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Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27/Mar/2002-03/Apr/2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The work reported was performed carefully, the results reasonable, and the conclusions conservative. Data was generated according to generally valid and internationally accepted testing guidelines.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))
Deviations:
yes
Remarks:
The total organic carbon concentration of the 20X-AAP media was greater than 2.0 mg/L. This deviation did not affect the outcome of the test.
Principles of method if other than guideline:
Although the cover of the report states "OECD 201" underneath the title, the body of the report clearly identifies the method followed as EPA OPPTS 850.4400. The OECD201 method is for determination of algal toxicity. It is believed that the phrase "OECD 201" on the report cover is an error is likely carried over from parallel algal studies conducted in the same period.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
PFPA (degradation product)
IUPAC Name:
PFPA (degradation product)
Details on test material:
- Name of test material (as cited in study report): T-7479
- Physical state: Clear colourless liquid
- Analytical purity: 99.93%
- Stability under test conditions: Hydrolyzes to perfluoropropionic acid (PFPA). PFPA is stable in water over the test period.
- Storage condition of test material: At room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Analysis was of perfluoropropionic acid (PFPA). The test substance hydrolyzes with a half life of less than 2.5 minutes to water-soluble perfluoropropionic acid (PFPA) and a volatile hydrofluorocarbon (heptafluoropropane). Therefore, the test solutions were monitored for PFPA concentrations during the study. It should be noted that the nominal concentrations presented in the report do not reflect the concentration of PFPA but that of the test substance prior to hydrolysis.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentration: 500 mg/L nominal concentration of T-7479.
- Sampling method: After preparation of the stock solution, two 60-mL aliquots were taken. The stock solution was then filter-sterilized (0.2 micrometer) and two additional aliquots were taken on 12March2002. Duplicate samples were taken of stock solutions at the beginning of the test (27March2002), and a single 60-ml aliquot was taken from each test vessel at the end of the test (03April2002). Samples from test vessels were glass fiber-filtered to remove plant material, whereas the blank sample was not.
- Sample storage conditions before analysis: Samples were stored in darkness under refrigerated conditions until shipment to sponsor for analysis. Samples were shipped in a cooler with ice by overnight courier and recieved 13March2002 and 04April2002. Sponsor lab stored samples at a nominal 4°C until analysis on 24March2002 and 16April2002.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 1.0020 g of test substance was added to 2,000 mL of enriched 20X AAP algal medium in an uncovered 1-gallon glass jar. The solution was mixed for approximately 24 hours on a magnetic stir plate which was isolated from the bottom of the mixing vessel to avoid excess heating of the solution. After mixing, the solution was allowed to settle for approximately 30 minutes. Insoluble test substance was observed as globules on the bottom of the mixing vessel prior to decanting the liquid fraction. The decanted liquid fraction was mixed for an additional 30 minutes. Two 60 mL aliquots were then taken and shipped to the sponsor for analysis. The remainder of the solution was then filtered through a 0.2 micrometer filter. Two 60 mL aliquots of the filtered stock solution were collected and also sent to the sponsor for analysis. The remainder was placed into a 2 liter Nalgene bottle and stored refrigerated in the dark for 14 days until test initiation. Prior to test initiation, stock solution pH was adjusted from 8.2 to 7.5 with 0.1N HCl.
The undissolved material from the initial stirring step was solublized with acetone and diluted with additional 20X AAP medium. These additional samples were shipped to the sponsor for possible analysis at the sponsor's discretion.
- Controls: negative control only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No insoluble material was observed during the definitive toxicity test.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed
- Strain: G3
- Source (laboratory, culture collection): Climate Stress Laboratory, USDA, Beltsville, MD
- Age of inoculum (at test initiation): 9 days, actively growing inoculum
- Method of cultivation: Transfer to fresh medium approximately every 7 days.
ACCLIMATION
- Acclimation period: 14 days
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
7 d
Post exposure observation period:
None

