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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 June 1993 - 15 June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EPA FIFRA Test Guidelines 122-2 and 123-2 (U.S. EPA, 1982) - Pesticide Assessment Guidelines, Subdivision J, Hazard Evaluation, Nontarget Plants. EPA 540/9-82-020, 27 October, U.S. EPA, Washington, DC.
Deviations:
yes
Remarks:
1. Drying fronds at 73ºC instead of 70ºC. 2. Additional chemical analysis newly prepared solutions on days 3 and 9 and from three-day-old solutions on day 12. 3. The three Quality Control samples were fortified at the single test concentration.
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:
- Concentrations: Single test concentration plus control.
- Sampling method: Samples taken from freshly prepared solutions, subsequent to division into replicate test vessels, at the start of day 0 and the start of day 3 and day 9 renewal periods. Also, composite samples from the three replicate test vessels taken from three day old solutions on day 3 and day 12. In addition three Quality Control samples were prepared at a nominal concentration equivalent to the concentration tested at each interval.
- Sample storage conditions before analysis: Samples collected on test days 9 and 12 were frozen until they were analysed 2 days after test termination.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 6.0 mg/ CGA 185072/mL stock solution was prepared by dissolving 0.3102 g of test material in 50mL of acetone. The test solution (6.0 mg CGA 185072/L) was prepared by diluting 50 µl of stock solution in Hoagland’s medium and made up to 500 ml.
- Controls: Hoagland’s solution and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 µl/L
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed
- Strain: G3
- Source (laboratory, culture collection): University of California, Los Angeles, California obtained 29 October 1992
- Age of inoculum (at test initiation): minimum of 5 days
- Method of cultivation: Cultivated in Hoagland's solution for a minimum of 5 days in 270 mL covered crystallizing dishes containing 100 mL of medium. Temperature 25 ± 2ºC and continuous illumination of 3800 to 5400 lux. Stock cultures transferred to fresh medium approximately weekly.

ACCLIMATION
- Acclimation period: 5 days
- Culturing media and conditions: same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
None
Hardness:
Not stated
Test temperature:
25 ± 2 ºC
pH:
5.0 to 6.1
Dissolved oxygen:
No details
Salinity:
Not applicable
Nominal and measured concentrations:
Range finding exposure, nominal concentrations; 0.0060, 0.060, 0.60 and 6.0 mg test substance/L. Based on data from this test a nominal concentration of 0.60 mg test substance/L was used for the Tier 1 definitive test. This concentration was selected because it is the water solubility limit of the substance in water. Mean measured concentration for definitive test was 0.42 mg/L. Blank and solvent control also included.
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: Sterile 270 mL crystallizing dishes
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 100 mL test solution used
- Type of cover: Sterile inverted glass Petri dishes
- Aeration: No details
- Agitation: No details
- Renewal rate of test solution (frequency/flow rate): Semi-static test. Test solutions and controls renewed every 3 days
- Control end cells density: 509 (SD 57)
- No. of colonies per vessel: 5
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes, Hoagland's medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water
- Total organic carbon: 2.0 to 2.7 mg/L
- Metals: Measured concentrations not considered toxic
- Pesticides: Measured concentrations not considered toxic
- Culture medium different from test medium: No
- Intervals of water quality measurement: Beginning of test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 5.0 ± 0.1 with 0.10 N sodium hydroxide
- Photoperiod: Continuous light
- Light intensity and quality: 3800 to 5400 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of frond number: Manual counting at 3 day intervals
- Determination of biomass: Fronds were dried at 73ºC for four days to determine dry weight


