Benzoic acid, 5-amino-2-chloro-, 1,1-dimethyl-2-oxo-2-(2-propen-1-yloxy)ethyl ester

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-(5-amino-2-chlorobenzoyloxy)-2-methylpropanoate

EC Number:

696-318-8

Cas Number:

174489-43-1

Molecular formula:

C14H16ClNO4

IUPAC Name:

prop-2-en-1-yl 2-(5-amino-2-chlorobenzoyloxy)-2-methylpropanoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: Samples kept at -18 to -22 °C until analysis.

- Sample preparation: The specimens were thawed in a microwave oven at ambient temperature. After intensively
shaking, the measured specimens were diluted with methanol in the ratio 1:1. The final
volume was measured to calculate the dilution factor. These solutions were filtered
through a 45 µm filter before injection.

- Reference solutions preparartion: 100.3 – 250.7 mg CA 2219 A were weighed, dissolved and made up to 100.0 ml with
methanol. From these stock solutions, reference solutions in the range of the test
concentrations (related to and extended to about 10 % below and above the expected
concentration) were diluted with methanol/test water = 1/1 (v/v). So references with
concentrations between 0.50 – 25.07 mg/L CA 2219 A were used.

Test solutions

Vehicle:

yes

Details on test solutions:

The nominal concentrations were blank, vehicle 1.25 – 40 mg/L CA 2219 A. The total content in the specimens of the test was found within 104.0 – 111.5 % of the nominal concentration at test begin and 82.0 – 106.0 % at test end. At test end, the total content present in the test vessels was also
determined. The total content was found within 96.0 – 113.3 % of the nominal concentrations. Theses indicate the correct dosing of the test item
into the vessels and show a slight inhomogeneous distribution in the test vessels at test end. No active ingredient could be detected in the blanks.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain: Clone 5
- Source: In house culture
- Age at study initiation: < 24 hours
- Food type/amount/frequency: No feeding was performed during the test
- Culturing: Culturing was performed in a constant environment room at 20 °C ± 2 °C and a photoperiod of 16 hours
light and 8 hours dark, with a transition period of 30 minutes. Three times a week the young daphnids
were separated from the original culture to maintain a homogenous stock of animals within the same age.

Study design

Test type:

static

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

244 mg CaCO3/L

Test temperature:

21 ± 1 °C

pH:

The pH was 8.6 – 8.6 and 8.0 – 8.1 at the start and at the end of exposure, respectively.

Dissolved oxygen:

Oxygen saturation 101 - 103 % and 99 - 104 % at the start and at the end of exposure, respectively.

Nominal and measured concentrations:

Nominal: 1.25, 2.5, 5.0, 10, 20 and 40 mg/L
Measured: 1.3, 2.7, 5.3, 11.0, 22.3 and 42.5 mg/L at the start of exposure and 1.1, 2.2, 4.7, 10.6, 20.4 and 32.8 mg test item/L
at the end of exposure.

Details on test conditions:

TEST SYSTEM
- Test vessels: 150 ml glass beakers containing 100 ml of test solution or blank test medium.
- Animals/concentration: 20 animals for each concentration and the controls.
- Animals/test vessel: 5 daphnids were exposed per test vessel.
- Test medium: M4-medium according to ELENDT (for details of composition refer to Table D).
- Photoperiod/light intensity: 16 hours light and 8 hours dark, with a transition period of 30 minutes. Fluorescent light (about 1200 lux).
- Replicates: Four replicates for the test concentrations and the control were performed.

TEST CONCENTRATIONS

- STOCK SOLUTIONS
- Preparation of stock solutions and test solutions:
4.0101 mg of CA 2219 A were mixed and made up to 10 ml with DMF (stock solution) 1). 2.5 ml of stock solution 1 were mixed and made up to 5 ml with DMF (stock solution 2). 2.5 ml of stock solution 1 were mixed and made up to 10 ml with DMF (stock solution 3). 1.25 ml of stock solution 1
were mixed and made up to 10 ml with DMF (stock solution 4). 0.625 ml of stock solution 1 were mixed and made up to 10 ml with DMF
(stock solution 5). 0.3125 ml of stock solution 1 were mixed and made up to 10 ml with DMF (stock solution 6). These stock solutions were used to prepare all test solutions with test concentrations of 1.25, 2.50, 5.0, 10, 20 and 40 mg/L.

- TEST CONCENTRATIONS
- Range-finding Test:
950.8 mg of CA 2219 A were mixed and made up to 10 ml DMF.
This stock solution was used to prepare all test solutions with test concentrations of 0.1, 1.0, 10.0 mg/L.

- Preliminary Test 1:
1.6075 g of CA 2219A were mixed and made up to 10 ml with DMF (stock solution 1). 5 ml of stock solution 1 were mixed and made up to 10 ml with DMF (stock solution 2). 5 ml of stock solution 2 were mixed and made up to 10 ml with DMF (stock solution 3). 5 ml of stock solution 3 were mixed and made up to 10 ml with DMF (stock solution 4). 5 ml of stock solution 4 were mixed and made up to 10 ml with DMF (stock solution 5). These stock solutions were used to prepare all test concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L.

