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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2016 - July 2016
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 15-TV09366 - BIN 5/2 BIS C607
- Expiration date of the batch: 31 October 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: 31 October 2020 (expiry date)
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Duplicate samples for possible analysis were taken from all test concentrations and the control.
2.0 ml from the approximate centre of the test vessels were taken at the start of the test and after 24 hours from the freshly prepared solutions (Fresh solutions) and at the first renewal (t=24h) and at the end of the test from the 24 hours old solutions (Old solutions).
The old test solutions were not pooled before sampling. Instead, duplicate samples were taken from the same replicate for each of the test 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..

- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling.
Vehicle:
yes
Remarks:
water
Details on test solutions:
Preparation of test solutions:
Preparation of test solutions started with the highest concentration of 100 mg/L applying 11-12 minutes of magnetic stirring in a closed glass vessel. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera)
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history.
- Feeding during test: none
- 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.

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 culture vessel.
- Maximum age of the cultures: 4 weeks
- Feeding frequency / Type and amount of food: Daily, a suspension of fresh water algae.
- Medium: M7, as prescribed by Dr. Elendt-Schneider (see composition in OECD guideline 202)
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3 (Adjusted ISO medium hardeness)
Test temperature:
ranged from 20 to 21°C over the test
pH:
ranged from 7.6 to 8.2 over the test
Dissolved oxygen:
ranged from 9.0 to 9.9 mg/L over the test
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L
Measured concentrations (average): 0.0026, 0.0069, 0.078 and 1.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 ml, all-glass, closed with no headspace
- Type: closed
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution: Semi-static with renewal of test solutions after 24 hours.
- No. of organisms per vessel: 5 per vessel
- No. of vessels per concentration (replicates):4 the highest (limit) concentration (20 daphnids) and 2 for the remaining concentrations (10 daphnids)
- No. of vessels per control (replicates):4 for the control (20 daphnids)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium (M7 medium)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

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

COMBINED RANGE-FINDING / LIMIT TEST
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: No; the Combined limit/range-finding test was performed as a definitve test due to the low solubility of the test item (loading rate)
Reference substance (positive control):
no
Remarks:
potassium dichromate (K2Cr2O7); commun to other studies; performed to check the sensitivity of the test system
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: water solubility of the test item < 0.1 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: water solubility of the test item < 0.1 mg/L
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: water solubility of the test item < 0.1 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: water solubility of the test item < 0.1 mg/L
Details on results:
1) Analytical measurements:
Initially measured concentrations in the freshly prepared solutions, at the beginning of the test and after 24 hours, were observed to be very low when compared to nominal, i.e. ranging between 0.51 and 2.6%. The test item appeared to be completely soluble at the loading rate initially prepared and did not undergo any filtration process, by which absorption of the test item on the filter could have occurred and caused the observed low initial concentrations. However, the test item was expected to be volatile and the solubility was determined to be very low, i.e. <0.01 mg/L (Charles River pr.512259). It is reasonable to suppose that the test item was subject to volatilization during the preparation of the test concentrations.
Analysis of the samples taken at 24 and 48 hours showed stable measured concentrations when comparing concentrations in the old and in the freshly renewed solutions (e.g., analysis of the samples for the highest concentration showed that measured concentrations at 24 hour and 48 hours for the old solutions were equal to 95-115% of the initial measured concentrations of the freshly prepared solutions).
The results of analysis of the samples taken during the test are described in Table 1 in "Any other information on results including tables"

2) Immobility:
No immobilization or other effects were observed in the control or in any of the tested concentrations.

