7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06/07/2009 - 27/08/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

The temperature of the main study lay in a range of 26.2 to 26.8 degree C and therefore higher than stated in the guideline. The temperature of the reference study lay in a range of 24.4 to 25.0 degree C and therefore higher than stated in the guideline.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

EC Number:

276-957-5

EC Name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Cas Number:

72869-86-4

Molecular formula:

C23H38N2O8

IUPAC Name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Details on test material:

Batch no.: 81106228
Purity: min. 96%, effective 96.99%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical Method
The concentrations of PC-Cure P-1001 were determined using a validated HPLC method. The validation report (written in German) and the corresponding raw data are archived following GLP regulations.

Parameters of Instrument
Test System: HPLC DIONEX with UV/VIS detector
Column: Reprosil 100 C18 4.6*150mm (Dr. Maisch)
Mobile phase: mixture of 75% acetonitrile and 25% water was used.
Flow rate: 1 mL/min.
Column temperature: 25°C
Detection λ: 210 nm
Injection volume: 50 µL
Retention time 4.0 – 4.1 min

Method Characterisation
Linear sector of method: 5 – 100 mg/L (see calibration curve, 6.6.4)
r of calibration function: 0.9997
Variation coefficient of method: ± 2.64 %
Limit of detection: 0.022 mg/L (based on the calibration curve equa-tion)
Limit of quantification 0.080 mg/L (based on the calibration curve equa-tion)

Stability of Solutions
A standard solution containing 10 mg/L of the test item was measured twice each after 0, 24 and 96 hours. The recovery rate after 24 hours storage at room temperature was de-termined as 98.8% and after 96 hours as 86.5% as can be taken from the following table.

Stability of Solutions
Time [h] Area recovery rate [%]
1 2 Mean
0 13,3799 13,3895 13,3847 -
24 13,2223 13,2197 13,221 98.8
96 11,5669 11,5815 11,5742 86.5

Test solutions

Vehicle:

yes

Details on test solutions:

The solubility in dilution water was tested before the start of the test. As solubility lies below 100 mg/L, the water-accommodated fraction was prepared for the test. This was done by weighing the nominal load, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 µm filters.

Dilution Water
Deionised water with an enrichment of certain minerals (as demanded in the guidelines) is used in the test.

Dilution water specification
Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80
Resulting hardness in mmol/L: 2.50
Resulting hardness in mg CaCO3/L: 250

After preparation, the dilution water is aerated, and the pH is adjusted to 7.8 +-0.2.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Specification
Species Daphnia magna
Variety STRAUS
Strain Berlin
Sex female
Age between 0 and 24 hours
Origin Umweltbundesamt Berlin
Arrival of Strain 27. Sep. 2007
Selection of the test system was made following the proposal of the guidelines.

Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The holding is performed similar to the method described in EN ISO 6341, following SOP 115 002 01 („Zucht und Hälterung von Daphnia magna STRAUS“), edition 9, adopted 27. Apr. 2007.
Vessels glass beakers, nominal volume 2 L
Medium M4-Medium (recipe of ELENDT), composition see annex
Food unicellular green algae (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16/8 hours, using neon tubes
Temperature 20 +- 2 °C

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no post exposure observation

Test conditions

Hardness:

250 mg CaCO3/l

Test temperature:

20 +- 2 °C

pH:

7.6 to 7.8

Dissolved oxygen:

7.6 to 7.7 mg/l

Nominal and measured concentrations:

Analytical Determinations
The recovery after 48 hours was 90 % of the start concentration. The measured concen-trations at the beginning and after 48 hours were 1 % of the nominal concentration. The measured values were not in the calibrated range but > LOQ. Therefore, the determination of the biological results was based on the mean of the measured concentrations.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 200 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Effect concentrations 2

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

1 200 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 1 200 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 200 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 1 200 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Effect concentrations 6

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 200 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: not determinable

Results with reference substance (positive control):

Biological Results Positive Control
Parameter Value 95%-confidence interval
NOEC 24 h 0.5 mg/L not determinable
24h EC50i 0.7 mg/L not determinable
24h EC100i 2.0 mg/L not determinable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

All validity criteria were met. For the estimation of the EC50 of the positive control, the fit showed sufficient statistical correspondence of the data with the dose-response-equation.
The 24h-EC50i of potassium dichromate was determined as 0.7 mg/L, lying within the demanded range of 0.6 – 1.7 mg/L.
The analytical determinations of the test item showed good recovery rates (comparison of measurements at the beginning and at the end of the experiment). Therefore, the test item PC-Cure P-1001 can be stated as stable under test conditions. Because of the poor water solubility of the test item in dilution water, the correlation between nominal and measured concentrations was weak. Therefore the determination of the results was based on the mean of the measured concentrations.
The result of the test can be considered valid.

Executive summary:

Three experiments were performed. Because of unexpected high inhibition in a first, invalid study, an additional range finding test without analytical determination was performed. In the study the result of the invalid first experiment and the range finding test aren’t reported.

The main study was performed as a limit test using the concentration 100 mg/L. Twenty daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

The concentration showed no toxicity. None of the animals was immobilised in the treatment and the control.

The 24h-EC50_iof potassium dichromate was tested in a current reference study. The value was determined as 0.7 mg/L, lying within the demanded range of 0.6 – 1.7 mg/L.

 

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-determination. The recovery after 48 hours was 90 % of the start concentration, the correlation between nominal and measured concentration was very poor. Therefore, the determination of the biological results was based on the mean of the measured concentration.

The following results were determined for the test itemPC-Cure P-1001(species:Daphnia magna).

 

24h-NOEC = 1.2 mg/L
48h-NOEC = 1.2 mg/L
24h-EC50_i: > 1.2 mg/L
48h-EC50_i: > 1.2 mg/L

Note: 1.2 mg/L should be considered as limit of water solubility under the conditions of the test.