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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018 to 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Chemicals Testing Guideline No.23 Guidance document on aquatic toxicity of difficult substances and mixtures.
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
EC Number:
219-094-1
EC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Cas Number:
2356-53-8
Molecular formula:
C3Cl2F6O
IUPAC Name:
1,2-dichloro-1,1,2-trifluoro-2-(trifluoromethoxy)ethane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D653 22_02_2018 (manufactured by the Sponsor)
- Expiration date of the lot/batch: Feburary 2023
- Purity test date: 2018/02/27

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All the tested concentrations (4.1, 9.1, 20.0, 45, 100% Saturated solution) and the control concentration were analytically verified.
For fresh test media samples (0 and 24 hours) additional analytical vessels were prepared at each media preparation occasion and sampled. This because the volatile nature of the compound, in this way it was avoided to sample test vessels reducing the media volume and creating a head space for the test substance to volatilise into.
Old media (24 and 48 hours) was sampled from two test vessels, selected at random per control and test concentrations, two vessels were sampled to allow us to compare differences in concentration between replicates.
- Sampling method: Analytical samples were then taken in duplicate using a gas tight syringe from the aqueous phase (mid-point) of the vessel. 1 mL samples were taken directly into 9 mL of methanol and rìtransferred to analytical department.
- Sample storage conditions before analysis: direct analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the difficoult nature of the test substance, extensive work (initial solubility trial + three media preparation trials) was conducted in order to define the best procedure for media preparation. The scope of the trials was to find a procedure for solubilizing the test item reducing as much as possible losses by evaporation.
The procedure described below is based on the results of the media preparation trials.

A saturated solution with an initial loading rate concentration of 10 g/L was prepared by adding 35.0 mL of test substance into ~5.5 L of Elendt media in a glass bottle, the bottle was completely filled with Elendt media and sealed with crimp cap lids. The stock solution was then stirred for ca 24 hours and then allowed to settle for ca 24 hours. Individual test vessels were pre-filled with an appropriate amount of Elendt media and then completely filled with saturate solution such that the appropriate concentrations were achieved. Stock solution was removed from the aqueous phase of the saturated solution (vessel mid-point) through a lid with gas tight tap reducing losses through volatilisation as much as possible.
Daphnia were added to each test and control replicate prior to stock solution along with vitamin stock solution in an effort to reduce losses through volatilisation as the vessels could not be opened once dosed.
The test vessels were approximately 118 mL glass bottles, completely filled and sealed with crimp cap lids.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Age at study initiation: less than 24 hours old.

CULTURING of DAPHNIA MAGNA
The Daphnia magna are cultured in 1000 mL glass beakers containing 1000 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of 15-20 daphnids per vessel. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures. The water in each culture was renewed or partially renewed at least twice a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 42 days. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore
pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old. All cultures, prior to and during toxicity tests, are maintained under fluorescent
lighting on a 16-hour light:8-hour dark photoperiod.
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity, the latest reference test was conducted on January 2019.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no

Test conditions

Hardness:
Elendt M4 medium.
Test temperature:
Temperature range over the test: 19.6 – 21.3°C
pH:
pH range over the test: 7.44 – 7.79
Dissolved oxygen:
Dissolved oxygen % ASV range over the test: 81.5 – 95.7%
Salinity:
//
Conductivity:
//
Nominal and measured concentrations:
Nominal : 4.1, 9.1, 20.0, 45, 100% of the Saturated solution
Time-weighted mean measured concentrations were calculated to be 1.65, 3.24, 5.83, 9.65 and 24.0 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 118 mL glass bottles, completely filled and sealed with crimp cap lids.
- Type (delete if not applicable): closed
- Material: glass
- Vessel size: 100 mL (nominal)
- Headspace: no
- Fill volume: 118 ca mL
- Volume of solution: 118 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicale

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used in the study was Elendt M4 medium prepared by the lab shortly before the usage:
Separate stock solutions (I) of individual trace elements are first prepared in reverse-osmosis (RO) water. From these separate stock solutions (I), a single second stock solution (II) is prepared which contains all trace elements as a combined solution. M4 medium is prepared using stock solution (II), the macro-nutrients and vitamin.

