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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:
22 January - 25 January 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guildeline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Protocols according to the ecotoxicological testing of petroleum products by CONCAWE 1992 and for aquatic toxicity testing of lubricants by ASTM (standard protocol 06081-97). These comply with the general principles of OECD guidelines.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Trade Name: Zonyl® PFBE

Chemical Name: 3;3,4,4,5,5,6,6,6-nonafluorohexene

CAS Number: 19430-93-4

Lot Number: 335

Purity: 96%

Water solubility: negligible

Vapour pressure: 0.2 bar (20 °C)

Chemical stability (water/light): stable

Specific density (20°C): 1.418 kg/L

Physical state and appearance: colourless, liquid

Boiling point: 58°C

Safety data sheet: yes

Waste disposal: aqueous organic waste

Storage conditions: 4°C in closed containers
Prevent spills and avoid open flames and keep away from heated surfaces above 200° C.

Expiry date: 2 years

Further information: none

Test material preparation and addition: WAF preparation

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test substance in the WAFs was analysed at the beginning and at the end of the exposure (48 h) of the Daphnia magna.

100 ml samples of each WAF were taken for the analytical measurements. At the end of the toxicity test periods, the WAF test media were filtered through a plastic sieve to remove any particles. The sieve was rinsed with 10 ml of the corresponding WAF before filtration.

Test solutions

Details on test solutions:
The WAF preparation was performed as described in the CONCAWE test protocol and in the ASTM standard 06081-97 (3) with the modifications given below. For each test concentration, a WAF was prepared.

The water-accommodated fractions (WAFs) were prepared by mixing the test substance with the dilution water at loading rates of 100 mg/L and 1000 mg/L in clean cylindrical mixing vessels. In contrast to the CONCAWE test protocol and to the ASTM standard 06081-97, the mixing vessels were not equipped with a drain port near the bottom for drawing off the WAF as the density of the test substance was above 1 gl/ml. So the aqueous WAF phase was drawn off through the upper orifice after WAF preparation.
The vessels were filled to a maximum without headspace. The containers were sealed with teflon covered screw caps. The vessels were tightly sealed to prevent loss of volatiles. The vessels were protected from light with tinfoil to prevent photochemical degradation of dissolved components.

A magnetic stirring bar was placed in each vessel. The test substance was added to the bottom of the vessels being careful not to contaminate the orifice and the side walls. Then the appropriate volume of water was added. Mixing was initiated with the vortex in the center, extending maximally 1/3 rd (10 - 35 % of vessel depth) from the top to the bottom of the vessel. It was as low as possible to maintain mixing of the water phase. Observations of the vortex-depth and mixture appearance were documented.
A mixing period of 24 hours was found to be sufficient for equilibration.

Following mixing, the contents of the vessels were allowed to stand undisturbed for 1 hour to allow separation of the aqueous and undissolved phases. The aqueous phase (the WAF) was then taken out of the orifice and filled into the test vessels for toxicity testing. The first portion of the WAF was used to rinse the vessels in order to saturate the surfaces. After filling, the vessels were sealed immediately and only opened again to introduce the test organisms and again at the end of the test.
Care was taken to ensure that any undissolved material was not transferred to the test vessels. The WAFs were not stored for more than 1 - 2 hours prior to testing. Storage was always in completely full sealed vessels, in the dark at 4°C if stored for periods longer than 2 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test organism

Specification: Daphnia magna STRAUS Cion 5, Crustacea, Cladocera.
Age: 4 - 24 hours old.
Origin: Daphnia magna (clone 5), Umweltbundesamt, lnstitut fur wasser-, Boden- und Lufthygiene, bred in the laboratory
Breeding/holding: Adult daphnids, at least 3 weeks old, were separated from the stock population by sieving and kept in groups of 30
organisms at room temperature in approximately 1800 ml of dilution water. The water was renewed at least once a week.
The daphnids were fed daily with an algal suspension (Scenedesmus subspicatus) and a suspension of solubilised algae (liquizeIIR; HOBBY, Germany). Newborn daphnids were separated by sieving, the first generation was discarded. Only healthy organisms free from disease were used in the study.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
enhanced limit test

