1,4-Methanonaphthalen-5(1H)-one, 9-(dichloromethylene)-2,3,4,6,7,8-hexahydro-, oxime, (5E)-

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2011 to 29 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study. Acceptable without restriction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control were sampled..
- Sampling method: All samples were taken from the approximate centre of the aquaria without mixing. Duplicate samples were taken from the test media of all test concentrations at the start and end of the test.
- Sample storage condition before analysis: Samples were deep-frozen immediately after sampling and stored at about -20˚C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in test water, a dispersion with the loading rate of 300 mg/L was prepared at the start of the test by dispersing 3.0010 g of the test item in 10 L of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. The test media were prepared just before the start of the test.
- Controls: Reconstituted test water, consisting of analytical grade salts dissolved in purified water. Potassium dichromate is tested as a positive control twice a year.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported after filtration.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Length at study initiation (length definition, mean, range and SD): 4.87 ± 0.18 cm (Mean ± SD)
- Weight at study initiation (mean and range, SD): 1.18 ± 0.11 g (Mean ± SD)
- Feeding during test: None

ACCLIMATION
- Acclimation period: One week
- Acclimation conditions (same as test or not): Same as test conditions
- Feeding frequency: During holding and acclimatization until two days before the start of the test
- Health during acclimation (any mortality observed): No mortalities and all fish were healthy

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

125 mg CaCO3/L

Test temperature:

14ºC

pH:

7.2 to 7.4

Dissolved oxygen:

9.5 to 9.9 mg/L (≥ 92% oxygen saturation)

Salinity:

Not applicable

Nominal and measured concentrations:

The following concentrations of test item were tested; undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2.2, 1:4.6, 1:10 and 1:22 of the undiluted filtrate with the loading rate of 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. Measured concentration at day 0 was 0.659 mg/L and at day 4 it was 0.452 mg/L for the undiluted filtrate with a loading rate of 100 mg/L, a reduction of 69% over the test period. The samples from the dilutions 1:22, 1:10, 1:4.6 and 1:2.2 were not analyzed, since these concentrations were below the NOEC determined in the test. The biological results were related to the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test (see table below).

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass test vessel (length: 35 cm; width: 23 cm; height 25 cm), height of water level: 18 cm, fill volume: 15 litres
- Aeration: Slightly aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable, static test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: 0.55 g fish wet weight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water, consisting of analytical grade salts dissolved in purified water:
- Alkalinity: 0.4 mmol/L
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were measured at the start of the test and once every day during the test at each test concentration and in the control. At the same occasions the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16:8 light/dark cycle with 30 minute transition period.
- Light intensity: Approximately between 180-480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed after approximately 2.5, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2 - Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The 96-hour LC50 value is above the solubility limit of the test substance.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Highest concentration tested.

Details on results:

- Behavioural abnormalities: None observed in any test concentration
- Mortality of control: None
- Other adverse effects control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed
- Effect concentrations exceeding solubility of substance in test medium: Yes, LC50 value could not be determined since no mortalities were observed at the solubility limit of the test substance.

Also see table below.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The 96-hour LC50 of the test item could not be calculated because no mortalities were observed at any test concentration. NOEC determined directly from the raw data.

Sublethal observations / clinical signs:

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test.

Treatment/Dilution	Mean measured concentration of the test item (geometric mean)
	mg/L	% of initially measured
Dilution 1:22	---	---
Dilution 1:10	---	---
Dilution 1:4.6	---	---
Dilution 1:2.2	---	---
Undiluted filtrate	0.55	83
---- not analysed (see above)

Table 1. Mortality and Visible Abnormalities in the Test Fish
Treatment/Dilutions	Mean measured concentration	Number of abnormal and dead fish / number of dead fish Observation time
	(mg/L)	2.5 hours	24 hours	48 hours	72 hours	96 hours
Control	---	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:22	---	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:10	---	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:4.6	---	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1.2.2	---	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
Undiluted filtrate (loading rate 100 mg/L)	0.55	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
	LC50* (mg/L)	>0.55	>0.55	>0.55	>0.55	>0.55
	95% C. I.*	n.d.	n.d.	n.d.	n.d.	n.d.
95% C .I.: 95% confidence interval of the LC50
---- : not analysed
n.d. : not determined
*: based on measured concentration

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid, since no mortality in the control was observed and the validity criterion on at least 60% oxygen saturation was fulfilled.

Conclusions:

The 96 hour-LC50 for Oncorhynchus mykiss exposed to the tets material could only be estimated as > 0.55 mg/L, because no mortalities were observed at the highest concentration tested, which was the solubility limit of the test substance in the test medium. The 96-hour NOEC was determined to be 0.55 mg/L.

Executive summary:

The sensitivity of Oncorhynchus mykiss to the test material was determined in a GLP-compliant test performed to standard guidelines. The 96 hour-LC50 for Oncorhynchus mykiss exposed to the test material could only be estimated as > 0.55 mg/L, because no mortalities were observed at the highest concentration tested, which was the solubility limit of the test substance in the test medium. The 96-hour NOEC was determined to be 0.55 mg/L. The fish were exposed for up to 96 hours to 5 concentrations of the test substance. As the test substance has low solubility in the test media a dispersion was formed by mixing the test substance in the test media for a prolonged period of time. After filtration this stock solution was used undiluted as the maximum exposure concentration and as a series of dilutions to form the other test concentrations. Analytical monitoring of the exposure concentrations was undertaken and the results are based on the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test.

Description of key information

The LC50 could only be estimated at >0.55mg/L as no mortalities were observed at the highest dose tested, which was the solubility limit of the test substance in the test medium.

Additional information

The sensitivity of Oncorhynchus mykiss to the test material was determined in a GLP-compliant test performed to standard guidelines. The 96 hour-LC50 for Oncorhynchus mykiss exposed to the test material could only be estimated as > 0.55 mg/L, because no mortalities were observed at the highest concentration tested, which was the solubility limit of the test substance in the test medium. The 96-hour NOEC was determined to be 0.55 mg/L. The fish were exposed for up to 96 hours to 5 concentrations of the test substance. As the test substance has low solubility in the test media a dispersion was formed by mixing the test substance in the test media for a prolonged period of time. After filtration this stock solution was used undiluted as the maximum exposure concentration and as a series of dilutions to form the other test concentrations. Analytical monitoring of the exposure concentrations was undertaken and the results are based on the mean measured test item concentration calculated as the geometric means of the concentrations measured at the start and end of the test. The study is considered reliable and adequate for the purposes of risk assessment and classification and labelling when considered alongside the other aquatic data generated for this substance.

The study is considered reliable and relevant for risk assessment. As no LC50 could be determined the result was not considered adequate for classification and labeling purposes.