2-({2-[4-(dimethylamino)benzoyloxy]ethyl}(methyl)amino)ethyl 4-(dimethylamino)benzoate

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:

29 January 2007 to 02 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

EU Commission Directive 92/69/EEC, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test article name : LFC 2098
Chemservice sample code : AA26964
Declared purity (according to CoA) : 100 %
Chemical class : Aminoester
Chemicalformula : C23H31O4N3
Molecularweight : 413
Preparation date : 05/29/2006
Solubility in water : Practically insoluble
Density : Not available
Physicalform at20°C : Solid (powder)
Colour : White
Storage conditions : Room temperature, protect from light
Safety precautions : Routinary hygienic procedures

Analytical monitoring:

yes

Details on sampling:

The study has been carried out at the only nominal concentration of 100 mg/L
Samples were taken at 0 and 96 hours
Since the samples could not be analysed immediately after sampling, they were stored at 4 °C and kept stored until analysis.

Vehicle:

no

Details on test solutions:

Given the low water solubility of the test item, a saturated solution of 100 mg/L has been prepared by direct weighting into 20 litres capacity glass bottle. The suspension was mixed by a magnetic stirrer for 48 hours to achieve the maximum solubilisation, then, it was filtered by a filtering system and poured into the test aquarium. The solution was let to reach the desired temperature before adding the fish.

The water temperature was manually measured in aquaria once per day during the exposure period. The measured values ranged from 21.9 to 22.8°C, thus complying with the guideline requisites.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with zebra fish (Danio rerio) obtained from a commercial supplier on January 11, 2007. Juvenile fish have been used and all of them were approximately the same age and size. In accordance with the test guidelines, the fish have
been held in the Chemservice laboratories for more than 18 days at the same conditions, regarding water quality, temperature and illumination, of the test.
During the holding period, the fish were fed commercial fish food until 48 hours before the test initiation. The used group of fish was accepted since there was no mortality during the acclimation period.
The fish were not fed during the test.

A representitive sample of the weight of the batch of fish used is given below
Specimen n. Weight (g)
1 0.69
2 0.62
3 0.65
4 0.56
5 0.6
6 0.62
7 0.52
8 0.53
9 0.5
10 0.54
Mean value 0.58

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol L-1 (= 250 mg L-1) as CaCO3

Test temperature:

21.9 to 22.8°C

pH:

7.50-7.88

Dissolved oxygen:

> 6 mg/L

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal concentration : 100 mg/L
Actual test concentration: 0.36 mg/L average [0.51 mg/L (start) to 0.21 mg/L (end)]

Details on test conditions:

For the only test concentration and for the negative control 60 L capacity glass aquaria have been used, filled with 20 L test medium. 7 fish were added to each aquaria, so that the loading rate did not exceed 0.8 g (fresh weight) of fish per litre of test solution per replicate at any time (OPPTS 850.1075).
One replicate per concentration was used.
The test was performed in single temperature-controlled aquaria and incubated in a temperature-controlled roomThe water temperature was manually measured in aquaria once per
day during the exposure period. The measured values ranged from 21.9 to 22,8°C, thus complying with the guideline requisites.
The test was performed under a 16 hours light and 8 hours darkness daily photoperiod; light intensity was 50 - 500 lux, with 2 daily 30-minutes transition periods (OPPTS 850.1075).
Test duration: 96 hours
Feeding: None
Exposure regime: static
Nominal concentration : 100 mg/L
Actual test concentration: 0.51 mg/L (start) to 0.21 mg/L (end)
Test water: Reconstituted test water: analytical grade salts will be dissolved in purified water to obtain the following nominal concentrations:
CaCl2 x 2H20: 2.0 mmol L-1 (= 294 mg L-1)
MgSO4 x 7H2O: 0.5 mmol L-1 (= 123 mg L-1)
NaHCO3: 0.75 mmol L-1 (= 65 mg L-1)
KCI: 0.075 mmol L-1 (= 5.8 mg L-1)
Water Hardness: 2.5 mmol L-1 (= 250 mg L-1) as CaCO3
Alkalinity: 0.8 mmol L-1
Ratio of Ca : Mg 4 : 1 (based on molarity)
Na : K 10 : 1 (based on molarity)
Dissolved oxygen: > 6 mg/L
The fish were checked at 2, 24, 48, 72 and 96 hours after test initiation for mortality and visible abnormalities.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 0.36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 0.36 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No mortality was observed in the test medium after 96 hours, therefore the NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours) of LFC 2098 to zebra fish was 0.36 mg/L or higher.
Based on the observed effects, the LOEC and the 96-h LC50 were determined to be greater than 0.36 mg/L, corresponding to 100 mg/L as nominal concentration.

