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

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-02-20 until 2011-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch number of test material: cC6O4 Sale di ammonio 3 ac.
- Purity test date: 21-03-2011
- Expiry date: 2013-12-31

Appearance: Liquid, pale amber – yellowish brown, clear
Stability under test conditions: The test item is an aqueous solution. Therefore, stability under test conditions is assumed.

Recommended storage: 18 ± 4 °C, humidity < 65 %
Storage at test facility: Room temperature, protected from moisture and light.
Retention of test item: At least 1 g was retained and stored at 6 ± 2 °C.

Radiolabelling:

no

Details on sampling:

Four fish samples were taken from each tank on days 0.5, 1, 2, 4, 6, 8, and 14 of the uptake phase as well as on days 0.33, 1, 2, 4 and 6 of the depuration phase. The fish were weighted and stored at ca. -18°C until analysis, if necessary. Fish from day 6 were not analysed, because the measured concentrations of cC6O4 ammonium salt in fish was below the limit of quantification throughout the study. Aqueous phase samples were taken and analysed on uptake phase days 0.5, 1, 2, 4, 6, 8, 14 and on days 0.33, 1, 2, 4 and 6 of the depuration phase.

The lipid content of the fish was determined at the start of the study (day 0), at the end of the uptake (study day 17) phase and at the end of the depuration phase (study day 24) from 3 fish (one fish per test group). The lipid content of the food is provided by the manufacturer: 15 % (raw fat).

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

A flow-through exposure design was carried out.
Solvent: with regard to the high solubility of the test item no organic solvent was needed. The stock solutions were prepared with demineralised water.
The test concentrations in terms of dry weight were 0.0361 and 0.361 mg/L, the latter one being ≤ 1 % of the acute LC50 (the LC50 of the test item (acute toxicity to fish) is > 100 mg/L.

One order of membrane piston pumps provided the water flow through. Application of the stock solutions was carried out via a multi channel tubing pump. The stock solutions were gently homogenized with a magnetic stirrer during application.

Test organisms (species):

other: Oncorhynchus mykiss (Rainbow trout), Gnathostoma, Pisces, Osteichthyes, Teleostei, Salmoniformes, Salmonidae

Details on test organisms:

All fish used in the test originated from the same delivery of the supplier.
103 fish were tested per group. The fish were taken from a pool larger than needed for the study to ensure quality of the organisms.
Age at test start : 12 weeks

The nominal loading did not exceed a range of 0.1 to 1.0 g of fish (wet weight) per liter per day.

Husbandry and acclimation: Holding was performed at the test facility at 13 - 17 °C, diffuse light (0.1 - 10 µmol*m-2 *s -1, diurnal light with 16 h light / 8 h dark) and under flow-through conditions. The dissolved oxygen concentration was more than 80 % of the air saturation value. Only rainbow trout with at least 12 days of acclimation and mortality < 5 % within the last 7 days before the study started were used in the test.

Water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Water parameters:
pH-value: 6.0 - 8.5
Hardness 10 - 250 mg CaCO3 / L

Feeding: Feeding was carried out working daily with an amount of 2 % of the initial fish weight six days before start of the study (uptake phase).
Food: F – 1,8 Pro Aqua Brut. The product is composed of fish products, oils and fats, cereal grains, oil seed products, byproducts, minerals and vitamins.

The fish were fed daily in two feedings. The given feed amount (2 % of body weight) was documented. The amount of food was adjusted accordingly to the weights of sacrificed fish to account for growth during the experiment.

Fish were introduced randomly to the test groups.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

17 d

Total depuration duration:

7 d

Hardness:

10 - 250 mg CaCO3 / L

Test temperature:

15 ± 2 °C

pH:

6.0 - 8.5

Dissolved oxygen:

Not less than 60 % of air saturation value.

TOC:

Two times before the uptake phase started: -48 and -24 h and weekly thereafter (in all vessels).
0.9-1.0 mg/L in the Test Vessels

Details on test conditions:

Test vessel Glass aquaria with a water volume of about 200 L and covered by glass tops were used.
Test volume About 120 L
Replicates One replicate per test group
Aeration The test vessels were aerated.
Test water Same as for holding
Photoperiod Diurnal light with 16 h light / 8 h dark was provided.
Light intensity 0.1 - 10 µmol x m x -2 x s -1

Three groups of fish were tested: two concentrations of the test item and one control group. Based on the chosen test concentrations any possible effects due to test item related toxicity could be excluded. Moreover, the chosen test concentrations allowed a safe determination of residues of the test item in the fish which could have detected any possible accumulation in fish.