- Intervals of water quality measurement: The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperatures were determined daily for the duration of the test. Continuous temperatures were measured using a digital (min/max) thermometer in an additional vessel maintained in the test area.
Water qualities were performed on media taken directly from the vessels housing the individual replicates.

OTHER TEST CONDITIONS
- Photoperiod: The test was conducted with a 16-hour light: 8-hour dark photoperiod.

VEHICLE CONTROL PERFORMED: Not relevant.

RANGE-FINDING STUDY
Based on the results of an initial solubility trial the range-finding test was conducted at nominal concentrations of 1.0, 10 and 100 mg/L, a control group was also included, media were prepared by direct addition and serial dilution prior to addition to test vessels. Two replicate test vessels (60 mL glass jars, completely filled and sealed with screw cap lids) were used for the control and each test substance concentration. The test was conducted for 48-hours. Media was prepared at 0-hours only. Analytical samples were taken to confirm concentrations of Methylic Adduct, samples were taken from preparation flasks at 0 hours and from pooled test vessels.
10% immobility was observed at 48h at the nominal concentration of 100 mg/L.
- Results used to determine the conditions for the definitive study: NO.
Chemical analysis showed that the measured concentrations during the range-finding test were not stable over the course of the 48-hour test period and concentrations were far from nominal, suggesting large losses through volatility during preparation as well as solubility issues. Therefore, media preparation trials were later conducted.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7.16 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits
Remarks:
(6.51 – 7.48)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
5.83 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Statistic
Remarks:
Fisher Exact/Bonferroni - Holm Test
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
9.65 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Statistic
Remarks:
Fisher Exact/Bonferroni - Holm Test
Details on results:
Considering the difficult nature of the substance, chemical analysis showed concentrations of Methylic Adduct remained relatively stable over the 48-hour test period, however, both increases and decreases in concentration were observed, likely caused by the volatile nature of the compound and due to dosing vessels individually.

At 24h 100% immbility was observed at the highest concentration of 100%SS (corresponding to 24 mg/L TWA).
At 48h, 100, 10, 10, 30% immobility was observed respectively at 45%SS, 20%SS, 9.1%SS, 4.1%SS (corresponding to 9.65, 5.83, 3.24, 1.65 mg/L TWA).
It could not be ascertained from the data what may have caused the immobility at the lowest concentration of 4.1%SS (1.65 mg/L), however, this was not considered to impact the integrity of the study as the NOEC was calculated to be 6.99 mg/L.
Results with reference substance (positive control):
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity, the latest reference test ws conducted on January 2019.
Test Substance: Potassium dichromate, Purity: 100%, expiry date: 31 Jan 2021
Test Medium: Elendt M4
Test Guideline: The study was designed in accordance with OECD Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004)
Objective: The objective of the study was to determine the 48-hour acute toxicity of the reference substance against the mobility of the freshwater planktonic crustacean, Daphnia magna.
Preparation method: An amount of reference substance (ca 5 mg) was dissolved in 500 mL of Elendt M4 Medium to give a 10 mg/L stock media, all other concentrations were then diluted from the stock.
Environmental Conditions: Temperature maintained between 18 and 22°C. pH between 6 and 9, oxygen was maintained > 3mg/L, 16:8 hours light:dark photoperiod.
Test Design: Static regime, with test media preparation at 0 hours only
Test Species: Daphnia magna (straus). Source of daphnia – MicroBioTest
Test Vessels: 100 mL glass beakers with petri dish lid.
Treatment Rates: Control, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
Observations: The Daphnia magna were observed after 24 and 48 hours exposure and the number immobilised in each vessel recorded
Study Duration: 48 hours
Test System: 5 < 24 hour old Daphnia magna per replicate
Statistical Analysis System: CETIS v1.8.6.8, CETIS – Comprehensive Environmetal Toxicity Information System. 2001-2012. Tidepool Scientific, LLC
Statistical analysis used:
24 and 48 hour EC50 = Linear Interpolation (ICPIN)
24 and 48 hour NOEC and LOEC = Derived empirically
Results:
EC50 24 hours: 1.6 mg/L; 95%Confidence Limits: (Lower: 1.3 mg/L ; Upper: 2.2 mg/L); NOEC: 1 mg/L; LOEC: 1.8 mg/L)
EC50 48 hours: 1.3 mg/L; 95%Confidence Limits: (Lower: 1.0 mg/L ; Upper: 1.4 mg/L); NOEC: 0.56 mg/L; LOEC: 1.0 mg/L)
Conclusion
The 48-hour EC50 value and NOEC were determined to be 1.3 and 0.56 mg/L, respectively. The EC50 result was within the expected range stated in the OECD 202 testing guideline (0.6 – 2.1 mg/L).
Reported statistics and error estimates:
STATISTICAL ANALYSIS
Statistical analysis was conducted on the data using the CETIS program v 1.8.6.8.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated using Fisher Exact/Bonferoni-Holm Test.
Linear interpolation analysis was performed in order to evaluate the EC50 values where appropriate.

