Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Feb - 15 Mar 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000)
Qualifier:
according to
Guideline:
other: ISO 10706, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the study duplicate samples for analyses were taken at the beginning and at the end of three intervals of 48hours (nominal days 0 and 2, 6 and 8, 14 and 16). In addition, samples were taken from freshly prepared solutions on days 2, 8, 16 and 20. Samples from the saturated stock solutions were taken and analysed on days 0, 2, 6, 8, 14, 16 and 20. From the test concentrations 10 mL and the saturated stock solutions 5 mL were taken. The samples were taken in a 20 mL GC-vial that already contained 0.5 mL acetone.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. 2,3,3,3-Tetrafluoroprop-1-ene HFO-1234yf is soluble in water (solubility = 160 mg/L at 25.5°C). Saturated stock solutions were prepared daily by loading two 10 litre Tedlar® gas sampling bags each with 7 or 7.5 litres of ISO medium followed by addition of ~3.5 or 3.75 grams gas (500 mg/L). The gas sampling bags were closed immediately after addition and then shaken carefully for 4 hours on a platform shaker. Subsequently, the bags were left to settle overnight. The two saturated media (expected concentration ~160 mg/L) were mixed and then used for preparing the test concentrations by serial dilution. The time frames for preparation were kept as short as practically possible and the stock and test solutions that were prepared were immediately closed as to prevent volatilisation as much as possible. The final test solutions were all clear and colourless. Note that preparation of the saturated stock solution was performed in combination with NOTOX Project 495933 „Fish juvenile growth test‟. A sufficient volume of the daily prepared stock solutions was used for the two-day renewals of the reproduction test with Daphnia. Trace elements, macro nutrients and vitamins were added to the ISO medium before exposure of the organisms as to make M7 medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: In-house laboratory culture with a known history
- Age of parental stock (mean and range, SD): at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Age of test individuals: neonate (<24h old), from parental daphnids more than two weeks old
- Feeding during test: yes
- Food type: Chlorella pyrenoidosa suspension
- Amount: 0.25 mL of suspension containing 1.46 x 10^8 cells. This daily ration corresponds to 0.2 mg C/Daphnia/day
- Frequency: daily

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week
- Temperature of medium: 18 - 22 °C
- Feeding: Daily, a suspension of fresh water algae
- Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in individual cultures and tested to meet the validity criteria for survival and reproduction
- Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
143 - 196 mg/L as CaCO3
Test temperature:
19.3 - 20.4 °C
pH:
7.4 - 9.1
Dissolved oxygen:
6.7 - 10.8 mg O2/L
Nominal and measured concentrations:
control, 1.5, 3.0, 6.0, 12 and 24 mg/L (nominal)
control, 1.1, 1.9, 4.1, 8.3 and 15.2 mg/L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: All-glass, 50 mL, no headspace and closed with a screw-cap, filled with 50 mL test medium
- Renewal rate of test solution (frequency/flow rate): renewed every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: 0.17 mg/L
- Intervals of water quality measurement: Temperature, oxygen and pH were checked at the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids. Hardness was checked once a week in fresh and old media from the control and the
highest test concentration, light at the start and the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: at the start: 652-700 lux (8.92-9.57µE x m-2 x s-1), at the end: 656-741 lux (9.46-10.27 µE x m-2 x s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Parental generation: Every workday and upon renewal on non-workdays, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed. The presence of eggs was checked every workday and upon renewal on non-workdays. Body lenght was measured at the end of the test.
F1 generation: Appearance of first brood when observed. Check for newborn daphnids upon renewals on work and non-workdays, the number of
newborn young was counted and the condition of the young recorded. Thereafter, the young were removed. When observed the presence of unhatched eggs and incidence of immobility were noted.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parental
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parental
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: parental body length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: parental body length
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: overall
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: overall
Details on results:
- Mortality of parent animals: Four out of the twenty parental daphnids died during the test period in the control. Hence, parental mortality did not exceed 20% in the controls. Mortality in the treatment groups was comparable to or below that of the control in 4 of the 5 groups tested (10 - 20%). Only at 8.3 mg/L a higher mortality of 40% was found. However, as the highest test concentration of 15.2 mg/L only induced 10% mortality this was considered not treatment related. Statistical analyses of the mortality data confirmed that the mortality in the treatment groups was not significant (Fisher's exact test, α= 0.05). Hence, the NOEC for survival was set at 15.2 mg/L, the highest concentration tested.
- Reproduction: There were no recordings of immobile young or appearance of unhatched (aborted) eggs at any of the test concentrations throughout the 21-day test period. Statistical analysis on the cumulative reproduction showed that the reproduction of the daphnids was not significantly reduced at any of the concentrations tested (Bonferroni t Test, Tukey's Test, α=0.05).Hence, the NOEC for reproduction was set at 15.2 mg/L, the highest concentration tested.
- Body length: No statistically significant reduction of body length was present in any of the concentrations tested according to the Tukey and Bonferroni t-test (α=0.05). Hence, the NOEC for growth was set at 15.2 mg/L, the highest concentration tested.
- Time to first brood release or time to hatch: In the controls, the presence of eggs in the brood pouch was generally recorded for the first time on day 6 and the first brood appeared on day 8 or 9. The first recording of the presence of eggs in the brood pouch and the appearance of the first brood was similar to the control in each of the treated solutions. Hence, no delay in appearance of the first brood was observed in the treatments when compared to the control.
Reported statistics and error estimates:
Total reproduction on day 21 was tested for normality and for homogeneity of variance and statistically tested using an ANOVA test followed by mean comparison tests (Bonferroni t Test and Tukey's Test). Body length was statistically tested using an ANOVA test followed by a Bonferroni t-test and a Tukey test). Survival of parental organisms was tested using the Fisher's exact test. All testing was performed using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

