3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

9 July 2008 - 30 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E1193-87: Standard Guide for Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

PFBA (degradation product)

IUPAC Name:

PFBA (degradation product)

Details on test material:

- Name of test material (as cited in study report): Perfluorobutyric acid, potassium salt; PFBA, K salt; TCR07-0013 potassium salt; L16149; CAS number 2966-54-3
- Molecular formula (if other than submission substance): C4F7O2K
- Molecular weight (if other than submission substance): 252.13
- Smiles notation (if other than submission substance): [K+].FC(F)(C(F)(F)C([O-])=O)C(F)(F)F
- InChl (if other than submission substance): InChI=1/C4HF7O2.K/c5-2(6,1(12)13)3(7,8)4(9,10)11;/h(H,12,13);/q;+1/p-1

- Substance type: pure active substance
- Physical state: white solid
- Analytical purity: 98.65%
- Stability under test conditions: Stable
- Storage condition of test material: Stored under frozen conditions
- Other: potassium salt of perfluorobutyric acid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Water samples were collected from each treatment and control group at test initiation, at the beginning and the end of the longest renewal cycle each week, and at test termination. Samples of "new" solutions were collected from the bulk preparations of each test solution. Samples of "old" solutions were collected from pooled solutions from two alternate replicates of each treatment and the control group (e.g. replicates A and B or C and D, etc.). All samples were collected from mid-depth in the test solutions and placed in glass scintillation vials.

- Sample storage conditions before analysis: Samples were analyzed immediately without storage.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared at a nominal concentration of 1000 mg a.i/L by dissolving a calculated amount of PFBA in dilution water. The stock solution was stirred with a stainless steel whisk to mix, and appeared clear and colorless. Proportional dilutions of the primary stock solution were made in dilution water to prepare test solutions at nominal concentrations of 63, 125, 250, and 500 mg a.i./L. The test solutions were stirred with a stainless steel whisk to mix, and approximately 200-mL aliquots were added to each test chamber. Test solution for the negative control group was untreated dilution water. The test solutions appeared clear and colorless at test initiation and termination. All reported nominal test concentrations were adjusted for the purity of the active ingredient in the test substance.

- Controls: Negative control (test dilution water) only

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test solutions appeared clear and colorless at test initiation and termination.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Wildlife International, Ltd. internal culture
- Age at study initiation (mean and range, SD): <24 hours
- Method of breeding: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.1 to 20.9°C, the pH of the water ranged from 8.3 to 8.8, and dissolved oxygen ranged from 7.0 to 8.9 mg/L (>78% of saturation). Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media, trout chow, and the green alga Pseudokirchneriella subcapitata. The adults were fed prior to test initiation. The seven adult daphnids used to supply neonates for the test were held for at least 21 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress during the holding period.
- Feeding during test: Yes
- Food type: Mixed, a mixture of yeast, cereal grass media, and trout chow with a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Frequency: Once daily

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

134 - 140 mg/L as CaCO3

Test temperature:

19.5 °C - 20.7 °C

pH:

8.1 - 8.5

Dissolved oxygen:

7.4 - 9.1 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: Negative control, 63, 125, 250, 500, 1000 mg/L
Measured concentrations: See Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Material, size, headspace, fill volume: Glass, 250 mL containing 200 mL of test solution. Solution depth was 7.7 cm.
- Type (delete if not applicable): open / closed : Loosely covered
- No. of organisms per vessel: One
- No. of vessels per concentration (replicates): Ten
- No. of vessels per control (replicates): Twenty

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water from a well approximately 40 meters deep was passed through a sand filter, pumped into a 37,800-L storage tank, and aerated. Prior to use, the water was filtered to 0.45 µm and passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: ≤1 mg/L
- Particulate matter: Not reported
- Metals: See Table 2
- Pesticides: See Table 3
- Chlorine: Not reported
- Alkalinity: 171 -182 mg/L as CaCO3
- Conductivity: 353-375 µmhos/cm

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod:16 hours of light and 8 hours of darkness, with 30-minute transitions periods of low light intensity.
- Light intensity: 391 lux at the surface of the water of one representative test chamber at test initiation. Light was provided by Colortone® 50 fluorescent bulbs which emit wavelengths similar to natural sunlight. Test chambers were indiscriminately positioned in a temperature-controlled environmental chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

First-generation daphnids: Observations were made daily during the test. The number of dead and immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming, lethargy, or opaque color). The presence of eggs in the brood pouch, aborted eggs, males or ephippia was recorded daily. The body length and dry weight of each surviving first-generation daphnid were measured at the end of the test.