- Definitive test concentrations:

- Test concentrations were 1.25, 2.50, 5.0, 10, 20 and 40 mg/L of CA 2219 A.

- Test Medium: Reconstituted water (M4-medium)
- Control: Blank (M4-medium)
Vehicle DMF

Remarks:
Calculated amounts of the stock solution to produce the desired test concentrations and vehicle were given into the reconstituted water and were homogenously distributed. Only in the two highest concentrations, some precipitation was observed.

- Water Quality: The oxygen content, pH and temperature were measured at test start and test end. Hardness and conductivity of
the test medium were determined from the freshly prepared M-4medium.

- Observations: Immobilization and other behavioural changes of the daphnids were recorded after 24 h and 48 h of exposure.
Those animals not able to swim within 15 seconds after gentle agitation of the test container were considered to
be immobile.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 34.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL

Details on results:

- Light Intensity: 1200 lux

- Additional water quality data: Conductivity: 687 µS/cm

- Analytical Results: The test item was homogeneous distributed in the test vessels at all test concentrations at the start of the
test. Only in the two highest concentrations, some precipitation was observed. The measured test
concentrations of CA 219 A (samples taken from the centre of the test vessels) were 1.3, 2.7, 5.3, 11.0, 22.3
and 42.5 mg/L at the start of exposure and 1.1, 2.2, 4.7, 10.6, 20.4 and 32.8 mg test item/L at the end of
exposure.
The total content of CA 2219 A was found 104.0, 108.0, 106.0, 110.0, 111.5 and 106.3 % of nominal
concentration at test start and 88.0, 88.0, 94.0, 106.0, 102.0 and 82.0 % at test end.
At test end, the total content present in the test vessels was also determined. The total content was found
within 96.0 – 113.3 % of the nominal concentration.

- Observations: No immobilization of daphnids was observed in the control (blank). In addition, during the biological phase
of the study, no unexpected phenomena were recorded.

Any other information on results incl. tables

 

Exposure period (h)	EC 50 (mg/L)	95 % confidence limit (mg/L)	Slope	No effect level (observed) (mg/L)
24 48	34.9 11.0	27.3 – 54.5 9.1 – 13.3	1.61 2.43	10.0 2.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (48h) of CA 2219 A was determined to be 11.0mg/L based on nominal concentrations. The highest concentration with no observed
effects after 48 hours was 2.5 mg/L based on nominal concentrations.

Executive summary:

 

Test concentrations of nominal 1.25, 2.5, 5.0, 10, 20 and 40 mg/L of CA 2219 A were selected based on results of a range-finding test and a preliminary test. Exposure started when the daphnids were placed in the test vessel containing the test solutions. 

Twenty daphnids were exposed to each of the desired test concentrations under static conditions in M4-medium. For the test concentrations and the control, four replicates were performed. Each replicate consisted of five specimens in 100ml test solution. The daphnids were observed for immobilization and behavioural changes after 24 h and 48 h of exposure. 

The water temperature was maintained constant throughout the duration of the test at 21±1 °C in all test concentrations. The oxygen saturation was measured to be 101 -103 % and 99 – 104 % at the start and at the end of exposure, respectively. The pH was 8.6 – 8.6 and 8.0 – 8.1 at the start and at the end of exposure, respectively. 

The test item was homogeneously distributed in the test vessels at all test concentrations at the start of the test. Only in the two highest test concentrations, some precipitation was observed. 

The measured test concentrations of CA 2219 A (samples taken from the centre of the test vessels) were 1.3, 2.7, 5.3, 11.0, 22.3 and 42.5 mg/L at the start of exposure and 51.1, 2.2, 4.7, 10.6, 20.4 and 32.8 mg test item/L at the end of exposure. 

 

The total content of CA 2219 A was found 101.0, 108.0, 106.0, 110.0, 111.5 and 106.3 % of nominal concentration at test start and 88.0, 88.0, 94.0, 106.0, 102.0 and 82.0 % at test end. 

At test end, the total content present in the test vessels was also determined. The total content was found within 96.0 – 113.3 % of the nominal concentration. 

Actual test concentrations were within 100 ± 20 % therefore following values are based on nominal concentrations.

 

Exposure period (h)	EC 50 (mg/L)	95 % confidence limit (mg/L)	Slope	No effect level (observed) (mg/L)
24 48	34.9 11.0	27.3 – 54.5 9.1 – 13.3	1.61 2.43	10.0 2.5

 

Conclusion:

The EC50 (48h) of CA 2219 A was determined to be 11.0mg/L based on nominal concentrations. The highest concentrations with no observed effects after 48 hours was 2.5 mg/L based on nominal concentrations.