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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity 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:

- Sampling method: Specimens of each test concentration were drawn from the approximate centre of test vessels.

- Sample storage conditions: The specimens were kept at -18°C to -22 °C until analysis.
The specimens were thawed in a microwave oven at ambient temperature.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Concentrations:
Reference solutions: 27.5 – 50.7 mg CA 2219 A were weighed, dissolved and made up to 25.00 ml with
N,N-Dimethylformamaide. 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
0.022 – 2.0 mg/L CA 2219 A were used.

Sample solutions: After intensively shaking, the measured specimens were diluted with methanol in the ratio 1:1.
The final volume wasmeasured to calculate the dilution factor.
Solutions were filtered through a 45 µm filter before injection.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: From own culture
- Age at study initiation:
- Length at study initiation: (mean) 49 mm (range 46 - 52 mm) based on 7 fish of the control
- Weight at study initiation: (mean) 0.93 g (range 0.80 - 1.10 g) based on 7 fish of the control
- Food type: Commercially prepared triut food diet, supplemented by living, frozen or dried organisms


ACCLIMATION
- Acclimation period: 95 days
- Acclimation conditions: In water of similar quality used in the test
- Feeding frequency: Not fed 24 hours prior to and during the exposure period
- Health during acclimation: Total mortality did not exceed 3 % during the 7 day period prior to the start of the test

Study design

Test type:

static

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

184 mg CaCO3/L

Test temperature:

13.4 - 13.6 °C

pH:

7.6 - 8.3

Dissolved oxygen:

93 - 105 %

Nominal and measured concentrations:

The nominal concentrations were 0.18, 0.32, 0.58, 1.0 and 1.8 mg test item/L.
The measured mean concentrations were 0.12, 0.19, 0.38, 0.78 and 1.5 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 20 liters filled with 15 liters of water (35/22/26 cm ; depthof the water: 20cm)
- Aeration: Gentle aeration during exposure (il free air)
- No. of organisms per vessel/replicates: 7 per concentration and control. 7 per aquairia. One test chamber per concentration and control.
- Biomass loading rate: 0.43 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated local tap water. Carbon fine filtered to eliminate chlorine and particulate material.
- Intervals of water quality measurement: Oxygen content measured 0, 24,48, 72 and 96 hours (as long as fish remained in the tank)
Hardness measured at start of test in control chamber
pH measured 0, 24,48, 72 and 96 hours (as long as fish remained in the tank)
Temperature measured 0, 24,48, 72 and 96 hours (as long as fish remained in the tank)

EFFECT PARAMETERS MEASURED:
- Intervals of biological observations: Mortality at 0, 24,48, 72 and 96 hours
Sublethal syptoms at 0, 24,48, 72 and 96 hours
Weight and length at start and end of test with controls only.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours daily with 30 minutes transition period
- Light intensity: Fluorescent light


TEST CONCENTRATIONS
- Stock solution:
- Preparation of a stock solution/test initiation:
Stock solution – 307 mg test item were mixed with and made up to 2000µl with diemthylformaide (DMF).
Calculated amounts of the stock solution to produce the desired test concentrations were given into the water. After an equilibration time of 6.25 hours, the fish were transferred into the aquaria (beginning of exposure).

- Test Concentrations:
- Range finding tests:
Prior to initiating definitive test, a static range-finding test was performed at concentrations 1.0, 10 and 100 mg/L.
Based on results of this range-finding test, the following nominal concentrations were selected for the definitive test.

- Definitive test:
Concentration(s) 0.18, 0.32, 0.58, 1.0 and 1.8 mg CA 2219 A/L (nominal).
Controls/Blank Dechlorinated tap water

- Sampling for test concentrations:
Specimens of each test concentration were drawn from the approximate centre of test vessels. They were taken immediately before exposure and after 96 hours, of all concentrations and the stock solution. If any of the concentrations showed 100 % mortality before the end of the test, specimens were taken when all fish had died. The specimens were kept at -18°C to -22 °C until analysis.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.82 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Water Quality:
The temperature was maintained constant at 13.4 – 13.6°C and the dissolved oxygen contents between 93 – 105 % of the saturation value by gentle aeration during exposure. The pH was determined to be 7.6 – 8.3 and the total hardness 184 mg/L.

