Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2012 to 13 MArch 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD Guideline-conform study conducted under GLP, however some limitations occurr due to technical difficulties in interpretation of results since the maximum concentration of GALDEN LMW achievable under the test conditions was lower than the water solubility value.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Minor deviations, consireded not able to affect the results.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L (nominal)
- Sampling method: 50 ml of test item solutions and negative controls were collected from freshly prepared solutions at time 0, 24, 48 and 72 hours and at time 24, 48, 72 and 96 hours from spent solutions.
- Sample storage conditions before analysis: Immediately after collection. If samples are stored at 4° in the dark, analysis can be performed within 24 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

The test medium was prepared as following:
Prior to the test start, two 5-liters flasks of 100.0 mg/L Galden LMW water solution were prepared by dosing with a micropipette 300 microliter of Galden LMW, corresponding to 501 mg considering ots density of 1.65 -1.68 g/cm3 (mean density = 1.67g/cm3) into 5010 mL of reconstituted water. The two flasks were tightly closed to prevent test item evaporation. The obtain solutions appeared transparent with oily droplets on the bottom of the flask, therefore they were mixed for two hours by the mean of a magnetic stirrer in dark conditions to achieve the maximum solubilisation of the test item. After stirring, they were settled down for two hours and then they were transferred into the test tank, by siphoning the 10 liters of solution with a peristaltic pump, avoinding to tranfert the unsolubilized test item present on the bottom of the flasks.

This procedure was repeated after 24, 49 and 72 hours to renew the test solution and the negative control.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Danio rerio)
- Source: Commercial supplier
- Age at study initiation (mean and range, SD): juvenile fishes of approximately the same age and size.
- Length at study initiation (length definition, mean, range and SD): the mean body lenth was 2,96 +- 0.07 cm
- Weight at study initiation (mean and range, SD): the mean body weight was 0.31 +- 0.02 cm
- Feeding during test: no

ACCLIMATION
- Acclimation period: 18 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food: Commercial fish food

At the start of the test, 7 fishes were randomly distributed to each aquarium and each 24 hours they were transferred in the fresh prepared media.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

140 -250 mg/L as CaCO3

Test temperature:

Water temperature in aquaria was monitored during the course of the study by a sensor connected to a PC.
The temperature during the test period was between 21.9 °C and 26.4 °C for the negative control tank and between 18.7 °C and 25.0 °C for the test item solution tank. The temperature recorded values were outside the OECD range (23 +- 2°C) for some periods, however this deviation was considered accettable as no mortality was observed during the test period.

pH:

During the test period, the pH value of the test item solutions and of the negative control solutions were measured with a portable pHmeter sampling from the middle of the tanks an aliquote of each solution (fresh and spent).
The pH of the fresh and spent solutions of the test item (nominal concentration = 100 mg/L ) was in the range 7.22 – 7.84.
The pH of the fresh and spent solutions for the negative controls was in the range 7.01 – 7.82. Therefore the pH of the test media was not adjusted.

Dissolved oxygen:

During the test period, the dissolved oxygen concentration in the test item solutions and in the negative control solutions were measured with a portable oxymeter sampling from the middle of the tanks an aliquote of each solution (fresh and spent).
The dissolved oxygen (%) in the test media and in the negative controls were always above the 70% of the air saturation value, in accordance with OECD No.203.

Salinity:

The following salts were dissolved in purified water in the following nominal concentrations:
CaCl2 x 2H2O = 294 mg/L
MgSO4 x 7H2O = 123 mg/L
NaHCO3 = 65 mg/L
KCl = 5.8 mg/L

Nominal and measured concentrations:

The main test was carried out at the only nominal test item concentration of 100.0 mg/L, corrected for its purity (99.9% ). The 100.0 mg/L test solution was an oversaturated solution, in order to obtain the maximum solubility of Galden LMW under the test conditions.

The analytical concentration of Galden LMW was measured at time 0, 24, 48, 72 hours in the freshy prepared solutions, and at time 24, 48, 72 and 96 hours in the spent solutions (i.e. after the 24 hour-exposure of the fish).
In the fresh solutions (time 0, 24, 48 and 72 hours) the analytical recoveries of the concentrations of Galden LMW were in the range 0.071 – 0.059 % of the nominal 100 mg/L, corresponding to the concentration range from 70.49 to 59.15 microgram/L.
In the spent test item solutions (time 24, 48, 72 and 96 hours) the test item concentration decreased; the analytical recoveries were in the range 0.012 – 0.003 % of the nominal values, corresponding to the concentration range from 2.66 to 12.21 microgram/L.