Table 1: Average exposure concentration versus nominal concentration

Nominal

concentration (mg/L)

Measured concentration (mg/L)

 

Average exosure

concentration

(mg/L)

 

t=0h

(fresh)

t=24h

(old)

t=24h

(old)

t=48h

(old)

 

0.10

0.0026

0.002

0.0024

0.0033

0.0026

1.0

0.0051

0.0046

0.0072

0.011

0.0069

10

0.075

0.068

0.0777

0.092

0.078

100

1.0

0.95

1.2

1.4

1.1

Table 2: Incidence of immobility in the test

Time (h)

Replicate

Average exposure concentration (mg/L)

 

 

control

0.0026

0.0069

0.078

1.1

0

A

5

5

5

5

5

 

B

5

5

5

5

5

 

C

5

 

 

 

5

 

D

5

 

 

 

5

24

A

0

0

0

0

0

 

B

0

0

0

0

0

 

C

0

 

 

 

0 (1)

 

D

0

 

 

 

0

48

A

0

0

0

0

0

 

B

0

0

0

0

0

 

C

0

 

 

 

0

 

D

0

 

 

 

0

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. This organism was

reimmersed into the respective solution before recording of mobility.

Table 3: Temperature, pH and oxygen concentrations during the test

average

exposure

concentration

(mg/L)

Start (t=0 h)

 

 

t=24h old

 

 

 

 

t=24h fresh

 

 

 

 

End (t=48 h)

 

 

 

 

pH

O2

pH

O2

T°C

pH

O2

T°C

pH

O2

control

7.6

9.1

7.6

9.3

20

7.7

9.3

20

8.2

9.9

1.1

7.6

9.0

7.7

9.2

20

7.7

9.3

21

8.1

9.9

Validity Criteria:

1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.

2. The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

Validity criteria fulfilled:
yes
Conclusions:

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as loading rate were as follows:
- 48h-EL50 > 100 mg/L
- 48h-NOELR ≥ 100 mg/L

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as the geometric mean of the measured concentrations were as follows:
- 48h-EC50 > 1.1 mg/L
- 48h-NOEC ≥ 1.1 mg/L
It should be noted that the measured concentration exceeded the test item solubility in water, which was determined to be <0.01 mg/L.
Executive summary:

The effect of the test item dichloro trifluoro methoxy dioxolane on the mobility of Daphnia magna was investigated in a 48hour semi-static test according to OECD Guideline 202 (2004), and the method C.2. of Commission Regulation (EC) No 440/2008. The study was compliant with the GLP principles.

 

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a nominal concentration of 100 mg/L in a limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L 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 exposure concentrations were taken at the start, after 24 hours in old and freshly renewed solutions and at the end of the test.

 

Analysis of the samples taken at 24 and 48 hours showed stable measured concentrations, both in the old and in the freshly renewed solutions (e.g., analysis of the samples for the highest concentration showed that measured concentrations at 24 hour and 48 hours for the old solutions were equal to 95-115% of the initial measured concentrations of the freshly prepared solutions). Nevertheless, initially measured concentrations in the freshly prepared solutions at the beginning of the test and after 24 hours were observed to be very low when compared to nominal, i.e. ranging between 0.51 and 2.6% of nominal. The test item appeared to be completely soluble at the loading rate initially prepared and did not undergo any filtration process, by which absorption of the test item on the filter could have occurred and caused the observed low initial concentrations. However, the test item was expected to be volatile and the solubility was determined to be very low, i.e. <0.01 mg/L. It is reasonable to suppose that the test item was subject to volatilization during the preparation of the test concentrations. Based on these results, the average exposure concentrations were calculated to be 0.0026, 0.0069, 0.078 and 1.1 mg/L.

The two validity criteria of the OECD guideline 202 were fulfilled, therefore this study is considered as valid.

Under the conditions of the present study, the 48h-EC50 was beyond the range tested, i.e. exceeded an average exposure concentration of 1.1 mg/L that was measured in a solution prepared at nominal 100 mg/L loading rate. Although, no NOEC value was reported in the study report and based on the results of the study, the 48-hour NOEC can be estimated to be the limit test concentration (48h-NOEC ≥ 1.1 mg/L and nominal 100 mg/L loading rate).

The reported biological results were related to the loading rate of 100 mg/L and to geometric mean of the measured concentrations of both 24h-periods.

