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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:
2014
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:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Q30420AAYB
- Expiration date of the lot/batch: 08 Aug 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (+5 to +30 °C), dark, dry
Analytical monitoring:
yes
Details on sampling:
The test medium samples (100 mL in a 250 mL glass bottle) were stored deep-frozen (below -18 °C) until analysis.
Vehicle:
no
Details on test solutions:
Due to low water solubility of the test item a saturated solution was prepared by weighing 600 mg FAT 93450/Z on a weighing scoop and transferring it to a volumetric flask. Test medium was added up to the bench mark (2 L). The solution was stirred for 48 hours in the dark. Afterwards the stock solution was withdrawn from the middle of the preparation vessel without transferring undissolved test item. Aliquots of the withdrawn phase were diluted sequentially with test medium to receive the lower test item concentrations.
Test organisms (species):
Lemna gibba
Details on test organisms:
The test organism was the aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae). The plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, USA. The plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 7000 lux. The temperature was 24 ± 2 °C. Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 5 fronds were transferred onto fresh medium and cultured for 3 weeks prior to testing, with two further transfers onto fresh medium before initiating the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Remarks on exposure duration:
daily water renewal
Test temperature:
22.0 – 24.0 °C
pH:
7.34 – 8.56
Nominal and measured concentrations:
see table below
Details on test conditions:
Test procedure: test with daily renewal of test solutions
Duration: 7 days
Temperature: 22.0 – 24.0 °C
pH-value of fresh solution: 7.34 – 8.56
Light intensity (mean): ~7200 lux
Light tubes: OSRAM light tubes (L 30 W77 and L 30 W25)
Exposure to light: continuously
Test vessels: 250 mL glass beakers filled with 150 mL
Number of fronds: 12 per test vessel
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
298 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate of dry weight
Remarks on result:
other: 155-26254
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
2.97 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Growth rate of dry weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
29.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate of dry weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.293 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: growth rate of dry weight
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield of frond number
Remarks on result:
other: 215-261
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
1.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield of dry weight
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate of frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Growth rate of frond numbers
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
126 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield of frond number
Remarks on result:
other: 90.7-153
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
93.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield of frond number
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
118 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate of frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
93.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate of frond number
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
58.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield of dry weight
Remarks on result:
other: 7.29-104
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
173 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield of dry weight
Remarks on result:
other: 90.7-267
Reported statistics and error estimates:
The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate of dry weight and yield of dry weight. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test). The calculation of EC10, EC20, EC50 values for yield of frond numbers, growth rate of dry weight and yield of dry weight was done using probit analysis following Gompertz procedure. Negative values were set to zero. Due to statistical reasons for yield of frond numbers the calculation was done only with the two highest loading rates of 93.8 and 300 mg/L. The calculation of EC50-values for growth rate of frond numbers was not indicated since inhibition was below 50 %. The calculation of EC10 and EC20 values for growth rate of frond numbers was done using probit analysis following Gompertz procedure. The evaluation of data was performed by SAS® (2002-2010).

 

FAT 93450/Z [mg/L]

actual3)

Endpoint

EC101)

95 % confidence limit

EC201)

95 % confidence limit

EC501)

95 % confidence limit

NOEC

LOEC

Yield of frond numbers

1.26

0.907-1.53

1.63

1.29-1.88

2.40

2.15-2.61

0.9382)

3.002)

Growth rate of frond numbers

1.24

-

2.06

-

> 3.00

-

0.9382)

3.002)

Yield of dry weight

0.594

0.441-0.737

0.910

0.732-1.07

1.73

1.52-1.95

0.2932)

0.9382)

Growth rate of dry weight

1.08

0.765-1.33

1.61

1.29-1.88

2.97

2.58-3.52

0.2932)

0.9382)

1)probit following Gompertz

2)following Dunnett`s-t-test

3)based on the geometric mean of the analysed concentration levels

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers is 2.40 mg/L, 1.73 mg/L for yield of dry weight and 2.97 mg/L for growth rate of dry weight. The actual EC50-value for growth rate of frond numbers is >3.00 mg/L.
Executive summary:

A Toxicity to the Duckweed Lemna gibba study under Laboratory Conditions with FAT 93450/Z was carried out according to OECD 221 guideline to assess the effect of the test item on the growth of Lemna gibba. A saturated stock dispersion was prepared by stirring 600 mg FAT 93450/Z in 2000 mL test medium for 48 hours in the dark. The stock solution was withdrawn from the middle of the preparation vessel. Aliquots of the withdrawn phase were diluted to receive the lower test item concentrations. A test with daily renewal of test solutions with the following loading rates was performed: undiluted solution (corresponding to 300 mg/L loading rate), 3.2-fold diluted solution (corresponding to 93.8 mg/L loading rate), 10.2-fold diluted solution (corresponding to 29.3 mg/L loading rate), 32.8-fold diluted solution (corresponding to 9.16 mg/L loading rate), 104.9-fold diluted solution (corresponding to 2.86 mg/L loading rate), 335.6-fold diluted solution (corresponding to 0.894 mg/L loading rate) and control. Vegetative growth was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. The following endpoints (NOEC/LOEC, EC10, 20, 50) were determined:

