Reaction mass of 2-{[(2-{3-[2-({[2-(methacryloyloxy)ethoxy]carbonyl}amino)propan-2-yl]phenyl}propan-2-yl)carbamoyl]oxy}propyl methacrylate and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxyethane-2,1-diyl) bis(2-methylacrylate) and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxypropane-2,1-diyl) bis(2-methylacrylate)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009 laying down test methods pursuant to REACH Regulation, 2009, C.26: Lemna sp. Growth Inhibition Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,3-phenylenebis(propane-2,2-diylcarbamoyloxyethane-2,1-diyl) bis(2-methylacrylate) and 2-{[(2-{3-[2-({[2-(methacryloyloxy)ethoxy]carbonyl}amino)propan-2-yl]phenyl}propan-2-yl)carbamoyl]oxy}propyl methacrylate and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxypropane-2,1-diyl) bis(2-methylacrylate)

Molecular formula:

C26H36N2O8, C27H38N2O8, C28H40N2O8

IUPAC Name:

Reaction mass of 1,3-phenylenebis(propane-2,2-diylcarbamoyloxyethane-2,1-diyl) bis(2-methylacrylate) and 2-{[(2-{3-[2-({[2-(methacryloyloxy)ethoxy]carbonyl}amino)propan-2-yl]phenyl}propan-2-yl)carbamoyl]oxy}propyl methacrylate and 1,3-phenylenebis(propane-2,2-diylcarbamoyloxypropane-2,1-diyl) bis(2-methylacrylate)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Preparation of Calibration Solutions: The test item (75.90 mg) was dissolved in methanol and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 1518 mg/L. A stock solution aliquot of 0.25 mL was diluted to 20 mL with methanol/water (v/v; 1/1) to obtain a working solution with a concentration of 19.0 mg/L. Defined volumes of the stock and the working solutions were further diluted with methanol/water (v/v; 1/1) to obtain calibration solutions of the test item in the range of 0.0949 to 30.4 mg/L. A second series of calibration solutions in the range of 0.0550 to 13.2 mg/L, based on a stock solution of 879 mg/L was prepared. The total calibration range used was 0.0550 to 30.4 mg/L. These solutions were used to calibrate the analytical system.
- Preparation of Spiked Recovery Solutions: To demonstrate the validity of the method, untreated test water was spiked with the test item. The test item (119.62 mg) was dissolved in methanol and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 2392 mg/L. A stock solution aliquot of 1 mL was made up to 20 mL with methanol to obtain a fortification solution with a concentration of 120 mg test item/L. Defined volumes of the stock and the fortification solutions were diluted with test water to obtain spiked samples with concentrations of 12.0 mg/L (from stock solution) and 0.359 mg/L (from fortification solution). Five spiked recovery samples were freshly prepared per concentration level, subjected to the same treatment as a test sample but without storage and subsequently analyzed. In addition, test water without the test item was analyzed after sample preparation (analytical blank).
- Preparation of Test Samples: The diluted test samples and control samples were thawed at room temperature for either 30 minutes or 1.5 hours and shaken manually to obtain homogeneous sample solutions for the sample analysis.
- Sample storage conditions before analysis: The samples were diluted with methanol by a factor of two before they were stored deep-frozen and protected from light until analysis was performed.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared:
loading rate 4.6 mg/L: 13.7050 mg test item in 3000 mL test water
loading rate 10 mg/L: 30.03 mg test item in 3000 mL test water
loading rate 22 mg/L: 66.11 mg test item in 3000 mL test water
loading rate 46 mg/L: 138.09 mg test item in 3000 mL test water
loading rate 100 mg/L: 301.67 mg test item in 3000 mL test water
The dispersions were subjected to ultrasonic treatment for 15 minutes and stirred for 24 hours at room temperature in the dark to dissolve a maximum amount of the different test item components in the dispersion. In order to separate undissolved test material from the test water (as far as possible), the dispersions were left to settle for about 60 minutes and the middle layers were drawn off with a Teflon® tube for filtration. The middle layers were filtered through a membrane filter (pore size 0.45 μm). The negative pressure of the filtration unit was reduced as much as possible to avoid passing of undissolved material of the liquid test item through the filter. The undiluted filtrates were tested as WAFs.
The stirring period of 24 hours was chosen based on the results of a pre-experiment (non-GLP).
Due to technical reasons, the WAF with the lowest loading rate of 2.1 mg/L was prepared as a dilution of the WAF with the loading rate of 4.6 mg/L. The test media were prepared just before the start of the exposure. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
- Controls: test water without test item; for evaluation of the sensitivity of the test system, the reference item 3,5-dichlorophenol is tested twice a year.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: Lemna gibba G3 (family Lemnaceae, Macrophyta)
- Source (laboratory, culture collection): The original culture was supplied by Bayer CropScience AG, 40789 Monheim, Germany in 2007.
- Method of cultivation: The plants were axenically cultivated in the laboratory for more than four weeks prior to the test and under standardised 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.
- Other: Lemna gibba is a preferred aquatic macrophyte species used to evaluate the toxicity of test items to higher aquatic plants. The test method and the test species are recommended by the test guideline. Commonly used and favoured tested species to coer the endpoints 6.1.5/6.1.6 are Pseudokirchneriella subcapitata (previously named Selenastrum capricornutum) Scenedesmus subspicatus and Chlorella vulgaris (OECD 201). All can be considered as equally accepted preferred species, but the OECD 221 study with Lemna gibba can be used as well: The Lemna test is a short-term test although it provides both acute and sub-chronic endpoints. The tests last for up to 14 days and are performed in nutrient enriched media similar to that used for algae, but may be increased in strength.)