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as loading rate were as follows:

- 48h-EL50 > 100 mg/L

- 48h-NOELR ≥ 100 mg/L

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as the geometric mean of the measured concentrations were as follows:

- 48h-EC50 > 1.1 mg/L

- 48h-NOEC ≥ 1.1 mg/L

It should be noted that the measured concentration exceeded the test item solubility in water, which was determined to be <0.01 mg/L. Therefore, it could be concluded that no toxicity was observed at the limit of solubility of the test item dichloro trifluoro methoxy dioxolane in the test medium.

Finally, according to the Guidance on the Application of the CLP Criteria, as the toxicity of the dichloro trifluoro methoxy dioxolane is above the limit of the solubility in the test medium and as the state-of-art was used to achieve the maximum dissolved concentrations,dichloro trifluoro methoxy dioxolane should not be classified in accordance with the criteria of the Regulation (EC) No 1272/2008.

Description of key information

One reliable study (klimisch 2) is available for the DICHLORO TRIFLUORO METHOXY DIOXOLANE for this endpoint. The effect of the test item DICHLORO TRIFLUORO METHOXY DIOXOLANE on the mobility of Daphnia magna was investigated in a 48‑hour semi-static test according to OECD Guideline 202 (2004), and the method C.2. of Commission Regulation (EC) No 440/2008. The study was compliant with the GLP.

Daphnids were exposed to nominal concentrations of 0.10, 1.0,10 and 100 mg/L in the combined range-finding/limit test.

The 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. Analytical measurement of exposure concentrations were performed at the start, after 24 hours in old and freshly renewed solutions and at the end of the test.

 

Analysis of the samples taken at 24 and 48 hours showed stable measured concentrations, both in the old and in the freshly renewed solutions. Nevertheless, initially measured concentrations in the freshly prepared solutions (T0 hour and T24 hours) were observed to be very low when compared to nominal, i.e. ranging between 0.51 and 2.6% of nominal. The test item appeared to be completely soluble at the loading rate initially prepared and did not undergo any filtration process, by which absorption of the test item on the filter could have occurred and caused the observed low initial concentrations. However, the test item was expected to be volatile and the solubility was determined to be very low, i.e. <0.01 mg/L. It is reasonable to suppose that the test item was subject to volatilization during the preparation of the test concentrations.

Current regulatory advice is that in cases where a decrease in measured concentrations is observed, geometric mean measured concentrations should be used for determining the ECx and NOEC value. Therefore based on the apporach, the average exposure concentrations were calculated to be 0.0026, 0.0069, 0.078 and 1.1 mg/L.

No immobilized test organisms were observed during the test period of 48 hours in the control and in all test solutions.

The 48-hour NOEC was therefore determined to be the limit test concentration.

 

The reported biological results were related to the loading rate of 100 mg/L and to geometric mean of the measured concentrations of both 24h-periods.

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as loading rate were as follows:

- 48h-EL50 > 100 mg/L

- 48h-NOELR ≥ 100 mg/L

The results obtained with the solutions prepared at nominal range from 0.1 to 100 mg/L and expressed as the geometric mean of the measured concentrations were as follows:

- 48h-EC50 > 1.1 mg/L

- 48h-NOEC ≥ 1.1 mg/L

 It should be noted that the measured concentration exceeded the test item solubility in water, which was determined to be <0.01 mg/L. Therefore, it could be concluded that no toxicity was observed at the limit of solubility of the test item dichloro trifluoro methoxy dioxolane in the test medium.

Finally, according to the Guidance on the Application of the CLP Criteria, as the toxicity of thedichloro trifluoro methoxy dioxolane is above the limit of the solubility in the test medium and as the state of art was used to achieve the maximum dissolved concentrations,dichloro trifluoro methoxy dioxolane should not be classified in accordance with the Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Additional information

Rational for classification

According to the Guidance on the Application of the CLP Criteria (June 2015; § I.4.2), as no acute toxicity was recorded at levels in excess of the limit of the solubility in the test medium and as the best technique was used to achieve the maximum dissolved concentrations, it is not deemed dichloro trifluoro methoxy dioxolane to be classified in accordance with the Regulation (EC) No 1272/2008 for environmental effects.