Test conditions

Test temperature:
20 ± 1 °C
pH:
8.99 - 8.64
Dissolved oxygen:
8.83 - 10.9 mg/l (98.1 - 121.1% saturation) at start of test
8.4 - 8.8 mg/l (93.3 - 97.4 % saturation) at end of test
Details on test conditions:
Performance of the Daphnia magna toxicity test

Test Vessels
Test vessels were 200 ml conical glass flasks with ground-in stoppers. The neck of the flasks were covered with tinfoil to exclude light from this area of the flask and to decrease the likelihood of the daphnids becoming trapped in a surface film of the test medium. Prior the test, the vessels were rinsed with the appropriate WAF solutions in order to saturate the surfaces.

Test procedure
The test organisms were exposed for 48 h to graded loading rates of the test substance and a control under static conditions without aeration. The daphnids were not fed during the test. Ten daphnids, less than 24 hours old, were added into every 200 mL flask which was completely filled with the test solution. The flasks were sealed by avoiding air bubbles. Each loading rate was tested in duplicate.
The pH value (pH-Meter, WTW 535) and the oxygen concentration (WTW Digitai­ Sauerstoff-MeBgerat Oxi Digi 550) were measured directly in an additional aliquot of the respective WAF after filling of the vessels. At the end of the test, a small aliquot of the test solution was gently decanted into a beaker to measure pH-values and oxygen concentration.
These parameters were not measured in the WAF samples for the immobilisation test due to possible absorption of the surface film on the electrodes. Sufficient oxygen supply for the daphnids was ensured by preparing the WAFs with aerated water.
The temperature (Digitalthermometer, Roth) was measured in the controls (without gas oil, treated like the samples) before adding the daphnids and at the end of the test.

The sealed flasks were subjected to a light/dark cycle of 16/8 hours. The test temperature during the test was 20 ± 1°(, the light intensity did not exceed 1000 lux.
The controls were held under the same conditions in dilution water.
The light intensity in the incubation chamber was documented at the start and at the end of the test (Illuminance-Meter, Minolta) .

Following loading rates were tested:
control (3 replicates)
1 00 mg/l (3 replicates)
1 000 mg/l (3 replicates)

The performance of the enhanced limit test was chosen according to CONCAWE 1992, because-a pre-test finding indicated that a WAF prepared with a loading of 1000 mg/l was not toxic.

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
See any other information on results incl. tables.



Any other information on results incl. tables

Stability of WAFs: Results of chemical analysis

The results of chemical analysis are given in Table 1. In the WAFs prior to test start, the measured concentrations of PFBE were 0.27 mg/L and 1 mg/L for loading rates of 100 mg/L and 1000 mg/L respectively. During the test period of 48 h, PFBE concentrations decreased to 70% of the initial concentration in the WAFs loaded with 100 mg/L and to 80% in WAFs loaded with 1000 mg/L. This concentration decline may be attributed to the high volatility of the test substance resulting in evaporation while preparing the test (e.g. transfer from mixing vessels into the test vessels, introducing the daphnids) and volatilization through the orifice, though ground-in stoppers were used. For generating concentration effect relationships, the measured concentrations at test start were used.

Table 1. Concentration of test substance in selected samples.

Sampling time

Loading mg/l

Replicate No.

Conc. Test substance

mean

% of start conc.

1

2

3

4

Day 0

control

 

<0.05

<0.05

 

 

<0.05

 

 

100

1

0.28

0.26

 

0.28

0.27

100

 

1000

1

-

0.83

1.07

1.10

1.00

100

 

 

 

 

 

 

 

 

 

Day 2

control

 

<0.05

<0.05

 

 

<0.05

 

 

100

1

0.14

0.20

0.21

 

0.18

66.7

 

100

2

0.21

0.25

0.23

 

0.23

85.2

 

100

3

0.15

0.17

0.16

 

016

59.3

 

 

mean

 

 

 

 

0.19

70.4

 

 

±sd

 

 

 

 

0.036

13.3

 

 

 

 

 

 

 

 

 

Day 2

1000

 

0.85

0.93

0.91

 

