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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:
supporting study
Study period:
From October to November 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The purpose of this non GLP study was to determine the effects of the test item (parent molecule) and its possible degradation products on the growth of the freshwater aquatic plant Lemna gibba (duckweed).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
yes
Remarks:
4 test item concentration levels, no analytical monitoring.
Principles of method if other than guideline:
- Principle of test: The purpose of this study was to determine the effects of the test item (parent molecule) and its possible degradation products on the growth of the freshwater aquatic plant Lemna gibba. In order to compare the toxic potential of the parent molecule with the toxic potential of its possible degradation products, the study comprised two experimental parts: In Experimental Part A, the parent molecule of the test item was tested. In Experimental Part B, the degradation products of the test item were tested.
- Short description of test conditions: For Experimental Part A, a semi-static test design with test medium renewal at Day 2 and Day 4 was applied. Experimental Part B was performed under static condition. The exposure periods of both experimental parts were run in parallel. The duration of the test was 7 days. The test item concentrations for both experimental parts were 100, 10, 1.0, and 0.10 mg/L. A control (test water without test item) was run in parallel. At the start of the test, Lemna colonies were transferred from the culture into the test vessels in a random order. A total of 12 fronds (3 colonies) was introduced into each of the two replicates per test concentration. Two control replicates were run in parallel (one replicate static and one replicate semi-static).
- Parameters analysed / observed: The Lemna colonies were inspected in each test vessel for changes in frond and colony number as well as appearance (discoloration, sinking, root length or other abnormalities) at the start of the test and at Days 2, 4 and 7. The number of fronds and colonies was counted. At test end the dry weight of the plants of each test vessel was determined.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I80561
- Expiration date of the lot/batch: 2 June 2017
- Purity test date: 3 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the fridge, protect from light and moisture or heat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: no

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Experimental Part A (Parent Molecule): The test medium of the highest nominal concentration of 100 mg/L was prepared at the start of the test and at the test medium renewals at Day 2 and Day 4 by mixing nominal 100 mg (effectieve range: 100.0 to 100.2 mg) of the test item into 1000 mL test water using intense stirring for 25 minutes at room temperature in the dark. The freshly prepared test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. Application and setup of the test was carried out as quickly as possible in order to avoid degradation of the test substance as far as possible.
Experimental Part B (Degradation Products): The test medium of the highest nominal concentration of 100 mg/L was be prepared by mixing 100.0 mg the test item into 1000 mL test water using intense stirring for 96 hours at room temperature in the dark. It was assumed, that during the long stirring time, the parent molecule was degraded to its degradation products. After stirring, the aged test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the appearance of the test media was recorded at Days 2, 4 and 7 (for Experimental Part A: in the freshly prepared and aged test media). See below the results

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Lemna gibba
- Strain: G3
- Source (laboratory, culture collection) / Method of cultivation: The plants was axenically cultivated at IES Ltd for at least 7 days prior to the test under the conditions of the test and in the same nutrient medium as used in the test. The plants were cultivated and tested in sterile synthetic test water, the "20×AAP growth medium" according to the OECD test guideline.

Study design

Test type:
other: Experiment A: semi-static, Experiment B: static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

Hardness:
Not reported
Test temperature:
The test vessels were incubated in a temperature-controlled growth chamber at a temperature of 24 °C.
pH:
The pH of the test water was adjusted to pH 7.5 ± 0.1.
During the exposure period, pH varied from 7.4 to 9.0 in the controls in exp. A and B. In exp. A, pH variations are in the range of 7.6 to 8.8 (pH increased once at 9.1 for 0.1 mg/L) for all test concentrations. In exp. B, the variations are in the range of 7.7 to 8.9 in all test concentrations.
Dissolved oxygen:
The dissolved oxygen concentrations were measured at all test concentrations at the start of the test and at Days 2, 4 and 7.
Nominal and measured concentrations:
Nominal concentrations: 100, 10, 1.0, and 0.10 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel / Material, size, headspace, fill volume: Glass vessels were used, filled with 150 mL of test medium (depth >20 mm). The test vessels were covered with glass dishes.
- Aeration: no
- Agitation: no
- Renewal rate of test solution (frequency/flow rate): For Experimental Part A, a semi-static test design with test medium renewal at Day 2 and Day 4 was applied. Experimental Part B was performed static.
- No. of colonies per vessel: 12 fronds (3 colonies)
- No. of fronds per colony: 4
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2 (one replicate static and one replicate semi-static)

GROWTH MEDIUM and TEST MEDIUM
- Standard medium used: yes; The plants were cultivated and tested in sterile synthetic test water, the "20×AAP growth medium" according to the OECD test guideline. Analytical grade salts were added to purified water at the following final nominal concentrations at least one day before use to allow the pH to stabilize. The pH of the test water was adjusted to pH 7.5 ± 0.1.

WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH values, dissolved oxygen concentrations and water temperatures were measured at all test concentration at the start of the test and at Days 2, 4 and 7 (for Experimental Part A: in the freshly prepared and aged test media). The appearance of the test media was recorded at the same dates. No analytical work was performed.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: The light intensity at the level of the test solutions was between 85-100 μE.m-2s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The Lemna colonies were inspected in each test vessel for changes in frond and colony number as well as appearance (discoloration, sinking, root length or other abnormalities) at the start of the test and at Days 2, 4 and 7 (test end). The number of fronds and colonies was counted. At test end the dry weight of the plants of each test vessel was determined (drying for 24 hours at 60°C).

