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Ecotoxicological information

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:
29 Oct - 27 Nov 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)
Version / remarks:
23 March 2006
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L (nominal)
- Sampling method: fresh test levels were sampled on day 0, 2 and 5 and aged sampled were taken after day 2, 5 and 7. All test levels, control and solvent control were analysed.
- Sample storage conditions before analysis: samples were stored in a freezer
Vehicle:
yes
Remarks:
Dimethylformamid (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution: 233 mg test substance and 2.00 mL dimethylformamide by intense stirring and ultrasonic treatment. Dilution series: an adequate amount of the stock solution was transferred to a dilution series; for detailed information see Table 1 in section "Any other information on materials and methods"
- Controls: yes, test medium and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 μL DMF (including the appropriate concentration of the test item) / 1000 mL medium
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Strain: G3
- Source (laboratory, culture collection): Eurofins Agroscience Services EcoChem GmbH, Eutinger Straße 24, 75223 Niefern-Öschelbronn, Germany
- Age of inoculum (at test initiation): age of pre-culture 7-10 d
- Method of cultivation: Stock cultures are kept in glass dishes with medium, illumination: 6500 – 7000 lux, temperature: 23 - 26 °C. Fresh medium: every 7-10 d; sterile conditions

ACCLIMATION
- Acclimation period: 7-10 d
- Culturing media and conditions (same as test or not): same
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
24.2 - 25.0 °C
pH:
7.5 - 8.9 (control)
7.5 - 8.9 (solvent control)
7.4 - 9.0 (test concentrations)
Nominal and measured concentrations:
control, solvent control, 0.0954, 0.305, 0.977, 3.13 and 10.0 mg/L (nominal)
control, solvent control, 0.0804, 0.268, 0.778, 2.05 and 6.64 mg/L (measured concentrations based on time weighted average)
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: no
Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass dishes diameter of 10 cm and a height of 6 cm (total volume of approx. 470 mL)
- Type of cover: lids of glass to permit gas exchange and illumination under sterile conditions
- Aeration: no
- Agitation: no
- Renewal rate of test solution (frequency/flow rate): on day 2 and 5
- Control end cells density: 215 fronds after 7 d, mean CV 5.6%
- No. of fronds per vessel: 12 fronds per vessel (3 – 4 fronds per plant)
- No. of vessels per concentration (replicates): 12
- No. of vessels per control (replicates): 12
- No. of vessels per vehicle control (replicates): 12

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: water source for deionised water, supplied by CURRENTA GmbH & Co. OHG, Leverkusen (also used for artificially prepared fish testing water according to ASTM Standard E 729-1008)
- Total organic carbon:< 2 mg/L
- Particulate matter: < 5 mg/L (total solids)
- Contaminants: 1 μg/L: Al, Zn; < 1 μg/L: As Cr; Cu ; Fe ; Co ; Pb ; Ni; < 100 μg/L: Bor; F- ; < 0.1 μg/L: Cd; Hg; Ag; < 0.03 mg/L: NH4+
- Organochlorine Contaminants: < 0.01μg/L: HCB, α-HCH, β-HCH, γ-HCH, Heptachlor, Heptachlorepoxid, Aldrin, Pentachlorbenzol, Quintozen, α –Endosulfan, β-Endosulfan, Dieldrin, 2,4-DDE, 4,4-DDE, 2,4-DDD, 4,4-DDD, 2,4-DDT, 4,4-DDT, Endrin, PCB 28, PCB 52, PCB 101, PCB 138, PCB 153, PCB 180
- Pesticides (residue):< 0.05 µg/L: 2,4-D, Lenacil; Anilazine; Linuron; Atrazine; MCPA; Azinphos Ethyl; Mecoprop; Azinphos Methyl; Mercaptodimethur; Bentazon; Metamitron; Bitertanol; Metazachlor; Bromacil; Methabenzthiazuron; Buturon; Metoxuron; Carbofuran; Metribuzin; Chlorphoxim; Monolinuron; Chlortoluron; Parathion Ethyl; Coumaphos; Parathion Methyl; Coumatetralyl; Phoxim; Cyanazin; Promecarb; Dichlorprop; Prometryn; Dinosep; Propanil; Dinosepacetat; Propazin; Disulfoton; Propoxur; Diuron; Simazin; Ethidimuron; 2,4,5-T; Fenitrothion; Tebuconazole; Fluopyram; Terbutylazin; Folpet; Thiram; Hexazinon; Triadimefon; Isofenphos; Triadimenol; Isomethiozin; Trichlorfon; Isoproturon;
- Chlorine (residual): < 0.01 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured on 0,2 and 7 d; temperature was measured on each day; the parameter of the dilution water were measured on July 24, 2014
- CSB: < 5 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 7.5 ± 0.1 with HCl
- Photoperiod: permanent light (repositioned and randomised after each observation)
- Light intensity and quality: 6500-7000 lux (± 15% variation from the mean) uniform overhead illumination was provided by a bank light containing fluorescent lamps