Second-generation daphnids: With the onset of reproduction, neonates were counted and then discarded every Monday, Wednesday and Friday during the test and at test termination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2-fold

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

239 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

Reproduction data summary in Table 4

NOEC for mortality, 962 mg/L
- Behavioural abnormalities: See Table 5
- Observations on body length and weight: No statistically significant differences (p > 0.05) were found between controls and test samples with respect to surviving 1st generation Daphnid length and dry weight. See Table 6
- Other biological observations: See Table 5
- Mortality of control: 15% mortality, within guideline criteria
- Other adverse effects control: See Table 5

Reported statistics and error estimates:

Survival data was considered to be discrete-variable data, while reproduction and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher's Exact test to identify treatment groups that showed a statistically significant difference (p ≤ 0.05) from the control. All continuous-variable data were evaluated for normality using the Chi-Square test and for homogeneity of variance using Levene's test (p = 0.01). The reproduction, length and dry weight data passed the assumptions of normality and homogeneity. Analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (p = 0.05). Those treatments that were significantly different from the control means were identified using Bonferroni's t-test or Dunnett's test (p≤0.05). All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

Any other information on results incl. tables

Table 4, Summary of Reproduction of Surviving First-Generation Daphnids
Mean Measured Test Concentration (mg a.i./L)	First Day of Reproduction	Number of Surviving Adults	Mean Number of Live Young Per Surviving Adult ± Std. Dev.
Negative control	8	17	196.9 ± 55.74
59	8	10	203.5 ± 73.32
120	8	9	205.2 ± 36.37
239	8	9	141.1 ± 83.15
476	8	9	111.3 ± 62.29*
962	8	9	108.9 ± 57.36*
* Statistically significant differences (p < 0.05) relative to negative control (Dunnett’s Test).

 

Table 5, Summary of Cumulative Percent Mortality, Immobility and Observations of First-Generation Daphnids
Mean Measured Test Concentration (mg/L)	Number exposed	Day 7			Day 14			Day 21			Percent survival
		Percent Dead	Percent Immobile	Obs^1	Percent  Dead	Percent Immobile	Obs	Percent  Dead	Percent Immobile	Obs
Negative Control	20	0	0	20 AN	5	0	1S; 18 AN	15	0	1S; 16 AN	85
59	10	0	0	1S; 9 AN	0	0	1S; 9 AN	0	0	1S; 9 AN	100
120	10	0	0	10 AN	0	0	10 AN	10	0	1D; 8 AN	90
239	10	0	0	2S; 9 AN	10	0	2S; 7 AN	10	0	2S; 7 AN	90
476	10	0	0	1S; 2D; 2C; 5 AN	0	0	1S; 1D; 8 AN	10	0	1S; 2D; 7 AN	90
962	10	0	0	1S; 4D; 2C,D; 3 AN	0	0	1S; 9 AN	10	0	1S; 8 AN	90
1, Observations: AN = appear normal; S = small in comparison to control organisms; C = lethargic; D = discoloration, daphnid appears pale in color.

 

Table 6, Summary of Length and Dry Weight of Surviving First-Generation Daphnids
Mean Measured Test Concentration (mg a.i./L)	Number of Surviving Adults	Mean Length^1 ± Std. Dev. (mm)	Mean Dry Weight^1 ± Std. Dev. (mg)
Negative control	17	4.5 ± 0.41	0.71 ± 0.16
59	10	4.4 ± 0.64	0.68 ± 0.20
120	9	4.5 ± 0.23	0.84 ± 0.22
239	9	4.1 ± 0.88	0.76 ± 0.32
476	9	4.1 ± 0.53	0.68 ± 0.22
962	9	4.0 ± 0.53	0.77 ± 0.24
1, No statistically significant differences (p < 0.05) relative to negative control (Dunnett’s Test).

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC (reproduction) for PFBA-K salt to Daphnia magna was 239 mg/L

Executive summary:

The chronic toxicity of PFBA to Daphnia magna was measured according to a method based on OPPTS 850.1300 and on OECD TG211. PFBA is a strong acid and is entirely deprotonated at environmental pH. The toxicity was measured using the potassium salt of PFBA. This procedure is equivalent to a toxicity test utilizing neutralized PFBA acid. Daphnid neonates were exposed to measured concentrations of < LOQ, 59, 120, 239, 476, or 962 mg/L with periodic renewal of test medium. The 21-day NOEC for reproduction was 239 mg/L.

This study was based on an internationally accepted test guideline and conducted in accord with GLP criteria. Therefore, this study is reliable without restrictions.

Results Synopsis

Test Type: semi-static

NOEC: 239 mg/L

Probit Slope: not applicable