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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:
Experimental schedule: 11/03/2008 - 11/07/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to the OECD Testing Guideline and GLP compliant. All validity criteria are fulfilled. (and no deviation)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SAMPLING:
Duplicate samples were taken from the limit test solution at the start and end of each test medium renewal period. From the control, also the duplicate samples were analyzed from each of the sampling times.

Test item concentration:
The concentration of the test item 1,6-Divinylperfluorohexane was analytically measured in the duplicate test medium samples of the single test concentration taken at the start and the end of the test.

All the samples were analyzed immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION OF THE LIMIT TEST SOLUTIONS:
The test medium with the loading rate of 100 mg/L was prepared prior to each test medium renewal as follows: 162.4 uL of the test item (corresponding to 234 mg due to the test item density of 1.506 g/mL) were dissolved as far as possible in 2340 mL of test water using intense stirring for 3 hours at room temperature in the dark in a completely filled and tightly closed stirring vessel. Following the stirring period of 3 hours, the test medium was
incubated in a separating funnel for an equilibration period of 15 minutes. During this period, different phases (a layer of test water with a maximum amount of dissolved test item and a layer of undissolved test item) were observable and were separately let out through the stopcock of the separating funnel. The test medium was prepared just before introduction of the daphnids (i.e., start of the test and prior to each test medium renewal).
The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnid
- Strain: Daphnia magna Straus
- Source: University of Sheffield / UK in 1992
- Age at study initiation: At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Feeding during test : The daphnids were not fed during the test

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see study design).

REFERENCE TEST:
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year.
The result of the latest positive control test in March 2008 (48-hour EC50: 0.73 mg/L, RCC Study No. B83744) indicated that the sensitivity of the test organisms was within the historical range of the RCC laboratory (48-hour EC50 from 1996 to 2008: 0.53-1.1 mg/L).
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
250 mg/L as CaCO3
Test temperature:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature during the test was 20 °C.
pH:
The pH values of the Control ranged from 7.7 to 7.8
The pH values of the Limit test solution ranged from 7.7 to 7.8
Dissolved oxygen:
The dissolved axygen concentration of the Control ranged from 8.4 to 8.6 mg/L O2
The dissolved axygen concentration of the Limit test solution ranged from 8.4 to 8.6 mg/L O2
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentration of the Limit test solution : loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM / STUDY DESIGN
- Test vessel: 50-mL glass tubes completely filled with about 50 mL of test medium
- Type: closed
- Renewal rate of test solution (frequency): each 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates):4


For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnia was 10 mL. Thus, the requirement of the test guidelines for the minimum volume of 2 mL test medium per daphnia was fulfilled. The daphnids were randomly distributed to the test vessels at initiation of the test.

Since the test item was determined to be volatile (according to the results of a pre-experiment,non-GLP), the test was performed in 50-mL glass tubes completely filled with about 50 mL oftest medium. The test tubes were made tight with glass stoppers to avoid losses of the volatile substance. The test tubes were labeled with the study number and all necessary additional information to ensure unmistakable identification.
A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium of the corresponding concentration.
The test was performed as far as possible in the dark to avoid a photolytic degradation of the test item.The test was performed in a temperature-controlled room with continuous monitoring of theroom temperature. The water temperature was maintained at 20°C.

TEST MEDIUM / WATER PARAMETERS:
Reconstituted test water according to ISO 6341 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:

Ingredients
/ Properties Concentration
mmol/L mg/L
CaCl2 x 2H20 2.0 294
MgS04 x 7H20 0.5 123
NaHC03 0.75 65
KC1 0.075 5.8
Water Hardnes 2.5 250 as CaC03
Alkalinity 0.8 —

The ratio of Ca.Mg and Na:K was 4:1 and 10:1, respectively, based on molarity
The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.

- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and end of each test medium renewal period, the pH values, dissolved oxygen concentrations, water temperature and were determined at the single test concentration and in the control.
- The appearance of the test medium was visually determined and recorded at the same time.

EFFECT PARAMETERS MEASURED :
The daphnids were observed for immobility after 24 and 48 hours of exposure (daphnids not being able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobilized).
The NOEC and ECo were determined directly from the raw data. The EC50 and the EC 100 could not be calculated due to the absence of a toxic effect of the test item in this test.