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: Counting of fronds
- Determination of frond area: determination of total frond area with LemnaTec Scanalyzer

RANGE-FINDING STUDY
- test concentrations was selected based on a pre experiment which was performed under the same biological conditions
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 6.64 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
other: mean growth rate of frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 6.64 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
other: mean growth rate of total frond area of plants
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
2.05 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
other: effect on mean growth rate of frond number and total frond area of plant
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
6.64 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
other: effect on mean growth rate of frond number and total frond area of plant
Details on results:
- Other: No sublethal effects on Lemna gibba were observed. Analytical and biological results are summarized in section "Any other information on results incl tables"
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Up to 0.977 (0.778 TWA) mg a.s./L no remarkable observations were recorded in the aged and freshly prepared test media. At 3.31 (2.05 TWA) mg a.s./L on day 5 and 7 in the aged test levels undissolved test item was observed at the surface. At 10.0 (6.64 TWA) mg a.s./L undissolved test item was recorded at water surface and the bottom of the test vessels in the aged test media on day 5 and 7.
Results with reference substance (positive control):
No reference substance was tested.
Reported statistics and error estimates:
ECx values and confidence intervals were calculated for the stated exposure period, using a commercial program. The LOEC determinations from the appropriate parameter (inhibition) were done, using the ANOVA procedure (p = 0.05, one sided) and properly selected multiple t-tests of a commercial program. Calculations were carried out using Microsoft Excel® spread sheets. All tabulated data are rounded. All further statistical evaluations were done using the commercial program ToxRat Professional.

Table 1: Biological results of the 7 d growth inhibition test

nominal concentration [mg a.s.]

time weighted average concentration [mg a.s./L]

frond number (day 7) mean values from 4 replicates

total frond area of plants (day 7) mean values from 4 replicates [mm2]

% inhibition

mean growth rate for frond number

mean growth rate for total frond area of plants

control

control

215

1916

--

--

solvent control

solvent control

216

1908

--

--

0.0954

0.0804

206

1847

2.4

1.6

0.305

0.268

213

1886

0.4

0.3

0.977

0.778

207

1888

1.5

0.0

3.13

2.05

203

1818

2.2

0.4

10.0

6.64

162

1375

10.1●

10.8

-%: inhibition: increase in growth relative to the control

●: Results which were significantly different (based on Welch-t-test Procedure) from the pooled controls

■: Results which were significantly different (multiple sequentially-rejective U-test) from the pooled controls

Table 2: Analytical findings

Day

nominal concentration in mg a.s./L

time weighted average concentration [mg a.s./L]

actual concentration of the substance

 

 

 

detection 1 [mg/L]

detection 2 [mg/L]

mean [mg/L]

% of nominal

0

 

 

< LOQ

< LOQ

< LOQ

-

2 new

 

 

< LOQ

< LOQ

< LOQ

-

2 aged

Control

Control

< LOQ

< LOQ

< LOQ

-

5 new

 

 

< LOQ

< LOQ

< LOQ

-

5 aged

 

 

< LOQ

< LOQ

< LOQ

-

7

 

