Cyclohexanecarboxylic acid, 1-[[2-(2,5-dimethylphenyl)acetyl]amino]-4-methoxy-, methyl ester, cis-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13,2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl cis-1-{[(2,5-dimethylphenyl)acetyl]amino}-4-methoxycyclohexanecarboxylate

EC Number:

606-524-1

Cas Number:

203313-47-7

Molecular formula:

C19H27NO4

IUPAC Name:

methyl cis-1-{[(2,5-dimethylphenyl)acetyl]amino}-4-methoxycyclohexanecarboxylate

Test material form:

solid: bulk

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

42.6 mg of the test item were dissolved in Acetonitrile and made up with Acetonitrile to the mark in a 100 mL volumetric flask to prepare a stock solution of 426 mg/L. Defined volumes of this stock solution were diluted with Milliporewater to obtain standard solutions in the range of 0.004 to 4.26 mg/L.
Although the overall method sensitivity would have been better, the concentration of the lowest used calibration solution was employed as the limit of quantification.
These solutions were used to calibrate the HPLC-system.

Sample storage conditions before analysis:
Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light

Test solutions

Vehicle:

no

Details on test solutions:

Culture and dilution water
Reconstituted water (so-called 'M4 medium') was used for the maintenance of the test animals and the preparation of stock and test solutions of the test item.


Pre-treatment of the test item
A stock solution was prepared to give the desired series of test concentrations.
To achieve this 120.3 mg of the test item were added to 1 litre of dilution water and treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer. Finally undissolved particles of the test item were removed by filtration using a folded filter of pore size 7-12 μm.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Common name: water flea
Source: Strain of Bundesgesundheitsamt Berlin
Maintenance and Acclimatisation:
A population of parthenogenetic females of synchronized age structure has been maintained for more than 15 years in the test facility under constant temperature conditions (20 +/- 1 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture water (so-called 'M4 medium') was partly renewed once a week.
The Daphnia were exclusively fed with unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily in a semiquantitative way. The neonates were separated from their parent Daphnia by filtration prior to the acute test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

341 mg/L CaCo3

Test temperature:

20.1-20.6 °C

pH:

7.8-7.9

Dissolved oxygen:

8.7-8.8 mg/L

Nominal and measured concentrations:

Nominal: control, 25, 50, 100 mg/L
Measured (48 h): < 0.004, 25.433, not measured, 100.911 mg/L

Details on test conditions:

Test vessels: 50 mL glass beakers holding 10 neonates in 20 mL of test medium

Experimental design:
3 test concentrations plus 1 control
10 neonates per vessel, 2 replicates per concentration/control
no feeding during the exposure period

Photoperiod: 16 h light : 8 h dark
Aeration: none

Criteria of effects: The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

mobility

Any other information on results incl. tables

No toxic effects were observed at the highest concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects were observed at the highest concentration. The results are expressed in terms of nominal concentrations at 24 h and 48 h. Recovery rates ranged from 97.8 – 98.7% of nominal values at 0 hours, and from 100.9 – 101.7% of nominal values at 48 hours, respectively.

Executive summary:

Analysis of the immobilisation rates (by probit analysis) gave the following results:

 

Time [h]	EC 50 [mg/L]
24	> 100
48	> 100

No toxic effects were observed at the highest concentration.

 

The results are expressed in terms of nominal concentrations at 24 h and 48 h.

Recovery rates ranged from 97.8 – 98.7% of nominal values at 0 hours, and from 100.9 – 101.7% of nominal values at 48 hours, respectively.