Lorenox™

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:

18 July to 05 August 2011

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 substance is adequately identified, but some data on composition is missing. - The treatment solutions were prepared with a solvent (acetone), which is not the best method at the time being but in acceptable quantity (0.1 mL/L). - The test item treatments were spaced by a factor of 2,5 instead of <=2,2. - Analytical monitoring was performed only on the two highest test item treatments as the three lowest were below the limit of quantification. - The test was performed under open conditions. - No dose-response curve was observed: the two successive test item treatments gave 0% and 86% for mortality, therefore the 96h-LC50 was not calculated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

The nominal values for the test item treatments were spaced by a factor of 2.5 instead of <=2.2, because the test item is known to be highly instable in water and care was taken to avoid overlapping between two successive concentrations.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 11 January 2011 / Date of signature: 11 March 2011

Specific details on test material used for the study:

- Physical state: Light yellow translucent liquid
- Storage condition of test material: Stored at room temperature protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Analytical verification of the test item was performed at the concentrations of 0.05, 0.12, 0.29, 0.72 and 1.80 mg/L

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For both the range-finding test and the definitive test, the test item was first diluted as stock solutions in acetone. Aliquot samples of 0.5 mL of the treatment solutions were added to the volume of the aquarium.
The test item treatments were adjusted afterwards as needed, using aliquot samples of the same stock solutions.
For the definitive test, the nominal values for the test item treatments were spaced by a factor of 2.5 instead of ≤2 .2, because the test item is known to be highly instable in water and care was taken to avoid overlapping between two successive concentrations.
- Controls: In each case a solvent control was prepared which received the same volume of acetone as the test item treatments.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 0.1 mL/L
- Analytical assessments proved that the test item treatments were not satisfactorily maintained within 80-120% of the nominal values throughout the test period. The calculations were based on mean measured concentrations throughout the exposure period.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Source: The batch of fish was received on June 2011 from a commercial breeder.
- Length at study initiation: Mean length was measured out from 15 % of the total population, taken at random: 2.3 ± 0.2 cm
- Fish were maintained at 23 ± 2 °C, with a 16 h/8 h light/dark cycle, in the same reconstituted water as used for testing.
- Feeding: The fish were kept unfed during the exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

Total hardness (measured value) = 240 to 250 mg/L

Test temperature:

22.5-23.5 °C

pH:

Start of the test: 7.52-7.56
End of the test: 7.19-7.38

Dissolved oxygen:

Start of the test: 8.07-8.38 mg/L
End of the test: 7.13-7.21 mg/L

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0.05, 0.12, 0.29, 0.72 and 1.80 mg/L
Measured concentrations: 0.03, 0.07, 0.19, 0.47 and 1.17 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 18 L capacity glass containers (30x20 cm x 30 cm height) filled with 5 L of reconstituted water.
- No. of organisms per vessel: Seven fish per aquarium
- No. of vessels per concentration (replicates): One replicate unit for the test item treatments
- No. of vessels per control (replicates): One replicate unit for the water control
- No. of vessels per vehicle control (replicates): One replicate unit for the solvent control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water (according to ISO 6341) was freshly reconstituted from analytical grade water using stock solutions of mineral components.
- Alkalinity: < 0.01 % NaOH
- pH (measured value): 7.74
- Na/K molar ratio (calculated value): 10/1
- Ca/Mg molar ratio (calculated value): 4/1

OTHER TEST CONDITIONS
- Photoperiod and light intensity: Test units were exposed to 16 h of light per day (150-700 lux) in a climatic chamber.

EFFECT PARAMETERS MEASURED
Mortality: The test units were inspected for mortality 24 h, 48 h, 72 hand 96 h after the test initiation. Any abnormal behavior or appearance, if any, was reported.
Water quality: The dissolved oxygen, temperature and pH were measured each day.

TEST CONCENTRATIONS
Range finding study :
- One replicate unit for each test item treatment and for the controls.
- Test concentrations: 1.80 and 12 mg/L (nominal concentrations); measured concentrations of 1.49 and 5.50 mg/L
- Results used to determine the conditions for the definitive study: All the fish had died after 48 h of testing for the 5.50 mg/L test item treatment. At 1.49 mg/L, two of them were still alive after 48 h of testing, but the swimming activity was affected. One of them died after 72 h of testing, and the last one after 90 h of testing. The test was not further continued.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 25, 50, 75, 100, 150 and 200 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.47 - < 1.17 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality: No mortality was observed in the controls and the test item treatments up to and including 0.47 mg/L. At 1.17 mg/L, two specimens had died after 24 hours of testing. On day 3, three additional specimens had died. At the end of the exposure period only one specimen out of 7 was still alive but its swimming activity was affected.

Physico-chemical parameters:
pH values were considered as similar to those in the controls for every test item treatment, ranging between 7.2 and 7.6. The dissolved oxygen concentrations were similar to those in the control and were > 85% of the saturation level in every case. The temperature remained within 22.5 - 23.5 °C for the test item treatments and for the controls.

Results with reference substance (positive control):

The LC50-24 h for Potassium dichromate was 112 mg/L; within the expected range of 50-250 mg/L.

Sublethal observations / clinical signs:

Analytical check of the test item treatments

The test item treatments were adjusted twice per day. The test item concentrations were checked in the old and in the new media, as long as one fish was still alive.