RANGE-FINDING STUDY
- Test concentrations: Nominal concentrations 0.0060, 0.060, 0.60 and 6.0 mg CGA 185072/L
- Results used to determine the conditions for the definitive study: Fronds densities, 588, 672, 608, and 631 respectively for each concentration, compared to control of 617 and 609 fronds for control and solvent . For definitive test nominal concentration of 0.60 mg CGA 185072/L (solubility limit of test material) used
Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: The 14 day frond density of the exposed solution was 5.2% higher than pooled control frond density.
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: The 14 day biomass of the exposed solution was 17% higher than pooled control frond biomass.
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: The 14 day frond density of the exposed solution was 5.2% higher than pooled control frond density.
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: The 14 day biomass of the exposed solution was 17% higher than pooled control frond biomass.
Details on results:
- Any visual signs of phytotoxicity (abnormalities):
- Necrosis / chlorosis: Fronds exposed to treatment level were slightly chlorotic on day 6 compared to the control.
- Any stimulation of growth found in any treatment: 5.2% increase in frond density and 17% based on biomass. Neither of these values is considered statistically significant.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance is reported to be hydrolytically stable for three days at pH 5, however the test substance may be photolytically degraded. Based on the observed decline in the test material concentration at the end of the two renewal periods, the results of this study are based on an average of the measured concentration for freshly prepared test solutions (0.42 mg/L).
- Effect concentrations exceeding solubility of substance in test medium: The water solubility of CGA 185072 at 25ºC was stated by the Study Sponsor to be 0.59 mg/L. Therefore, testing above this concentration was not carried out.

Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
A t-Test (Stokal and Rohlf, 1981, Biometry 2nd Edition, W. H. Freeman and Co., New York, NY, pp 859) was used to statistically compare 3, 6, 9, 12 and 14 day control frond density with solvent control frond density. When data was not significantly different, data from the two controls sets was pooled for further analysis. When a significant difference was found data from the solvent control was used for statistically comparison with the treatment data. A t-Test was used to compare the 14 day pooled control and treatment data for frond density and biomass.
Validity criteria fulfilled:
yes
Conclusions:
In a 14-day semi-static renewal toxicity test which assessed the impact of cloquintocet-mexyl on the growth of the duckweed Lemma gibba the EC50 based on frond density and biomass was estimated to be >0.42 mg test substance/L. The 14-day NOEC based on both frond density and biomass was determined to be 0.42 mg/L.
Executive summary:

The toxicity of cloquintocet-mexyl to the duck weed Lemna gibba was studied under GLP in a 14-day sem-static test conducted to FIFRA guideline 122-2 and 123-2. The test was conducted as a limit test with renewal of the test solution every three days. The exposure concentration was verified by analysis and results are reported based on the mean measured concentration. No statistically significant adverse effect was observed based on frond density or biomass. The EC50 based on frond density and biomass was estimated to be > 0.42 mg mg/L. The 14-day NOEC based on both frond density and biomass was determined to be 0.42 mg/L

Description of key information

14-day NOEC >0.42 mg/L (mean measured), Lemna gibba (EPA 122-2, 123-2)
14-day NOEC for main metabolite >11 mg/L, (mean measured), Lemna gibba (EPA 122-2, 123-2)

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
0.42 mg/L

Additional information

Hoburg (1993) investigated the toxicity of cloquintocet-mexyl to duckweed (Lemna gibba) in a GLP compliant study performed according to EPA FIFRA Test Guidelines 122-2 and 123-2, with minor deviations. The test was conducted as a limit test with renewal of the test solution every three days. No statistically significant adverse effect was observed based on frond density or biomass at the highest rate tested (mean measured concentration of 0.42 mg/L). Therefore, the EC50 based on frond density and biomass was estimated to be >0.42 mg/L. The 14-day NOEC based on both frond density and biomass was determined to be 0.42 mg/L.


Hoburg (1993) also investigated the toxicity of the main environmental metabolite of cloquintocet-mexyl to duckweed (Lemna gibba) under GLP and to the EPA FIFRA test guidelines. No statistically significant adverse effect was observed based on frond density or biomass at the highest rate tested (mean measured concentration 11 mg/L). Accordingly, the EC50 based on frond density and biomass was estimated to be >11 mg/L, whilst the NOEC was determined to be 11 mg/L.


Both studies are considered to be reliable without restrictions and demonstrate that cloquintocet-mexyl exhibits stronger effects to duckweed than its main metabolite. 


The 14-day study with duckweed is considered to be a short-term endpoint (ECHA, 2008: Chapter R.7b: Endpoint specific guidance). Therefore, the key value used in the Chemical safety assessment is the 14-day EC50 for cloquintocet-mexyl of >0.42 mg/L.