[No public or meaningful name is available]

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:

From December 10, 2012 to December 21, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 203 with GLP certificate. All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons: - The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0.125 mL/L). - The test was performed under open conditions. - At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised and therefore physical effects are expected. However, approximately 50% mortality was observed at 2,1 mg/L (concentration lower than the water solubility; analytically confirmed nominal concentration), the LC50 value is acceptable but possible overestimation of the toxicity as the test solutions were not prepared individually.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

July 1992

Deviations:

yes

Remarks:

Solvent was used and visual observation showed incomplete solubilisation of the test item at the highest concentration (5,5 mg/L). Possible overestimation of the toxicity.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on January 10, 2013

Specific details on test material used for the study:

- Physical state: white crystalline powder
- Storage condition: Room temperature protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

The test item concentrations were measured in the old and in the new media in additional volumes without fish.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The treatment solutions were prepared in acetone. The different treatment solutions are shown in Table 1 in "Any other information on materials and methods incl. tables", as they were prepared (nominal concentrations).Taking into account the results of the analytical validation study, the test solutions were set a 120% of the nominal values, and they were renewed each day. The controls were renewed accordingly. The nominal concentrations were thus considered as 0.8, 1.3, 2.1, 3.4 and 5.5 mg/L.
- Controls: water control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.125 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Strain: Danio rerio
- Source: Not communicated
- Age at study initiation (mean and range, SD): no details
- Length at study initiation (length definition, mean, range and SD): no details
- Weight at study initiation (mean and range, SD): no details
- Method of breeding: no details
- Maintenance of the brood fish: Fish were maintained at 23 +e/- 2 °C, with a 16h/8h light/dark cycle, in the same reconstituted water as used for testing.
- The fish were kept unfed during the exposure period.

Test type:

semi-static

Water media type:

other: ISO test water (according to ISO 6341) was freshly reconstituted by dilution of mineral stock solutions in pure water.

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 +/- 2°C

pH:

Between 7.6 - 8.5 throughout the test period for the controls and the test item treatments.

Dissolved oxygen:

More than 80% of the saturation level for every test item treatment and for the controls in the new media and the old media.

Nominal and measured concentrations:

Definitive test : 0,8 - 1,3 - 2,1 - 3,4 and 5,5 mg/L (nominal)
Test solutions were set at 120 % of the nominal values and were renewed each day. The test item concentrations were measured in the old and in the new media in additional volumes without fish.
At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised (physical effects can be expected).

Details on test conditions:

TEST SYSTEM
- Test vessel: 8.0 L capacity glass containers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with 4L of reconstituted water
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 7 fish per aquarium
- No. of vessels per concentration (replicates): one replicate unit for each the five test treatments with 7 fish per aquarium
- No. of vessels per control (replicates): one replicate unit for the controls with 7 fish per aquarium
- No. of vessels per vehicle control (replicates): one replicate unit for the controls with 7 fish per aquarium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water(according to ISO 6341) was freshly reconstituted by dilution of mineral stock solutions in pure water.

OTHER TEST CONDITIONS
- Photoperiod: 16h light per day
- Light intensity: 200-260 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test units were inspected for mortality 24h, 48h, 72h and 96h after initiation. Any abnormal behavior or appearance, if any, was reported.


TEST CONCENTRATIONS

- Range finding study : yes
- Test concentrations: 0.01 - 0.1 - 1.0 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: At 10.0 mg/ L all the specimens had died within 24 hours of testing.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 1.3 - 3.4 mg/L. based on analytically confirmed concentrations

Duration:

96 h

Dose descriptor:

other: Highest tested concentration without observed effect

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on analytically confirmed concentrations

Details on results:

- The calculations for the LC50-24h and -48h were based on the three intermediate test item treatments. The two extreme test item treatments were excluded becaused of the plateau phase. Calculations gave 3.8 mg/L and 2.7 mg/L respectively. On day 3 and 4 the calculation involved the five test item treatments, and the LC50 stabilized at 2.2 mg/L and 2.1 mg/L, respectively.
- Mortality of control: no mortality was observed in the water control and the solvent control.
- For the test item treatments no specimen had died at 0,8 mg/L. The NOEC-96h was thus taken as 0.8 mg/L.
At higher concentrations mortality was observed (moribund specimens were considered as dead).
All the specimens has died on day 3 at 5,5 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured values proved that part of the test item was retained by the fish : recovery of the test item concentrations was regularly 10-25 % lower than in the un-populated units (data not shown). The hypothesis was that the test item was part sorbed onto the fish.
At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised (physical effects can be expected).

