO3)
- Conductivity: 310 (μS/cm)
- Culture medium different from test medium:
- Intervals of water quality measurement: 4-Week Period

OTHER TEST CONDITIONS
- Adjustment of pH:
no
- 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:
562 lux at the surface of the water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED:no

RANGE-FINDING STUDY
- Test concentrations: 0.16, 0.63, 2.5 and 10 mg/L
- Results used to determine the conditions for the definitive study: see attached document

POST-HATCH DETAILS
- Begin of post-hatch period: When hatching reached > 90% in the control group on Day 4 of the test, the larvae were released to their respective test chambers and the post-hatch period began.
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20
- Release of alevins from incubation cups to test chamber on day no.: 4

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

Post-Hatch and Overall Survival
Post-hatch larval survival in the negative control and the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups was 98.8, 97.5, 97.4, 93.8, 97.5 and 88.7%, respectively. Overall survival at test termination in the negative control and the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups was 97.5, 97.5, 95.0, 93.8, 97.5, and 87.5%, respectively. According to the Mann Whitney U-test, there was a statistically significant decrease in both post-hatch survival and overall survival in the 11 mg a.i./L treatment group in comparison to the negative control group (p ≤ 0.05). Consequently, the NOEC and LOEC for both post-hatch survival and overall survival were determined to be 5.0 and > 11 mg a.i./L, respectively. The LC10 and LC20 values for post-hatch and overall survival could not be estimated, since the calculated LCx values were extrapolated beyond the data range used in the calculation and/or the 95% confidence intervals were overly wide.

Biological Observations
In general, the majority of the fish in the control group and the 0.68, 1.2, 2.5 and 5.0 mg a.i./L treatment groups appeared normal throughout the test. Observations of unusual behavior or appearance in the control and all treatment groups included appearing small, weak, discolored, morphologically deformed (e.g. curved, curved spine, hemorrhage on top of head), loss of equilibrium, and lying on the bottom of the tank with little motion other than minor gill movement. There was an increased number of unusual behaviors or appearances in the 11 mg a.i./L treatment group in comparison to the negative control.

Total Length
Mean total length at test termination in the negative control and the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups was 23.7, 23.8, 23.7, 23.9, 23.5 and 22.8 mm, respectively. According to Dunnett’s test, there was a statistically significant decrease in the 11 mg a.i./L treatment groups in comparison to the negative control group (p ≤ 0.05). Consequently, the NOEC and LOEC for total length were determined to be 5.0 and 11 mg a.i./L, respectively. Since there was a less than a 10% inhibition for total length between the treatment group means and the negative control mean, the IC10 and IC20 values were empirically estimated to be greater than the highest treatment group.

Wet Weight
Mean wet weight at test termination in the negative control and the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups was 110.7, 111.2, 115.8, 117.1, 112.1 and 101.2 mg, respectively. According to Dunnett’s test, there were statistically significant differences in the 2.5 and 11 mg a.i./L treatment groups in comparison to the negative control group (p ≤ 0.05). However, the increase in the 2.5 mg a.i./L treatment group was not dose-responsive. Consequently, the NOEC and LOEC for wet weight were determined to be 5.0 and 11 mg a.i./L, respectively. Since there was a less than a 10% inhibition for wet weight between the treatment group means and the negative control mean, the IC10 and IC20 values were empirically estimated to be greater than the highest treatment group.

Dry Weight
Mean dry weight at test termination in the negative control and 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups was 24.1, 24.1, 24.5, 25.4, 24.1 and 21.3 mg, respectively. According to Dunnett’s test, there was a statistically significant decrease in the 11 mg a.i./L treatment groups in comparison to the negative control group (p ≤ 0.05). Consequently, the NOEC and LOEC for dry weight were determined to be 5.0 and 11 mg a.i./L, respectively. The IC10 and IC20 values for dry weight 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.

Validity criteria fulfilled:

yes

Conclusions:

Fathead minnows (Pimephales promelas) were exposed to FR-513 at mean measured concentrations of 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./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 hatching success at concentrations ≤ 11 mg a.i./L. While there were statistically significant increases observed in mean time to hatch in the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups, these increases were not considered to be biologically meaningful since the increases were less than one day and the data were not dose-responsive. There were statistically significant decreases in post-hatch survival, overall survival, total length, wet weight and dry weight in the 11 mg a.i./L treatment group. There was a statistically significant increase in wet weight in the 2.5 mg a.i./L treatment group. However, the increase was not dose-responsive. Consequently, the NOEC and LOEC were 5.0 and 11 mg a.i./L, respectively.

Lethal concentrations (LC10 and LC20) for the hatching success endpoint was empirically estimated to be greater than the highest test concentration, since there was less than a 10% reduction in any treatment group when compared to the negative control group. Lethal concentrations (LC10 and LC20) for post-hatch and overall survival could not be calculated since the estimated LCx values were extrapolated beyond the data range used in the calculation. Inhibition concentrations (IC10 and IC20) for the total length and wet weight endpoints were empirically estimated to be greater than the highest test concentration, since there was a less than a 10% inhibition between the treatment group means and the negative control mean. Inhibition concentrations (IC10 and IC20) for dry weight not be calculated since the estimated ICx values were extrapolated beyond the data range used in the calculation.

Executive summary:

Fathead minnows (Pimephales promelas) were exposed to FR-513 at mean measured concentrations of 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L under flow-through conditions for 32 days.  There were no statistically significant treatment-related effects on hatching success at concentrations ≤ 11 mg a.i./L.  While there were statistically significant increases observed in mean time to hatch in the 0.68, 1.2, 2.5, 5.0 and 11 mg a.i./L treatment groups, these increases were  not considered to be biologically meaningful since the increases were less than one day and the data were not dose-responsive.  There were statistically significant decreases in post-hatch survival, overall survival. NOEC and LOEC were 5.0 and 11 mg a.i./L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5 mg/L