Ethane, 1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoro-

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:

September 2016 - January 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Accurate measured concentrations could not be determined due to variability in analytical results. The source of such variability was not clarified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: • Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethane

EC Number:

618-014-6

Cas Number:

874288-98-9

Molecular formula:

C4F8O2Cl2

IUPAC Name:

1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Manufacuring plant
- Lot/batch No.of test material: 15-TV10790
- Expiry date: 30 November 2020
- Purity : 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule:
Frequency:
1. At the start of the test and after 24 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
Volume:
2.0 ml from the approximate centre of the test vessels
Storage
Samples were stored in a freezer (≤ 15°C) until analysis

Samples were taken from the remainder of the bulk solution immediately after filling the replicates, but before addition of daphnids.
For sampling of the old solutions the replicates were not pooled before sampling.
Sample s were taken from one replicate per concentration for all tested concentrations. Due to the volatile nature of the test item, all samples were transferred to a vessel containing 100 µL of Methanol immediately after sampling to prevent any loss of test item from the sample before analysis. During method development and validation it was proven that samples are stable in the freezer applying this procedure.
Additionally, single reserve samples of 2.0 ml were taken for possible analysis using the same procedure. If not used, these samples were stored in a freezer (≤ 15°C) for a maximum of three months after delivery of the draft report.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A Saturated Solution (SS) was prepared with a loading rate of 100 mg/L applying an overnight period of magnetic slow stirring (~ 100 rpm) in a closed vessel to ensure maximum dissolution of the test item in test medium. The resulting aqueous mixture was clear and colourless with undissolved test item visible at the bottom of the flask. The mixture was left to stabilize for approximately 2.75 hours after which the clear soluble part was collected. This procedure was repeated for the renewal on day 1 of the study.
The entire preparation and equilibration procedure of the Saturated Solution occurred in a completely closed vessel with very limited headspace in order to avoid losses by volatilization of the test item from the aqueous media.
The obtained Saturated Solution (SS) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. One dilution was prepared for each concentration in a measuring flask and the content was divided over the replicate test vessels immediately thereafter. Subsequently, samples were taken and daphnids were added directly thereafter. All final test solutions were clear and colourless and were renewed after 24 hours.

The tested batch of MOVE3 ADDUCT had a purity of 98.0%. No correction was made for the purity/composition of the test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test young daphnids with an age of < 24 hours were selected from parental daphnids older than two weeks.

Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C). Additionally, temperature was also measured at 24 hours in the old and freshly prepared test solutions, for the control and the highest test concentration showing temperature 20 or 21°C.

pH:

Limits prescribed by the study plan : pH: 6.0-8.5, not varying by more than 1.5 unit

Measured value:
Control > pH range: 7.9 - 8.1. mean = 8, SD = 0.08
Saturated Solution > pH range: 7.8 - 8.0. mean = 7.9, SD = 0.08

Dissolved oxygen:

Limits prescribed by the study plan: dissolved oxygen > 3 mg/L at the end of the test

Measured value:
Control > range: 8.4 - 9.2 MgO2/L
Saturated Solution > range: 8.2 - 8.7 MgO2/L

Salinity:

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Conductivity:

n.a.

Nominal and measured concentrations:

A saturated solution (SS) was prepared with a 100 mg/L loading rate.
Lower test concentrations were prepared by subsequent dilutions of the 100% Saturated Solution in test medium, resulting in 1 % SS, 10% SS.
Accurate measured concentrations could not be determined due to variability in analytical results.

Details on test conditions:

Test system: COMBINED LIMIT / RANGE-FINDING TEST
Test duration : 48 hours
Test type: Semi-static with renewal of test solutions after 24 hours
Test vessels : 60 ml, all-glass, air-tight closed with no headspace
Medium: Adjusted ISO medium
Number of daphnids : 20 each for the control and the highest (limit) test concentration, 10 each for the remaining concentrations.
Loading: 5 daphnids per vessel containing 60 ml of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnids : Within 26 minutes after preparation of the test solutions

EFFECT PARAMETERS MEASURED :
Immobility (including mortality):
At 24 hours and at 48 hours.

pH and dissolved oxygen:
At the beginning, after 24 hours of exposure and at the end of the test from the old and the freshly prepared solutions, for the highest concentration and the control.

