Ammonium undecafluorohexanoate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to fish, other

Remarks:

Fecunditiy/Fertility; Vitellogenin

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 - 23 Oct 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 229, October 2, 2012, "Fish Short Term Reproduction Assay"

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 10, 100 mg/L

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Required test sample was diluted in dechlorinated tap water to prepare the x20 stock solution of each test concentration. A certain amount of the stock solution adjusted by a metering pump and dilution water adjusted by diluter system was admixed to prepare the each test solution, continuously.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: medaka
- Source: in-house culture
- Age at study initiation (mean and range, SD): sexually dimorphic adult fish (18 weeks after hatching) were used
- Weight at study initiation (mean and range, SD): 313 ± 26 mg (male), 351 ± 53 mg (female)
- Feeding during test: Fish were fed ad libitum with live Artemia nauplii three times a day.

ACCLIMATION
- Acclimation period: 14 d
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Fish were fed ad libitum with live Artemia nauplii.
- Feeding frequency during acclimation: three times a day
- Health during acclimation (any mortality observed): Fish were healthy; no medicine for external disinfection was used.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

42.1 - 45.1 mg CaCO3/L

Test temperature:

24.8 - 25.1 °C

pH:

7.6 - 7.8

Dissolved oxygen:

7.5 - 8.1 mg O2/L

Nominal and measured concentrations:

nominal: control, 10, 100 mg a.i./L
measured (0 h): < LOD, 9.99, 104 mg a.i./L
measured (7 d): < LOD, 10.1, 105 mg a.i./L
measured (14 d): < LOD, 10.2, 104 mg a.i./L
measured (21 d): < LOD, 10.3, 102 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tanks
- Size of vessel: 3 L (diameter: 16 cm, depth: 17 cm)
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): constant flow using a metering pump and capillary tubes
- Renewal rate of test solution (frequency/flow rate): renewal rate: 16 times/day
- No. of organisms per vessel: 6 (3 males; 3 females)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Total organic carbon: < 0.5 mg/L
- Metals: < LOQ
- Pesticides: < LOQ
- Chlorine: < LOQ
- Alkalinity: 29 mg/L
- Conductivity: 11 mS/m
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h darkness (room light)

EFFECT PARAMETERS MEASURED
- Mortality: daily
- Fecundity and fertility: A one-week pre-exposure was performed with medaka placed in vessels similar to the actual test before the exposure. Egg production was assessed during last four days of pre-exposure by counting the number of eggs and fertility per day. Then the respective number of eggs and fertility per vessel were calculated, and the vessels were assigned to each test level as there were no statistically significant differences among test levels on their number of eggs and fertility rate. As well as pre-exposure, egg production was assessed four days a week to calculate total number of eggs and fertility per test vessel during exposure.
- Vitellogenin determination: After 21 days of exposure, all living fish were euthanized in ice water and dissected for the excision of their liver. Excised liver was stored at -80 °C until pretreatment of analysis for vitellogenin. As the pretreatment, the liver was homogenized with 50 µL of ELIZA dilution buffer per mg liver weight, and centrifuged at 13000xg for 10 minute at 5 °C (High-speed refrigerated centrifuge, CR21 Gil, Hitachi Koki). Then the supernatant was collected and stored at -80 °C until analysis for vitellogenin. VTG concentration was measured by using Medaka Vitellogenin ELISA kit (EnBio Medaka Vitellogenin ELISA system, Fujikura Kasei).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

fertility

Remarks:

fecundity

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: vitellogenin

Details on results:

- Cumulative mortality at each concentration and for each recommended observation time if possible: 5.6% at 10 mg/L (one male on day 10)
- Mortality in the controls: 0%
- Behavioural observation of the fish: No abnormal behaviour of fish was noticed.
- Effect concentrations exceeding solubility of substance in test medium: The test solution in all test levels was colorless and clear at the start and end of exposure.

Reported statistics and error estimates:

Statistical analysis on the results of mortality, total number of eggs and fertility were assessed on the basis of replicate vessel, and vitellogenin concentration was assessed on the basis of individual fish.
Statistical analysis on the results of mortality, total number of eggs and fertility were performed by Dunnett's multiple comparison test. Statistical analysis on the results of vitellogenin concentration was performed by the Mann-Whitney U test. All statistical treatments were performed with IBMID SPSS® version 22 (International Business Machines Corporation), and statistical difference was considered to be significant at p < 0.05.