ACCLIMATION
- Acclimation period: not applicable
- Culturing media and conditions (same as test or not): cultivation conditions same as test

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:

3.0 mmol/L (= 300 mg/L as CaCO3)

Test temperature:

22-23°C

pH:

7.5 - 9.2

Nominal and measured concentrations:

WAFs with the following loading rates of the test item were tested: 2.1, 4.6, 10, 22, 46 and 100 mg/L. Corresponding mean measured concentrations (except for 2.1 mg/L loading rate: the samples from the 2.1 mg/L-WAF were not analysed since this treatment was not relevant for the interpretation of the biological results): 2.0, 0.65, 7.4, 0.83, 7.6 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes; the test vessels were incubated in a temperature-controlled water bath with a temperature of 22–23 °C.
- Test vessel: 250-mL glass dish (diameter of 9.5 cm)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume; type of cover: Each replicate consisted of a 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 and incubated in a water bath in a randomized order. The test vessels were repositioned at each counting date and were labeled with the study number and all necessary additional information to ensure unique identification.
- No. of colonies per vessel: At the start of the test, Lemna colonies were transferred aseptically from the pre-culture into the different test vessels in a randomised order. The test was started with three randomly selected colonies per vessel.
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes; reconstituted test water (20X AAP growth medium prepared according to the OECD test guideline)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (20X AAP growth medium prepared according to the OECD test guideline) was used for cultivation and testing. The test water was prepared one day prior to use.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and at the end of the test. The water temperature was measured in a vessel filled with water (incubated under the same conditions as the test vessels) at each working day. Additionally, the appearance of the test media was recorded on the counting dates of the plants.

OTHER TEST CONDITIONS
- Adjustment of pH: pH of the test water was adjusted to 7.5 ± 0.1 with a 1 M hydrochloric acid solution
- Photoperiod / light intensity and quality: continuously illuminated using fluorescent tubes (Philips TLD 36W-1/840) installed above the test vessels in order to achieve a light intensity of 6300–8000 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: The EC50 values for the inhibition of the growth rate and yield based on frond numbers and dry weight were determined.
On Days 2 and 5 and at the end of the test on Day 7, the number of fronds and colonies of the Lemna plants were counted. Fronds visibly projecting over the edge of the mother frond were counted as separate fronds. At the same dates, the plants were inspected for changes in appearance (e.g., discoloration, sinking, root length, or other abnormalities).
The dry weight of a sample of twelve fronds was determined at the start of the test. At test termination, the dry weight of the plants of each test vessel was determined. The plants were dried at about 60 °C in a laboratory vacuum oven for 48 hours (sufficient to reach a constant weight).

