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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition 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:

2 samples (2 times about 30 ml per concentration and control) were taken at the start and 2 samples (2 times about 30 ml per concentration and control) at the end of exposure. Samples were taken from the freshly prepared test solutions immediately before exposure and from two composite test vessels after 72 hours exposure. All samples were kept at -18 to -22 °C until analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

Refer to anlaytical methods section.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Strain: Selenastrunm capricornutum ATCC 2262 ≡ Pseudokirch subcapiata SAG 61.81
- Source: Collection of algae cultures, Pflanzenphysiologisches Institut, University, Nikolausberger Weg 180, D-37073 Göttingen, Germany.
- Initial cell density: 9770 cells/ml
- Pre-culture: 4 days under test conditions
- Culture: The algal culture used was demonstrated to be adequately sensitive. The ErC 50 (0 – 72 h) and the EbC 50
(0 – 72 h) of potassium dichromate were 0.97 and 0.51 mg/L.
- Culture medium: Refer to Table 2

Study design

Test type:

static

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

24°C ± 1°C

pH:

The pH of the test solutions was 7.9 – 8.0 at the start and between 7.4 – 7.7 at the end of exposure

Nominal and measured concentrations:

The nominal test concentrations were: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L.
The measured test concentrations of CA 2219 A were 3.0, 6.1, 11.1, 23.8, 49.5 and 85.8 mg/L at the start of exposure and 1.4, 3.3, 7.0, 16.3, 35.0 and 63.7 mg test item/L at the end of exposure.
Actual mean concentrations were 2.20, 4.70, 9.05, 20.15, 42.55 and 74.75 mg/L corresponding to 70.4, 75.2, 72.4, 81.0, 84.5 and 74.8 % of nominal concentrations.

Details on test conditions:

TEST SYSTEM:

Vessels: 100 ml Erlenmeyer flasks (total volume 125 ml), cotton stoppers, on Lab shaker, 30ml test solution per flask.

Temperature: 24 ± 1°C

Lighting: Continuous illumination, cold white fluorescent light (approx. 8000 lux)

Shaking rate: 150 rpm

Duration: 72 hours

PREPARATION OF STOCK SOLUTIONS (test Item):

10.335 g of CA 2219 A were mixed and made up to 10 ml with DMF. This first stock solution was diluted with DMF (2.5 ml of SL 1 ad 5 ml DMF 2.5, 1.25, 0.625 and 0.312 ml of SL 1 ad 10 ml DMF, respectively) to produce stock solutions 2 to 6. Stock solutions 1-6 were used to prepare test solutions with test concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 mg/L, respectively.

TEST CONCENTRATIONS:

A range-finding test (test concentrations 0.1, 1.0 and 10 mg/L) was conducted to determine a concentration range of CA 2219 A to use in the definitive test. The cell concentrations were measured at the start, after 24 and 48 hours and at the end of exposure. No inhibition of the algae growth was seen at test concentration of 0.1 and 1.0 mg/L.

Based on the results of the range-finding test the following nominal concentrations were selected for the definitive test:
Nominal: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L.
Control: Reconstituted water without (blank) and with DMT (vehicle control)
Replicates: Each test concentration was tested in 3 replicates, the blank control in 6.

Remark: Calculated amounts of the stock solution to produce the desired test concentrations were given into the water and were homogeneously distributed. The algae were then transferred to the flasks.

MEASUREMENTS:

Cell densities were measured at 24, 48 and 72 hours of exposure using CytoFluor II (a Fluorescence Multi-Well Plate Reader).
Temperature was continuously measured in the room and daily in water in a separate test vessel.
The pH of the test solutions was measured at 0h and 72 hours of exposure.
Light intensity was measured at the start of the exposure using a Quantum Radiometer/Photometer.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 20.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 9.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Cell densities were measured at 24, 48 and 72 hours exposure.
Changes in mean density indicated that exponential growth occurred in the negative control replicates.
No significance difference in mean cell density between the controls and the actual mean concentrations of 2.20, 4.7 and 9.05 mg/L treatment groups on the basis of growth rate (0 – 16.4 % inhibition) were observed.

