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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23/09/2013 (signature of the study protocol) to 02/04/2014 (date of the final test report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 211 compliant
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
During the definitive exposure, a sample of newly prepared test solutions (days 0, 2, 16 and 19) was removed from each treatment level, and control prior to division into replicate test vessels and analyzed for trifluoromethanesulfonic acid concentration. Additionally, aged test solutions were sampled and analyzed on test days 2, 5, 19 and 21 for trifluoromethanesulfonic acid concentration. Samples from aged test solutions were removed from a composite of all available replicate vessels.
Vehicle:
no
Details on test solutions:
Prior to exposure initiation and again prior to each renewal interval, a 100 mg/L primary stock solution was prepared by adding approximately 0.35 g (range of 0.3483 to 0.3514 g) of trifluoromethanesulfonic acid to 3.5 L of dilution water. Each primary stock solution was observed to be clear and colorless with no visible undissolved test substance following mixing with a stir plate and magnetic stir bar for approximately five minutes. The pH of each of the primary stock solutions was adjusted using 1.0 N sodium hydroxide or 0.10 N hydrochloric acid to a final pH between 8.0 and 8.3. Exposure solutions were prepared at exposure initiation and at each renewal interval by dilution of the primary stock solution.
Exposure solutions were mixed for approximately one minute using a glass rod prior to division into replicate exposure vessels. Following mixing, all exposure solutions were observed to be clear and colorless with no visible undissolved test substance.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magne
- Source: laboratory cultures maintained at Smithers Viscient
- Parental stock : The adult daphnids used to produce offspring for this exposure were derived from a healthy culture stock, were not from the first brood of the culture and showed no signs of stress such as high mortality, presence of males or ephippia.
- Feeding during test: yes
- Food type: Daphnids were fed unicellular green algae, Ankistrodesmus falcatus (4 x 107 cells/mL) in addition to a suspension of YCT (yeast, cereal leaves and digested flaked fish food).
- Amount and Frequency: During the exposure, the food was introduced at a rate of 200 μL of algal suspension (Ankistrodesmus falcatus; 4 x 10E7 cells/mL), and 50 μL of a yeast, cereal leaves and digested flaked fish food (YCT) suspension, once daily. This quantity of algal suspension (200 μL/daphnid/day) is equivalent to approximately 0.25 mg carbon/daphnid/day.

ACCLIMATION
No acclimatation because the culture conditions were similar to the test conditions.

QUARANTINE (wild caught)
none

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
From 170 to 180 mg/L CaCO3
Test temperature:
from 19 to 21°C.
pH:
From 7.9 to 8.6
Dissolved oxygen:
From 7.2 to 9.4 mg O2/L (from 80 to 100% of saturation)
Salinity:
Not applicable
Nominal and measured concentrations:
nominal concentrations were 6.3, 13, 25, 50 and 100 mg/L. The corresponing mean measured concentration with standard deviation in parenthesis were respectively 6.6 (0.56), 14 (0.71), 25 (1.3), 55 (4.6 and 110 (7.8) mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel:100 mL clear glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: each beaker contained 80 mL of test solution
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): evry two or three day interval.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
Culture and test dilution water were prepared in 1900-L batches by fortifying well water according to the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. The source well water is from a 100-meter bedrock well supplemented with dechlorinated Town of Wareham well water.
Fortified water was discarded if not used within 14 days of preparation. The water prepared during the definitive exposure was characterized as having a total hardness and total alkalinity range as calcium carbonate (CaCO3) of 170 mg/L and 84 to 92 mg/L, respectively, a pH range of 7.7 to 8.2, a dissolved oxygen concentration range of 9.5 to 11 mg/L, and a conductivity range of 530 to 630 microsiemens per centimeter (μS/cm). Water quality parameters were measured on each batch of fortified water prior to use. The total organic carbon (TOC) of the dilution water for November 2013 was 0.87 mg/L. Representative samples of the dilution water were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice. Daphnid cultures are maintained in water from the same source as the dilution water utilized in this study and have successfully survived and reproduced over several generations. The acceptable performance of the daphnid cultures, in combination with the previously mentioned analyses, confirmed the acceptability of this dilution water for use during the conduct of bioassays.

OTHER TEST CONDITIONS
- Adjustment of pH: see description of the stock solution preparation
- Photoperiod: The photoperiod during testing was the same as that provided in the culture area (16 hours of light, 8 hours of darkness).
- Light intensity: Light at an intensity range of 3.0 to 18 μE m-2 s-1 at the surface of the exposure solutions was provided by fluorescent bulbs. Light intensity was measured using a Licor Model LI-189 photometer. Sudden transitions from light to dark and vice versa were avoided.