TEST CONCENTRATIONS: Limit test
- Test concentration: loading rate of 100 mg/L and control
- Results used to determine the conditions for the definitive study: The limit test was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water (non-GLP).

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on Daphnia magna at a loading rate of 100 mg/L. The limit test was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water (non-GLP).

The analytically determined test item concentration in the analyzed test medium was between 0.48 and 1.1 mg/L at the start and between 0.2 and 0.21 mg/L at the end of the test medium renewal periods (see analytical results and Table 2 in the attached analytical part report). The reported biological results are related to the loading rate of 100 mg/L and to the arithmetic mean of the geometric means of both 24h-periods (0.40 mg/L).

In the control and at the limit test solution (loading rate of 100 mg/L) , no immobilized test organisms were observed during the test period of 48 hours.
Therefore, the 48-hour NOEC and the 48-hour ECo of 1,6-Divinylperfluorohexane to Daphnia magna were determined to be at least 100 mg/L (mean measured concentration: 0.40 mg/L). The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L (mean measured concentration: 0.40 mg/L). These values could not be quantified due to the absence of toxicity of 1,6-Divinylperfluorohexane up to the water solubility limit of the
test item.

No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration

At the beginning and end of the test medium renewal periods, the pH values of the test medium and control were between 7.7 and 7.8. The dissolved oxygen concentration in the test medium and control was at least 8.4 mg/L . The water temperature during the test was 20 °C
Results with reference substance (positive control):
Results with reference substance valid? : yes
The result of the latest positive control test in March 2008 was : 48-hour EC50: 0.73 mg/L.

Table 1      Effect of 1,6-Divinylperfluorohexane on the Mobility of Daphnia magna

Loading rate (mg/L)

No. of

daphnids

tested

Immobilized

daphnids after

24 hours

Immobilized

daphnids after

48 hours

 

 

No.

%

No.

%

Control

20

0

0

0

0

limit test solution: 100 (mean measured

concentration: 0.40)

20

0

0

0

0

Table 2     Results Obtained for the Concentrations of the Treatment Samples

Age (day/h)

Loading rate of 100 mg test item/L

Cnom

Measured concentration of test item

Sample preparation

factor F

Determined average concentration of test item

(mg/L)

Sample 1

(mg/L)

Sample 2

 (mg/L)

Average |(mg/L)

0/0

Control undiluted

<LOQana

1.01

<LOQana

1.17

n.a. 1.09

1.01

1.01

n.a.

1.10

1/24

Control undiluted

<LOQana

0.261

<LOQana

0.161

n.a. 0.211

1.01

1.01

n.a.

0.213

1/0

Control

undiluted

<LOQana

0.361

<LOQana

0.590

n.a. 0.476

1.01

1.01

n.a.

0.480

2/48

Control undiluted

<LOQana

0.246

<LOQana

0.158

n.a. 0.202

1.01

1.01

n.a.

0.204

LOQana = 0.134 mg test item/L

n.a. = not applicable

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:
yes
Remarks:
according to the criteria of the OECD TG 202
Conclusions:
The results obtained with loading rates were as follows:
48h-EL50 < 100 mg/L
48h-NOELR ≥ 100 mg/L

The results obtained with the geometric mean of the measured concentrations were as follows:
48h-EC50 < 0.40 mg/L
48h-NOEC ≥ 0.40 mg/L
Executive summary:

The effect of the test item 1,6-Divinylperfluorohexane on the mobility of species Daphnia magna was investigated in a 48‑hour semi-static limit 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.

Pre-experiments (non-GLP) were performed in order to determine the volatility, the solubility in test medium and the optimum application method. The test item 1,6-Divinylperfluorohexane was determined to be volatile and poorly soluble in the test medium.

Since the test item is volatile, the test was performed in closed system i.e.glass tubes were completely filled with test medium.

Since the test item is poorly soluble, the limit test was performed using the loading rate method. The limit test solution with a loading rate of 100 mg/L was prepared in the test medium, stirred for 3 hours in the dark to dissolve a maximum amount of the test item then incubated in a separating funnel. The layer of test water with maximum dissolved test item was used as limit test concentration.

 

Based on results of a pre-experiments and on the results of a preliminary range-finding test, Daphnia magna were exposed to the loading rate of 100 mg/L (limit test concentration) and a control for 48 hours, under semi-static conditions i.e. the test medium was renewed after 24 hours.

