Benzoic acid, 2-chloro-5-nitro-, 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-(2-chloro-5-nitrobenzoyloxy)-2-methylpropanoate

EC Number:

696-317-2

Cas Number:

174489-76-0

Molecular formula:

C14H14ClNO6

IUPAC Name:

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Specimens of each test concentration were drawn from the approximate centre of the test vessels. They were taken immediately before the exposure, after 48 hours and 96 hours exposure. 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.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Stock solution

Preparation of a stock solution/test initiation:
Stock solution – 1.0 g test item was mixed with and made up to 2000ml with water (additional ultrasonification for approx. 5 min.). The stock
solution was filtered over filter paper (Ederol). The CA 2218 A concentration in the filtered stock solution was determined by HPLC. Calculated amounts of the filtered saturated stock solution t produce desired test concentrations were given into the water. After an equilibration time of
4 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 0.1, 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) 0.081, 0.16, 0.33, 0.65, 1.3 and 2.6 mg CA 2218 A/L (nominal).
Controls/Blank Dechlorinated tap water

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. Hohloer / CH-4314 Zeiningen / Switzerland
- Length at study initiation: (mean) 50 mm (range 43 - 54 mm) based on 7 fish of the control.
- Weight at study initiation: (mean) 1.05 g (range 0.73 - 1.24 g) based on 7 fish of the control.

ACCLIMATION
- Acclimation period/conditions: Held for 21 days prior to testing in water of similar quality used in the test.
- Type and amount of food: Commercially prepared trout food diet, supplemented by living , frozen or dried organism
eg., daphnia and larvae of mosquitoes until 24 hours prior to the test.
- 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:

semi-static

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

196 mg CaCO3/L

Test temperature:

13.4 – 13.6°C

pH:

7.5 - 8.4

Nominal and measured concentrations:

The nominal concentrations were 0.081, 0.16, 0.33, 0.65, 1.3 and 2.6 mg test item/L.
The measured mean concentrations were 0.050. 0.10, 0.22, 0.43, 0.99 and 2.6 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel/fill volume: Glass aquaria of 20 litres (35/22/26 cm) filled with 15 litres of water, depth of water 20 cm.
- Aeration: The water was gently aerated during exposure.
- No. of organisms per vessel: 7 per aquarium
- Replicates: One test chamber per concentration and control
- Biomass loading rate: Fish biomas to solution ratio was 0.49 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap waterof the town of Basel (carbon filtered to eliminate chlorine and particulate
material).

OTHER TEST CONDITIONS
- Light intensity/Photoperiod: Fluorescent light, 16 hours daily with 30 min transistion period.
- Temperature : 13.4 - 13.6 °C maintained by a temperatutere controlled water bath (all tanks were kept in the same water bath).

EFFECT PARAMETERS MEASURED:
The oxygen content (mg/L), pH and temperature (°C) were measured in each test chamber at 0, 24, 48, 72 and 96 hours (as long as there remained any fish in the tank). In addition, total hardness (as CaCO3) was measured in the blank control chamber immediately before the start of the test using a complexometric titration. (Mettler memo titrator DL40GP) with Titriplex III and a mixed indicator. The temperature and the dissolved oxygen were measured with a WTW OXI 323 dissolved oxygen-751 meter with a combined glass probe of Metrohm.
Characteristics of the test solutions (such as precipitated materials, cloudiness, etc) were recorded in daily intervals.

At 2, 24, 48, 72 and 96 hours, observations of mortality, and sublethal symptoms, such as abnormal behavioural activity and stress were made (swimming behaviour, loss of equilibrium, repratory functions, exophthalmus, 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 measurements of weight and length.

TEST CONCENTRATIONS
- Range finding study:
Range finding tests:
Prior to initiating definitive test, a static range-finding test was performed at concentrations 0.1, 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:
- Test concentrations: 0.081, 0.16, 0.33, 0.65, 1.3 and 2.6 mg CA 2218 A/L (nominal).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL

Effect concentrations 4

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % CL

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 0.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 6

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

ca. 0.43 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Lethal effects:
A decrease of the LC50-value from 0.65 to 0.31 mg/L was observed during the period 24 to 48 h exposure. The LC 50 then remained constant until the end of exposure. The LC 0 (96h) was 0.22 mg/L and the LC 100 (96h) 0.43 mg/L.

