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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
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 211 (Daphnia magna Reproduction Test)
Version / remarks:
2 October 2012.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Test solution samples were collected from one replicate test chamber of each treatment and control group six, five and four days prior to the start of exposure to confirm concentrations after conditioning the diluter system for two, three and four days, respectively. Samples of the stock solutions being delivered to the test system were collected for analysis six days prior to the start of the test.
- Sampling method: 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. Additional samples were collected on Day 16 after a power outage interrupted toxicant flow to the mixing chambers. Samples were also collected on Day 17 to indicate concentrations returned to nominal after interruption. Samples (8.0 mL) were collected from mid-depth, placed in glass vials.
- Sample storage conditions before analysis:Samples (8.0 mL) were collected from mid-depth, placed in glass vials containing 2.0 mL of 0.5% formic acid in methanol and processed immediately for analysis. Stock solution samples (approximately 2 mL) were collected from the syringes, placed in glass vials and processed immediately for analysis.
Vehicle:
yes
Remarks:
dimethylformamide (DMF)
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested and were prepared every three or four days during the test. Test solution concentrations were not adjusted for the main component of the test substance during preparation and are based on the test substance as received. The primary stock solutions were prepared by mixing a calculated amount of test substance into HPLC grade dimethylformamide (DMF) at a nominal concentration of 2.4 mg/mL. The primary stock solution was sonicated for 10 minutes and inverted to mix. The primary stock solutions appeared clear and colorless with no visible precipitates. Four secondary stock solutions were prepared in DMF at nominal concentrations of 0.15, 0.30, 0.60 and 1.2 mg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by inversion and appeared clear and colorless with no visible precipitates.



Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: The cladoceran, Daphnia magna,
- Strain/clone: Parental-generation daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by Eurofins-Easton.
- Age at study initiation (mean and range, SD): Neonates (< 24 hours old at initiation)
- Method of breeding: The seven adult daphnids used to supply neonates for the test were held for at least 21 days prior to collection of the juveniles for testing and had each produced at least four previous broods. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7 day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. Prior to initiation, all parental-generation daphnids were verified to be female using a dissecting microscope.
- Source:cultures maintained by Eurofins-Easton.
- Age of parental stock (mean and range, SD): Parental-generation daphnid neonates used in the test were less than 24 hours old.
- Feeding during test During culture and testing, daphnids were fed three times per day through Day 7 of the test and then were fed three or four times per day until the last day of the test where they were fed once.
- Food type: mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata.
- Amount: At each feeding, each test chamber was fed 0.80 mL of YCT and 1.5 mL of algae. At one feeding per day, each test chamber was dosed with 0.50 mL of vitamin solution. This amount of feed is equal to approximately 0.6 to 0.7 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.
- Frequency: During culture and testing, daphnids were fed three times per day through Day 7 of the test and then were fed three or four times per day until the last day of the test where they were fed once.

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of immobile parental-generation daphnids were recorded, along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding), abnormal behavior or appearance, and the onset of reproduction (the time in days to the release of the first brood in each test vessel). Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized (even if they could still move their antennae). Immobilization was used as a surrogate for death. Therefore, dead animals were counted as immobile. The presence of eggs in the brood pouch, males or ephippia also was recorded daily. With the onset of reproduction, neonates and aborted eggs produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday, and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test. Daphnids were placed in an oven at approximately 60oC for approximately 24 hours to obtain dry weight data.
Hardness:
136 - 148 mg/L as CaCO3
Test temperature:
19.0 - 20.3°C
pH:
7.9- 8.1
Dissolved oxygen:
≥ 69%; no aeration
Salinity:
Alkalinity 168- 178 mg/L as CaCO3
Conductivity:
304 - 356 uS/cm
Nominal and measured concentrations:
Nominal Concentration (μg/L): 7.5, 15, 30, 60 120
Mean Measured Concentration (μg/L) : 4.4, 13, 25, 46, 88

Details on test conditions:
The target test temperature during the test was 20 ± 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. Temperature also was monitored continuously in one negative control test chamber using a minimum/maximum thermometer, which was calibrated prior to exposure initiation with a digital thermometer.

Dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately three times per week during the test, and at the end of the test. Additional measurements were taken on Day 16 to monitor of dissolved oxygen concentrations after an electrical power outage that lasted approximately 20 minutes.. Measurements of pH were made in one replicate test chamber 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 typically rotated between the four replicates in each treatment or control group at each measurement interval. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter, and measurements of pH were made using a Thermo Scientific Orion DUAL STAR pH/ISE meter.