The 20 daphnids per test solution, divided into four replicates of 5 daphnids each, were observed for immobility after 24 and 48 hours of exposure.

Analysis of the limit test solution at 0 hours and 24 hours (start of the renewal periods) showed measured test concentrations of 1.1 and 0.48 mg/L for the loading rate of 100 mg/L respectively.

Analysis of the limit test solution at 24 hours and 48 hours (end of the renewal periods) showed a decrease of test item concentration in the test media since the test item concentrations were 0.2 and 0.21 mg/L for the loading rate of 100 mg/L respectively.

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 the reported biological results were related to the loading rate of 100 mg/L and to the arithmetic mean of the geometric means of both 24h-periods (0.40 mg/L) and to the loading rate of 100 mg/L.

No immobilized test organisms were observed during the test period of 48 hours in the control and at limit test solution (loading rate of 100 mg/L).The limit test concentration of 100 mg/L (loading rate of 100 mg/L and mean measured concentration: 0. 40 mg/L) was, therefore, determined to be the 48-hour NOEC.

 

The results obtained with loading rates were as follows:

48h-EL50 > 100 mg/L

48h-NOELR ≥ 100 mg/L

The results obtained with the geometric mean of the measured concentrations were as follows:

48h-EC50 > 0.40 mg/L

48h-NOEC ≥ 0.40 mg/L

 

It could be concluded that no toxicity was observed at the limit of solubility in the test medium. Limit of solubility in this test medium is 1.1 mg/L (result of the T0 analytical measurement of the stock solution of a loading rate of 100 mg/L).

 

The two validity criteria of the OECD guideline 202 were fulfilled, therefore this study is considered as reliable without restrictions.

 

According to the Guidance on the Application of the CLP Criteria, as the toxicity of the1,6-Divinylperfluorohexane is above the limit of the solubility in the test medium and as the best technic was used to achieve the maximum dissolved concentrations, 1,6-Divinylperfluorohexane should not be classified in accordance with the Regulation (EC) No 1272/2008

Description of key information

According to the results of the study compliant with the GLP and with the OECD TG 202, the 1,6-Divinylperfluorohexane is considered as not toxic for the aquatic organisms at its limit of solubility.

Key value for chemical safety assessment

Additional information

One reliable key study is available for 1,6-Divinylperfluorohexane for this endpoint.

In this acute toxicity study (S. Hoger, 2010), the acute immobilization (EC50 and NOEC) of the test item 1,6-Divinylperfluorohexane to daphnia magna (STRAUS) was determined according to the method C.2 of the Commission Regulation (EC) No 440/2008 and the OECD Guideline 202. The study was compliant with the GLP.The study was conducted under semi-static conditions over a duration of 48 hours.

 

Following pre-experiments (non GLP), the test item 1,6-Divinylperfluorohexane was determined to be volatile and poorly soluble in the test medium, these pre-experiments were also performed to determine the optimum application method.

Therefore, based on results of a pre-experiments and a preliminary range-finding test, a limit test was performed in closed system (i.e. glass tubes completely filled with test medium) due to its volatility and the limit test solution was prepared from a solution with a loading rate of 100 mg/L due to its poor solubility.

20 test organisms, divided into four replicates of 5 daphnids each, were exposed to the limit test concentration (loading rate of 100 mg/L) and control and were observed for immobility after 24 and 48 hours of exposure. The test solutions were renewed after 24 hours.

 

The measured concentrations of the limit test solution (loading rate of 100 mg/L) were 1.1 and 0.48 mg/L at 0 and 24 hours (i.e. start of the renewal periods) and 0.2 and 0.21 mg/L at 24 and 48 hours (end of the renewal periods) respectively.

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

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 the arithmetic mean of the geometric means of both 24h-periods (0.40 mg/L).

 

The results obtained with loading rates were as follows:

- 48h-EL50 > 100 mg/L

- 48h-NOELR100 mg/L

The results obtained with the geometric mean of the measured concentrations were as follows:

- 48h-EC50 > 0.40 mg/L

- 48h-NOEC ≥ 0.40 mg/L

 

It could be concluded that no toxicity was observed at the limit of solubility of the test item 1,6-Divinylperfluorohexane in the test medium.

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