The measured concentrations of the test item remained within 80 - 120% of nominal. Thus, the effect concentrations were based on the nominal concentrations.

Table 1: Total number of eggs

Concentration	Replicate	Total number of eggs	Mean	S.D.
Control	A	621	574	41
	B	546
	C	556
10 mg/L	A	633	586	73
	B	623
	C	501
100 mg/L	A	653	586	64
	B	525
	C	581

Table 2: Fertility

Concentration	Replicate	Fertility [%]	Mean	S.D.
Control	A	94.1	94.9	0.7
	B	95.3
	C	95.2
10 mg/L	A	98.7	95.6	3.2
	B	92.2
	C	95.7
100 mg/L	A	82.6	91.2	7.6
	B	97.1
	C	93.9

Table 3: Vitellogenin in female medaka after 21 d.

Fish No.	Vitellogenin concentration [ng/mg liver weight] in females after 21 d of exposure
	Control	10 mg/L	100 mg/L
A-1	44.0	831	333
A-2	50.2	601	432
A-3	48.4	2060	809
B-1	506	496	496
B-2	42.7	379	252
B-3	642	24.4	463
C-1	42.7	424	681
C-2	345	594	250
C-3	49.1	355	34.5
Mean	197	641	417
S.D.	238	575	235

Table 4: Vitellogenin in male medaka after 21 d.

Fish No.	Vitellogenin concentration [ng/mg liver weight] in males after 21 d of exposure
	Control	10 mg/L	100 mg/L
A-1	n.d.	n.d.	n.d.
A-2	n.d.	n.d.	n.d.
A-3	n.d.	-	n.d.
B-1	n.d.	n.d.	n.d.
B-2	n.d.	n.d.	n.d.
B-3	n.d.	n.d.	n.d.
C-1	n.d.	n.d.	n.d.
C-2	n.d.	n.d.	n.d.
C-3	n.d.	n.d.	n.d.
Mean	n.d.	n.d.	n.d.
S.D.	-	-	-

n.d.: not detected (< 1.00 ng/mg liver weight)
‘-‘: fish was dead

Validity criteria fulfilled:

not applicable

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling procedure: Duplicate samples (20 mL) of test media from each of the test and control vessels were taken for analysis on days 0, 1, 8, 14, 22 and 28 during the pre-hatch period, and on days 0, 7, 11, 14, 17, 21, 27 and 28 during the post-hatch period.
Samples of freshly prepared concentrated aqueous stock solutions were taken for analysis at -24 hours, Day 0, 4, 8, 12, 16, 20, 24 and 28 pre-hatch and on Day 3, 7, 11, 14, 15, 19, 23 and 27 during the post-hatch period.
Samples of expired concentrated aqueous stock solutions were taken for analysis on Day 4, 8, 12, 16, 20, 24 and 28 pre-hatch and on Day 3, 7, 11, 19, 23 and 27 during the post-hatch period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Concentrated aqueous stocks were prepared by direct addition of approx. 48, 154, 492, 1574 and 5040 mg and made to a final volume of 15 L with treated mains water at each treatment level. All media were stirred for at least 24 hours to aid dissolution of the test substance and to ensure that the test was conducted using the active moiety. This assumption was based on the known kinetics of the test substance. Complete dissociation to the active moiety is considered to occur between 12 and 24 hours following addition to water. The concentrated aqueous stock vessels were covered with black plastic to comply with the storage conditions of the test substance.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Approximately 6000 Oncorhynchus mykiss eggs were supplied, these were washed with a 0.9 g/L sodium chloride solution. Milt was added to the eggs and gently mixed. The eggs were then washed with treated mains water and transferred to an egg holding tray with continuous renewal of water at a rate of 500 mL/minute.
- Subsequent handling of eggs: The fertilised eggs were less than 24 hours old on addition to the test system. Egg addition was achieved by careful addition of the required number of eggs directly to the incubation chambers as soon as possible after receipt to avoid handling stress.

