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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:
2009-01-16 till 2009-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with guidelines
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling: at the start and end of the test
- Sample storage conditions before analysis:The samples were stored deep-frozen and protected from light until analysis was performed.
- Preparation of Samples:Treatment samples and control samples were thawed at room temperature for 1.5 hours and shaken manually to obtain homogeneous sample solutions. Aliquots of the samples were analyzed by HPLC with UV/VIS detection.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Reconstituted Test Water
Ingredients / Properties Concentration
mg/L
Macro-Nutrients CaCl2 x 2H20 88.2
MgSO4 x 7 H2O 294.0
NaHCO3 300.0
K2HPO4 20.9
NaNO3 510.0
MgCl x 6H2O 243.3
Trace elements H3BO3 3.7
MnCl2 × 4 H2O 8.3
ZnCl2 0.06
CoCl2 × 6 H2O 0.03
CuCl2 × 2 H2O 0.2 μg/L
Na2MoO4 × 2 H2O 0.14
FeCl3 × 6 H2O 3.2
Na2EDTA × 2 H2O 6.0
Water Hardness: 3.0mmol/L (=300 mg/L as CaCO3)
The test water was prepared at least one day prior to use. At this stage, the pH of the test water
was adjusted to 7.5 ± 0.1 with a 1 M hydrochloric acid solution.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
The test organism used for the study was the duckweed Lemna gibba G3 (family Lemnaceae,
Macrophyta). The original culture was supplied by Bayer CropScience AG, 40789 Monheim,
Germany in 2007. The plants were axenically cultivated in the laboratories of Harlan
Laboratories Ltd. for more than four weeks prior to the test under standardized conditions in the
same nutrient medium as used in the test.
The pre-culture was maintained under the conditions of the test (nutrient medium, light
conditions and temperature) for more than seven days prior to the start of the test. The test was
started with plants from an exponentially growing culture. Only young, rapidly growing colonies
without visible lesions were used.


At the start of the test, Lemna colonies were transferred aseptically from the pre-culture into the
test vessels in a randomized order. The test was started with three randomly selected colonies per
vessel

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
none

Test conditions

Hardness:
Water Hardness: 3.0mmol/L (=300 mg/L as CaCO3)
Test temperature:
The water temperature was 22-23 °C during the test period.
pH:
At the start of the test, the pH of the test media and the control was 7.5. At the end of the test, pH values between 8.8 and 9.2 were measured. The increase of the pH during the test was attributed to the CO2 consumption of the plants due to their growth.
Nominal and measured concentrations:
1, 3.2, 10, 32 and 100mg/L
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes, water bath
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 2 250-mL glass dish (diameter of 9.5 cm) filled with 150 mL of test medium, resulting in a water depth of approximately 21 mm. The test vessels were covered with glass dishes. The test vessels were labeled with the study number and all necessary additional
information to ensure unique identification.

- Agitation: yes/no
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Control end cells density:
- No. of colonies per vessel:
- No. of fronds per colony:
- No. of vessels per concentration (replicates):three replicates per test concentration
- No. of vessels per control (replicates):three replicates per control.

The test vessels were incubated in a temperature-controlled water bath in a random order at a
temperature of 22-23 °C (Table 10). The test item was colored and light absorbing. To avoid
undesired influence of illumination and reflection on the growth of the control and less colored
dilutions of the test item, the test vessels were placed into a water bath which was covered with a
plate at the surface and with black foil on each side and the bottom. To minimize the influence of
reflections on the growth, the plants were continuously illuminated at a light intensity of about
9600 Lux (mean value), range: 9000 to 9950 Lux (minimum and maximum value of
measurements before the start of the test at nine places distributed over the test area at the
surface of the test media) and thus, at the upper limit of the recommended range (6500 – 10000
Lux). The light intensity over the test area did not exceed the range of ±15% of the mean value.
The illumination was achieved by fluorescent tubes (Philips TLD 36W-1/840) installed above
the test vessels. The test vessels were repositioned at each counting date.

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
-see "Details on Test Solutions"

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: prior to the start of the test, see "Details on Test Solutions"


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: [manual counting / computerized image analysis]
- Determination of biomass: [dry weight / fresh
weight]
- Determination of frond area:
- Other:


RANGE-FINDING STUDY
- yes, non GLP study
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol tested 2x/year. Result latest positive control test in 2008: sensitivity within historical range (7-day EC50 for growth rate based on frond numbers: 10.0 mg/L, study C05854; internal historical range from 2003-08: 8.0-10.8 mg/L).