0.90

90.0

 

1000

 

0.70

0.79

0.78

 

0.76

76.0

 

1000

 

0.67

0.85

0.74

 

0.75

75.0

 

 

mean

 

 

 

 

0.80

80.3

 

 

±sd

 

 

 

 

0.084

8.4

Results of Daphnia magna toxicity test

Table 2 shows the cumulative immobile daphnids during the test period. The loading rates of 100 mg/l and 1000 mg/l had no significant effect compared to the controls. According to the OECD Test Guideline 202, an immobilisation rate of 10 % at the end of the test is accepted at the control level. In no replicates had immobilisation of >10% been observed. For correlating observed effects to analytical concentrations, the concentrations at the start of the test (day 0) were used. Because no immobilisation was observed, the effect values are as follows: NOEC, LOEC and EC50 > 1000 mg/l (loading rate) NOEC, LOEC and EC50 >1.0 mg/l (measured concentration)

Table 2. Acute immobilisation: cumulative mortality (number of immobile daphnids per vessel, 10 daphnids per vessel = 30 daphnids per loading) n.a. not analysed.

Time

Loading rate mg/l

Vessel

Measured conc. Mean mg/l

Immobile daphnids

∑immobile

% immobilie

24 hr

control

1

 

0

 

 

 

 

2

 

1

 

 

 

 

3

 

0

1

3.3

 

100

1

n.a

0

 

 

 

 

2

n.a

0

 

 

 

 

3

n.a

0

0

0

 

1000

1

n.a

0

 

 

 

 

2

n.a

0

 

 

 

 

3

n.a

0

0

0

48 hr

control

1

 

0

 

 

 

 

2

 

1

 

 

 

 

3

 

0

1

3.3

 

100

1

0.18

1

 

 

 

 

2

0.23

0

 

 

 

 

3

0.16

0

1

3.3

 

1000

1

0.9

1

 

 

 

 

2

0.76

1

 

 

 

 

3

0.75

0

2

6.6

Validity of the test

The conditions of the OECD guideline 202 for the validity of the tests are met:

(1) There was no mortality>10% of the daphnids in the controls.

(2)The dissolved oxygen value did never fall below 60% of the air saturation value at the temperature used.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see any other information on results incl. tables
Conclusions:
Up to the highest nominal loading rate of 1000 mg/l PFBE (3,3,4,4,5,5, 6,6,6,-nonafluorohexene), no significant immobilisation of daphnids was observed after 24 h and 48 h compared to controls.

NOEC >/=1000 mg/L (loading rate)
LOEC > 1000 mg/L (loading rate)
EC50 > 1000mg/L (loading rate)

NOEC >/= 1.0 mg/L (measured concentration)
LOEC > 1.0 mg/L (measured concentration)
EC50 > 1.0 mg/L (measured concentration)
Executive summary:

The study was performed to evaluate the effects of PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) on the mobility of the freshwater crustacean, Daphnia magna, after an exposure time of 24 and 48 h. The objective of the study was the assessment of the acute toxicity of PFBE to Daphnia magna. Due to the low water solubility and high volatility of the test substance, the study was performed using water accommodated fractions (WAF) of PFBE as the test medium, as recommended for ecotoxicological testing of petroleum products by CONCAWE 1992 and for aquatic toxicity testing of lubricants by ASTM (standard protocol 06081-97). These protocols are designed to comply with the general principles outlined in the respective OECD guidelines. The mobility of the

freshwater crustacean, Daphnia magna, under static conditions was assessed after 24 and 48 hours. The validity criteria of the OECD Test Guideline 202 are fulfilled under the conditions of the modified procedures.

Summary of the test results

Up to the highest nominal loading rate of 1000 mg/l PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene), no significant immobilisation was observed after 24 h and 48 h compared to controls.

NOEC >/=1000 mg/L (loading rate)

LOEC > 1000 mg/L (loading rate)

EC50 > 1000mg/L (loading rate)

NOEC >/= 1.0 mg/L (measured concentration)

LOEC > 1.0 mg/L (measured concentration)

EC50 > 1.0 mg/L (measured concentration)