POST-HATCH FEEDING
- Start date: between days 10 and 13
- Type/source of feed: 40 g/L solution of ground Nutrafry 01; From day 14 onwards the fish were fed with Nutrafry 00 ad libitum, in excess, three times per day.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

post-hatch (pre-hatch period: 28 d)

Hardness:

50 - 75 mg/L as CaCO3

Test temperature:

11.6 - 12.9 °C

pH:

6.9 - 7.8

Dissolved oxygen:

77 - 105% ASV

Nominal and measured concentrations:

nominal: control, 0.20, 0.641, 2.05, 6.56, 21.0 mg test item/L; corresponding to control, 0.0949, 0.304, 0.972, 3.11, 9.96 mg a.i./L
measured (mean, pre-hatch): < LOQ, 0.104, 0.312, 0.929, 3.19, 10.3 mg a.i./L
measured (mean, post-hatch): < LOQ, 0.100, 0.306, 0.892, 3.04, 9.59 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 4 L glass tanks (fill volume: 3 L, sides were covered with black plastic to reduce disturbance to embryos and fry); Egg incubation chambers used in the test were 17 cm long glass cylinders (6.5 cm internal diameter) with a 0.5 – 1.0 mm non-reactive mesh attached to one end (bottom) using silicon adhesive.
- Aeration: yes
- Type of flow-through: peristaltic pump
- Renewal rate of test solution (frequency/flow rate): Following preparation each bulk test medium was pumped, using a peristaltic pump, at a rate of 1 mL/minute into a mixing vessel. Dilution water (treated mains water) was pumped at a rate of 15 mL/minute into each mixing vessel. The test medium and dilution water were mixed using a magnetic stirrer and then continually added to the appropriate test vessels. A total volume replacement is achieved every 24 h.
- No. of fertilized eggs/embryos per vessel: approx. 40 eggs
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used for conducting the definitive test was dechlorinated mains water that had been passed through particulate and activated charcoal filters and treated with ozone (O3) to remove residual algal cells and fungal spores for improved water quality.
- Pesticides: < LOQ
- Chlorine: < LOQ - 0.08 mg/L
- Culture medium different from test medium: no, same as test
- Intervals of water quality measurement: Water quality parameters of the dilution water were checked regularly; Water temperatures (°C) were measured in freshly prepared and old test media on each test media renewal occasion and were monitored continuously in two vessels (V1 (control) and V12 (nominal 21 mg/L)) using minimum / maximum digital thermometers. The pH and dissolved oxygen concentration, expressed as a percentage of the air saturation value (% ASV) and as mg/L O2, were measured in freshly prepared and old test media at each renewal. In addition, the total hardness (mg/L CaCO3) and residual chlorine were measured in fresh control media at the start of the test and at each renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h darkness

EFFECT PARAMETERS MEASURED
- Hatching success
- Survival
- Fish total lengths and dry weights

POST-HATCH DETAILS
- Begin of post-hatch period: 28 d after fertilization
- No. of hatched eggs (alevins)/replicate released to the test chamber: 28/31 (control), 31/33 (0.20 mg/L), 28/26 (0.641 mg/L), 23/27 (2.05 mg/L), 27/25 (6.56 mg/L), 22/30 (21 mg/L)
- Release of alevins from incubation cups to test chamber on day no.: Once all viable eggs had hatched across all treatments and fish were actively feeding and mobile, the egg chambers were removed (day 10 post-hatch).

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

larvae

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

larvae

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks:

dry weight

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 9.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks:

dry weight

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: Larval mortality: 4 (control), 3 (0.20 mg/L), 0 (0.641 mg/L), 2 (2.05 mg/L), 0 (6.56 mg/L), 1 (21 mg/L)
- Days to hatch or time to release of young: 25-26 d
- Numbers hatched (pooling of two replicates): 59 (control), 64 (0.20 mg/L), 54 (0.641 mg/L), 50 (2.05 mg/L), 52 (6.56 mg/L), 52 (21 mg/L)
- Percentage of surviving larvae at 28 d post-hatch: 93% (control), 96% (0.20 mg/L), 100% (0.641 mg/L), 96% (2.05 mg/L), 100% (6.56 mg/L), 99% (21 mg/L)
- Observations on body length and weight of young and/or exposed parents at one or more time periods: Body length and dry weight of fish did not differ significantly between controls and treatments.
- Effect concentrations exceeding solubility of substance in test medium: no, test solutions were clear and colorless