Test conditions

Hardness:
Not reported
Test temperature:
24.4-24.9°C
pH:
7.5 initial, 9.2 final (controls)
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
500 mg/L T-7479 were stirred with 20X-AAP medium 24 hours, but did not completely dissolve. Undissolved test substance was removed by decanting and filtration. Dissolved test substance hydrolyzes completely with a half life of less than 2.5 minutes to water-soluble perfluoropropionic acid (PFPA) and a volatile hydrofluorocarbon (heptafluoropropane). Mean measured concentration of PFPA was 17.7 mg/L in the stock solution (Table 2)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks capped with inverted beakers
- Material, size, headspace, fill volume: 200 mL of test solution, depth approximately 4 cm in 500 mL flask
- Aeration: daily swirling
- No. of organisms per vessel: five, three fronds per plant (15 fronds in total)
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): three
GROWTH MEDIUM
- Standard medium used: 20X-AAP
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution was in 20X-AAP medium
- Total organic carbon: 4.0 mg/L
- Particulate matter: Filter sterilized at 0.2 micrometer
- Metals: See Table 1
- Pesticides: See Table 1
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: biannual
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 24 hour (continuous)
- Light intensity and quality: 500 footcandles (5220-5440 lux), cool white fluorescent. Flasks randomly repositioned daily.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: Total number of fronds (parental fronds and fronds that visibly projected beyond the edge of the parental frond). Chlorotic fronds. Dry weight of fronds. Presence of flowers. Abnormalities.
-Observation intervals: observations after three, five and seven days. Frond dry weight measured after day seven only.
TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study: none
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 17.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: hydrolysis product
Basis for effect:
other: growth rate, EC50 based on nominal concentration of CAS# 756-13-8 = > 500 mg/L
Details on results:
Concentration of hydrolysis product, PFPA. Dissolved test substance hydrolyses completely with a half life of less than 2.5 minutes to water-soluble perfluoropropionic acid (PFPA) and a volatile hydrofluorocarbon (heptafluoropropane). No insoluble material observed during the definitive test.

- Exponential growth in the control: yes
- Observation of abnormalities: none observed
- Other: No flowering was observed in either controls or test plants. Two chlorotic fronds in total among controls and test plants. Average dry mass of fronds in test chambers was 89% of control. This difference was not statistically significant according.
- Any stimulation of growth found in any treatment: No
- Effect concentrations exceeding solubility of substance in test medium: The test substance is of very low water solubility. Dissolved test substance hydrolyzes completely to PFPA in water.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
All statistical analyses were performed using the number of normal, non-chlorotic duckweed fronds or the dry weight of fronds and the nominal concentrations of test substance. The 7-day EC50 values and 95% confidence intervals could not be calculated because there was greater than 50% of control growth at the limit concentration. The 7-day NOEC values for duckweed growth (number of normal, non-chlorotic fronds and total dry weight) were determined using a t-test.

Any other information on results incl. tables

The study report concludes that the NOEC based on frond number is 17.7 mg/L. Average specific growth rates were not presented in the study report. Growth rates were calculated by the sponsor after the report was issued, using methods described in OECD TG211, Lemna sp. Growth Inhabition Test (adopted 23 March 2006) using Microsoft Excel. Reanalysis of the data using Student's t-test indicates that the 16% reduction in total number of fronds relative to controls on day seven is statistically significant (P=0.03). Comparison of average specific growth rates on day seven shows a 7.4% reduction by day 7 (P=0.03). The reported NOEC is therefore not valid.

Tables 4 & 5, Plant biomass and average specific growth rate data from study report.

TABLE 4:

Table 4. Frond growth data from the toxicity test with test substance and the freshwater plantLemna gibba.
Nominal Concentration of test substance (mg/L) Measured Concentration of
PFPA (mg/L)		 Rep Number of fronds Total dry weight of fronds (mg)
Day of Exposure
0 3 5 7
Total Chlorotic Total Chlorotic Total Chlorotic Total Chlorotic
0 below LOQ1 1 15 0 54 0 118 0 189 1 19.2
(control) (control) 2 15 0 49 0 121 0 194 1 19.3
    3 15 0 51 0 98 0 176 0 17.1
    mean 15   51   112   186   18.5
    std. dev. 0   2.5   12.5   9.3   1.24
                       
