2-Propenoic acid, 2-chloro-, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl ester

Long-term toxicity to aquatic invertebrates

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:

11 February - 03 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to recognised OECD Guideline (OECD 211) and GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0080, 0.014, 0.024, 0.044 and 0.079 mg/L.

- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 1, 6, 7, 13, 14, 20 and 21

- Sample storage conditions before analysis: Water samples were taken from the control, solvent control and each surviving test group for immediate quantitative analysis, with the exception of the Day 1 samples which were stored frozen prior to analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 6, 13 and 20 and of the expired test preparations (replicates pooled) on Days 1, 7, 14 and 21. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (50 mg) was dissolved in DMF and the volume adjusted to 25 mL to give a 2.0 mg/mL stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 0.16, 0.28, 0.48, 0.88 and 1.58 mg/mL. The solvent stock solutions were prepared on Days 0, 6, and 13 of the test and were stored in a refrigerator between use. The stock solutions were inverted several times to ensure adequate mixing and homogeneity. An aliquot (100 μL) of each solvent stock solution was separately dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to give the required test concentrations of 0.0080, 0.014, 0.024, 0.044 and 0.079 mg/L.
- Controls: untreated control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: not specified
- Age at study initiation (mean and range, SD): <24 h old (1st instar)
- Method of breeding: reproduction by parthenogenesis
- Source: In-house laboratory cultures
- Feeding during test: yes
- Food type: Each daphnid received approximately 5 to 20 μL of an algal suspension (Pseudokirchneriella subcapitata) daily and approximately 20 μL of GM300 fish food suspension on Days 0, 1, 2 and 3.
- Amount: Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

ACCLIMATION
- Acclimation period: not reported
- Acclimation conditions (same as test or not): unkown
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Pseudokirchneriella subcapitata) and GM300 fish food suspension.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt M7

Limit test:

no

Total exposure duration:

21 d

Hardness:

252-260 mg/L as CaCO3

Test temperature:

Target: 18-22°C, Actual: 18.9-20.9°C

pH:

Target: 6-9, Actual: 7.6-8.2

Dissolved oxygen:

Target: >3 mg O2/L, Actual: 5.7-9.3 mg O2/L

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations: 0.0080, 0.014, 0.024, 0.044 and 0.079 mg/L. The geometric mean measured concentrations were determined to be 0.00035, 0.00046, 0.00065, 0.00086 and 0.0012 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels
- Type (delete if not applicable): closed, sealed, as recommended for volatile substances in OECD Guidance Document No. 23
- Material, size, headspace, fill volume: 100 mL test solution in sealed glass vessel
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Ingredient Final concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

- Culture medium different from test medium: no, both Elendt M7
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark with 20 min dawn dusk cycle
- Light intensity: less than 1500 lux (actual during the study: 811-1034)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : survival of parent daphnia (p1) generation daily, survival of offspring (f1) generation daily, number of discarded unhatched eggs daily, general condition/size, length of parent daphnia at end of test. dissolved oxygen, pH and temperature before and after each media renewal, temperature logged every hour in one control replicate. Light intensity recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.75
- Range finding study
- Test concentrations: 0.00010, 0.0010, 0.010 and 0.079 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parent daphnia

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: body length (parent Daphnia)

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parent daphnia

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: length measured at test end
- Other biological observations: one adult daphnia at 0.014 mg/L was observed to be pale on Days 13 and 14
- Mortality of control: 0% mortality in untreated control, 10% mortality in solvent control
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

All data were compared to a pooled control group. Parental mortality data was analysed using the Fisher’s Exact Binomial Test with Bonferroni Correction. Analysis of the number of live young produced and length at Day 21 were compared using the Williams Multiple Sequential t-test Procedure incorporating Levene’s test on variance homogeneity and Shapiro-Wilk’s test on Normal Distribution. No significant differences (P≥0.05) were found between the pooled controls and any test groups. ECx values were determined using Logit analysis with linear maximum-likelihood regression, 3-parameter normal cumulative distribution function and probit analysis with linear maximum-likelihood regression, for the parental mortality, parental length and reproduction data, respectively.

