Registration Dossier

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 28 wt%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling: Test solutions (including the well water control) and carbon filter effluent were sampled on days –2, 0, and 6, and weekly thereafter until the end of the definitive study (day 90).
- Concentrations: Each test solution concentration (including the well water control).
- Sampling method: A primary sample plus a back-up sample was collected from each replicate test chamber at all test concentrations including the well water control. In addition, one sample plus a back-up sample was collected from the carbon filter effluent.
- Sample storage conditions before analysis: Samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock solution was prepared at a nominal concentration of approximately 15 mg total fluorinated solids/L on a daily basis and was added to dilution water in a stainless steel stock tank housed in the waterbath to prepare the high test substance concentration. After mixing for approximately 20 to 30 minutes, the primary stock solution was transferred to a second stock tank or diluter supply tank housed in the waterbath. This stock solution was used for proportional dilution to yield the remaining test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Throughout the test, the stock solution was clear and colourless with no visible precipitate present. During the pre-thinning time period, settled test substance was observed on the bottom of the test chamber at all test substance concentrations. During the post-thinning time period, settled test substance was observed on the bottom of the test chamber at all test substance concentrations and, beginning on Day 71, bubbles appeared at the surface of the two highest test substance concentrations.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow trout, Oncorhynchus mykiss, unfertilized eggs and sperm were purchased from Troutlodge, Inc., Sumner, Washington. Eggs and sperm (collected from 5 female and 2 male brood fish) were received in separate plastic bags which were shipped on ice (approximately 2.8 - 6.1°C at receipt). Eggs and sperm were allowed to equilibrate to approximately 12°C in bags placed in a water bath. Approximately 200 mL of the buffer solution containing glycine, TRIS, and sodium chloride was added to the bag containing the eggs. The purpose of the buffer was to remove any ovarian fluid that could impede fertilization. The bag was placed back into the water bath for approximately 20 minutes. The bag of eggs was taken out of the water bath and the buffer solution was removed. The bag of sperm was removed from the water bath and its contents were added to the bag of eggs. The bag was gently rocked back and forth to evenly distribute the sperm. Buffer solution (50 mL) was also added to the bag to help activate the sperm. The bag was again gently rocked back and forth and returned to the water bath for about 20 minutes. The embryos were removed from the waterbath and the buffer solution and sperm poured off of the embryos. The embryos were then rinsed with cold test facility well water to remove residual sperm and other debris before being transferred to 2 stainless steel tanks, each containing 10 L of aerated test facility well water, in a water bath (12.3 and 12.4°C, respectively) under darkness. Approximately 24 hours later, embryos were randomly placed into embryo cups to begin the test. The embryonic stage at the beginning of exposure was verified by selecting a representative sample of fertilized eggs, photographing, and comparing with literature sources.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
90 d

Test conditions

Hardness:
100-140 mg/L as CaCO3
Test temperature:
12.4 to 13.4°C (mean 12.9°C)
pH:
7.6 to 8.3
Dissolved oxygen:
Dissolved oxygen concentrations ranged between 6.1 and 9.9 mg/L (56 and 92% of air saturation at 12.0°C) over the duration of the study. Test solutions were unaerated on days 0-69 and were aerated on days 70-90 as a result of dissolved oxygen levels below 60% of the saturation value at 12°C. The deviation resulting from the addition of aeration had no effect on the scientific integrity of the study or the maintenance of the test substance concentrations as demonstrated by the analytical measurements conducted during the study.
Nominal and measured concentrations:
Nominal Test Concentrations: 3.23, 6.46, 12.9, 25.5, and 51.0 mg/L test substance (corresponding to nominal total fluorinated solids concentrations of 0.95, 1.9, 3.8, 7.5, and 15 mg/L), dilution water control.
Measured Test Concentrations: 0.648, 1.21, 2.69, 5.86, and 13.4 mg total fluorinated solids/L, none detected in dilution water control (LOD, 0.001 mg total fluorinated solids/L).

