Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jan 2001 - 2 Feb 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline 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 203 (Fish, Acute Toxicity 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

Saftey 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:
Sampling
The concentration of the test substance in the WAFs was analyzed at the beginning and the end of the exposure of the zebra fish toxicity test. Following samples were analyzed:

0 h start
24 h end of exposure
24 h renewal, start
48 h renewal start
72 h renewal start
96 h end of exposure

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

Test solutions

Details on test solutions:
Test solution: Preparation of WAF
The WAF preparation was performed according to the CONCAWE test protocol and the ASTM standard D6081-97 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 D6081-97, 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 g/ml. So the aqueous WAF phase was drawn off after WAF preparation through the upper orifice.
The 5 L 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 (1 0- 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. The liquid test substance formed a drop on the bottom of the bottle, partly enfolding the stirring bar.
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 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. Another portion was filled in a separate vessel for chemical analysis.
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):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Test organism:
Specification: Dania rerio (Hamilton-Buchanan, 1822; Teleostei, Cyprinidae)
Length: 2-3 cm
Source: laboratory bred
The fish were originally obtained from: West Aquarium GmbH
PB 146
D-37431 Bad Lauterberg
Germany

Holding: The fish were acclimated for 14 days (minimum) in water of the same quality as used in the test (purified drinking water). They were fed daily with TetraMinR Hauptfutter (Tetra Werke, Melle, Germany).

Mortality: The criteria of the test guideline were followed. Only healthy fish without diseases and abnormalities were used in the study.






Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Test temperature:
22 ± 1°C
pH:
Control (vessel0):7.6 - 8.16
100 mg/l (vessel 1): 7.62 - 8.23
1000 mg/l (vessel 2): 7.51 - 8.22
1000 mg/l (vessel 3): 7.48 - 8.21
Dissolved oxygen:
Mean dissoved O2 across all test concentrations
8.0 mg/l (88.6% saturation)(24 hr test medium)
7.9 mg/l (89.2% saturation)(48 hr test medium)
7.9% (86.8% saturation) (72 hr test medium)
7.2mg/l (79.4% saturation) (96 hr test medium)

Nominal and measured concentrations:
At a loading rate of 100 mg/L, measured concentrations of PFBE in the WAFs prior to the start of the test (set of WAF preparations) were found between 0.11-0.75 mg/L, with a mean of 0.31 ±0.29 mg/L. At a loading rate of 1000 mg/L, PFBE concentrations were analysed between 1.56-2.57 mg/L, with a mean of 1.86±0.35 mg/L in the WAFs prior to the start of the test (set of WAF preparations). During the exposure period of 24 h, concentrations decreased between 27-19% in WAFs loaded with 100 mg/L and between 34-44% 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 fish) and volatilization through the orifice, though ground-in stoppers were used.
The measured concentrations at the start of the test (WAF preparations) were used for generating concentration-effect relationships.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 1.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
See other information on results. incl. tables

Any other information on results incl. tables

Table 2 shows the cumulative mortality of fish during the test period. The highest loading rate of 1000 mg/L had no significant lethal effect compared to the control. According to the OECD Test Guideline 203, mortality of 10 % at the end of the test is acceptable at the control level. In the two replicates, 10% and 0% mortality, respectively, were observed. To ensure complete filling of the test vessels, marbles had been introduced into the flasks. The mortality observed can be explained by damage to the fish during changes of the test medium.

A concentration-effect relationship could not be established. The highest loading rate of 1000 mg/L revealed no toxicity different from controls. Therefore, in the acute toxicity test with Danio redo (zebra fish), the effect values are as follows:

NOEC, LOEC and EC 50 > 1000 mg/L (loading rate)

NOEC, LOEC and EC 50 > 1.86 mg/L (measured concentration)

Table 2. Cumulative mortality. Total number of fish per loading during the test period of 96hr (10 fish per vessel). Measured concenrations are the mean values of WAFs at test start.

Nominal Loading (mg/l)

Measured Conc.

(mg/l)

Cumulative mortality

24 hr

48 hr

72 hr

96 hr

% mortality

Control

0

0

0

0

0

0

100

0.31

1

1

1

1

10

1000(1)

1.96

0

1

1

1

10

1000(2)

0

0

0

0

0

0

Validity of the test

The conditions for the validity of the test are complied with:

1)The mortality in the controls did not exceed 10 per cent at the end of the test.

2)The oxygen concentrations did not fall below 60 per cent of air saturation value throughout the test.

3)Constant exposure had been maintained by semi-static conditions with renewal periods of 24 hours. The stability and concentration of the test substance in the WAF had been verified analytically.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The conditions for the validity of the test are complied with. See other information on results incl. tables
Conclusions:
A concentration-effect relationship could not be established. The highest loading rate of 1000 mg/L PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) revealed no toxicity different from controls. Therefore, in the acute toxicity test with Danio redo (zebra fish) the effect values are as follows:
NOEC >/= 1000 mg/L (loading rate)
LOEC >/= 1000 mg/L (loading rate)
EC 50 > 1000 mg/L (loading rate)

NOEC >/= 1.86 mg/L (measured concentration)
LOEC > 1.86 mg/L (measured concentration)
EC 50 > 1.86 mg/L (measured concentration)
Executive summary:

The objective of the study was the assessment of the acute toxicity of PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) on the zebra fish, Danio rerio. 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 acute mortality of zebra fish (Danio rerio) under semi-static conditions was assessed after 24, 48, 72 and 96 hours. The validity criteria of the OECD Test Guideline 203 were fulfilled under the conditions of the modified procedures.

A concentration-effect relationship could not be established. The highest loading rate of 1000 mg/L PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) revealed no toxicity different from controls.

Therefore, in the acute toxicity test with Danio redo (zebra fish), the effect values are as follows:

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

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

EC 50 > 1000 mg/L (loading rate)

NOEC >/= 1.86 mg/L (measured concentration)

LOEC > 1.86 mg/L (measured concentration)

EC 50 > 1.86 mg/L (measured concentration)