Table 1                       Summary of Findings Following Exposure of D. magna for 21 days

 

Geometric Mean Measured Concentration (mg/L)	Parental (P1) Generation Mortalities (Initial Population = 10)			Total Number of Live Young	Number of Live Young from Parents Surviving to the End of the Test		Number of Live Young at the End of the Test Excluding Parents that were Accidental or Inadvertent Mortalities
	Total	Accidental	Inadvertent		Total	Average per Parent	Total	Average per Parent
Control	0	0	0	1509	1509	151	1509	151
Solvent Control	1	0	1	1499	1378	153	1378	153
0.00035	0	0	0	1402	1402	140	1402	140
0.00046	2	0	2	1283	1244	156	1244	156
0.00065	3	0	3	1373	1124	161	1124	161
0.00086	0	0	0	1597	1597	160	1597	160
0.0012	1	0	1	1312	1312	146	1312	146

 

Table 2           Body Length of Surviving Adults at Day 21

 

Geometric Mean Measured Concentration(mg/L)	Individual Daphnia Lengths (mm)
	1	2	3	4	5	6	7	8	9	10	Average	St Dev
Control	4.4	4.5	4.6	4.7	4.2	4.4	4.6	4.7	4.7	4.6	4.54	0.17
Solvent Control	4.3	4.5	4.5	4.5	4.6	4.5	4.1	4.3	4.2	-	4.39	0.17
0.00035	4.6	4.5	4.3	4.5	4.2	4.2	4.0	4.2	3.8	4.2	4.25	0.24
0.00046	4.3	4.6	4.5	4.3	-	-	4.4	4.4	4.1	4.1	4.34	0.18
0.00065	4.4	4.5	4.3	-	4.6	4.5	-	4.7	-	4.6	4.51	0.14
0.00086	4.6	4.7	4.3	4.6	4.1	4.4	4.5	4.2	4.5	4.6	4.45	0.20
0.0012	4.3	4.6	4.0	4.5	4.2	4.3	4.5	4.4	4.4	-	4.36	0.1

Validity criteria fulfilled:

yes

Conclusions:

For the test to be valid, the following criteria should be met:
Mortality of parent test animals in the controls should not exceed 20% at the end of the test (0% in untreated control), the mean number of living offspring in the control should be at least 60 per surviving adult daphnia at the end of the test (152 per control parent), the coefficient of variation around the mean number of living offspring produced per parent animal in the control should be equal to or less than 25% (10%), no ephippia are produced in the controls (achieved), dissolved oxygen concentration should remain greater than 3 mg O2/L throughout the test (>5.7 mg O2/L throughout the test) and pH of the controls should be within the range 6 to 9 pH units and not vary by greater than 1.5 units throughout the test (7.6 to 8.2 with a deviation of no more than 0.7 units).

All criteria were met therefore the test is considered valid.

Although the validity criteria were fulfilled, showing the environmental conditions were appropriate for testing, the study report does not show data or evidence of optimisation of the test design so that the toxicological properties of the test item can be properly investigated. Additionally, calculation of exposure concentrations is not consistent with OECD 211, or OECD GD 23.

Executive summary:

A study was performed according to OECD 211 to assess the effects of test item on reproduction in Daphnia magna. Daphnia magna <24 h old were exposed to the test item at nominal concentrations of 0.0080, 0.014, 0.024, 0.044 and 0.079 mg/L for a period of 21 days under semi-static conditions (daily renewal of solutions). As the test substance is poorly soluble in water, dimethylformamide was used as a co-solvent to aid test solution preparation. Sealed test vessels were employed, as recommended in OECD Guidance Document 23, as the test item is volatile. Reproduction and mortality was measured daily; length of adult daphnia was measured at the end of the test. Geometric mean measured test concentrations during the test period were found to be 0.00035, 0.00046, 0.00065, 0.00086 and 0.0012 mg/L, respectively. No statistically significant effects on reproduction compared to the pooled control were identified during statistical analysis of the data, therefore the NOEC was determined to be 0.0012 mg/L.

Description of key information

Reproduction EC10: >0.0012 mg/L, Reproduction NOEC: 0.0012 mg/L; OECD 211;

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.001 mg/L

Additional information

A study was performed according to OECD 211 to assess the effects of test item on reproduction in Daphnia magna.Daphnia magna<24 h old were exposed to the test item at nominal concentrations of 0.0080, 0.014, 0.024, 0.044 and 0.079 mg/L for a period of 21 days under semi-static conditions (daily renewal of solutions). As the test substance is poorly soluble in water, dimethylformamide was used as a co-solvent to aid test solution preparation. Sealed test vessels were employed, as recommended in OECD Guidance Document 23, as the test item is volatile. Reproduction and mortality was measured daily; length of adult daphnia was measured at the end of the test. Geometric mean measured test concentrations during the test period were found to be 0.00035, 0.00046, 0.00065, 0.00086 and 0.0012 mg/L, respectively. No statistically significant effects on reproduction compared to the pooled control were identified during statistical analysis of the data, therefore the NOEC was determined to be 0.0012 mg/L.