EFFECT PARAMETERS MEASURED :
The number of immobilized adult daphnids and observations of abnormal behavior were recorded daily. Immobilization is defined as the inability of daphnids to swim within 15 seconds of gentle agitation of the test vessel. Numbers of offspring were determined upon the first brood release in any vessel (day 7) and daily throughout the remainder of the test. Offspring were removed, counted and discarded at each observation interval. In addition, the number of immobilized offspring and the time to first brood release were recorded for each treatment level and control. Observations of physical characteristics of test solutions (e.g., precipitate, cloudy solution) were recorded whenever the test organisms were observed, if applicable.
At exposure termination (day 21), the total body length of each surviving adult daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the shell spine using an Olympus SZ40 dissecting scope in combination with a calibrated ocular.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
A 15-day preliminary range-finding exposure was initiated at Smithers Viscient under static-renewal conditions exposing daphnids (< 24 hours old, ten replicates, one daphnid per replicate vessel) to nominal trifluoromethanesulfonic acid concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/L, and a control. Solutions were renewed every two to three days during the preliminary exposure.
Biological endpoint results from this exposure are presented below (Standard deviation presented in parentheses) :
Nominal Concentration (mg/L) : Control, 0.01 mg/L , 0.1 mg/L, 1 mg/L, 10 mg/L, 100 mg/L
Mean Percent Survival (%) : 100, 100, 100, 100, 100, 100
Mean Number of Offspring Released per Female : 78 (20), 105 (18), 102 (20), 76 (28), 99 (15), 103 (23)
Mean Total Body Length (mm) : 4.52 (0.11), 4.59 (0.15), 4.56 (0.12), 4.60 (0.14), 4.72 (0.10), 4.54 (0.13)
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Evaluation of Test Conditions:
The results of water quality determinations made during the 21-day exposure period demonstrated that the dissolved oxygen concentration, temperature, pH, total hardness, total alkalinity and conductivity of the test solutions were unaffected by the concentrations of trifluoromethanesulfonic acid tested.

Analytical Results:
Measured concentrations were generally consistent between sampling intervals and maintained the expected concentration gradient. The concentration of trifluoromethanesulfonic acid in new solutions ranged from 93 to 120% of nominal concentrations while the concentration of the test substance in aged solutions ranged from 95 to 120% of nominal concentrations. Mean measured concentrations ranged from 100 to 110% of nominal concentrations and defined the treatment levels tested as 6.6, 14, 25, 55 and 110 mg/L. As the concentration of trifluoromethanesulfonic acid has been satisfactorily maintained within ± 20 per cent of the nominal concentration throughout the test, the results are based on nominal values.
Analysis of 17 of the 18 QC samples for trifluoromethanesulfonic acid resulted in measured concentrations that were consistent with the predetermined recovery range and ranged from 90.1 to 105% of the nominal fortified levels (3.27 to 109 mg/L). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of the exposure solutions. One of the eighteen QC samples was measured at a concentration below the predetermined recovery range of 80 to 120% (51.3%). The low recovery of this single QC sample can be attributed to spurious baseline noise at the retention time of the peak of interest which interfered with its quantitation.

Biological results:
After 21 days of exposure, survival among the control organisms averaged 100%. The mean number of offspring released per female organism in the control during the 21-day test was 184 offspring per female. As demonstrated by the performance of the control, the exposure system provided conditions that were appropriate for promoting acceptable survival and reproduction of the test species and met the minimum standard criteria established by OECD guidelines (i.e., ≥ 80% survival and ≥ 60 offspring per female). Control daphnids released their first brood of offspring on test day 7. Time of the first brood release was within normal performance expectation for this species. The coefficient of variation for the control fecundity was 7.5%.
Survival among daphnids exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels all averaged 100%. No significant reduction was determined in survival among daphnids exposed to any of the treatment levels tested compared to the control (100%). The NOEC for survival was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.
Following 21 days of exposure, the organisms exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels had released a mean cumulative offspring per female of 193, 197, 201, 206 and 187, respectively. First brood release by daphnids occurred on day 7 for all treatment levels, with the exception of the 100 mg/L treatment level where first brood release occurred on day 8. Throughout the exposure, no aborted eggs, dead offspring or epiphia were observed. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in offspring per female among daphnids exposed to any of the treatment levels compared to the control (184 offspring per female). Therefore, the NOEC for reproduction was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.
Total body length at exposure termination among control daphnids averaged 4.89 mm. Total body length among daphnids exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 4.94, 4.98, 4.92, 4.90 and 4.98 mm, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in total length among daphnids exposed to any of the treatment levels compared to the control (4.89 mm). Therefore, the NOEC for total body length was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.
Reported statistics and error estimates:
All statistical analyses were conducted at the 95% level of certainty except in the case of the Shapiro-Wilks’ Test (U.S. EPA, 2002) and Bartlett's Test (U.S. EPA, 2002), in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests.