See table 1 for details on Recoveries.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 L capacity glass aquaria
The test was performed in 4 temperature-controlled aquaria closed with glass covers and incubated in a temperature-controlled room.
- Type (delete if not applicable): closed
- Renewal rate of test solution: 24 hours

TEST MEDIUM / WATER PARAMETERS
For reconstituted test water preparation, analytical grade salts were dissolved in purified water until obtaining the following nominal concentration:
CaCO2 x 2 H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O : 0.5 mmol/L ( = 123 mg/L)
NaHCO3 : 0.075 mmol/L ( = 65 mg/L)
KCl : 0.075 mmol/L ( = 5.8 mg/L)
- Ca/Mg ratio: 4:1 (based on molarity)
- Na/K ratio: 10:1 (based on molarity)

The pH values and dissolved oxygen concentrations in the test media and in the negative controls were daily measured in fresh and spent solutions.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16 hours light / 8 hours darkness with 30-min transition period
- Light intensity: 195 -251 Lux (measured values)


TEST CONCENTRATIONS
- Test concentrations: 100mg/L (nominal)
GALDEN LMW has a low water solubility. The nominal concentration of 100 mg/L was selected in order to obtain the maximum solubility of the test item.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

20.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: The reported value is the geometric mean deriving from the maximum concentrations of GALDEN LMW achievable under test conditions. No biological effects were observed.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 20.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: The reported value is the geometric mean deriving from the maximum concentrations of GALDEN LMW achievable under test conditions. No mortality was observed.

Details on results:

No biological effects were observed during the entire test period.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The CETIS v.1.026D software was used to carry out the statistical analysis: it automatically chooses the most appropriate analysis for the pool of data. The IC0 and IC100 values were determined directly from Raw Data at each observation time.
The LC 50 value at 24, 48, 72 and 96 hours was determined by statistical analysis with Linear Interpolation analysis.
The NOEC value at 24, 48, 72 and 96 hours was determined by comparison with the control by Fisher's Exact method (alfa = 0.05)
As input data, the measured geometric mean of test item Galden LMW was used.

Sublethal observations / clinical signs:

Table 1. Analytical recovery of the measured test concentrations reffered to the nominal ones.

Time (hours)	Nominal test item concentration (mg/L)	Actual test item concentration (microgram/L)	Analytical Recovery ( % )
0	100	62.39	0.062
24 fresh	100	2.66	0.003
24 spent	100	59.15	0.059
48 fresh	100	5.44	0.005
48 spent	100	70.49	0.071
72 fresh	100	10.93	0.011
72 spent	100	69.66	0.070
96	100	12.21	0.012

Table 2. Results in terms of measured geometric mean concentration and of nominal test item concentration.

Time (hours)	NOEC	LC 0	LC 50
24	12.87 microgram/L * 100 mg/L **	12.87 microgram/L* 100 mg/L **	> 12.87 microgram/L * > 100 mg/L **
48	15.20 microgram/L * 100 mg/L **	15.20 microgram/L* 100 mg/L **	> 15.20 microgram/L * > 100 mg/L **
72	18.58 microgram/L * 100 mg/L **	18.58 microgram/L* 100 mg/L **	> 18.58 microgram/L * > 100 mg/L **
96	20.80 microgram/L * 100 mg/L **	20.80 microgram/L* 100 mg/L **	> 20.80 microgram/L * > 100 mg/L **
* Geometric mean between the start of the experiment and the time point
** Nominal concentration

The LC50 and NOEC values were determined from the Raw Data by the statistical comparison with the negative control.

LC0 values at any observation time were directly extracted from raw data.

Validity criteria fulfilled:

yes

Conclusions:

The test was conducted in order to obtain the maximum concentration of GALDEN LMW in water. Due to the substance properties (low water solubility and high volatility) the analytical concentration in water was very lower than the nominal one (100 mg/L). No biological effects occurred at the maximum concentration of test item achievable under test conditions.