Results with reference substance (positive control):

- Results with reference substance valid?
- LC50: 96h-LC50 between 50 - 250 mg/L
Raw data not provided in the study report

Sublethal observations / clinical signs:

Table 6.1.1/2: Definitive test - % recovery of the test item treatments throughout the test period

	Nominal treatment concentrations (mg/L)
	0,8	1,3	2,1	3,4	5,5
Day 0	1,01 122,0 %	1,53 116,0 %	2,52 117,4 %	3,99 116,2 %	6,68 121,7 %
Day 1, Old media	0,67 81,3 %	1,15 87,3 %	1,86 86,8 %	2,81 81,9 %	4,29 78,2 %
Day 1, New media	1 121,8 %	1,58 120,0 %	2,59 120,7 %	3,94 114,9 %	6,23 113,4 %
Day 2, Old media	0,71 86,1%	1,17 88,9 %	1,9 88,7 %	2,93 85,2 %	4,23 76,9 %
Day 2, New media	1,01 122,6 %	1,52 115,5 %	2,44 114,1 %	3,92 114,1 %	6,11 111,2 %
Day 3, Old media	0,78 94,6%	1,16 87,9 %	1,91 89,2 %	3,00 87,5 %	4,85 88,3 %
Day 3, New media	0,96 116,9%	1,6 121,2 %	2,48 115,8 %	3,92 114,2 %	6,32 115,0 %
End of test	0,69 84,2 %	1,14 86,6 %	1,87 87,2 %	2,96 86,2 %	4,76 86,7 %

 

Table 6.1.1/3: Definitive test - Observed mortality

	Mortality %
	24h	48h	72h	96h
Water control	0,0	0,0	0,0	0,0
Solvent control	0,0	0,0	0,0	0,0
Ambramone
0,8 mg/L	0,0	0,0	0,0	0,0
1,3 mg/L	0,0	0,0	14,3	14,3
2,1 mg/L	28,6	28,6	42,9	57,1
3,4 mg/L	42,9	71,4	85,7	85,7
5,5 mg/L	42,9	71,4	100	100

Remarks concerning the calculations :

The data reported herein are rounded values but the calculations were based on un-rounded values. The cumulative percentage mortality for each exposure period was plotted against concentrations. The LC50 values and related 95%-confidence intervals were derived from regression analysis of the concentration response-curves (full definitive test only).

Validity criteria fulfilled:

yes

Remarks:

Mortality in the controls did not exceed one fish at the end of the test; Constant environmental conditions were maintained throughout the test; The dissolved oxygen concentration was at least 60 per cent of saturation value throughout the test

Conclusions:

The acute toxicity of the test item to Danio rerio has been investigated and gave a 96-hour LC50 value of 2.1 mg/L with 95% confidence limits of 1.3 - 3.4 mg/L; based on nominal concentrations (analytically confirmed). The highest concentration without observed effect was 0.8 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of the test material to Danio rerio. The method followed is the OECD Guideline No. 203.

Following a preliminary range-finding study, fish were exposed, in groups of 7 fish per aquarium, to an aqueous dispersion of the test material (with solvent) over a range of concentrations of 0,8 - 1,3 - 2,1 - 3,4 and 5,5 mg/L (analytically confirmed nominal concentrations) for a period of 96 hours under semi-static test conditions.

The 96 hours LC50 for the test material to Danio rerio was 2.1 mg/L with 95% confidence limits of 1.3 - 3.4 mg/L. The highest concentration without observed effect was 0.8 mg/L.

All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons:

- The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0.125 mL/L).

- The test was performed under open conditions.

- At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised and therefore physical effects are expected. However, approximately 50% mortality was observed at 2,1 mg/L (concentration lower than the water solubility; analytically confirmed nominal concentration), the LC50 value is acceptable but possible overestimation of the toxicity as the test solutions were not prepared individually.

Description of key information

OECD Guideline 203, GLP, key study, validity 2:

96h-LC50 (Danio rerio) = 2.1 mg/L (95% CL: 1.3 - 3.4 mg/L), based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.1 mg/L

Additional information

One experimental study is available to assess the short-term toxicity of the registered substance to fish.

This study (Phytosafe, 2012), performed according to OECD Guideline 203 with GLP statement, assess the acute toxicity of the registered substance to Danio rerio. Following a preliminary range-finding study, fish were exposed, in groups of 7 fish per aquarium, to an aqueous dispersion of the test material (with solvent) over a range of concentrations of 0,8 - 1,3 - 2,1 - 3,4 and 5,5 mg/L (analytically confirmed nominal concentrations) for a period of 96 hours under semi-static test conditions.

The 96 hours LC50 for the test material to Danio rerio was 2.1 mg/L with 95% confidence limits of 1.3 - 3.4 mg/L. The highest concentration without observed effect was 0.8 mg/L.

All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons:

- The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0.125 mL/L).

- The test was performed under open conditions.

- At the highest tested concentration (5,5 mg/L) visual examination proved that part of the test item was not solubilised and therefore physical effects are expected. However, approximately 50% mortality was observed at 2,1 mg/L (concentration lower than the water solubility; analytically confirmed nominal concentration), the LC50 value is acceptable but possible overestimation of the toxicity as the test solutions were not prepared individually.