 

< LOQ

< LOQ

< LOQ

-

0

 

 

< LOQ

< LOQ

< LOQ

-

2 new

 

 

< LOQ

< LOQ

< LOQ

-

2 aged

solvent control

solvent control

< LOQ

< LOQ

< LOQ

-

5 new

 

 

< LOQ

< LOQ

< LOQ

-

5 aged

 

 

< LOQ

< LOQ

< LOQ

-

7

 

 

< LOQ

< LOQ

< LOQ

-

0

 

 

0.133

0.134

0.134

140

2 new

 

 

0.129

0.130

0.130

136

2 aged

0.0954

0.0804

0.0769

0.0765

0.0767

80

5 new

 

 

0.106

0.105

0.106

111

5 aged

 

 

0.0388

0.0390

0.0389

41

7

 

 

0.0368

0.0372

0.0370

39

0

 

 

0.417

0.402

0.410

134

2 new

 

 

0.408

0.405

0.407

133

2 aged

0.305

0.268

0.230

0.229

0.230

75

5 new

 

 

0.421

0.421

0.421

138

5 aged

 

 

0.129

0.129

0.129

42

7

 

 

0.153

0.153

0.153

50

0

 

 

1.18

1.18

1.18

120

2 new

 

 

1.15

1.15

1.15

118

2 aged

0.977

0.778

0.717

0.717

0.717

73

5 new

 

 

1.18

1.18

1.18

120

5 aged

 

 

0.384

0.384

0.384

39

7

 

 

0.432

0.431

0.432

44

0

 

 

2.90

2.89

2.89

92

2 new

 

 

3.15

3.16

3.15

101

2 aged

3.13

2.05

1.84

1.83

1.84

59

5 new

 

 

3.09

3.09

3.09

99

5 aged

 

 

1.08

1.05

1.06

34

7

 

 

1.09

1.19

1.14

37

0

 

 

9.71

9.67

9.69

97

2 new

 

 

9.90

9.87

9.89

99

2 aged

10.0

6.64

6.02

6.00

6.01

60

5 new

 

 

10.2

10.2

10.2

102

5 aged

 

 

3.25

3.25

3.25

33

7

 

 

3.94

3.97

3.95

40

 

Validity criteria fulfilled:
yes

Description of key information

EC50 (7 d): > 6.64 mg a.s./L (M-532733-02-1, for frond number an frond area; Lemna gibba, OECD 221, based on time weighted average values)

NOEC (7 d): 2.05 mg a.s./L (M-532733-02-1, for frond number an frond area; Lemna gibba, OECD 221, based on time weighted average values)

Key value for chemical safety assessment

EC50 for freshwater plants:
6.64 mg/L
EC10 or NOEC for freshwater plants:
2.05 mg/L

Additional information

One study (M-532733-02-1) is available investigating the short-term toxicity of the test item to duckweed (Lemna gibba). The study was performed according to OECD 221 and GLP. Cultures of Lemna gibba G3 with an initial density of 12 fronds per vessel were exposed in a chronic multi-generation test for 7 d under semi-static exposure conditions to the nominal concentrations of 0.0954, 0.305, 0.977, 3.13 and 10 mg a.s./L, corresponding to time weighted average concentrations (TWAC) of 0.0804, 0.268, 0.778, 2.05 and 6.64 mg a.s./L, respectively. Due to low solubility the test item was dissolved in dimethylformamide (DMF). Plant frond numbers and total frond area of plants were recorded on days 0, 2, 5 and 7. Frond numbers and total frond area at each occasion were used to determine the effect concentrations. The analytical findings of the test item found in all freshly prepared test levels on day 0, 2, and 5 ranged between 92 and 140% of nominal concentrations and in aged test levels on days 2, 5, and 7 between 33 and 80% of nominal, respectively. Therefore all results based on time weighted average values. The ErC50 (7 d) for frond number and frond area was determined to be > 6.64 mg a.s./L. The NOErC (7 d) for frond number and frond area was determined to be 2.05 mg a.s./L.