Equilibration period: test solutions flowed through the test vessels for 17 days prior to exposure.
Flow through conditions: a continuous water flow of 25 L/h was provided, resulting in 5 water exchanges per day.

Nominal and measured concentrations:

Two nominal concentrations of 0.10 and 1.0 mg/L were tested, corresponding to dry salt concentrations of 0.0361 and 0.361 mg/L.
The measured concentrations were respectively 0.036 mg/L (SD+- 0.02, CV% 5.6) and 0.37 (SD+- 0.2, CV% 5.4).

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Fish growth rates have been calculated. No uptake of cC6O4 ammonium salt in fish was observed. Therefore, no uptake / depuration rates and no bioconcentration factors (steady state and kinetic, respectively) have been calculated. Bioconcentration factors at any sampling date during the uptake phase have been calculated based on fish body weight.

Lipid content:

2.06 %

Time point:

start of exposure

Lipid content:

3.03 %

Time point:

end of exposure

Lipid content:

3.02 %

Time point:

other: end of depuration phase

Key result

Conc. / dose:

0.036 mg/L

Type:

BCF

Value:

<= 2.8 dimensionless

Basis:

whole body w.w.

Time of plateau:

17 d

Calculation basis:

other: a concentration of LOQ/2 was assumed in fish tissues

Remarks on result:

not determinable

Remarks:

BCF could not be determined because of the lack of uptake

Conc. / dose:

0.361 mg/L

Type:

BCF

Value:

<= 0.25 dimensionless

Basis:

whole body w.w.

Time of plateau:

17 d

Calculation basis:

other: a concentration of LOQ/2 was assumed in fish tissues

Remarks on result:

not determinable

Remarks:

BCF could not be determined because of the lack of uptake

Details on results:

- Fish Observations, Mortality and non-lethal Effects of test organisms:
One dead fish was observed and removed on study day 8 in the nominal concentration level of 0.10 mg/L (corresponding to 0.0361 mg/L dry salt concentration). In other concentration and control no dead organisms were observed. However this mortality was considered not significant.
Therefore no significant mortality was observed throughout the study and no significant non lethal effects (morphological and behavioural) were found at the test item groups and at the control group.
- Feeding Behaviour: No anomalous feeding behaviour was observed throughout the study in any one of the test groups.
- Growth Rates: Initial mean wet weight and length of 10 representative fish sampled from the stock fish at test start was 1.018 g and 4.47 cm. At each sampling date wet weight and length of the sampled fish were determined. The mean wet weight of the control group increased to 1.3104 g, the mean length increased to 5.23 cm.
Weight data of the control and test item group were converted to natural logs and plotted vs. day. A linear correlation was calculated for the ln(fish wet weight) vs. day. The variances in the slopes of the test and control lines were calculated and used to evaluate the statistical significance of the difference in the slopes (growth rates). Since there was no significant difference, the test and control data were pooled and an overall fish growth rate for the study (kgrowth) calculated as the overall slope of the linear correlation (Table 1).

-Depuration phase: No depuration rates were calculated since no uptake was observed.
-BCF: The bioconcentration factors at any time during the uptake phase were determined. No uptake was observed. The BCF any time was below 1 throughout the whole study. No kinetic BCF and no steady state BCF have been calculated (Table 2).

Table 1: Growth rates for the control and the test item concentrations (referred on dry salt)

 

	Control	0.0361 mg/L	0.361 mg/L	Overall
Growth rate  (=Slope)	0.0165	0.0264	0.0245	0.0225
Stat. significance	No			n.a.
Intercept	0.0470	-0.00359	-0.0223	0.00705
CI	0.00439 – 0.0287	0.0129 – 0.0398	0.0128 – 0.0362	0.0155 – 0.0294

CI = Confidence interval, p = 95 %  n.a. = not applicable

Analytical Results

Water

The concentrations ofcC₆O₄-NH₄in the aqueous phase were measured during the uptake and depuration phase. A mean value of 0.036 ± 0.002 mg/L (100% of the nominal value) was obtained in the exposure group with nominal0.0361 mgcC₆O₄-NH₄/L. All concentrations stayed within ± 20% of the mean value fulfilling therefore one validity criterion of the corresponding test guideline.A mean value of 0.37 ± 0.02 mg/L (102%) was obtained in the exposure group with nominal0.361 mgcC₆O₄-NH₄/L. Also all concentrations stayed within ± 20% of the mean value.During the depuration phase samples from day 1, 2, 4 and 6 confirmed the absence ofcC₆O₄-NH₄in the test vessels. The concentrations ofcC₆O₄-NH₄in the test vessels during the uptake phase are summarised intable(all referred to dry salt).