500 17.7 1 15 0 40 0 82 0 150 1 15.8
    2 15 0 48 0 101 0 162 0 15.9
    3 15 0 46 0 89 0 141 0 15.5
    4 15 0 42 0 79 0 125 1 14.0
    5 15 0 54 0 107 0 182 0 18.5
    6 15 0 50 0 103 0 175 0 18.7
    mean 15   47   94   156   16.4
    std. dev. 0   5.2   11.8   21.4   1.84
    % control 100   92   84   84   89
    P-value     0.10   0.03   0.03   0.06
1. LOQ was 0.209 mg/L

TABLE 5:

Table 5, Average and segment specific growth rates based on total number of fronds (1/day) from the toxicity test with test substance and the freshwater plantLemna gibba.1
Nominal Concentration of test substance (mg/L) Measured Concentration of
PFPA (mg/L)		 Rep Average specific growth rate (1/day) Segment-specific growth rate (1/day)
Day of Exposure Day of Exposure
3 5 7 3 5 7
0 below LOQ1 1 0.427 0.413 0.362 0.427 0.391 0.236
(control) (control) 2 0.395 0.418 0.366 0.395 0.452 0.236
    3 0.408 0.375 0.352 0.408 0.327 0.293
    mean 0.410 0.402 0.360 0.410 0.390 0.255
    std. dev. 0.016 0.023 0.007 0.016 0.063 0.033
                 
500 14.2 1 0.327 0.340 0.329 0.327 0.359 0.302
    2 0.388 0.381 0.340 0.388 0.372 0.236
    3 0.374 0.356 0.320 0.374 0.330 0.230
    4 0.343 0.332 0.303 0.343 0.316 0.229
    5 0.427 0.393 0.357 0.427 0.342 0.266
    6 0.401 0.385 0.351 0.401 0.361 0.265
    mean 0.377 0.365 0.333 0.377 0.347 0.255
    std. dev. 0.043 0.033 0.030 0.043 0.023 0.021
    % inhibition 8.1 9.3 7.4 8.1 11.1 0.022
    P-value 0.10 0.04 0.03 0.10 0.08 0.50
1. Values were calculated by the sponsor according to methods in OECD TG221 (adopted 23March2006) after issue of the final study report.
2. LOQ was 0.209 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Lemna gibba to CAS# 756-13-8 resulted in 7 day EC50s (based on number of normal fronds and frond dry weight) that were greater than the nominal loading rate. CAS# 756-13-8 was added to the stock solutions at 500 mg/L, which is in excess of water solubility. The dissolved material hydrolyzed completely with a half life of less than 2.5 minutes to water-soluble perfluoropropionic acid (PFPA) and a volatile hydrofluorocarbon (heptafluoropropane). PFPA was quantified at 17.7 mg/L in the stock solution. This concentration of perfluoropropionic acid had a statistically significant effect on growth rate at five and seven days but did not have a significant effect on average frond dry mass or number of chlorotic fronds. No NOEC can be determined from this study
Executive summary:

The 7-day toxicity of CAS# 756-13-8 to Lemna gibba was studied under static conditions. Lemna gibba was exposed to a negative control and CAS# 756-13-8 at a nominal concentration of 500 mg/L for seven days.  Dissolved CAS# 756-13-8 hydrolyzes completely with a half-life of 2.5 minutes to perfluoropropionic acid (PFPA) and a volatile hydrofluorocarbon (heptafluoropropane). PFPA concentration was monitored by HPLC-MS. The measured exposure concentration of PFPA was 17.7 mg/L. The 7-day EC50 based on average specific growth rate were >17.7 mg/L as PFPA.  The 7-day NOEC based on average specific growth rate could not be calculated because there was statistically significant inhibition at the only concentration tested.

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with algae.

 

Results Synopsis

 

Test Organism Age: nine days

Test Type: Static

                         

EC50:  >17.7 mg/L as PFPA                      95% C.I.:  not applicable

NOEL:  not calculated                                 Probit Slope:  not applicable

Endpoint(s) Effected:  Average specific growth rate