Reported statistics and error estimates:

Day 28 total lengths and dry weights were analysed using analysis of variance, fitting fixed effects for group and vessel within group. The test concentrations were compared with the water control using Dunnett's test. Linear contrasts were performed to compare the two vessels within each group. All tests were interpreted with two-sided risk. The number of hatched larvae and hatched larvae surviving at Day 28 post-hatch were analysed using a Fisher's exact test for pairwise comparisons of test concentrations with the control. The tests were interpreted for decreasing incidence with increasing concentration. The Lowest Observed Effect Concentration (LOEC) is defined as the lowest test concentration, which produces a statistically significant adverse effect (P<0.05) when compared with the control(s). All test concentrations above the LOEC were required to show an effect that is statistically different from the control. The No Observed Effect Concentration (NOEC) is defined as the highest test concentration, immediately below the LOEC, which does not produce a statistically significant adverse effect (P<0.05) when compared with control(s).

Measured concentrations in the concentrated stock solutions ranged between 98 and 100% of the nominal concentrations, test media concentrations ranged between 94 and 108% of nominal corresponding to geometric mean measured concentrations of 0.103, 0.310, 0.916, 3.14 and 10.1 mg/L.

Validity criteria fulfilled:

yes

Description of key information

NOEC (28 d post-hatch): ≥ 9.59 mg a.i./L (EPA OPPTS 850.1400)
NOEC (21 d): ≥ 100 mg a.i./L (OECD 229)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

9.59 mg/L

Additional information

Two experimental studies are available investigating the chronic effects of ammonium undecafluorohexanoate (CAS 21615-47-4) to fish. The key study was performed according to EPA OPPTS 850.1400 investigating the toxicity of the test item to early-life stages of Oncorhynchus mykiss. In the supporting study performed according to OECD 229 the effects of the test item on fertility/fecundity of Oryzias latipes as well as the induction of vitellogenin in male fish were investigated.
In the key study fertilised eggs of O. mykiss were exposed to the test item at concentrations of 0.0949, 0.304, 0.972, 3.11, 9.96 mg a.i./L including a control under flow-through conditions. Nominal concentrations were monitored by LC/MS-MS analysis confirming that the substance remained stable during exposure. (mean measured pre-hatch: < LOQ, 0.104, 0.312, 0.929, 3.19, 10.3 mg a.i./L; mean measured post-hatch: < LOQ, 0.100, 0.306, 0.892, 3.04, 9.59 mg a.i./L. Approximately 40 fertilized eggs were placed in egg incubation chambers and exposed in 4 L glass tanks. Once all viable eggs had hatched across all treatments and fish were actively feeding and mobile, the egg chambers were removed (day 10 post-hatch). The post-hatch phase started once all of the viable eggs were considered to have hatched in the control groups (day 28 pre-hatch). During exposure the effects of the test item on hatching success as well as survival and fish total lengths and dry weight was analysed. The statistical analysis of the data did not reveal any significant effects on all parameters. Thus, an overall NOEC (28 d) of ≥ 9.96 mg a.i./L based on mean measured concentrations was derived.
In the supporting study O. latipes was exposed to nominal concentration of 10 and 100 mg a.i./L including a control under flow-through conditions for 21 d. Sexually dimorphic adult fish (18 weeks after hatching) were used for testing (three males and three females per replicate). The total number of eggs and fertility were analysed during exposure. At the end of the study all living fish were euthanized and dissected for the excision of their liver. Subsequently the vitellogenin concentration in the liver was analysed in males and females. Total number of eggs and fertility were not significantly reduced compared to the control. Moreover, no induction of vitellogenin production was recorded in male fish indicating that the substance does not express the vitellogenin gene. Thus, this result indicates that the substance does not mimic endogenous estrogens. 
In conclusion both available studies, investigating different life stages and endpoints do not indicate any adverse chronic effects on freshwater fish.