Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

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:

15 Feb 2021 to 11 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations: All concentrations (10, 3.2, 1, 0.32, 0.1 mg/L) and the control.
The freshy prepared solutions were sampled on day 0, 7, and 16.
The old solutions were sampled on day 2, 9, and 19.

Sampled volume: 1.2 mL (in silanized glass vials)
Storage Samples were stored in a freezer (set to maintain -20°C) until analysis (stability of samples under these conditions was experimentally demonstrated)

At the end of the renewal period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.2 mL were taken from all test concentrations and the control as per the scheme above for possible analysis.

Vehicle:

no

Details on test solutions:

The test item tested was a colourless liquid with a purity of 39% w/w ammonium salt in water. A correction factor of 2.564 was used to correct for the water content of the test item in the way that the end concentrations refer to the test chemical CYCLIC C6O4 AMMONIUM SALT. The test
item was completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest concentration (100 mg/L in the pre-test and 10 mg/L in the full test) applying a 15-minute period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: To initiate the test, young daphnids < 24 hours old were selected.

Breeding
Start of each batch: With new-born daphnids, i.e. less than 3 days old, by placing them individually in 50 mL M7-medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Monitoring of the individual cultures: Three times a week the young are counted and the parental daphnids are transferred to new media.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of freshwater algae (Chlorella sp.)
Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
Medium: M7

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no

Hardness:

> 140 mg/L (as CaCO3)
Actual values during the full test: 196 - 303 (as mg CaCO3)

Test temperature:

18-22°C, constant within 2°C
Actual values during the full test: 19-21°C

pH:

Between 6.0 to 9.0, constant within 1.5 units
Actual values during the full test: 7.7-8.3

Dissolved oxygen:

> 3 mg/L
Actual values during the full test: 7.6 -9.6 mg/L

Nominal and measured concentrations:

Nominal: 0.10, 0.32, 1.0, 3.2 and 10 mg /L
The measured test item concentrations remained within 20% of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels. Volume: 60 mL (6 x Ø 3.5 cm), all-glass covered with a Perspex plate.
- Fill volume: 50 mL
- Renewal rate of test solution (frequency/flow rate): Three times a week (i.e. on day 2, 5, 7, 9, 12, 14, 16, 19 of exposure)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Chemicals (analytical grade) were dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
- Total organic carbon: Preferably < 2 mg C per litre
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of temperature, pH and dissolved oxygen occurred at the start and at the end of the exposure. In addition, in both fresh and old medium at each renewal.

OTHER TEST CONDITIONS
- Photoperiod: Daily photoperiod of 16 h.
- Light intensity: The intensity did not exceed 1000- 1500 lux which corresponded with a range not exceeding 15-20 μE/m2/s.

EFFECT PARAMETERS MEASURED :
Survival
Presence of eggs in the brood pouch
Number of (immobile) offspring

Frequency of observations:
Parental daphnids
Condition: 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.
Body length: At the end of the test.

Offspring:
Appearance first brood: When observed.
New-born daphnids: Every workday, the number of new-born young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.
Incidence of entrapment: When observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125

- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg /L
- Results used to determine the conditions for the definitive study: yes
[No mortality was recorded in the controls or any of the concentrations tested throughout the exposure period in the range finding test. In all test groups including the control, the first brood in the controls and at the various test item treatments appeared on day 8. There was no delay in offset of reproduction observed at any test concentration when compared to the controls. In the control, 68 offspring were produced throughout the 10-day exposure period. Comparable numbers were recorded for the test item treated groups (59-76 living young) without correlation with test item concentration.]

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

(parental survival)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

(parental growth)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Age at first reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: immobile offspring

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: aborted eggs

Details on results:

- Survival and Condition of Parental Daphnids
None out of the ten parental daphnids died during the test period in the control (see Table 1).
Hence, parental mortality did not exceed 20% in the control. In the test item treated groups mortality did not exceed 10%. Mortality was not correlated with applied concentration. Since up to 20% mortality are accepted for the control group, the observed mortality in the test item treated groups was concluded to be biologically irrelevant and likely inadvertent. Trend analysis revealed no significant increase in mortality, hence, those replicates with parental mortality were excluded from the analysis of the test results and the effects on reproduction were calculated per surviving parental daphnid. Statistical analysis showed that mortality in test item treated groups was not significantly different from the control.
Since ≤ 20% mortality is accepted in the control group, and mortality did neither exceed 20% in any test group, nor was statistically significant compared to the control, the NOEC for parental survival was 10 mg/L. The LOEC was beyond the range of concentrations tested.

- Age at First Reproduction
The first brood was observed at day 8 in all groups and all replicates. Therefore, the test item
did not cause delay in onset of reproduction.

- Reproduction
Neither dose-related nor statistically significant reduction in offspring was recorded at any of the concentrations tested. The NOEC for reproduction was 10 mg/L. The LOEC was beyond the range of concentrations tested.
There were only incidental recordings of immobile young or appearance of unhatched (aborted) eggs at the concentrations tested during the 21-day test period. The occurrences were considered to not be treatment related. Numbers of immobilised offspring or aborted eggs observed during the study are summarized in Table 3.