Calculations of EC50 and NOEC are based on actual mean concentrations of CA 2219 A – refer to Table 1

ErC 50 (0 - 72h) and EbC 50 (0 - 72h) of CA 2219 A were determined to be 20.7 and 3.15 mg/L based on actual mean concentrations.

The NOErC and NOEbC growth inhibition (0 - 72h) were 9.05 and 2.20 mg/L based on actual mean concentrations of CA 2219 A.

Results with reference substance (positive control):

The algal culture used was demonstrated to be adequately sensitive.
Refer to table 3.

Any other information on results incl. tables

Table 1:

Test item results	Inhibition (growth rates)	Inhibition (areas under growth curve)
EC 50 (0 – 72 h): 95 % confidence limit: Slope: NOEC (0- - 72 h): (significant on 5% level)	ErC 50 : 20.7 mg/L None 21.679 ± 0.163 NOErC : 9.05 mg/L	EbC 50: 3.15 mg/L 0.40 – 5.9 mg/L 1.205 ± 0.477 NOEbC : 2.20 mg/L

Table 3:

Reference item results:	Inhibition (growth rates)	Inhibition (areas under growth curve)
EC 50 (0 – 72 h): 95 % confidence limit: Slope: NOEC (0- - 72 h): (significant on 5% level)	ErC 50 : 0.97 mg/L None 4.288± 2.982 NOErC : 0.50 mg/L	EbC 50: 0.51 mg/L 0.44 – 0.57 3.952 ± 0.999 NOEbC : 0.25 mg/L

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC 50 (0 – 72 h) of CA 2219 A was determined to be 20.7 mg/L based on actual mean concentrations.

The NOErC growth inhibition (0 – 72 h) was 9.05 mg/L based on actual mean concentrations of CA 2219 A.

Executive summary:

Range-finding tests showed that the ErC50 (72h) would be between 1 – 100 mg/L. Based on the range-finding test, the following nominal concentrations of CA 2219 A were chosen for the main test: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L. 10.335 g of CA 2219 A were mixed and made up to 10 ml with DMF. This first stock solution was diluted with DMF to produce stock solutions 2 to 6. Stock solutions 1-6 were used to prepare test solutions 1-6, respectively. The prepared test solutions were distributed in the test vessels before the algae were placed in the test vessels. Exposure of Selenastrum capricornutum was started at an initial cell density of 9770 cells/mL after pre-culture period of three days. 

 

The test with CA 2219 A, was conducted at a temperature of 24 ± 1°C. Continuous illumination with cold white fluorescent light at 117 µE/m²(approx. 8000 lux) was used. The pH of the test solutions was 7.9 – 8.0 at the start and between 7.4 – 7.7 at the end of exposure. The multiplication factor of the algae cell density in the control within 72 hours was 61 in the control. 

The nominal test concentrations were: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L. The measured test concentrations of CA 2219 A were 3.0, 6.1, 11.1, 23.8, 49.5 and 85.8 mg/L at the start of exposure and 1.4, 3.3, 7.0, 16.3, 35.0 and 63.7 mg test item/L at the end of exposure. Actual mean concentrations were 2.20, 4.70, 9.05, 20.15, 42.55 and 74.75 mg/L corresponding to 70.4, 75.2, 72.4, 81.0, 84.5 and 74.8 % of nominal concentrations. 

 

Following calculations of EC50 and NOEC are based on nominal concentrations. 

 

ErC 50 (0 – 72 h):                20.7 mg/L

95% confidence limit:           none

Slope:                                  21.68 ± 0.163

NOEC (0 – 72 h):                NOErC : 9.05 mg/L

(significant on 5% level)

 

ErC 50 (0 – 72 h) of CA 2219 A was determined to be 20.7 mg/L based on actual mean concentrations. 

 

The NOErC growth inhibition (0 – 72 h) was 9.05 mg/L based on actual mean concentrations of CA 2219 A.