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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:
March - November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
As per ECHA Decision number: CCH-D-2114449848-30-01/F
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples (8.0 mL) were collected from mid-depth
- Sampling method: Test solution samples were collected from one replicate test chamber of each treatment and control group four, three and one day prior to the start of exposure to confirm concentrations after conditioning the diluter system for three, four and six days, respectively. Samples of the stock solutions being delivered to the test system were collected for analysis one day prior to the start of the exposure. Stock solution samples (~2.0 mL) were collected from the syringes, placed in glass vials and processed immediately for 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
- Sample storage conditions before analysis:processed immediately for analysis

Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared every three to five days during the test. The primary stock solutions were sonicated for 10 minutes, inverted to mix and appeared clear and colorless. Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations of 0.15, 0.30, 0.60 and 1.2 mg/mL. The secondary stock solutions were mixed by inversion and appeared clear and colorless. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every one to four days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 10.00 µL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 7.5, 15, 30, 60 and 120 µg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethyl foramide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 2.4 mg/mL. The concentration of DMF in the solvent control and all tBuTPP low TPP treatment groups was 0.050 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow, Pimephales promelas,
- Strain:
- Source: Fathead minnow embryos used in the test were obtained from cultures maintained by Eurofins-Easton.
- Age at study initiation (mean and range, SD): < 24 hours old
- Method of breeding:
- Feeding during test : yes, 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. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
- Food type: Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct of Ogden, Utah.
- Amount: The concentrations of selected pesticides and organic and inorganic constituents in the Artemia cysts are measured annually and the results from the most recent analysis
- Frequency: 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.

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
33 Days (5-Day Hatch and 28-Day Post-Hatch)
Hardness:
128-140 (mg/L as CaCO3)
Test temperature:
24.2-25.6
pH:
7.8-8.0
Dissolved oxygen:
7.1-8.2
Salinity:
Alkalinity 166-184 (mg/L as CaCO3)
Conductivity:
328-352 (µS/cm)
Nominal and measured concentrations:
Mean Measured
Nominal (total product)
Negative Control Solvent Control 7.5 µg/L 7.5 µg/L
15 µg/L 12 µg/L
30 µg/L 23 µg/L
60 µg/L 47 µg/L
120 µg/L 100 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass
- Size of vessel: The test chambers were 9-L glass aquaria. The depth of the test water in a representative test chamber was 15.9 cm.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with approximately 7 L of test solution.
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): The stock solutions were delivered to the diluter mixing chambers (at a rate of 10.00 L/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 7.5, 15, 30, 60 and 120 µg/L.
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle 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 0.0376 g of fish per liter of test solution that passed through the test chamber during a 24-hour period.

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 Eurofins-Easton site. The well water was passed through a sand filter 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 um to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.

- Total organic carbon: 1.239 (mg C/L)
- Chlorine: 307 mg/L
- Alkalinity: 166-184 (mg/L as CaCO3)
- Conductivity: 328-352 (µS/cm)
- Intervals of water quality measurement: periodically

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 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: 642 lux at the surface of the water of one representative test chamber


TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2

RANGE-FINDING STUDY
- Test concentrations: 1.9, 7.5, 30 120 µg/L
- Results used to determine the conditions for the definitive study:
Nominal Hatching Number Post-Hatch Mean Wet
Concentration 1 Number Number Success Surviving to Survival Weight
(µg/L) Replicate Exposed Hatched (%) 3 Day 18 (%) 3 (mg) 3

Negative Control 1 A 25 24 96 23 96 28.8
B 25 24 96 24 100 28.7
96 98 28.7

Solvent Control 2 A 25 25 100 24 96 28.2
B 25 23 92 21 91 27.2
96 94 27.7

1.9 A 25 24 96 24 100 28.8
B 25 25 100 25 100 27.2
98 100 28.0

7.5 A 25 24 96 23 96 25.1
B 25 25 100 25 100 27.4
98 98 26.2

30 A 25 25 100 23 92 25.4
B 25 25 100 25 100 27.0
100 96 26.2

120 A 25 25 100 25 100 23.9
B 25 25 100 25 100 23.2
100 100 23.6
1 Appearance of test solutions: clear and colorless with no evidence of precipitates.
2 Solvent concentration: 0.05 mL DMF/L
3 Calculations performed using Excel 2010.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
time to hatch
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
weight
Details on results:
In general, the majority of the fish in the control groups and all treatment groups appeared normal throughout the test. Observations of unusual behavior or appearance included appearing small, weak, curled, morphologically deformed (e.g. crooked spine, swollen abdomen), erratic swimming, and lying on the bottom of the tank with little motion other than minor gill movement. However, these observations were infrequent, did not follow a dose-responsive pattern, and were comparable in the controls.
Reported statistics and error estimates:
The data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01). Proportions derived from hatching success, post-hatch survival and overall survival were transformed by arc-sin square root prior to the test for normality and homogeneity of variance. The hatching success, post-hatch survival and overall survival data passed assumptions of homogeneity of variance but failed assumptions of normality. The hatching success, post-hatch survival and overall survival data were visually evaluated for monotonicity. The data appeared not to be monotonic, so the Mann-Whitney U-test was used to determine which treatment means were significantly different from the pooled control groups (p ≤ 0.05). For the mean time to hatch, total length, wet weight and dry weight data, the treatment means that were significantly different from the pooled control means were identified using Dunnett’s test (p ≤ 0.05). The statistical tests were performed using a personal computer with SAS software.
Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to tBuTPP low TPP at mean measured concentrations of 7.5, 12, 23, 47 and 100 µg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success, post-hatch larval survival or overall survival at concentrations ≤ 100 µg/L. There were statistically significant effects on time to hatch and growth, however, these were not biologically meaningful. Consequently, the NOEC and LOEC, were 100 µg/L and > 100 µg/L, respectively.

Lethal concentrations (LC10 and LC20) for the hatching success and survival endpoints were 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 pooled control group. Inhibition concentrations (IC10 and IC20) for total length were empirically estimated to be > 100 µg/L. The IC10 and IC20 values for wet and dry weight endpoints could not be estimated, since the estimated ICx values were extrapolated beyond the data range used in the calculation.

Executive summary:

Fathead minnows (Pimephales promelas) were exposed to tBuTPP low TPP at mean measured concentrations of 7.5, 12, 23, 47 and 100 µg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period).  There were no statistically significant treatment-related effects on hatching success, post-hatch larval survival or overall survival at concentrations ≤ 100 µg/L.

Description of key information

No statistically significant treatment-related effects on hatching success, post-hatch larval survival or overall survival

identified up to maximum attainable concentration in aquatic medium.

Key value for chemical safety assessment

Fresh water fish

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

Additional information