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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
yes
Remarks:
The deviation had no adverse impact on the results or interpretation of the study.
Qualifier:
according to
Guideline:
other: EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
The deviation had no adverse impact on the results or interpretation of the study.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test initiation (0 hour), and at day 3 and day 5 of the exposure, a sample was removed from each test concentration, the blank control and the solvent control and analyzed for the test substance. The aged solution for the abiotic stability control (1000 μg a.s./L) was sampled and analyzed at day 3 and 5. Samples analyzed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels. Samples analyzed at the end of each renewal period were removed from the composited replicate solutions of each test concentration and control. Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations of the test substance approximating the test concentration range and remained with the exposure solution samples throughout the analytical process.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 20 mg a.s./mL primary stock solution was prepared prior to test initiation by adding 0.5035 g of the test substance (0.5000 g a.s.) to a 25-mL volumetric flask and bringing it to volume with dimethylformamide. Secondary stock solutions were prepared by adding variying volumes of the primary stock solution with 10 mL of DMF. The 0.1 mL of the resultant secondary stock solutions were made to a final volume of 1000 mL with 20X AAP nutrient medium.
- Controls: a dilution water control, an abiotic control and a solvent control
- Chemical name of vehicle: dimethylformamide
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Strain: Lemna gibba
- Source: Canadian Phycological Culture Centre (CPCC), Toronto, Canada
- Stock cultures were grown in 270-mL covered crystallizing dishes containing 100 mL of 20X AAP medium. Approximately 50 fronds were transferred weekly into fresh 20X AAP medium to maintain a continuous culture under testing conditions. The cultures were maintained in an environmental chamber with the following conditions: a temperature of 24 ± 2ºC and continuous illumination of approximately 650 to 880 footcandles (7000 to 9500 lux).
Test type:
static
Water media type:
saltwater
Total exposure duration:
7 d
Test temperature:
23-24°C
pH:
7.7-9.4
Nominal and measured concentrations:
Nominal: 0, 10, 26, 64, 160, 400, 1000 μg a.s./L
Measured: 0, 7.9, 20, 49, 160, 340, 1000 μg a.s./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 270-mL crystallizing dishes containing approximately 100 mL of test solution and covered with an inverted, sterile glass Petri dish
- Renewal rate of test solution: Test solutions were renewed on days 3 and 5.
- No. of organisms per vessel: 12
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per solvent control (replicates): 7
GROWTH MEDIUM
- Standard medium used: yes (20X AAP medium)
OTHER TEST CONDITIONS
- Photoperiod and Light intensity: Continuous fluorescent lighting at an intensity of 7000 to 8800 lux
EFFECT PARAMETERS MEASURED:
- Fronds were counted and observations were made on days 3 and 5, and at test termination (day 7). Frond density determinations were completed at test termination and the fronds from three of the four
replicates (six of the seven solvent control replicates) were removed from each vessel, blotted dry, and transferred to pre-weighed aluminum pans. Fronds were dried in a Labline Imperial II oven at 72 to 73°C
for five days prior to dry weight determination.

TEST CONCENTRATIONS
- Range finding study: A range-finding exposure was conducted with a blank control and nominal concentrations of 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L. Test solutions were renewed on days 3 and
5. Two exposure vessels were used for each concentration and the blank control. Following 7 days of exposure, fronds exposed to the 1.0 mg a.s./L treatment were observed to be curled. Fronds exposed
to all remaining treatment levels (0.10, 0.010, 0.0010, and 0.00010 mg a.s./L) and the blank control were observed to be normal. Frond densities in the 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L treatment
levels averaged 391, 381, 386, 425 and 123 fronds, respectively. Mean frond density in the blank control was 437 fronds. The preliminary range-finding test with this test substance was conducted without the use of a co-solvent. Analytical sampling performed in conjunction with this exposure resulted in recoveries of approximately 20% of nominal concentrations. Additional solubility trials with this test substance were conducted to evaluate the use of a co-solvent (i.e., dimethylformamide, DMF). Based on these results, and in consultation with the Study Sponsor, DMF was used to solubilize the test substance for the definitive testing. An initial definitive test was conducted at nominal concentrations of 12, 31, 77, 190, 480, 1200, and 3000 μg a.s./L, a blank control and a solvent (DMF) control. However, due to variable analytical results related to the solubility of the test substance at nominal concentrations >1000 μg a.s./L, the definitive test was repeated at nominal concentrations of 10, 26, 64, 160, 400, and 1000 μg a.s./L.
Reference substance (positive control):
no
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
260 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density
Remarks on result:
other: 95% confidence limits 160-340 μg a.s./L
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
230 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield based on frond density
Remarks on result:
other: 95% confidence limits 160-330 μg a.s./L
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: growth rate based on frond density
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield and growth rate based on dry weight
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
49 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density; yield and growth rate (based on frond density)
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
49 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr. (total fraction)
Basis for effect:
other: dry weight; yield and growth rate (based on dry weight)
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
160 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density; yield and growth rate (based on frond density)
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
160 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight;yield and growth rate (based on dry weight)
Details on results:
A summary of inhibition for each endpoint following exposure of Lemna gibba to the test substance for 7 days is presented in the table (refer 'Any other information on results including tables').
Reported statistics and error estimates:
Frond density, dry weight biomass, and growth rate and yield based on frond density and dry weight data were evaluated for normality and homogeneity of variance (α = 0.05) using the Shapiro-Wilks’ and Bartlet t’s Tests, respectively. Since all data sets were normal with homogeneous variances, the treatment groups were compared to the pooled control using Williams’ Test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC and LOEC relative to each parameter at 7 days. All calculations and statistical analyses were conducted using TOXSTAT Version 3.5.