- Body Length
Neither dose-related nor statistically significant reduction of parental body length was found
at the range of concentrations tested. The NOEC for parental growth was 10 mg/L. The
LOEC was beyond the range of concentrations tested. (Table 4)

Reported statistics and error estimates:

Statistical analysis

- Trend in parental mortality: Step-down Cochran-Armitage Test Procedure (onesided greater, alpha = 0.05)
- Parental survival: Fisher`s Exact Binomial Test with Bonferroni correction (one-sided greater, alpha = 0.05), preceded by a qualitative trend analysis by contrasts (monotonicity of the dose-response curve).
----------------------------
Determination of the NOEC for reproduction, immobile offspring, aborted eggs and growth

- Pretesting:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)

- Reproduction: Williams Multiple Sequential t-test Procedure (onesided smaller, alpha = 0.05).
- Age at first reproduction: No statistical comparison was performed as first offspring was observed on the same day in all replicates of the control and all test concentrations.
- Parental growth: Dunnett`s Multiple t-test (one-sided greater, alpha = 0.05), preceded by a trend analysis by contrasts (monotonicity of the dose-response curve).


EC-values for reproduction: No EC10 values could be calculated as no effects were observed.

Table 1_Mortality (Immobility) of Parental Daphnids at the End of the Full Test

Test concentration (mg/L)	Introduced	Mobile	% immobility
control	10	10	0
0.10	10	9	10
0.32	10	9	10
1	10	9	10
3.2	10	10	0
10	10	10	0

 

Table 2_Cumulative Mean Number of Living Offspring per Surviving Parent and Reduction of Reproduction at the End of the Full Test

Test concentration (mg/L)	Mean	St. Dev.	n	% change
control	168.0	9.5	10	-
0.10	174.6	13.8	9	3.9
0.32	177.9	15.8	9	5.9
1	172.0	10.9	9	2.4
3.2	181.5	8.7	10	8.0
10	184.4	8.3	10	9.8

 

Table 3_Incidence of Immobilised Offspring, Offspring Trapped at the Surface and Aborted Eggs at the End of the Full Test

Test concentration (mg/L)	Number of immobile spring	Number of aborted eggs
control	0	0
0.10	0	1
0.32	1	2
1	0	0
3.2	1	1
10	0	2

 

Table 4_Group Mean Body Length (mm) and Reduction of Growth of Parental Daphnids at the End of the Full Test

Test concentration (mg/L)	Mean	St. Dev.	n	% change
control	5.23	0.127	10	-
0.10	5.35	0.085	9	2.2
0.32	5.37	0.140	9	2.5
1	5.29	0.174	9	1.0
3.2	5.27	0.156	10	0.59
10	5.39	0.104	10	3.0

Validity criteria fulfilled:

yes

Remarks:

1. The mortality of the parent animals (female Daphnia) in the control did not exceed 20% at the end of the test (0%). 2. The average cumulative number of young per female in the controls after 21 days was ≥ 60 (168 ± 5.6%4).

Conclusions:

The test item did not affect reproduction, growth and survival of Daphnia magna at 10 mg/L after 21 days of exposure (NOEC).

Executive summary:

The effects of the substance on the reproductive capacity output of Daphnia magna was evaluated in a OECD TG No.211 study conducted in accordance to GLP without deviations.

Test organisms (young female Daphnia, aged less than 24 hours at the start of the test) were exposed to aqueous solutions containing the test chemical at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication observed were compared with corresponding parameters in the controls.
The test chemical was available in the form of 39% aqueous solution, all the test
solutions were generated considering the water content of the test item in the way that the end concentrations refer to the test chemical.
The definitive reproduction test was performed based on the results of a range-finding test.
The study met the acceptability criteria prescribed by the study plan and was considered valid.
An analytical method for determination of the test substance in M7 medium was developed and validated for use in the study over a concentration range 0.1 to 100 mg/L. The measured concentrations were at the level of nominal throughout

the test and therefore, effect parameters are expressed in terms of analytically confirmed nominal concentrations.

Under the conditions of the study the test item did not affect reproduction, growth and survival of Daphnia magna at 10 mg/L after 21 days of exposure (NOEC).

The NOEC for reproduction, parental mortality, parental growth, and age at first reproduction was 10 mg/L, while the LOEC of these endpoints exceeded the highest concentration tested (>10 mg t.c./L).

Due to the absence of effects, no EC10 values could be calculated.

Description of key information

The long-term toxicity of the test item cC6O4 ammonium salt to aquatic invertebrates was determined in a Daphnia magna reproduction study according to the OECD Guideline for Testing of Chemicals, No. 211 (2012).

The study was performed under GLP without deviations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Additional information

The long-term toxicity of the test item cC6O4 ammonium salt to aquatic invertebrates was determined in a Daphnia magna reproduction study according to the OECD Guideline for Testing of Chemicals, No. 211 (2012).

The study was performed under GLP without deviations.

 

The test demonstrates that the test item cC6O4 ammonium salt has no effect on the tested organisms at the maximum tested concentration of 10 mg/L.

Because of the absence of effects the EC10 could not be determined.

NOEC = 10 mg/L.