Temperature of medium:
Continuously in a temperature control vessel, beginning at the start of the test. Additionally, temperature was also measured at 24 hours in the old and freshly prepared test solutions, for the control and the highest concentration.

Reference substance (positive control):

not required

Remarks:

Reference tests with Potassium DIchromate are periodically conducted at test facilities in order to check the sensitivity of the test system. The result of the more recent reference test (July 2016) are reported.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: > 100% Saturated Solution

Remarks:

No effects observed at the limit of solubility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: = 100% saturated solution

Remarks:

No effects observed at limit of solubility

Details on results:

No biological relevant immobility was observed during the test. No immobility was observed at any test concentration ( 0/10 immobilized daphnids at 1% SS; 0/10 immobilized daphnids at 10% SS; 0/20 immobilized daphnids at 100% SS).
10% effect (2/20 immobilized daphnids) was observed in the control. It should be noted that the guideline allows for 10% effect in the control treatment. Hence, effects below or equal to 10% are not considered significant.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Relevant effect levels: Immobilization

Daphnids were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The 48h-EC50 was 0.39 mg/L with a 95% confidence interval ranging from 0.33 to 0.44 mg/L.
The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses.
The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L.

Any other information on results incl. tables

DETAILS ON MEASURED CONCENTRATIONS.

The results of analysis of the samples taken during the combined limit/range-finding test are described in the following table:

Time of sampling [days]	Date of sampling	Date of analysis	Percentage of SS.  [%]	Analysed concentration [mg/L]
0	27-Sep-2016	06-Oct-2016	0	< lowest calibration solution (i.e. < 0.0025 mg/L)
0	27-Sep-2016	06-Oct-2016	100	0.805
0	27-Sep-2016	13 -Oct-2016 (reserve sample)	100	0.817
1 (old)	28-Sep-2016	06-Oct-2016	0	< lowest calibration solution (i.e. < 0.0025 mg/L)
1 (old)	28-Sep-2016	06-Oct-2016	100	0.413
1 (old)	28-Sep-2016	13-Oct-2016 (reserve sample)	100	0.0437
1 (fresh)	28-Sep-2016	06-Oct-2016	0	< lowest calibration solution (i.e. < 0.0025 mg/L)
1 (fresh)	28-Sep-2016	06-Oct-2016	100	0.137
1 (fresh)	28-Sep-2016	13-Oct-2016 (reserve sample)	100	0.0243
2 (old)	29-Sep-2016	06-Oct-2016	0	< lowest calibration solution (i.e. < 0.0025 mg/L)
2 (old)	29-Sep-2016	06-Oct-2016	100	0.377
2 (old)	29-Sep-2016	13-Oct-2016 (reserve sample)	100	0.311

Samples taken from the highest concentration were analysed. Analysis of the sample taken at the start of the test showed a measured concentration of 0.81 mg/L, being at (or very close to) the limit of water solubility of the test item (i.e. 0.84 mg/L).

The measured concentration measured after 24 hours had decreased to 0.41 mg/L (51% of initial), probably due to the high volatility of the test item. Unexpected results were obtained in the 2^ndrenewal period (t=24-48 h). The measured concentration in the freshly prepared solution at 24 hours of exposure was 0.14 mg/L and, at the end of the test, it was 0.38 mg/L (276% of initial).

