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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-20 to 2017-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
The test temperatures recorded during the test were outside the range 21-25°C controlled at ± 2°C, as recommended in the test guideline. This deviation was considered to have not affected the outcome or the achievement of the study objectives.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
aliquots were removed from each prepared concentration every day on fresh and old solutions.
Vehicle:
no
Details on test solutions:
Due to the test item characteristics test solutions were prepared from a saturated stock solution prepared using slow-stirring conditions described in the OECD 123 guideline. This method used a special temperature controlled glass-jacketed test vessel (around 8000 mL of capacity), with a sampling tap at the lower end and a magnetic stir bar at its bottom: a known volume of test medium (around 7000 mL) was first poured into the test flask. Then a known quantity of test item (753 µL which corresponds to a loading rate of 1 g/L) was gently added at the surface of water. The solution was continuously stirred during at least 24h with the magnetic stir bar at a speed avoiding formation of an emulsion (i.e. 300 rpm). The speed was set up in order to create a vortex depth of around 0.5 cm.

As the definitive was conducted with semi-static conditions a new test item stock solution was prepared every day as described above.

The pH of the test solutions were not adjusted.

Test solutions were renewed every day throughout the 96h test period (semi-static test).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The fish used (Danio rerio (Teleostei, Cyprinidae)) was obtained on the 28 July 2016 from “La Grande Rivière France” (69490 Saint-Forgeux, FRANCE). All fish were in good health and free from any apparent malformation. Mortality of the batch was less than 5% in the week preceding the start of the study. No disease treatments were administered throughout holding and testing.

Holding was maintained within the laboratory at a temperature of 21-25¿C in glass tanks. A light cycle of 12 h light and 12 h dark was applied, illumination being provided by fluorescent tubes (intensity between 400 and 800 lux at the surface of the tanks). Tanks were aerated to ensure that the dissolved oxygen concentration is at least 60% of air saturation value in holding tanks and in test tanks. During holding, fish were fed twice per day with ground flake food TetraMin®.

The fish were not fed for a period of 24h prior to test commencement and up to the end of the test

Fish were added to the test tanks within 30 min of the completion of test solution preparation. All test fish were weighed prior to the test and a representative number of test fish batch (10 at random) was measured after the test to assess compliance with guideline criteria.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
according to standard NF EN ISO 6341
Test temperature:
the temperature was maintained in a range from 21°C to 25°C but not remaining constant within ± 2°C during the test:
¿ mean measured temperature: 21.5°C
¿ min.: 20.2°C
¿ max.: 22.9°C.
pH:
7.6 - 8.0
Dissolved oxygen:
> 83% saturation
Conductivity:
according to standard NF EN ISO 6341
Nominal and measured concentrations:
Concentrations were measured on fresh and old (24h) solutions. Geometric means were used for calculations:

(Geometric means, mg/L)
0 (control)
0.47
0.65
0.88
1.40
2.74
5.53
Details on test conditions:
Test tanks (filled with 2.5 L of test solution) containing fish were incubated with a photo period of 9 hours of darkness per 24 h, at a temperature ranging from 21°C to 25°C and remaining constant within ± 2°C during the test.

All test tanks were made of clear glass and covered by plates with ventilation holes to minimise evaporation and avoid the entry of dust into the solutions.

Test solutions were prepared using the synthetic medium. Chemical reagents used for the preparation of the dilution water was of “analytical grade”.

The control treatment was maintained under the same conditions, with no test item.

The test was conducted under semi-static conditions at the following series of dilutions: 5.3, 9.5, 17.1, 30.9, 55.6 and 100%. The range of concentrations was obtained from dilutions of the saturated solution. One tank was prepared at each test concentration and the control group. Each tank contained 7 fish.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
96 d
Dose descriptor:
LC10
Effect conc.:
1.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Details on results:
Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.
Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated with every batch of fish by performing an acute toxicity test on potassium dichromate. The 24h-LC50 value obtained with the batch used was as follows:

Batch 16/Br/01/2: 24h-LC50 = 173.6 mg/L (obtained on 11 August 2016)

This result is consistent with the laboratory control quality chart.
Sublethal observations / clinical signs:

Cumulative lethality (Definitive test)

Test solution

(Geometric means, mg/L)

Cumulative dead fish

Mortality at the end of the period

T2h

T24h

T48h

T72h

T96h

Control

0

0

0

0

0

0.0%

0.47

0

0

0

0

0

0.0%

0.65

0

0

0

0

0

0.0%

0.88

0

0

0

0

0

0.0%

1.40

0

0

0

0

0

0.0%

2.74

0

0

0

4

7

100.0%

5.53

0

0

7

7

7

100.0%

Visible abnormalities (Definitive test)

Test solution

(Geometric means, mg/L)

Visible abnormalities

T0

T24h

T48h

T72h

T96h

Control

-

-

-

-

-

0.47

-

-

-

-

-

0.65

-

-

-

-

-

0.88

-

-

-

-

-

1.40

slight discoloration

-

-

-

red gills

2.74

-

-

Low mobility, discoloration, red gills

Low mobility, red gills

ND

5.53

discoloration - difficult swimming

slight discoloration

ND

ND

ND

-: nothing to report

ND: Not Concerned

Validation criteria

The definitive test met the validity criteria of the test guideline detailed as follows:

- The mortality in the control did not exceed 10% at the end of the test (0%),

- The dissolved oxygen in the test tanks remained above 60% of the air saturation value at the end of the test (>87,5% of ASV),

- The pH did not vary by more than 1 unit.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to Danio rerio exposed for 96h was assessed according to the OECD 203 Guideline. Fish were exposed to a series of dilutions of saturated stock solutions in dilution water (renewed every day) throughout the test period.

Test item concentrations were determined at each observation time. Results have shown an important loss of the test item; as a consequence the exposure concentrations were based on the geometric mean of measured concentration. EC10 and EC50 values were calculated to be as follows:

96h-EC10 = 1.41 mg/L (95% CI: 1.07 - 1.68 mg/L)
96h-EC50 = 1.90 mg/L (95% CI: 1.59 - 2.35 mg/L)
Executive summary:

This study was designed to determine the effects of the test item to Danio rerio over a 96h period with semi-static conditions, according to the OECD 203 Guideline.

 

The test item being suspected to be unstable in water (due to its chemical nature), tests solutions were prepared from dilutions of a saturated solutionprepared using slow-stirring conditionsdescribed in the OECD 123 guideline (a new test item stock solution was prepared every day).

 

As the analytical determination of the test item concentrations throughout the test period have shown unstability of the substance, geometric means of measured concentrations were used to determine 96h-EC10 and 96h-LC50 (all results are mg/L):

 

 

 

 

95% confidence level

 

 

 

Lower limit

Upper limit

Definitive test

(semi-static test)

96h-LC10

1.41 mg/L

1.07

1.68

96h-LC50

1.90 mg/L

1.59

2.35

 

The definitive test met the validity criteria of the test guideline detailed as follows:

 

§ The mortality in the control did not exceed 10% at the end of the test (0%),

§ The dissolved oxygen in the test tanks remained above 60% of the air saturation value (ASV) at the end of the test (>87,5% of ASV),

§ The pH did not vary by more than 1 unit.

Description of key information

A study assessing the acute toxicity of 3,3,5-TRIMETHYLCYCLOHEXYL ACRYLATE (CAS 86178-38-3) to Danio rerio was conducted in accordance with the OECD 203 Test Guideline and GLP requirements.

The 96h-LC50 was determined to be 1.90 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
1.9 mg/L

Additional information