TEST CONCENTRATIONS
- Test concentrations: WAFs with the following loading rates of the test item were tested: 2.1, 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control group (test water without test item) was tested in parallel. Loading rates of the test item exceeding 100 mg/L were not tested in accordance with the test guidelines.
The selection of the test concentrations was based on the results of a range-finding test and on results of pre-experiments to determine the solubility of the test item (non-GLP).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol is tested as a positive control twice a year, result of the latest positive control test in Apr 2011 (7-d EC50 growth rate, frond no.: 11 mg/L) showed that the sens. of test system was within the historical range (7-d EC50: 8-11 mg/L).

Results and discussion

Effect concentrations

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 7.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Any visual signs of phytotoxicity (abnormalities): No abnormalities in appearance of the test plants were recorded in the control and at all loading rates.
- 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 throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: EC50 of the test item was above the solubility limit of the test item in the test water under the conditions of the test.

Results with reference substance (positive control):

- Results with reference substance 3,5-dichlorophenol valid? yes
- EC50: 11 mg/L (7d)
- Other: internal historical range (7-d EC50 from 2003 to 2011: 8.00-11 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on loading rates and mean measured concentrations of the test item the biological results of the study were as follows:
7-day EC50 > 100 mg/L (loading rate; based on frond number, growth rate and yield)
7-day EC50 > 7.6 mg/L (mean measured concentration; based on dry weight of the plants, growth rate and yield)
In conclusion, the EC50 of the test item was above the solubility limit of the test item in the test water under the conditions of the test.

Executive summary:

The influence of the test item on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7-day static test (GLP), according to OECD Guideline 221 and EU Method C.26. Commonly used and favoured tested species to cover the IUCLID endpoint 6.1.5 / 6.1.6 are Pseudokirchneriella subcapitata (previously named Selenastrum capricornutum) Scenedesmus subspicatus and Chlorella vulgaris (OECD 201). All can be considered as equally accepted preferred species, but the OECD 221 study with Lemna gibba can also be used: The Lemna test is a short-term test although it provides both acute and sub-chronic endpoints. The tests last for up to 14 days and are performed in nutrient enriched media similar to that used for algae, but may be increased in strength.

In order to assess the toxicity of the test item containing different components to Lemna gibba, water accommodated fractions (WAFs) with the loading rates of 2.1, 4.6, 10, 22, 46 and 100 mg/L were tested. Additionally, a control group was tested in parallel.

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 24 hours to dissolve a maximum amount of the different test item components in the dispersion. After stirring each dispersion was left to settle for about one hour and the middle layer was drawn off with a Teflon® tube for filtration (pore size 0.45 μm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAF with the lowest loading rate of 2.1 mg/L was prepared as a dilution of the WAF with the loading rate of 4.6 mg/L.

The preparation of the test media was based on "OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures".

At the start of the test, the measured test item concentrations (based on the analysis of the three main components of the test item) in the test media with the loading rates of 4.6, 10, 22, 46 and 100 mg/L were 3.5, 1.2, 12, 1.5 and 12 mg/L, respectively. At the end of the test, the measured concentrations were 1.1, 0.34, 4.8, 0.47 and 4.9 mg/L, respectively.

Based on loading rates and mean measured concentrations of the test item the biological results of the study were as follows:

7 -day EC50 value      (mg/L)	Parameter based on
	Frond numbers		dry weight of the plants
	Growth rate	Yield	Growth rate	Yield
Loading rate	> 100	> 100	> 100	> 100
Mean measured concentration	> 7.6	> 7.6	> 7.6	> 7.6

In conclusion, the EC50 of the test item was above the solubility limit of the test item in the test water under the conditions of the test [it is important to recognise that the maximum achievable dissolved concentration of the substance in the test medium is not the same as the water solubility of the substance as determined by OECD Guideline 105 (as typically, the concentration is less). This is due to the fact that the water solubility measurements made for regulatory purposes are usually made in distilled water (pH=6-9; here 6.3) and not test media (pH=7-8; here: 7.9-8) and that differences in pH of the test media and distilled water affects the solubility].