Table 1: Inhibition summary for each endpoint following exposure

of Lemna gibba to the test substance for 7 days

Mean,

measured

concentration

(μg a.s./L)

% Inhibition relative to pooled control

Frond

Density

Yield (Frond

Density)

Growth Rate

(Frond

Density)

Dry Weight

Yield (Dry

Weight)

Growth Rate

(Dry Weight)

Untreated

Control

-

-

-

-

-

-

Solvent

Control

-

-

-

-

-

-

7.9

-7

-8

-2

0

-2

0

20

3

4

2

4

3

2

49

9

9

2

2

0

0

160

42

44

16

24

24

10

340

55

57

24

43

44

20

1000

70

73

37

38

39

17

Negative values indicate stimulation; positive values indicate inhibition.

Validity criteria fulfilled:
yes
Conclusions:
The most sensitive parameter was yield based on frond density with an EyC50 value of 230 μg a.s./L, a NOEC of 49 μg a.s./L, and a LOEC of 160 μg a.s./L based on mean, measured concentrations.
Executive summary:

The acute toxicity of the test substance to duckweed, Lemna gibba (L. gibba), under static-renewal conditions was determined in a 7-day exposure test. Test solutions were renewed on days 3 and 5. The test was conducted in accordance with the OECD Guideline 221 (2006) and U.S. EPA OPPTS Draft Guideline 850.4400 (1996). The study was conducted with six concentrations of the test substance, a dilution water (twenty strength

synthetic algal-assay-procedure nutrient medium, 20X AAP) control, an abiotic control and a solvent (dimethylformamide, DMF) control at a temperature of 23 to 24°C. Four replicates with 12 fronds per replicate were initiated for each test substance concentration and the dilution water control. Seven replicates with 12 fronds per replicate were initiated for the solvent control. A single test vessel containing no L. gibba was initiated at the highest test concentration for the abiotic stability control.

Nominal exposure concentrations of the test substance were 10, 26, 64, 160, 400, and 1000 μg active substance (a.s.)/L. Mean, measured concentrations were 7.9, 20, 49, 160, 340, and 1000 μg a.s./L, respectively, and ranged from 77 to 100% of nominal. The mean, measured concentration of the 1000 μg a.s./L abiotic control was 960 μg a.s./L.

Inhibition of frond density (number of fronds/100 mL) of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L as compared to the pooled control was -7, 3, 9, 42, 55, and 70%, respectively, at the end of the 7-day exposure period. The 7-day “effective concentration” producing a 50% inhibition of growth (EC50), based on mean, measured concentrations of the test substance and frond density, was 260 μg a.s./L.

Inhibition of frond dry weight (biomass) of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was 0, 4, 2, 24, 43, and 38%, respectively, at the end of 7 days. The 7-day EbC50, based on mean, measured concentrations of test substance and dry weight, was > 1000 μg a.s./L.

Inhibition of yield based on frond density of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -8, 4, 9, 44, 57, and 73%, respectively, at the end of 7 days. The 7-day EyC50, based on mean, measured concentrations of the test substance and yield based on frond density, was 230 μg a.s./L.

Inhibition of growth rate based on frond density of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -2, 2, 2, 16, 24, and 37%, respectively, at the end of 7 days. The 7-day ErC50, based on mean, measured concentrations of the test substance, was > 1000 μg a.s./L.

Inhibition of yield based on dry weight of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -2, 3, 0, 24, 44, and 39%, respectively, at the end of 7 days. The 7-day EyC50, based on mean, measured concentrations of the test substance and yield based on dry weight, was > 1000 μg a.s./L.

Inhibition of growth rate based on dry weight of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was 0, 2, 0, 10, 20, and 17%, respectively, at the end of 7 days. The 7-day ErC50, based on mean, measured concentrations of the test substance and growth rate based on dry weight, was > 1000 μg a.s./L.

Recovery was assessed for all mean, measured test concentrations (7.9, 20, 49, 160, 340, and 1000 μg a.s./L). Based on a > 7X increase in healthy frond count in 7 days, the effects upon growth and reproduction of L. gibba were found to be phytostatic within 7 days at mean, measured concentrations less than or equal to 1000 μg a.s./L.

Description of key information

7-day ErC50 (Lemna gibba): 0.230 mg/L; OECD 221; Reliability = 1

7-day NOEC (Lemna gibba): 0.049 mg/L; OECD 221; Reliability = 1

Key value for chemical safety assessment

EC50 for marine water plants:
0.23 mg/L
EC10 or NOEC for marine water plants:
0.049 mg/L

Additional information

The ErC50 from the 7-day Lemna gibba test is 0.230 mg/L, based on growth rate. The ECx value for aquatic plants is less than 1.0 mg a.s./L.