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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-08 to 2017-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Aliquots were removed from each prepared concentration every day for the range finding test and every day on fresh and old solutions for the definitive test.
Vehicle:
no
Details on test solutions:
For both range-finding test and definitive tests, as the test item was suspected to be unstable in water (due to its chemical nature), saturated stock solutions were prepared using slow-stirring conditions described in the OECD 123 guideline. The method used a special temperature controlled glass-jacketed test vessel (around 1000 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 was first poured into the flask then a known quantity of test item (107.5 µL) was gently added at the surface of medium. The solution was stirred at room temperature with a magnetic stir bar at a speed avoiding formation of an emulsion. The speed was set up in order to create a vortex depth of around 0.5 cm. The aqueous phase was kept under stirring during approximately 24 hours, and then drawn off as a clear solution after a rest phase of 1h without agitation. Tests solutions were then prepared from dilutions of this saturated solution.

The pH of the test solutions were not adjusted.

Test solutions were not renewed throughout the 48h test period for the range-finding test (static test) and renewed every day for the definitive test (semi-static test).
Test organisms (species):
Daphnia magna
Details on test organisms:
The test organisms used for this study was Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. D. magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny.

Test animals were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The medium was prepared as described in standard NF EN ISO 6341.
Sum of Ca and Mg ions in dilution water is of 2.5 mmol/L and ratios of Ca/Mg et Na/K are 4 and 10 respectively.
Test temperature:
from 19.7 to 20.1°C (mean 19.9°C)
pH:
7.7 - 8.0
Dissolved oxygen:
8.5 - 8.8 mg/L
Nominal and measured concentrations:
Range finding test showed that the concentrations could not be maintained during 48h. Therefore a semi-static design was selected for the definitive test with solutions renewal at 24h.
Nominal concentrations in the definitive test as % of stock saturated solution were: 0 (control) - 9.5 - 17.1 - 30.9 - 55.6 - 100.0%
Mean measured concentrations are reported in the table below.
Details on test conditions:
Culture and test vessels containing D. magna (20 daphnids per tested concentration for the definitive test) were incubated with a photo period of 8 hours of darkness per 24h at a temperature ranging from 18°C to 22°C but remaining constant within ± 1°C during the test.

Test vessels were made of clear glass tubes (around 20 mL capacity) and covered to minimise evaporation and avoid the entry of dust into the solutions. At least 3 mL of exposure solution was provided for each neonate.

The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item.
Reference substance (positive control):
yes
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
14.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
7.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
Test item concentration results are presented in table above. Chemical analysis of test samples taken indicated that measured concentrations of the test item were not maintained over the 48h test period (i.e. deviation >20%). As a consequence, the stability of the substance was not confirmed over the test period and geometric means of measured concentrations were thus used to determine EC50.
Table below reports immobilised daphnia according to exposure concentration.
Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated monthly by performing an acute Daphnia magna toxicity test on potassium dichromate. The nearest value of 24h-EC50 obtained on August 2015 was 0.72 mg/L.

For information, ISO 6341 has a validity criteria specifying that EC50 must be ranged from 0.6 to 2.1 mg/L.

Cumulative immobilisation (Definitive test)

Test solution

(Geometric means, mg/L)

Total daphnids

at T0

Immobile daphnids

Mobility inhibition at 48h(%)

24h

48h

Control

20

0

0

0.0%

1.30

20

0

0

0.0%

2.18

20

0

0

0.0%

3.87

20

0

0

0.0%

7.76

20

0

3

15.0%

13.79

20

0

9

45.0%


 

Validity criteria fulfilled:
yes
Conclusions:
This study was designed to determine the effects of the test item to Daphnia magna over a 48h period with a semi-static test (Definitive test), according to the OECD 202 Guideline.

Daphnids were exposed to serial dilutions of a saturated stock solution of the test item prepared with slow-stirring conditions due to the characteristics of the substance.

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 48h-EC10 and 48h-EC50:

48h-EC10 = 7.24 mg/L (95% confidence interval: 3.63 - 9.20 mg/L)
48h-EC50 = 14.43 mg/L (95% confidence interval: 11.42 - 27.03 mg/L)
Executive summary:

This study was designed to determine the effects of the test item to Daphnia magna over a 48h period under semi-static test, according to the OECD 202 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 solution prepared using slow-stirring conditions described in the OECD 123 guideline. Due to results obtained from the preliminary range-finding test, the definitive test was conducted under semi-static conditions. The test solutions were renewed 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 48h-EC10 and 48h-LC50 (all results are mg/L):

 

 

 

 

95% confidence level

 

 

 

Lower limit

Upper limit

Definitive test

(semi-static test)

48h-EC10

7.24 mg/L

3.63

9.20

48h-EC50

14.43 mg/L

11.42

27.03

 

 

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

 

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

 

§ The dissolved oxygen concentrations remained above 3 mg/L over the test period (above 7.7 mg/L in the definitive test).

Description of key information

A study assessing the acute toxicity of 3,3,5-TRIMETHYLCYCLOHEXYL ACRYLATE (CAS 86178-38-3) to Daphnia magna was conducted in accordance with the OECD 202 Test Guideline and GLP requirements.

The 48h-EC50 was determined to be 14.43 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
14.43 mg/L

Additional information

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