Measured concentrations:

The results of analysis of the duplicate samples taken during the reproduction test are given in Tables 1 - 3 below. The results of analyses of the saturated stock solutions showed that measured concentrations generally ranged between 160 and 190 mg/L. This was slightly higher than expected based on the solubility as provided by the sponsor, being 160 mg/L. Measured concentrations in freshly prepared solutions generally ranged between 70 and 90% relative to the target concentrations. On day 0 of the test recoveries were slightly lower with approximately 20 to 50% relative to the targets, while another few concentrations showed slightly lower recoveries on various days. It was unknown why these concentrations were lower than what was generally measured during the 21-day test period. Concentrations measured in the spent solutions were generally maintained within 80% of the initial measured concentrations with only a few exceptions. The exceptions with somewhat lower recoveries were not unexpected considering the fact that the substance tested was a highly volatile gas and that food was provided on a daily basis which required opening the test vessels for a short period. Table 3 below shows the mathematical means of the concentrations measured in the samples taken during the various renewal intervals. The means of all the renewal periods were used to calculate the mean measured concentrations per test group. The means were calculated to correspond to 1.1, 1.9, 4.1, 8.3 and 15.2 mg/L, i.e. 63 to 72% relative to the target concentrations.

Table 1: Procedural recovery samples

Date of preparation and analysis [dd-mm-yy]

Target concentration [mg/L]

Nominal concentration [mg/L]

Analysed concentration [mg/L]

Recovery [%]

Mean recovery [%]

22-02-11

0.1

0.100

0.0864

86

87

 

 

0.100

0.0881

88

 

22-02-11

100

100

101

101

100

 

 

100

98.9

99

 

24-02-11

0.1

0.100

0.0804

80

80

 

 

0.100

0.0799

80

 

24-02-11

100

100

111

111

114

 

 

100

116

116

 

24-02-11

100

100

122

122

119

 

 

100

115

115

 

28-02-11

0.1

0.100

0.0815

81

81

 

 

0.100

0.0808

81

 

28-02-11

100

100

109

109

111

 

 

100

113

113

 

02-03-11

0.1

0.100

0.0989

99

96

 

 

0.100

0.0921

92

 

02-03-11

100

100

100

100

101

 

 

100

103

103

 

08-03-11

0.1

0.100

0.0914

91

91

 

 

0.100

0.0907

91

 

08-03-11

100

100

107

107

108

 

 

100

109

109

 

10-03-11

0.1

0.100

0.0932

93

94

 

 

0.100

0.0957

96

 

10-03-11

100

100

103

103

104

 

 

100

105

105

 

14-03-11

0.1

0.100

0.0855

86

85

 

 

0.100

0.0854

85

 

14-03-11

100

100

102

102

102

 

 

100

101

101

 