- Concentration of Test Item in water:
The test item appeared homogeneously distributed in the test vessels at all test concentrations over the period of the test. No precipitation was observed in the testing solutions throughout the duration of the test.
The nominal concentrations were 0.18, 0.32, 0.58, 1.0 and 1.8 mg test item/L.
The measured mean concentrations were 0.12, 0.19, 0.38, 0.78 and1.5 mg test item/L.

- Lethal Effects:
After 96 hours exposure, mortality occurred at concentrations of 0.78 and 1.5 mg/L with 100% mortality, each.
A decrease of the LC50-value from 0.82 to 0.54 mg/L was observed during the period 24 to 48 h exposure. The LC 0 (96h) was 0.38 mg/L and the LC 100 (96h) 0.78 mg/L.
The mortality in the blank control was 0 %.

- Sublethal Effects:
Sublethal effects were observed at concentrations ≥ 0.78 mg/L, such as change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal and lethal effects was 0.38 mg/L.

- Conclusion:
The LC50 (96 h) of CA 2219 A was determined to be 0.54 mg/L based on measured mean concentrations. The highest concentration with no sublethal effects was 0.38 mg/L

Reported statistics and error estimates:

The LC-50 values were calculated according to the maximum likelihod method, probit model (Finney, D.J. 1971) and were graphically determined.

Any other information on results incl. tables

Sublethal observations / clinical signs:

 

Exposure period	LC50	95% confidence	Slope	Goodness of fit
(h)	(mg/L)	(mg/L)	(mg/L)	X^2	df	p
96 72 48 24	0.54 0.54 0.54 0.82	none none none none	23.0 23.0 23.0 11.7	0.00 0.00 0.00 0.00	3 3 3 3	1.00 1.00 1.00 1.00

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The LC50 (96 h) of CA 2219 A was determined to be 0.54 mg/L based on measured mean concentrations. The highest concentration with no sublethal effects was 0.38 mg/L

Executive summary:

Static pre-tests showed that the LC50 (96h) was 1 mg/L. Therefore, the following nominal concentrations were chosen for the main test: 0.18, 0.32, 0.58, 1.0 and 1.8 mg test item/L. 

After the preparation of the test concentrations and an equilibrium time of 6.3 hours, 7 fish were randomly placed in each of the 20 L glass aquaria containing 15 L of test solution (beginning of exposure). Fish were exposed to CA 2219 A for 96 hours and were observed for adverse effects at 2, 24, 48, 72 and 96 hours. 

 

Findings:

The temperature was maintained constant at 13.4 – 13.6°C and the dissolved oxygen contents between 93 – 105 % of the saturation value by gentle aeration during exposure. The pH was determined to be 7.6 – 8.3 and the total hardness 184 mg/L. 

The test item appeared homogeneously distributed in the test vessels at all test concentrations over the period of the test. No precipitation was observed in the testing solutions throughout the duration of the test. 

The nominal concentrations were 0.18, 0.32, 0.58, 1.0 and 1.8 mg test item/L. 

The measured mean concentrations were 0.12, 0.19, 0.38, 0.78 and1.5 mg test item/L. 

 

The following calculations and values are based on measured mean concentrations. 

 

Exposure period	LC50	95% confidence	Slope	Goodness of fit
(h)	(mg/L)	(mg/L)	(mg/L)	X^2	df	p
96 72 48 24	0.54 0.54 0.54 0.82	none none none none	23.0 23.0 23.0 11.7	0.00 0.00 0.00 0.00	3 3 3 3	1.00 1.00 1.00 1.00

 

 

After 96 hours exposure, mortality occurred at concentrations of 0.78 and 1.5 mg/L with 100% mortality, each. 

A decrease of the LC50-value from 0.82 to 0.54 mg/L was observed during the period 24 to 48 h exposure. The LC 0 (96h) was 0.38 mg/L and the LC 100 (96h) 0.78 mg/L. 

 

Sublethal effects were observed at concentrations ≥ 0.78 mg/L, such as change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal and lethal effects was 0.38 mg/L.  

 

Conclusion:

The LC50 (96 h) of CA 2219 A was determined to be 0.54 mg/L based on measured mean concentrations. The highest concentration with no sublethal effects was 0.38 mg/L