Total fluorinated solids includes 28.0% amphoteric fluorinated surfactant, 1.4% impurity 1, and < 0.2% impurity 2.
Mean, measured concentration as mg total fluorinated solids/L was calculated as the average of daily mean measured values for centrifuged sample
preparations from day 0 through day 90 within a test concentration.
The level of fluorinated solids in the dilution water control was below the limit of quantitation of 0.0400 mg/L after application of a 4x dilution factor.
Details on test conditions:
TEST SYSTEM
- Embryo cups (if used, type/material, size, fill volume): 212-mL glass embryo cups (5.5-cm diameter) with screen mesh bottoms attached with silicone adhesive.
- Test vessel: Test chambers (2 replicate chambers per concentration) were 13-L stainless steel aquaria (30 [length] x 14.5 [width] x 30 [height] cm), each holding approximately 6 litres of solution (15-cm liquid depth) and fitted with a screen mesh-covered overflow pipe. A recirculating waterbath was used to maintain temperature in the test chambers at 12 ± 2°C.
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter system
- Renewal rate of test solution (frequency/flow rate): Test solutions were supplied to each replicate test chamber at a rate of approximately 1.25 L of test solution per test chamber each hour, resulting in approximately 5 test solution volume additions of 6 L (30 L total) every 24 hours.
- No. of fertilized eggs/embryos per vessel: Two embryo cups were suspended in each replicate. Twenty embryos were placed into each embryo cup (total of 40 embryos per replicate and 80 embryos per test concentration).
- No. of vessels per concentration (replicates): Two embryo cups were suspended in each replicate.
- No. of vessels per control (replicates): Two embryo cups were suspended in each replicate.
- On day 45, after swim-up had begun in the dilution water control, the fingerlings were thinned to a total of 30 fish per concentration (15 fish per replicate, 2 replicates per concentration). On day 45, feeding was initiated. The fingerlings were fed newly-hatched brine shrimp (days 45-52) or newly-hatched brine shrimp and trout chow (days 53-89).
- Biomass loading rate: Loading in the combined replicates of the dilution water control at the end of the test was 0.662 g fish per litre passing through the test chamber in 24 hours (g/L/day), based on a daily turnover rate of 30 L or approximately 5, 6-L test solution volumes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test facility well which is 480-feet deep and is cased and sealed to bedrock.
- Total organic carbon: No data
- Metals and Particulate matter: After adjustment for hardness, the water is aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 0.45-μm cartridge filters to remove particulates.
- Pesticides: Not detected at the method detection limit.
- Chlorine: Not detected at the method detection limit.
- Alkalinity: 61 - 92 mg/L as CaO3
- Ca/mg ratio: The hardness of the dilution water was adjusted to approximately 100-140 mg/L as CaCO3 by the flow-proportioned addition of CaCl2.
- Conductivity: 220 - 295 μmhos/cm
- Intervals of water quality measurement: Twice yearly for major anions and cations, metals, total organochlorine and organophosphate pesticides, and polychlorinated biphenyls

OTHER TEST CONDITIONS
- Photoperiod: Embryos and alevins were held in relative darkness until the completion of hatching on day 30. A photoperiod of 16 hours light and 8 hours darkness, which included 30 minutes of transitional light between the light and dark intervals, was used throughout the remainder of the study.
- Light intensity: 221 - 495 Lux and approximately 3 - 219 Lux between the light and dark intervals.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily observations were made for assessment of number of dead eggs, first and last day of hatching, first day of swim-up, survival, and abnormalities from hatching to thinning, and survival and abnormalities from thinning to test end. Standard length and blotted wet weight of surviving fingerlings were determined at test end.