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Details on results:
- Any visual signs of phytotoxicity (abnormalities):
No abnormalities in growth and appearance of the test plants were recorded in the control and the test concentrations of 1.0 and 3.2 mg/L. At the end of the test and at the concentrations of 10 to 100 mg/L the single roots of the plants were blue colored by the test item.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:No remarkable observations were made concerning the appearance of the test media. All test media were clear and the solutions were colored by the test item throughout the test period




Results with reference substance (positive control):
- Results with reference substance valid?yes
For evaluation of the sensitivity of the test system, the reference item 3,5-dichlorophenol is tested twice a year. The result of the latest positive control test performed in 2008 showed that the sensitivity of the test system was within the historical range (7-day EC50 for the growth rate based on frond numbers: 10.0 mg/L, study C05854; internal historical range from 2003 to 2008: 8.0-10.8 mg/L).

Any other information on results incl. tables

The biological results can be summarized as follows (based on nominal concentrations of the test item):

7-day EC values (mg/L)

Parameter based on

 

frond numbers

dry weight of the plants

 

Growth rate

Yield

Growth rate

Yield

 

EC10

 

93

1.3

>100

0.10

EC20

 

>100

35

>100

25

EC50

 

>100

>100

>100

>100

NOEC

 

3.2

3.2

3.2

3.2

LOEC

 

10*

10

10*

10

The 95% confidence intervals could not be determined

* LOEC << EC10: caused by flat concentration/response curves (see. Fig. 2 and 4)

Average Growth Rates Based on Frond Numbers

Nominal test item

Concentration (mg/L)

Average Growth rateμ(day-1) and inhibition ofμ(Ir)

Days 07

μ

Ir(%)

Control

0.355

0.0

1.0

0.336

5.5

3.2

0.345

3.0

10

0.334*

6.1

32

0.328*

7.6

100

0.317*

10.7

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided,α= 0.05)

Yield Based on Frond Numbers

Nominal

test item

concentration (mg/L)

Yield Y and inhibition of Y (Iy)

Days 07

Y

Iy(%)

Control

132.7

0.0

1.0

114.0

14.1

3.2

122.3

7.8

10

112.0*

15.6

32

108.0*

18.6

100

98.7*

25.6

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided,α= 0.05)

Average Growth Rates Based on Dry Weights

Nominal test item

Concentration (mg/L)

Average Growth rateμ(day-1) and inhibition ofμ(Ir)

Days 07

μ

Ir(%)

Control

0.338

0.0

1.0

0.319

5.5

3.2

0.320

5.3

10

0.308*

8.9

32

0.313*

7.3

100

0.304*

10.2

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided,α= 0.05)

Yield Based on Dry Weights

Nominal

test item

concentration (mg/L)

Yield Y and inhibition of Y (Iy)

Days 07

Y

Iy(%)

Control

17.5

0.0

1.0

15.0

14.1

3.2

15.2

13.4

10

13.7*

21.6

32

14.4*

18.0

100

13.3*

24.1

*: mean value significantly lower than in the control

(according to Dunnett’s tests, one-sided,α= 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The doubling time (Td) of frond number in the control was calculated to be 2.0 days (Td = ln 2 / μ). According to the test guideline, the validity criterion for the study (Td < 2.5 days corresponding to an average growth rate of 0.275 day-1) was fulfilled
Conclusions:
The EC50 >100mg/l (7 days) indicates that test material is not acute toxic to Lemna gibba and is therefore not classified.,
The test item test material had a statistically significant inhibitory effect on the growth of Lemna gibba (growth rate and yield based on frond number and dry weight) after the exposure period of 7 days at the concentration of 10 mg/L and at all higher test concentrations. At all lower test item concentrations, the growth rate and yield based on frond number and dry weight were not statistically significantly lower than in the control.
Executive summary:

The influence of the test item test material on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7-day static test, based on the OECD Guideline No 221 “Lemna sp. Growth Inhibition Test” (2006).

The following nominal concentrations of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel.

The analytically determined concentrations of the test item in the test media of the nominal concentrations from 10 to 100 mg/L were between 106 and 115% of the nominal values. The determined concentrations for 3.2 mg/L (NOEC) were 127 and 112 % of the nominal values at the start and the end of the test, respectively. The analytically determined concentrations of all treatments which result in a biological effect (10 to 100 mg/L) confirmed the correct dosage and the stability of the test item during the test period of seven days under the conditions of the test. Therefore the biological results are related to the nominal concentrations of the test item.

The concentration of 10 mg/L was determined to be the 7-day LOEC. The average growth rate and the yield based on frond numbers and dry weight after the exposure period of 7 days were statistically significantly lower than in the control and symptoms of toxicity were determined at this test concentration. The 7-day NOEC was determined to be 3.2 mg/L. Up to and including this concentration, the growth of the plants was not inhibited and no abnormalities in growth and appearance of the plants were determined after the exposure period of 7 days.