Polyhaloalkene

Long-term toxicity to aquatic invertebrates

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ISO 10706, 2000

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

468-710-7

EC Name:

-

Cas Number:

754-12-1

Molecular formula:

C3H2F4

IUPAC Name:

2,3,3,3-tetrafluoroprop-1-ene

Sampling and analysis

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.

Test solutions

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

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)

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

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

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Effect concentrations 3

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

Effect concentrations 4

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

Effect concentrations 5

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

Effect concentrations 6

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

Effect concentrations 7

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

Effect concentrations 8

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

Effect concentrations 9

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.

Any other information on results incl. tables

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