PROTOCOL DEVIATIONS

The protocol states that parental daphnids will be measured to the nearest 0.01 mm. At test termination, parental daphnids were measured to the nearest 0.05 mm. Since the 0.01 mm value in the protocol was a typographical error and since it is our standard practice to measure to the nearest 0.05 mm, this deviation is not considered to have a negative impact on the exposure.

Validity criteria fulfilled:
yes
Remarks:
≥ 80% survival and ≥ 60 offspring per female in control
Conclusions:
No effect on the reproduction to daphnia has been observed during this study. The 21d NOEC for reproduction of daphnia was set at the highest tested concentration, i.e. 100 mg/L.
Executive summary:

The purpose of the study was to estimate the effect of the test item Trifluoromethanesulfonic acid on the reproductive output of Daphnia magna according to the OECD 211 guideline and performed under GLP conditions.

Daphnia magna were exposed in a semi-static test to aqueous test media containing the test item for 21 days.

 

Ten replicates of 10 daphnids each were tested for each treatment and control group. The Daphnids were exposed to the test item at the following nominal exposure concentrations: 6.3, 13, 25, 50 and 100 mg/L. Mean measured concentrations ranged from 100 to 110% of nominal concentrations therefore the results are based on nominal values.

 

The chronic effect of trifluoromethanesulfonic acid was evaluated based on the survival, reproduction and growth (mean total body length) of the daphnids.

 

No mortality has been observed during the study. Therefore, the NOEC for survival was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.

 

Following 21 days of exposure, the organisms exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels had released a mean cumulative offspring per female of 193, 197, 201, 206 and 187, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in offspring per female among daphnids exposed to any of the treatment levels compared to the control (184 offspring per female). 

 

Control daphnids released their first brood of offspring on test day 7. First brood release by daphnids occurred on day 7 for all treatment levels, with the exception of the 100 mg/L treatment level where first brood release occurred on day 8. Throughout the exposure, no aborted eggs, dead offspring or epiphia were observed.

 

Therefore, the NOEC for reproduction was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.

 

Total body length at exposure termination among control daphnids averaged 4.89 mm. Total body length among daphnids exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 4.94, 4.98, 4.92, 4.90 and 4.98 mm, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in total length among daphnids exposed to any of the treatment levels compared to the control (4.89 mm). Therefore, the NOEC for total body length was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.

 

Description of key information

In an OECD TG211 study, the 21-day NOEC value was determined to be >= 100 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
100 mg/L

Additional information

One reliable study is available for this endpoint (Urann K., 2014). The purpose of this study was to estimate the effect of the test item Trifluoromethanesulfonic acid on the reproductive output of Daphnia magna according to the OECD 211 guideline and performed under GLP conditions.

Daphnia magna were exposed in a semi-static test to aqueous test media containing the test item for 21 days.

 

Ten replicates of 10 daphnids each were tested for each treatment and control group. The Daphnids were exposed to the test item at the following nominal exposure concentrations: 6.3, 13, 25, 50 and 100 mg/L. Mean measured concentrations ranged from 100 to 110% of nominal concentrations therefore the results are based on nominal values.

 

The chronic effect of trifluoromethanesulfonic acid was evaluated based on the survival, reproduction and growth (mean total body length) of the daphnids.

 

No mortality has been observed during the study. Therefore, the NOEC for survival was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.

 

Following 21 days of exposure, the organisms exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels had released a mean cumulative offspring per female of 193, 197, 201, 206 and 187, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in offspring per female among daphnids exposed to any of the treatment levels compared to the control (184 offspring per female). 

 

Control daphnids released their first brood of offspring on test day 7. First brood release by daphnids occurred on day 7 for all treatment levels, with the exception of the 100 mg/L treatment level where first brood release occurred on day 8. Throughout the exposure, no aborted eggs, dead offspring or epiphia were observed.

 

Therefore, the NOEC for reproduction was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.

 

Total body length at exposure termination among control daphnids averaged 4.89 mm. Total body length among daphnids exposed to the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 4.94, 4.98, 4.92, 4.90 and 4.98 mm, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in total length among daphnids exposed to any of the treatment levels compared to the control (4.89 mm). Therefore, the NOEC for total body length was determined to be 100 mg/L and the LOEC was determined to be > 100 mg/L.