TEST SYSTEM
- Test vessel:glass
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Test chambers were 7 L glass aquaria filled with approximately 6.5 L of test water. Test compartments were 300 mL glass beakers, approximately 6 cm in diameter and 12 cm in height. The depth of the test solution in a representative compartment was 8.5 cm, while the depth of water in a representative test chamber was 15.2 cm.
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous-flow diluter used to deliver
- Renewal rate of test solution (frequency/flow rate): The stock solutions were pumped into the diluter mixing chambers assigned to the treatment groups at a target rate of 9.00 µL/minute and were mixed with dilution water in the mixing chambers, delivered at a target rate of 180 mL/minute to achieve the desired nominal test concentrations.
- No. of organisms per vessel:five neonate daphnids (a total of 20 daphnids per test concentration)
- No. of vessels per concentration (replicates):Four replicate test chambers
- No. of vessels per control (replicates):Four replicate test chambers
- No. of vessels per vehicle control (replicates):Four replicate test chambers
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Freshwater obtained from a well approximately 40 meters deep located on the Eurofins - Easton site
- Total organic carbon:<1 (mg C/L)
- Alkalinity:166 - 178 (mg/L as CaCO3
- Chlorine: 3.7 mg/L
- Conductivity:313 - 352 (µS/cm)
- Intervals of water quality measurement: periodic analyses

OTHER TEST CONDITIONS
- Adjustment of pH:
- 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:879 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Nominal test concentrations selected were 7.5, 15, 30, 60 and 120 µg tBuTPP low TPP/L.
- Results used to determine the conditions for the definitive study:

Results of a 14-Day Non-GLP Range-finding Test with tBuTPP low TPP

Nominal Day 14 Mean Number of Mean Number of
Concentration 1 Adult Survival (%) Young Produced Per Young Produced Per
(μg/L) (Observations 3) Reproductive Day Parent Animal Alive at the Beginning of the Test
Negative Control 100 (10 AN) 12.0 ± 0.19 96 ± 1.6
Solvent Control 2 90 (1 S; 8 AN) 11.7 ± 1.82 88 ± 21.9
1.9 90 (9 AN) 12.1 ± 0.34 92 ± 9.5
7.5 100 (1 S; 9 AN) 10.9 ± 0.88 87 ± 7.1
30 100 (10 AN) 11.6 ± 1.57 93 ± 12.6
120 100 (10 AN) 9.8 ± 0.42 78 ± 3.4

1 Appearance of test solutions: all clear and colorless with no evidence of precipitates in the test chambers and mixing chambers at initiation and termination.
2 Solvent concentration: 0.05 mL DMF/L
3 Observations: AN = appears normal; S = small.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
88 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
88 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
88 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth
Details on results:
Samples of the stock solutions being delivered to the diluter system had measured concentrations that ranged from 97.8 to 104% of nominal concentrations, confirming the concentrations being delivered to the diluter system. The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 66.1 to 136% of nominal concentrations. Samples of the test solutions collected during the test had measured concentrations that ranged from 37.2 to 166% of nominal concentrations. Even though there was an extended pretest in an attempt to achieve equilibrium, DMF was used as the vehicle and there was no evidence of precipitation during the study, stable concentrations could not be obtained. A non-GLP study was conducted and indicates that the test substance adheres to glassware. The low recoveries from pretest samples with nominal recoveries from stock solutions support the Sponsor’s claim that the test substance is hydrophobic. When the measured concentrations of test solution samples collected during the test were averaged for each treatment group, the mean measured test concentrations were 4.4, 13, 25, 46 and 88 μg/L, which represented 58.4, 89.9, 83.5, 76.0 and 73.0% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.
Reported statistics and error estimates:
Samples of the stock solutions being delivered to the diluter system had measured concentrations that ranged from 97.8 to 104% of nominal concentrations, confirming the concentrations being delivered to the diluter system (Table 2). The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 66.1 to 136% of nominal concentrations (Table 3). Samples of the test solutions collected during the test had measured concentrations that ranged from 37.2 to 166% of nominal concentrations (Table 4). Even though there was an extended pretest in an attempt to achieve equilibrium, DMF was used as the vehicle and there was no evidence of precipitation during the study, stable concentrations could not be obtained. A non-GLP study was conducted and indicates that the test substance adheres to glassware (Appendix 8). The low recoveries from pretest samples with nominal recoveries from stock solutions support the Sponsor’s claim that the test substance is hydrophobic. When the measured concentrations of test solution samples collected during the test were averaged for each treatment group, the mean measured test concentrations were 4.4, 13, 25, 46 and 88 μg/L, which represented 58.4, 89.9, 83.5, 76.0 and 73.0% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.
Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to tBuTPP low TPP at mean measured concentrations of 4.4, 13, 25, 46 and 88 μg/L under flow-through conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤ 88 μg/L in comparison the pooled control.

Consequently, the NOEC was determined to be 88 μg/L. The LOEC was determined to be > 88 μg/L. Since there was less than a 10% decrease in survival in the highest treatment group in comparison to the pooled control, the 21-day EC10, EC20, and EC50 values based on survival were empirically estimated to be > 88 μg/L, the highest concentration tested. Since there was less than a 10% decrease in reproduction or growth in the highest treatment group in comparison to the pooled control, the 21-day IC10 and IC20 values were empirically estimated to be > 88 μg/L, the highest concentration tested.
Executive summary:

The cladoceran,Daphnia magna,was exposed to tBuTPP low TPP at mean measuredconcentrations of4.4, 13, 25, 46 and 88μg/L under flow-through conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤ 88 μg/L in comparison the pooled control.

Consequently, the NOEC was determined to be 88 μg/L. The LOEC was determined to be > 88 μg/L.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
88 µg/L

Additional information