Benzoic acid, 2-chloro-5-isocyanato-, 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:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

yes

Remarks:

No replicates made (one aquaria /concentration). 7 organisms exposed at different concentrations. Temperature measured every 24 hours.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-(2-chloro-5-isocyanatobenzoyloxy)-2-methylpropanoate

EC Number:

695-953-8

Cas Number:

204918-22-9

Molecular formula:

C15H14ClNO5

IUPAC Name:

prop-2-en-1-yl 2-(2-chloro-5-isocyanatobenzoyloxy)-2-methylpropanoate

Sampling and analysis

Analytical monitoring:

no

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: Commercial supplier (P. Holer / CH-4314 Zeiningen / Switzerland)
- Length at study initiation: (mean) 45 mm (range 40 - 50 mm) based on 7 fish of the control.
- Weight at study initiation: (mean) 0.74 g (range 0.54 - 1.02 g) based on 7 fish of the control.
- Feeding during test: No feeding 24 hours prior to and during the test.


ACCLIMATION
- Acclimation period/conditions: Fish wre gradually acclimated to the test conditions, and were held for 13 days prior to testing in water of
similar quality used in the test (activ carbon filtered).
- Type and frequency of feeding: Daily (except for weekends) commercially prepared trout food diet.
- 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

Test conditions

Hardness:

142 mg CaCO3/L

Test temperature:

12.9 - 13.7°C

pH:

7.6 - 8.2

Dissolved oxygen:

93 - 102%

Nominal and measured concentrations:

Nominal: 0.13, 0.23, 0.40, 0.60, 1.0 and 1.7 mg CA 2814 B/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 20 litres filled with 15 litres of water (35/22/26 cm; depth of water approx. 20 cm).
- No. of organisms per vessel: 7 per aqarium.
- No. of vessels per concentration: One test chamber per concentration and control.
- Biomass loading rate: 0.35 g/L

TEST MEDIUM / WATER PARAMETERS
- Water: Well water of 'Lange Erlen' and 'Hard' was used as dilution water. Prior to its use, the water was passed
through an activated carbon filter. The water was gently aerated during exposure (oil free air).

OTHER TEST CONDITIONS
- Photoperiod: Fluorescent light, 16 hours light and 8 hours dark (30 minute transistion period).


EFFECT PARAMETERS MEASURED
- Water Quality Observation Intervals: Oxygen content (mg/L): 0, 24, 48, 72 and 96 hours
pH: 0, 24, 48, 72 and 96 hours
Temperature (°C): 0, 24, 48, 72 and 96 hours
- Total hardness: Monthly in representative specimens of the dilution water.
- Biological Observations: 2-4, 24, 48, 72 and 96 hours (observations and sublethal symptoms, such as abnormal behavioural
activity and stress were made (swimming behaviour, loss of equilibrium, exophthalmos, respiratory
function, pigmentation and other observations). Dead fish, if any, were removed from the test solutions
at least at the above mentioned intervals. At test termination, the fish in the control were retained for
measurement of weight and length).

TEST CONCENTRATIONS
- Stock solution:
200.0 mg test item were dissolved in 10 ml DMF. To obtain the treatment concentrations of 0.13, 0.23, 0.40, 0.60, 1.0 and 1.7 mg test item/L, the following amounts of the stock solution were added to respective test vessels: 0.1, 0.17, 0.30, 0.45, 0.75 and 1.3 ml. For the solvent control, 1.3ml DMF were added in the 15L test water. After an equilibration time of 5-15 minutes, the fish were transferred into the aquaria (beginning of
exposure).
- Test concentrations:
- Range-finding test: Prior to initiating the definitive test, a static range-finding test was performed at concentrations of
1 and 10 mg/L.
Based on the results of this range-finding test, the following nominal concentrations were selected
for the definitive test:
- Definitive test: Concentration(s): 0.13, 0.23, 0.40, 0.60, 1.0 and 1.7 mg CA 2814 B/L (nominal).
Blank control: Dilution water without test item.
DMF: Dilution water with 1.3 ml/L DMF

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: none

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: none

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: none

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: none

Details on results:

Lethal Efects: After 24 hours, no mortality was recorded at any of the test item concentrations (Table 1). Only the highest test
concentration of 1.7 mg/L showed effects (mortality after 96 H). The LC50 was nearly constant during the exposure window 48h – 96h; just a slight decrease from 1.6 to 1.4 mg/L was observed. The highest concentration with no
sublethal and lethal effects after 96 hours was 1.0mg/L. None of the test concentrations showed 100 % mortality.
Therefore, the lowest concentration with 100 % mortality after 96 hours was greater than 1.7 mg/L.