Sublthal effects:
No sublethal effects were observed at all concentrations tested, such as a change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal effects was 0.22 mg/L.

Reported statistics and error estimates:

Mortality data derived from the test were used to statistically estimate a median lethal concentration (LC50) and it's 95% confidence interval, where
possible.
Procedures:
Results of the toxicity test were interpreted by standard statistical techniques usung SAS Version 6.11. (SAS 1990 a) and the procedure "ECOS" by Fisch and Strehlau, 1998 using SAS PROC GLM and PROC NLIN (SAS 1990b).
LC-50:
The LC-50 values were calculated according to the maximum likelihood method, robit odel (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	0.31 0.31 0.31 0.65	none none none none	18.0 18.0 18.0 18.0	0.00 0.00 0.00 0.00	4 4 4 4	1.00 1.00 1.00 1.00

 

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The LC50 (96 h) of CA 2218 A was determined to be 0.31 mg/L based on measured mean concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, CA 2218 A is “very toxic to Rainbow Trout” based on the inherent toxicity of CA 2218 A in this test.
The highest concentration with no sublethal and lethal effects was 0.22 mg CA 2218 A/L.

Executive summary:

After 96 hours exposure, mortality occurred at concentrations of 0.43, 0.99 and 2.6 mg/L with 100% mortality, each. 

A decrease of the LC50-value from 0.65 to 0.31 mg/L was observed during the period 24 to 48 h exposure. The LC 50 then remained constant until the end of exposure. The LC 0 (96h) was 0.22 mg/L and the LC 100 (96h) 0.43 mg/L. 

Static pre-tests showed that the LC50 (96h) was between 1 and 10 mg/L. Therefore, the following nominal concentrations were chosen for the main test: 0.081, 0.16, 0.33, 0.65, 1.3 and 2.6 mg test item/L. 

After the preparation of the test concentrations and an equilibrium time of 4 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 2218 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 89 – 99 % of the saturation value by gentle aeration during exposure. The pH was determined to be 7.5 – 8.4 and the total hardness 196 mg CaCO₃/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 stock solutions or the testing solutions throughout the duration of the test. 

The nominal concentrations were 0.081, 0.16, 0.33, 0.65, 1.3 and 2.6 mg test item/L. 

The measured mean concentrations were 0.050. 0.10, 0.22, 0.43, 0.99 and 2.6 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	0.31 0.31 0.31 0.65	none none none none	18.0 18.0 18.0 18.0	0.00 0.00 0.00 0.00	4 4 4 4	1.00 1.00 1.00 1.00

 

 

After 96 hours exposure, mortality occurred at concentrations of 0.43, 0.99 and 2.6 mg/L with 100% mortality, each. 

A decrease of the LC50-value from 0.65 to 0.31 mg/L was observed during the period 24 to 48 h exposure. The LC 50 then remained constant until the end of exposure. The LC 0 (96h) was 0.22 mg/L and the LC 100 (96h) 0.43 mg/L. 

 

No sublethal effects were observed at all concentrations tested, such as a change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal effects was 0.22 mg/L. 

 

The LC50 (96 h) of CA 2218 A was determined to be 0.31 mg/L based on measured mean concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, CA 2218 A is “very toxic to Rainbow Trout” based on the inherent toxicity of CA 2218 A in this test. 

The highest concentration with no sublethal and lethal effects was 0.22 mg CA 2218 A/L. 

 

sublethal effects was 0.22 mg/L. 

 

The LC50 (96 h) of CA 2218 A was determined to be 0.31 mg/L based on measured mean concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, CA 2218 A is “very toxic to Rainbow Trout” based on the inherent toxicity of CA 2218 A in this test. 

The highest concentration with no sublethal and lethal effects was 0.22 mg CA 2218 A/L.