Table 2: Concentrations of the test substance in the stock solutions - final test

Time of sampling [days]

Date of sampling and analysis [dd-mm-yy]

Nominal concentration [mg/L]

Analysed concentration [mg/L]

0

22-02-11

500

142

 

 

500

169

2

24-02-11

500

192

 

 

500

186

6

28-02-11

500

163

 

 

500

162

8

02-03-11

500

187

 

 

500

174

14

08-03-11

500

182

 

 

500

171

16

10-03-11

500

183

 

 

500

168

20

14-03-11

500

199

 

 

500

187

Table 3: Target and mean measured exposure concentrations

Target Concentration (mg/L)

Mean exposure concentration during refreshment periods (mg/L)

Mean day 0-21# (mg/L)

Day 0-2 mean

Day 2-4 mean*

Day 6-8 mean

Day 8-10 mean*

Day 14-16 mean

Day 16-18 mean*

Day 20-21 mean*

1.5

0.73

1.23

0.82

1.29

1.14

1.34

1.05

1.1 (72%)

3.0

0.64

2.01

1.62

1.97

2.22

2.30

2.76

1.9 (64%)

6.0

2.00

5.06

3.81

4.33

4.33

4.95

4.28

4.1 (68%)

12

4.98

8.54

8.18

9.63

9.31

8.66

8.74

8.3 (69%)

24

8.57

16.27

16.50

16.50

18.76

18.25

11.75

15.2 (63%)

#: Between brackets: % recovery relative to nominal

*: Calculated with 24- or 48-hour concentrations set at 85% relative to the freshly analysed concentrations

Table 4: Cumulative mortality of parental daphnids during the 21-day exposure period

Concentration (mg/L)

Number exposed

Cumulative number of dead parental daphnids on day:

Mortality %

2

4

6

7

8

10

12

14

16

18

20

21

Control

20

0

0

0

0

1

1

1

2

2

2

3

4

20

1.1

10

0

0

0

0

0

0

0

0

0

0

1

1

10

1.9

10

0

0

0

0

0

1

1

1

1

1

2

2

20

4.1

10

0

0

0

0

0

0

0

0

0

0

2

2

20

8.3

10

0

1

1

1

1

3

3

3

4

4

4

4

40

15.2

10

0

0

0

0

0

1

1

1

1

1

1

1

10

Table 5: Average body lengths (mm) of parental daphnids at the end of the test and % reduction of body length relative to the control

Concentration (mg/L)

Average length (mm)

SD

% Reduction

Control

4.06

0.08

-

1.1

4.06

0.08

0

1.9

4.09

0.09

0

4.1

4.09

0.09

0

8.3

4.08

0.09

0

15.2

4.02

0.11

1

Table 6: Cumulative mean number of living young per parent at the various mean concentrations (in mg/L)

 

Day 7

Day 8

Day 10

Day 12

Day 14

Day 16

Day 18

Day 20

Day 21

Control

0.0

0.4

13.2

21.2

33.7

53.2

56.9

76.6

76.9

1.1

0.0

3.4

11.1

26.4

35.6

48.0

73.8

81.9

91.2

1.9

0.0

0.9

11.2

25.2

30.0

49.6

64.6

79.4

83.8

4.1

0.0

1.0

12.3

26.1

32.5

51.8

68.4

76.9

84.1

8.3

0.0

0.3

12.0

23.6

29.4

50.8

69.3

75.7

87.2

15.2

0.0

0.2

15.1

27.0

33.1

51.7

65.9

71.2

77.1

Description of key information

NOEC reproduction (21 d): 15.2 mg/L (measured; OECD 211)

Key value for chemical safety assessment

Additional information

The long-term toxicity of 2,3,3,3-Tetrafluoropropene to Daphnia magna was tested according to OECD guideline 211 in compliance with OECD Guidance Document No. 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (Daikin Industries Ltd., 2011). The Daphnids were exposed to mean measured test substance concentrations of 1.1, 1.9, 4.1, 8.3 and 15.2 mg/. The test was performed in closed test vessels due to the high volatility of the test substance. The exposure period was 21 days. During the exposure time the mortality and reproduction of the test organisms was recorded. No effect on parental survival and/or reproduction rate was observed. Thus, a NOEC(21 d) of 15.2 mg/L based on the reproduction rate was reported.