Subletal Effects: In the test item concentration, i.e. 1.7 mg/L, a slight change of the behaviour was observed since the fish calm.
Sublethal effects, such as a change in pigmentation, loss of equilibrium, change in swimming behaviour,
in the respiratory unction were not observed at any of the test item concentrations.

Any other information on results incl. tables

Sublethal observations / clinical signs:

 

Table 1

Nominal Conc. (mg/L)	Cumulative Mortality					96 h (%)
	Number of dead fish
	2-4 h	24 h	48 h	72 h	96 h
Blank	0	0	0	0	0	0
Control-blank	0	0	0	0	0	0
0.13	0	0	0	0	0	0
0.23	0	0	0	0	0	0
0.40	0	0	0	0	0	0
0.60	0	0	0	0	0	0
1.0	0	0	0	0	0	0
1.7	0	0	4	5	6	86

 

Applicant's summary and conclusion

Conclusions:

The LC50 (96h) of CA 2814 B was determined to be 1.4 mg/L based on the nominal concentrations. The highest concentration with no sublethal and lethal effects was 1.0 mg/L.

Executive summary:

Because of the poor solubility of the test item, N,N-Dimethyl formamide (DMF) was used as solubilising agent for the preparation of the stock solution. Static pre-tests showed that the LC50 (96 h) was between 0.1 and 1 mg/L. Therefore, the following nominal concentrations were chosen for the definitive test: 0.13, 0.23, 0.40, 0.60, 1.0 and 1.7 mg test item/L in addition to a control blank and a DMF-control, both without test item. 

After the preparation of the test concentrations and an equilibrium time of 5 – 20 minutes, 7 fish were randomly placed in each of the 2 L glass aquaria containing 15 L of test solution (beginning exposure). 

Fish were exposed to CA 2814 B for 96 hours and were observed for adverse effects at 2-4, 24, 48, 72 and 96 hours. 

 

The temperature was maintained constant at 12.9 – 13.7 °C and the dissolved oxygen content between 93 – 102 % of the saturation value by gentle aeration during exposure. The pH was determined to be 7.6 – 8.2 and total hardness 142 mg CaCO₃/L. 

Due to the instability of the test item in test water , the actual test concentrations of CA 2814 V were not determined analytically. Hence, the results were calculated with the nominal concentrations. 

 

 

Exposure period	LC50	95% confidence
(h)	(mg/L)	(mg/L)
96 72 48 24	1.4 1.5 1.6 >1.7	none none none none

 

 

After 24 hours, no mortality was recorded at any of the test item concentrations. Only the highest test concentration of 1.7 mg/L showed effects (mortality after 96 H). The LC50 was nearly constant during the exposure window 48h – 96h; just a slight decrease from 1.6 to 1.4 mg/L was observed. The highest concentration with no sublethal and lethal effects after 96 hours was 1.0mg/L. None of the test concentrations showed 100 % mortality. Therefore, the lowest concentration with 100 % mortality after 96 hours was greater than 1.7 mg/L. In the test item concentration, i.e. 1.7 mg/L, a slight change of the behaviour was observed since the fish calm. Sublethal effects, such as a change in pigmentation, loss of equilibrium, change in swimming behaviour, in the respiratory unction were not observed at any of the test item concentrations. 

 

The LC50 (96h) of CA 2814 B was determined to be 1.4 mg/L based on the nominal concentrations. The highest concentration with no sublethal and lethal effects was 1.0 mg/L.