Only the two highest test item treatments were checked as the three lowest were below the quantification level reported for the GC-FID determination of test item in water.

The results showed that the test item treatments regularly decreased to less than 80 % of the nominal values. The test item concentrations are reported as mean measured values throughout the exposure period.

The results showed that the mean measured value represented approximately 65% of the nominal value in both cases. The three lowest test item treatments were thus extrapolated as 65% of their nominal value, respectively: 0.03, 0.07 and 0.19 mg/L.

For the two highest test item treatments, the measured concentrations were presented in the table 6.1.1/2 below.

Table 6.1.1/2: Definitive test - Measured concentrations for the two highest test item treatments

Hours		0.72 mg/L	1.80 mg/L
0 18 18 24 24 42 42 48 48 66 66 72 72 90 90 96	- Old New Old New Old New Old New Old New Old New Old New Old	0.64 0.21 0.60 0.44 0.63 0.26 0.61 0.43 0.70 0.25 0.70 0.45 0.59 0.20 0.50 0.40	1.52 0.42 1.46 0.93 1.50 0.52 1.39 0.91 1.50 0.71 1.68 1.26 1.62 0.72 1.49 1.01
Mean		0.48	1.16
% Nominal		65.98	64.63

 

Table 6.1.1/3: Mortality – results

 

Groups	Mortality
	24 h	48 h	72 h	96 h
Water control	0	0	0	0
Solvent control	0	0	0	0
Test item 0.03 mg/L	0	0	0	0
Test item 0.07 mg/L	0	0	0	0
Test item 0.19 mg/L	0	0	0	0
Test item 0.47 mg/L	0	0	0	0
Test item 1.17 mg/L	2	5	5	6

 

VALIDITY OF THE TEST RESULTS

The tests were considered as valid:

Mortality in the control unit 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 the air saturation value (approximately 9 mg/L) throughout the test,

The test item concentrations were difficult to maintain throughout the exposure period because of volatilization, even though the test item treatments were adjusted twice per day. The test item treatments used for the analysis were based on mean measured concentrations at the two highest test item treatments.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 96-hour LC50 of the test item was between 0.47 and 1.17 mg/L based on mean measured concentrations.

Executive summary:

One experimental study is available to assess the 96h-acute toxicity of the registered substance to fish Brachydanio rerio (now Danio rerio).

This study was performed according to OECD Guideline 203 with GLP statement. Fish were exposed to the test item (prepared with acetone as solvent, at 0.1 mL/L)at the following concentrations: 0,0.05, 0.12, 0.29, 0.72 and 1.80 mg/L (measured concentrations of <LOQ,0.03, 0.07, 0.19, 0.47 and 1.17 mg/L) for 96 hours, under semi-static conditions. Water and solvent controls were included. There wasone replicate unit for the test item treatments and for the controlswithseven fish per replicate, which provided a total of seven fish per treatment and control group at test initiation. The test temperature was 22.5 -23.5 °C. Prior to a definitive test, a range-finding test was conducted at 1.49 and 5.50 mg/L.

No mortality was observed in the controls and the test item treatments up to and including 0.47 mg/L. At 1.17 mg/L, two fish died after 24 hours of testing. On day 3, three additional specimens died. At the end of the exposure period only one fish out of 7 was still alive although the swimming activity of this animal was affected. Two successive test item treatments gave 0% and 86% for mortality, respectively. Thus the 96h-LC50value was not calculated but reported as an interval: 96h-LC50between 0.47 and 1.17 mg/L.

The LC₅₀-24 h for Potassium dichromate was 112 mg/L; within the expected range of 50-250 mg/L.

Under the test conditions, the 96-hour LC₅₀of the test item wasbetween 0.47 and 1.17 mg/L based on mean measured concentrations.

Description of key information

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

96h-LC50 (Brachydanio rerio) comprised between 0.47 and 1.17 mg/L, based on mean measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.47 mg/L

Additional information

One experimental study is available to assess the 96h-acute toxicity of the registered substance to fish Brachydanio rerio (now Danio rerio).

This study was performed according to OECD Guideline 203 with GLP statement. Fish were exposed to the test item (prepared with acetone as solvent, at 0.1 mL/L) at the following concentrations: 0, 0.05, 0.12, 0.29, 0.72 and 1.80 mg/L (measured concentrations of <LOQ, 0.03, 0.07, 0.19, 0.47 and 1.17 mg/L) for 96 hours, under semi-static conditions. Water and solvent controls were included. There was one replicate unit for the test item treatments and for the controls with seven fish per replicate, which provided a total of seven fish per treatment and control group at test initiation. The test temperature was 22.5 -23.5 °C. Prior to a definitive test, a range-finding test was conducted at 1.49 and 5.50 mg/L.

No mortality was observed in the controls and the test item treatments up to and including 0.47 mg/L. At 1.17 mg/L, two fish died after 24 hours of testing. On day 3, three additional specimens died. At the end of the exposure period only one fish out of 7 was still alive although the swimming activity of this animal was affected. Two successive test item treatments gave 0% and 86% for mortality, respectively. Thus the 96h-LC50 value was not calculated but reported as an interval: 96h-LC50 between 0.47 and 1.17 mg/L.

The LC₅₀-24 h for Potassium dichromate was 112 mg/L; within the expected range of 50-250 mg/L.

Under the test conditions, the 96-hour LC₅₀of the test item was between 0.47 and 1.17 mg/L based on mean measured concentrations.