Any other information on results incl. tables

DETAILS on CHEMICAL ANALYSIS RESULTS

The 0 hour analytical batch met all acceptance criteria, the recoveries that were re-run alongside the re-injected samples met acceptance criteria at 24 hours also, showing frozen stability. Therefore, storing the samples without a previous storage stability check was not considered to impact on the integrity of the study. Both biological and analytical samples were stored and re-run.

The 24-hour analytical batch mean recovery criteria were met (80 – 120% of nominal), C of V (coefficient of variation) = 20.8 % (<20% being the acceptability

criteria), this was considered to have been caused by chromatographic peak shape. Some variation (15.2%) was seen in method validation, which was considered to be

caused by method performance at differing concentration levels. Therefore, this small deviation is not considered to have any impact on the outcome or integrity of the

analytical batch and therefore the study.

The 48-hour analytical batch mean recovery criteria were not met (80 – 120% of nominal), value seen was 120.6%, however, this was considered to be caused by method performance at differing concentration levels. The C of V was 13% which passes acceptance criteria. Therefore, this small deviation is not considered to have any impact on the outcome or integrity of the analytical batch and therefore the study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on time-weighted mean measured concentrations, the 48-hour EC50 of Methylic Adduct was calculated to be 7.16 mg/L.
Executive summary:

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test (adopted April 13 2004) and OECD Chemicals Testing Guideline No.23 Guidance document on aquatic toxicity of difficult substances and mixtures and in compliance with GLP requirements.

 

Methylic Adduct is volatile and difficult to solubilise in water. Preliminary work in order to define the best procedure for solubilizing the substance avoiding losses by volatilization was conducted. Based on the results of the range-finding test and three media preparation trials the definitive test was conducted at nominal concentrations of 4.1, 9.1, 20, 45 and 100% saturated solution. A control group was also included. The test was conducted using a semi-static design with renewal of the test media performed at both 0 and 24 hours.

Because of the volatile nature of the test item, completely filled and sealed crimp cap test vessels with a nominal volume of 100 mL and a total volume of ca 118 mL were used.

For fresh test media samples, additional analytical vessels were prepared at each media preparation occasion in the same way as the test vessels (but without adding daphnia) and immediately sampled. This allowed to avoid sampling from the test vessels, reducing the losses by volatilization due to the increased headspace.

Old media was sampled from two test vessels, selected at random per control and test concentration, two vessels were sampled to allow to compare differences in concentration between replicates.

 

Considering the difficult nature of the substance, chemical analysis showed concentrations of Methylic Adduct remained relatively stable over the 48-hour test period, however, both increases and decreases in concentration were observed, this variability was considered to be caused by the volatile nature of the test substance and due to dosing vessels individually. Losses were likely to have happened during media preparation and analytical sampling. Therefore, results were based on time-weighted mean measured concentration as this best represents what the daphnia were exposed over the 48-hour test period. The time-weighted mean measured concentrations were calculated to be 1.65, 3.24, 5.83, 9.65 and 24.0 mg/L.

 

Based on time-weighted mean measured concentrations, the 48-hour EC50 was calculated to be 7.16 mg/L.

The corresponding NOEC value was determined to be 5.83 mg/L.

The validity criteria for control immobility (10%) and dissolved oxygen (3 mg/L) were both satisfied. The test is therefore considered valid.