Executive summary:

The acute toxicity of Galden LMW to zebra fish (Danio rerio) was determined in a limit 96 -h semi-static test according to OECD guideline No. 203 and OPPTS 850.1075 EPA guideline.

For this purpose, 7 juvenile fishes of the speciesDanio reriowere exposed, after an acclimatation period of 18 days, to an aqueous test medium containing the test item at the only nominal concentration of 100 mg/L.

The 100 mg/L test solution was an oversaturated solution, in order to reach the maximum solubility of the test item in the water media.

According to the semi-static method, the test solution was renewed every 24 h.

The test organism were checked for mortality end-point after 2, 24, 48, 72 and 96 hours from test initiation.

The actual test concentration of Galden LMW were analytically determined measured at the beginning of the test, after 24, 48 and 72 hours (in the freshly prepared and in the spent solutions) and at the end of the test period (96 hours). Details of analytical recoveries are reported in the following table:

Time (hours)	Nominal test item concentration (mg/L)	Actual test item concentration (microgram/L)	Analytical Recovery ( % )
0	100	62.39	0.062
24 spent	100	2.66	0.003
24 fresh	100	59.15	0.059
48 spent	100	5.44	0.005
48 fresh	100	70.49	0.071
72 spent	100	10.93	0.011
72 fresh	100	69.66	0.070
96	100	12.21	0.012

In the fresh solutions (time 0, 24, 48 and 72 hours) the analytical recoveries of the concentrations of the test item were in the range 0.071 – 0.059 % of the nominal 100 mg/L, and in the spent test item solutions (time 24, 48, 72 and 96 hours) the analytical recoveries were in the range 0.012 – 0.003 % of the nominal values.(The analytical recoveries were referred to the test item content corrected for its purity >= 99.9% )

The low concentration obtained in test solutions were mainly due to:

- test item low solubility in the test item;

- test item high volatility.

The low % analytical recoveries (obtained compared to nominal test item concentration) were due to the preparation of an oversatured solution, in order to reach the maximum test item concentration in reconstituted water, as suggested by OECD No. 23(2000) on testing difficult substances.

Test solutions preparation was strictly standardized in order to get the maximum reproducibility and to reduce the test item evaporation. The toxicity on zebra fish was conducted in tanks closed with glass covers.

As recommended by OECD Guidance Document on testing of difficult substances No. 23 (2000), the biological results were referred to the geometric mean of the test item measured concentrations, since the analytical recoveries of test item was lower than the range 80-120 %.

In the negative control no mortality was observed during the test period. This value complies with the validity of the criterion of OECD guideline No. 23 (1992), that required a maximum mortality of one fish on seven exposed fish at the end of the test.

The results, in terms of measured geometric mean concentration and of nominal test item concentration, were as following:

Time (hours)	NOEC	LC 0	LC 50
24	12.87 microgram/L * 100 mg/L **	12.87 microgram/L* 100 mg/L **	> 12.87 microgram/L * > 100 mg/L **
48	15.20 microgram/L * 100 mg/L **	15.20 microgram/L* 100 mg/L **	> 15.20 microgram/L * > 100 mg/L **
72	18.58 microgram/L * 100 mg/L **	18.58 microgram/L* 100 mg/L **	> 18.58 microgram/L * > 100 mg/L **
96	20.80 microgram/L * 100 mg/L **	20.80 microgram/L* 100 mg/L **	> 20.80 microgram/L * > 100 mg/L **
* Geometric mean between the start of the experiment and the time point
** Nominal concentration

 

The LC50 and NOEC values were determined from the Raw Data by the statistical comparison with the negative control.

In conclusion, no biological effects were observed at the maximum concentrations of GALDEN LMW achievable under test conditions.

Description of key information

No biological effects occurred at the maximum concentration of GALDEN LW  achievable under test conditions.

Additional information

An OECD Guideline-conform study was conducted in order to define the acute toxicity of GALDEN LMW to fish. The test was performed according to OECD guideline No. 203 to zebra fish (Danio rerio) in a limit 96 -h semi-static test.

The test was conducted in order to obtain the maximum concentration of GALDEN LMW in water, however due to the substance properties (low water solubility and high volatility) the maximum concentration achievable under test condition was much lower than the nominal one (100 mg/L).

At the maximum concentration of GALDEN LMW achievable under test conditions, no biological effects were observed.