Results and discussion

Effect concentrationsopen allclose all
Duration:
90 d
Dose descriptor:
NOEC
Effect conc.:
5.86 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: measured total fluorinated solids concentrations
Basis for effect:
other: first and last day of hatching
Duration:
90 d
Dose descriptor:
LOEC
Effect conc.:
13.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: measured total fluorinated solids concentrations
Basis for effect:
other: first and last day of hatching
Duration:
90 d
Dose descriptor:
NOEC
Effect conc.:
13.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total fluorinated solids
Basis for effect:
other: egg hatching, first day of swim-up, larval survival, and abnormalities at thinning and test end, and length and wet weight at test end
Remarks on result:
other: the highest test concentration
Duration:
90 d
Dose descriptor:
LOEC
Effect conc.:
> 13.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total fluorinated solids
Basis for effect:
other: egg hatching, first day of swim-up, larval survival, and abnormalities at thinning and test end, and length and wet weight at test end
Details on results:
- See Table 1: Summary Of Hatching, Survival, Abnormalities, And Swim-Up From Hatching To Thinning (Overall Mean Values)
- See Table 2: Summary Of Survival, Abnormalities, And Growth From Thinning To Test End (Overall Mean Values)

The nominal 0.95 mg total fluorinated solids/L replicate A2 was excluded from all statistical analyses due to 0% hatching after the eggs were inadvertently exposed to 70% ethanol in water during an embryo cup transfer.

Based on mean, measured fish tissue results on days 0-45 and days 0-90 and mean, measured test solution concentrations from day 0-48 (due to routine test solution sampling) and day 0-90, bioconcentration factors ranged from 0.637 to 3.01 L/g and from 0.410 to 1.18 L/g, respectively.
Reported statistics and error estimates:
Analyses are reported based on mean, measured total fluorinated solids concentrations and were conducted using SAS Version 8.2. All statistical tests were calculated at a significance level of p = 0.05.
The data for egg hatching and larval survival and abnormalities at thinning and test end were checked for a decreased response and a decreased trend (one-way test) relative to the dilution water control using Fisher exact comparisons with Bonferroni-Holm adjusted p values and the Cochran-Armitage test, respectively. No statistically significant differences were observed between the dilution water control and any test concentrations. The Cochran-Armitage test was used to determine the NOEC values for those parameters.
The data for first and last day of hatching and first day of swim-up were analysed for decreased or increased response (two-way test) relative to the dilution water control. The data were determined not to be normally distributed (Shapiro-Wilk test), and a non-parametric analysis was performed (Kruskal-Wallis test). Massive ties, as determined by exact permutation data analysis methods, were present and consequently an exact version of the Jonckheere-Terpstra test was used to determine the NOEC values for those parameters.
The data for length and wet weight at test end were evaluated for decreases relative to the control and were analysed grouped by replicate means. The data were found to be normally distributed (Shapiro-Wilk test) and equal variances were assumed (Levene test for variances was not possible since there were no groups with 3 or more observations, as the dilution water control and all test concentrations were tested as 2 replicates). No outliers were identified (Tukey outlier rule) for length. Outliers were identified for wet weight but were included in the analysis since the data was normally distributed with equal variances. The Jonckheere-Terpstra trend test was used to determine the NOEC values for those parameters.

Any other information on results incl. tables

Table 1: Summary of Hatching, Survival, Abnormalities, and Swim-Up from Hatching to Thinning

 

Hatching to Thinning

 

Meana, Measured Concentration (mg total fluorinated solids/L)

Hatching Day

Percent Hatch

Survival

Abnormalities

First Day of Swim-Up

 

First

Last

Number Hatching

Total

Percent

Number Alive

Number Hatching

Percent

Number Affected

Number Alive

Percent

 

Dilution Water Control

23.25

25

19.25

20

96.3

19

19.25

98.8

2

19

10.6

37.25

0.648

23.33

25

18.67

20

93.3

18.33

18.67

98.2

2.33

18.33

12.8

37.67

1.21

23.25

25

19.25

20

96.3

19

19.25

98.8

2.25

19

11.8

37

2.69

23

25.25

19.25

20

96.3

19

19.25

98.7

1.25

19

6.6

37

5.86

23.75

25.5

19.5

20

97.5

19.25

19.5

98.8

0.25

19.25

1.3

37.25

13.4

24

25.5

18

20

90.0

18

18

100.0

0.5

18

3.0

37.25

a Calculated as mean of replicate values, percent is based on mean values.