As a result, it was decided to analyse the reserve samples taken from the highest test item concentration. Analysis of the reserve sample taken at the start of the test showed a measured concentration of 0.82 mg/L, also in this case at (or very close to) the limit of water solubility of the test item. At the end of the 1^strenewal period, the measured concentration had decreased to 0.044 mg/L (5.4% of initial). Unexpectedly, the concentration measured in the freshly prepared solution at 24 hours of exposure was only 0.024 mg/L. At the end of the test, the measured concentration was 0.31 mg/L (1280% of initial).

It was unclear why measured concentrations were so variable. Possible reasons could be losses by volatilization during sampling or handling (e.g. for the observed concentration in the freshly prepared solution on the 2^ndrenewal period lower than the concentration in the corresponding old solutions), losses by volatilization during storage due to sample vials that might not have been completely sealed (e.g. for the two outlying low results in the reserve samples), solutions not fully homogeneous which could result in differences when taking small volume samples.

It was concluded that based on the obtained analytical results it was not possible to accurately determine the actual exposure concentration during the test period.

 

However, considering the procedure used for the preparation of the saturated solution (i.e. a loading rate (100 mg/L) much higher than the solubility value (0.84 mg/L) of the test item, closed system, prolonged stirring period (~16 hours), ca. 3h equilibration period in a closed system) it is considered that the concentrations achieved in the initial solution were at or close to the saturation in test medium.

Part of the analyses results in the fresh saturated solutions show concentrations close to the water solubility value.

In the old saturated solutions, most of the analytical results show concentrations in the range of 0.31 – 0.41 mg/L.

Assuming that the unexpected lower concentrations observed in the fresh and old solutions are outlier values due to an unknown source of variability or error, and discharging the supposed outlier values from the analytical results, figures would be on line with a hypothetical pattern of actual concentrations of ca. 0.8 mg/L in the fresh solution and a decrease in concentration of ca. 50 - 60% over the 24h renewal period. This pattern would be consistent with a maximum concentration in the saturated solutions close to the water solubility value and with a decrease due to volatilization during the 24h exposure period as expected based on the properties of this substance.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the 48h-EC50 exceeded the concentration present in the saturated solution (SS) prepared at a loading rate of 100 mg/L.

Executive summary:

An Acute Toxicity Study in Daphnia magna (semi-static exposure) was conducted in order to assess the effect of 1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethane on the mobility of Daphnia magna during an exposure period of 48 hours.

1,2-dichloro-1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethane is a colourless liquid, poorly soluble and highly volatile. Because of its chemical and physical properties the test substance falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures).

The study was conducted under GLP, and it was based on the OECD guideline No. 202, 2004, EU Method C.2, 2008, the ISO International Standard 6341, 1996 and the OECD series on testing and assessment number 23, 2000.

In order to ensure the maximum dissolution of the test item in aqueous medium, a Saturated Solution (SS) was prepared with a loading rate of 100 mg/L applying magnetic stirring for an overnight period. The resulting aqueous mixture was left to equilibrate for approximately 2.75 hours. The supernatant was removed to give the 100% Saturated Solution (SS). The SS was clear and colourless. In order to avoid volatilization of the test item from the aqueous media all the preparation procedure of the SS, including the equilibration period, occurred in a completely closed vessel with very limited headspace. The obtained Saturated Solution (SS) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 100% of the SS in a limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed to 1.0 and 10% of the SS in the combined range-finding test. The total exposure period was 48 hours and test solutions were renewed after 24 hours. The test was performed in closed vessels without headspace due to the expected volatility of the test item. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours in old and freshly renewed solutions and at the end of the test.

No effects on mobility of Daphnia magnawere observed during the test period in any of the concentrations tested including the highest.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

An accurate measured concentration over the test period could not be calculated due to variable analytical results.

Anyway, basing on the procedure used for the preparation of the saturated solution, it is considered that the concentrations achieved in the saturated solutions was at or close to the saturation in the test media.

Under the conditions of the present study, the 48h-EC50 exceeded the concentration present in the saturated solution (SS) prepared at a loading rate of 100 mg/L.