Reported statistics and error estimates:

The NOEC and LOEC were determined directly from the raw data without any statistical analysis.

Sublethal observations / clinical signs:

Test concentration [mg/L]	n. of exposed fish	n. dead fish
		2/4 h	24 h	48 h	72 h	96 h
0 (control)	7	0	0	0	0	0
0.36	7	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The test item LFC 2098 caused 0% mortality to zebra fish after 96-hours exposure under static conditions at the nominal concentration of 100 mg/L, corresponding to an actual concentration of 0.36 mg/L, which represents the maximum water solubility of the test item under the test conditions.

Given the nature of limit test, the 96-hour L050 could not be determined, but, according to the binomial theory, using 7 fish per test the absence of mortality provides at least 99% confidence that the L050 is greater than the concentration tested (i.e., L050 > 100 mg/L).

The NOEC and the LOEC were determined directly from the raw data.
The results are therefore as follows:
96 h NOEC 2 100 mg/L
96 h LOEC > 100 mg/L
96 h LC50 > 100 mg/L

Corresponding to the following values in terms of analytical concentration:
96 h NOEC 2 0.36 mg/L
96 h LOEC > 0.36 mg/L
96 h LC50 > 0.36 mg/L

Executive summary:

The acute toxicity of the test item LFC 2098 to zebra fish (Danio rerio) was determined in a limit, 96-hour static test according to the OECD Guideline for Testing of Chemicals, N. 203. “Fish, acute toxicity test”, 1992, the EU Commission Directive 92/69/EEC, C.1. "Acute Toxicity for Fish", 1992 and the OPPTS 850.1075 guideline “Fish acute toxicity test, freshwater and marine”, EPA Ecological effects Test guidelines, 1996.

For this purpose, juvenile fish of the species Danio rerio were exposed, after an acclimation period, to the test item at the only concentration of 100 mg/L under defined conditions. The exposed organisms were checked for mortality after 2, 24, 48, 72 and 96 hours from test initiation.

The actual test concentration was analytically measured at the beginning and at the end of the test. The test item concentration has shown a large decrease already short after the test solution was prepared (0.51 mg/L) at time 0, and was quite stable during the following days (0.21 mg/L) at the end of test period. This behaviour could be due to the low water solubility of the test item and the method of preparation of the test solutions.

Therefore, the biological results are not referred to the nominal concentration but to the median of the measured concentrations, according to the OECD Guidance Document on testing of difficult substances (2000).

The pH of the test medium after preparation of the test solution was 7.88. Therefore, the pH of the test medium was not adjusted before the start of the test. The dissolved oxygen was all the time greater than 6 mg/L either in the control and in the test solution. The temperature was for all the time between 21.9 and 22.8°C in both the aquaria.

In the control 0% mortality was observed during the test period. This value complies with the validity criteria of the test, according to the mentioned guidelines.

No mortality was observed in the test medium after 96 hours, therefore the NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours) of LFC 2098 to zebra fish was 0.36 mg/L or higher. Based on the observed effects, the LOEC and the 96-h LCso were determined to be greater than 0.36 mg/L, corresponding to 100 mg/L as nominal concentration.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Additional information

LC50 96 hour > 100 mg/L