- frond numbers and dry weight

- growth rates of frond numbers and dry weight

- yield of frond numbers and dry weight

- doubling times of frond numbers

Significant inhibitory effects were determined for yield and growth rate of frond numbers at 300 mg/L loading rate and 3.00 mg/L (actual). Significant inhibitory effects were determined for yield and growth rate of dry weight at 93.8 mg/L loading rate and 0.938 mg/L (actual). The overall LOEC was based on the most sensitive parameter and was determined to be 93.8 mg/L loading rate and 0.938 mg/L (actual). The corresponding NOEC was determined at 29.3 mg/L loading rate and 0.293 mg/L (actual). The nominal EC10-value for yield of frond numbers is 126 mg/L loading rate, 58.2 mg/L loading rate for yield of dry weight and 106 mg/L loading rate for growth rate of dry weight. The nominal EC10-value for growth rate of frond numbers is 118 mg/L loading rate. The test is valid as the doubling time of control frond numbers was calculated to be 33.6 h, corresponding to 1.401 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

Toxicological endpoints for FAT 93450/Z:

 

FAT 93450/Z

[mg/L loading rate]

Endpoint

EC101)

EC201)

EC501)

NOEC

LOEC

Yield of frond numbers

126

163

240

93.82)

3002)

Growth rate of frond numbers

118

204

> 300

93.82)

3002)

Yield of dry weight

58.2

89.8

173

29.32)

93.82)

Growth rate of dry weight

106

160

298

29.32)

93.82)

 

FAT 93450/Z TE [mg/L]

actual3)

Endpoint

EC101)

EC201)

EC501)

NOEC

LOEC

Yield of frond numbers

1.26

1.63

2.40

0.9382)

3.002)

Growth rate of frond numbers

1.24

2.06

> 3.00

0.9382)

3.002)

Yield of dry weight

0.594

0.910

1.73

0.2932)

0.9382)

Growth rate of dry weight

1.08

1.61

2.97

0.2932)

0.9382)

1)probit following Gompertz

2)following Dunnett`s-t-test

3)based on the geometric mean of the analysed concentration levels

Based on the geometric mean of the analysed concentration levels, the EC10-value for yield of frond numbers is 1.26 mg/L, 0.594 mg/L for yield of dry weight and 1.08 mg/L for growth rate of dry weight. The actual EC10-value for growth rate of frond numbers is 1.24 mg/L. The nominal EC20-value for yield of frond numbers is 163 mg/L loading rate, 89.8 mg/L loading rate for yield of dry weight and 160 mg/L loading rate for growth rate of dry weight. The nominal EC20-value for growth rate of frond numbers is 204 mg/L loading rate. Based on the geometric mean of the analysed concentration levels, the EC20-value for yield of frond numbers is 1.63 mg/L, 0.910 mg/L for yield of dry weight and 1.61 mg/L for growth rate of dry weight. The actual EC20-value for growth rate of frond numbers is 2.06 mg/L. The nominal EC50-value for yield of frond numbers is 240 mg/L loading rate, 173 mg/L loading rate for yield of dry weight and 298 mg/L loading rate for growth rate of dry weight. The nominal EC50-value for growth rate of frond numbers is >300 mg/L loading rate. Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers is 2.40 mg/L, 1.73 mg/L for yield of dry weight and 2.97 mg/L for growth rate of dry weight. The actual EC50-value for growth rate of frond numbers is >3.00 mg/L.

Description of key information

The aquatic plant duckweed, Lemna gibba G3 was used to determine the toxicity effect of FAT93450 in a growth inhibition test according to OECD guideline 221. Significant inhibitory effects were determined for yield and growth rate of frond numbers at 300 mg/L loading rate and 3.00 mg/L (actual). Significant inhibitory effects were determined for yield and growth rate of dry weight at 93.8 mg/L loading rate and 0.938 mg/L (actual). Based on the geometric mean of the analysed concentration levels, the EC50-value for yield of frond numbers was 2.40 mg/L, 1.73 mg/L for yield of dry weight and 2.97 mg/L for growth rate of dry weight, NOEC value was 0.293 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:
2.97 mg/L
EC10 or NOEC for freshwater plants:
0.293 mg/L

Additional information