 

Table 2: Summary of Survival, Abnormalities, and Growth from Thinning to Test End

Meanb, Measured Concentration (mg total fluorinated solids/L)

Thinning to Test End

Growth of Surviving Fingerlings

 

Number

Alive

Total

Percent

Number Affected

Number Alive

Percent

Mean Standard Length ± Std. Dev. (cm)

Mean Wet Weight ± Std. Dev. (cm)

Dilution Water Control

13

15

86.7

0

13

0.0

4.9 ± 0.3

1.527 ± 0.260

0.648

14

15

93.3

0

14

0.0

4.9 ± 0.2

1.488 ± 0.157

1.21

12

15

80.0

0

12

0.0

5.0 ± 0.2

1.565 ± 0.252

2.69

13.5

15

90.0

0

13.5

0.0

5.0 ± 0.2

1.508 ± 0.197

5.86

14

15

93.3

0.5

14

3.6

4.8 ± 0.3

1.403 ± 0.269

13.4

11.5

15

76.7

0

11.5

0.0

4.9 ± 0.2

1.494 ± 0.193

b Calculated as mean of replicate values, percent is based on mean values.

Based on mean, measured fish tissue results on days 0-45 and days 0-90 and mean, measured test solution concentrations from day 0-48 (due to routine test solution sampling) and day 0-90, bioconcentration factors ranged from 0.637 to 3.01 L/g and from 0.410 to 1.18 L/g, respectively.

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The 90-day NOEC and LOEC values for first and last day of hatching based on mean, measured total fluorinated solids concentrations were 5.86 and 13.4 mg total fluorinated solids/L, respectively. The NOEC and LOEC values for all other parameters, including egg hatching, first day of swim-up, larval survival, and abnormalities at thinning and test end, and length and wet weight at test end, were 13.4 (the high test concentration) and greater than 13.4 mg total fluorinated solids/L, respectively. Day 45 and day 90 bioconcentration factors were less than or equal to 3.01 and 1.18, respectively, corresponding to log BCF values of less than or equal to 0.479 and 0.072, respectively.
Executive summary:

The effect of the test substance on hatching, growth and survival of rainbow trout, Oncorhynchus mykiss, embryos, alevins, and fingerlings was assessed in a flow-through 90-day early life-stage toxicity test. Nominal test concentrations were 3.23, 6.46, 12.9, 25.5, and 51.0 mg/L test substance (corresponding to nominal total fluorinated solids concentrations of 0.95, 1.9, 3.8, 7.5, and 15 mg/L) and dilution water control. Measured test substance concentrations were 0.648, 1.21, 2.69, 5.86, and 13.4 mg total fluorinated solids/L; none detected in dilution water control. A total of 80 embryos were exposed per concentration (20 embryos per embryo cup, 2 cups per replicate, 2 replicates per concentration) at test start. On day 45, after swim-up had begun in the dilution water control, the fingerlings were thinned to a total of 30 fish per concentration (15 fish per replicate, 2 replicates per concentration). Observations were made for assessment of number of dead eggs, first and last day of hatching, first day of swim-up, survival and abnormalities from hatching to thinning, and survival and abnormalities from thinning to test end. Standard length and blotted wet weight of surviving fingerlings were determined at test end. The No Observed Effect Concentration (NOEC) for the test substance was 5.86 mg total fluorinated solids/L based on mean, measured total fluorinated solids concentrations and first and last day of hatching. The NOEC values for other test endpoints were 13.4 mg total fluorinated solids/L, the highest tested concentration. Day 45 and day 90 bioconcentration factors were less than or equal to 3.01 and 1.18, respectively, corresponding to log BCF values of less than or equal to 0.479 and 0.072, respectively.