RANGE-FINDING STUDY: no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/growth rate
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
4.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/yield
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond nummber/growth rate
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/yield
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/growth rate
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/yield
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/growth rate
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/yield
Remarks on result:
other: Exp. part A
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/growh rate
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/yield
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
9.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number/growth rate
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Fronf number/yield
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/growth rate
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/yield
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/growth rate
Remarks on result:
other: Exp. part B
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight/yield
Remarks on result:
other: Exp. part B
Details on results:
- Any visual signs of phytotoxicity (abnormalities): see below for details
Reported statistics and error estimates:
EC10, EC20, and EC50-Values for lemna growth (yield and growth rate) after the 7 days test period were estimated by Probit Analysis (statistical analysis was performed using ToxRat
Professional®).

Any other information on results incl. tables

Number of fronds during the 7-day exposure period

Experimental part A (parent molecule):

 Nominal test item concentration (mg/L)  Vessel No.  day 0  day 2  day 4  day 7
 Control  1  12  23  48  196
   2  12  23  43  172
   mean  12  23 45.5  184
 0.10  1  12  25  48  196
 2  12  22  47 185
   mean  12  23.5  47.5  190.5
   % of control*  100  102  99  97
 1.0  1  12 21 38 128
   2  12  25  47  160
   mean  12  23  42.5  144
   % of control*  100  100  89  73
 10  1  12  21  28  66
   2  12  21  31  63
   mean  12  21  29.5  64.5
   % of control*  100  91  61  33
 100  1  12  16  20  25
   2  12  19  24  30
   mean  12  17.5  22  27.5
   % of control *  100  76 46   14

*: For calculation, replicate 1 with semi-static test design was taken into account.

Experimental part B (degradation products):

 
 Nominal test item concentration (mg/L)  Vessel No.  day 0  day 2  day 4  day 7
 Control  1  12 23  48  196 
   2  12 23  43  172 
   mean  12 23  45.5  184 
 0.10  1  12 23  45  180 
 2  12 23  46  157 
   mean  12 23  45.5  168.5 
   % of control*  100 100  106  98 
 1.0  1  12 23  43  174 
   2  12 22  44  165 
   mean  12 22.5  43.5  169.5 
   % of control*  100 98  101  99 
 10  1  12 23  48  168 
   2  12 21  41  117 
   mean  12 22  44.5  142.5 
   % of control*  100 96  103  83 
 100  1  12 19  28  48 
   2  12 19  32  45 
   mean  12 19  30  46.5 
   % of control *  100 83  70  27 

*: For calculation, replicate 2 with static test design was taken into account.

Dry weight of the Lemna plants after the 7-day exposure period

Experimental part A (parent molecule)

 Nominal test concentration (mg/L)  No. replicate  Dry weight per replicate (mg)   Mean dry weight per treatment(mg)  % of control*
 control  1  25 24   
   2  23    
0.10  1  24 26  104 
   2  28    
 1.0  1  17 20  80 
   2 22     
 10  1  7  6.5  26
   2  6    
 100  1  2  2.5  10
   2  3  

 

  *: For calculation, replicate 1 with semi-static test design was taken into account.                                                                                                         

Experimental part B (degradaion products)

 Nominal test concentration (mg/L)  No. replicate  Dry weight per replicate (mg)   Mean dry weight per treatment(mg)  % of control*
 control  1  25    
   2  23  24  
0.10  1  25  22  96
   2  19    
 1.0  1  23  21  91
   2  19    
 10  1  23  20  87
   2  16    
 100  1  6  6  26
   2  6  

 

*: For calculation, replicate 2 with static test design was taken into account.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The doubling time of frond number in the control was less than 2.5 days
Conclusions:
The results obtained during this 7-days growth test indicate, that the parent molecule of the test item is more toxic to Lemna gibba compared to its degradation products.
Executive summary:

The purpose of this non GLP study based on OECD 221 guideline, was to determine the effects of the test item (parent molecule) and its possible degradation products on the growth of the freshwater aquatic plant Lemna gibba (duckweed).

In order to compare the toxic potential of the parent molecule with the toxic potential of its possible degradation products, the study comprised two experimental parts: In Experimental Part A, the parent molecule of the test item was tested. In Experimental Part B, the degradation products of the test item were tested. For Experimental Part A, a semi-static test design with test medium renewal at Day 2 and Day 4 was applied. Experimental Part B was performed under static condition.

The exposure period of both experimental parts did run in parallel. The test duration was 7 days. The test item concentrations for both experimental parts were 100, 10, 1.0, and 0.10 mg/L. A control (test water without test item) was run in parallel. At the start of the test, Lemna colonies were transferred from the culture into the test vessels in a random order. A total of 12 fronds (3 colonies) was introduced into each of the two replicates per test concentration. Two control replicates were run in parallel (one replicate static and one replicate semi-static). The Lemna colonies were inspected in each test vessel for changes in frond and colony number as well as appearance (discoloration, sinking, root length or other abnormalities) at the start of the test and at Days 2, 4 and 7 (test end). The number of fronds and colonies was counted. At  test end the dry weight of the plants of each test vessel was determined.

For experimental part A (parent molecule), ErC50-7d=24 mg/L (Frond number), ErC10=0.91 mg/L (Frond number), ErC50-7d=23 mg/L (Dry weight), ErC10=1.1 mg/L (Dry weight)

For experimental part B (degradation products), ErC50-7d=99 mg/L (Frond number), ErC10=9.7 mg/L (Frond number), ErC50-7d>100 mg/L (Dry weight), ErC10=14 mg/L (Dry weight)

The results obtained during this 7-days growth test indicate, that the parent molecule of the test item is more toxic to Lemna gibba compared to its degradation products. The validity criteria were fulfilled according to the OECD guideline 221.