 

 

Study Day	Control	0.0361 mg/L	0.361 mg/L
Uptake 0.5	< LOQS	0.0343	0.383
Uptake 1	< LOQS	0.0361	0.352
Uptake 2	< LOQS	0.0331	0.349
Uptake 4	< LOQS	0.0394	0.401
Uptake 6	< LOQS	0.0357	0.355
Uptake 8	< LOQS	0.0372	0.376
Uptake 14	< LOQS	0.0368	0.379
Mean	-	0.036	0.37
SD ±	-	0.002	0.02
CV [%]	-	5.6	5.4

 

Fish

The mean concentrations of cC6O4 ammonium salt in relation to body weight were throughout the uptake phase below the LOQ_M(=0.181 µg/g body weight) in both exposure groups (per sampling day, n=4). Fish extracts (n=4) from the control vessel were measured in parallel to confirm the absence ofcC₆O₄-NH₄.All obtained data were < LOQ_S.

  

Food

Three food samples were analysed to determine ifcC₆O₄-NH₄was present in the fish food. The results demonstrated that the food as source for the uptake ofcC₆O₄-NH₄can be ruled out.

BCF

The bioconcentration factors at any time during the uptake phase were determined. No uptake was observed. The BCF any time was below 1 throughout the whole study. No kinetic BCF and no steady state BCF have been calculated.

Table 2. log BCF Body weight based any time (BCFat)

Test Item Concentration	0.0361 mg/L	0.361 mg/L
Calculated from Uptake Day	Log BCFat*)
0.5	0.42	-0.62
1	0.40	-0.59
2	0.44	-0.59
4	0.36	-0.64
6	0.40	-0.60
8	0.39	-0.62
14	0.39	-0.62

*) All calculations were done using ½ LOQ (= 0.091 mg dry salt/L) as concentration in fish since for all
  analysed fish the measured concentration was below the LOQ.

Validity criteria fulfilled:

yes

Conclusions:

cC6O4 ammonium salt did not bioaccumulate in rainbow trout (whole) over a period of 17 days when tested with two concentrations.
The log BCF any time did not exceed 0.44.

Executive summary:

A bioaccumulation study with rainbow trout (Oncorhynchus mykiss) was conducted to determine the bioconcentration potential of cC₆O₄-NH₄ .

The study consisted of two phases: the exposure (uptake phase) and post exposure (depuration) phase. The definitive study was conducted according to OECD 305 (1996). A flow-through test with 3 groups (one control group and two exposure groups) was carried out. Two nominal concentrations of 0.10 and 1.0 mg/L were tested, corresponding to dry salt concentrations of 0.0361 and 0.361 mg/L. The study was conducted over 24 days. The uptake phase lasted 17 days and the depuration phase lasted 7 days. All validity criteria required in the guideline were fulfilled.

The aqueous concentrations were stable throughout the uptake phase.

Fish growth rates have been calculated. No uptake of cC6O4 ammonium salt in fish was observed (Test item concentrations were below the limit of quantification in all fish samples). Therefore, no uptake / depuration rates and no bioconcentration factors (steady state and kinetic, respectively) have been calculated. Bioconcentration factors at any sampling date during the uptake phase have been calculated based on fish body weight.

The calculation were done using 1/2 LOQ (=0.091 mg dry salt/kg) as concentration in fish. The max calculated Log BCF was 0.44 corresponding to a BCF <= 2.8.

Description of key information

A bioaccumulation study with rainbow trout (Oncorhynchus mykiss) was conducted to determine the bioconcentration potential of cC6O4 ammonium salt. The study was conducted according to OECD 305 (1996). All validity criteria required in the guideline were fulfilled. No uptake of cC6O4 ammonium salt in fish was observed (Test item concentrations were below the limit of quantification in all fish samples) therefore, the uptake / depuration rates and the bioconcentration factors (steady state and kinetic, respectively) could not be calculated. However, bioconcentration factors were calculated based on fish body weights and considering 1/2 LOQ (=0.091 mg dry salt/kg) for the concentration in the fish tissues. The max calculated Log BCF was 0.44 corresponding to a BCF <= 2.8.

BCF = 2.8 is considered as key value for risk characterization purpose.

Key value for chemical safety assessment

BCF (aquatic species):

2.8 dimensionless