Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC analysis, UV/vis-detection

Details on sampling:

Sampling schedule:
Control: on day 0, 5, 7, 12 and 17 in the freshly prepared media; on day 3, 7, 10, 14 and 19 in the old media
Test concentrations: 1, 3.16 and 10 mg/L on day 0, 5, 7, 12 and 17 in the freshly prepared media and on day 3, 7, 10, 14 and 19 in the old media

Vehicle:

no

Remarks:

auxiliaries used were an ultrasonic bath, a magnetic stirrer and a folded filter

Details on test solutions:

Pre-treatment of the test item
- the glass beakers for holding the daphnids were saturated with the test item
- 50 mg of the test substance were added to 5 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer and filtered through a folded filter (pore size 7 - 12 µm)
- individual dilutions were prepared from the filtrate in 2 L volumetric flasks and were distributed to the corresponding beakers
- after a saturation time of 24 hours the solutions were poured out again
- beakers were not rinsed over the entire test period of 21 days

Preparation of stock solution
- 50 mg of the test item were added to 5 litres of dilution water, treated for 1 hour in an ultrasonic bath and stirred for 24 h on a magnetic stirrer
- undissolved particles were removed by filtration using a folded filter (pore size 7 - 12 µm)
- pH was measured and ranged between 7.8 and 8.0

Test organisms (species):

Daphnia magna

Details on test organisms:

- Name: Daphnia magna STRAUS, parthenogenetic females
- Source: Strain of Bundesgesundheitsamt Berlin
- Maintenance and Acclimatisation: A population of parthenogenetic females of synchronized age structure has been maintained for more than 20 years in the test facility under constant temperature conditions (20 +/- 2 °C) at a 16 : 8 hour light-dark photoperiod (light intensity:
< 20 µE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily in a semi-quantitative way. The neonates were separated from their parent Daphnia by filtration prior to the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

14.3 - 15.8 °dH (= 255 - 282 mg/L CaCO3)

Test temperature:

20.0 - 20.7 °C

pH:

7.7 - 8.1

Dissolved oxygen:

8.4 - 9.2 mg/L

Nominal and measured concentrations:

nominal (time-weighted mean measured concentration) [mg/L]: 0.1, 0.316, 1.0 (0.190), 3.16 (0.255) and 10 (0.642)

Details on test conditions:

Exposure conditions
- Test vessels: 150 mL preadapted glass beakers covered with watch glasses, holding 1 neonate in 100 mL of test medium
- Experimental design: 5 test concentrations plus 1 control; 1 neonate per vessel, 10 replicates per concentration/control
- Semi-static system (renewal of the test media on Monday, Wednesday and Friday)
- Method of initiation: neonates were placed in prepared media
- Photoperiod: 16 h light: 8 h dark
- Light intensity: not exceeding 15 - 20 µE x m-2 x s-1
- Temperature of incubation unit: 19.2 to 20.0 °C
- Aeration: none
- Test item concentration/s: 0.1, 0.316, 1.0, 3.16 and 10 mg/L (nominal)
- Food source and feeding rate: Desmodesmus subspicatus; day 0 - 7: 0.1 mg C / Daphnia / day, day 8 - 21: 0.2 mg C / Daphnia / day
- Duration of exposure: 21 days
- Criteria of effects: item-induced inhibitory effect on the parthenogenetic reproduction rate and the mobility behaviour/mortality rate of parent Daphnia, all effects being assessed at least three times a week

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.255 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.642 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.642 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.642 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The results are expressed in time-weighted mean measured concentrations.
Effective concentrations ranged from 7.2 % to 15.8 % of the nominal values in the freshly prepared media and from 2.6 % to 11.0 % in the media after 48 hours or 72 hours of exposure.
The reproductive output of the Daphnia exposed to the test item was compared to that of the control in order to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) according to Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment.
All values were calculated using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

An acute Daphnia toxicity test preceded the test of this report. It provided information about the range of concentrations to be used in the reproduction test. The following nominal concentrations of the test item were tested in the acute test: 1, 10 and 100 mg/L. No toxic effects were observed in the acute test up to the highest tested concentration of 100 mg/L (nominal).

Validity criteria fulfilled:

yes

Remarks:

mortality rate (controls) <= 20 % at test end; mean number of live offspring produced per parent Daphnia surviving at test end ≥ 60 in controls; dissolved oxygen concentration > 3 mg/L; pH within 6 - 9; Water hardness > 140 mg/L (as CaCO3)

Conclusions:

A 21d-NOECreproduction of 0.255 mg/L was determined for Macrolex Orange R towards Daphnia.

Executive summary:

A study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.20 'Daphnia magna Reproduction Test'' (2008), which equals in all relevant parts to the OECD Test guideline 211 (Daphnia magna Reproduction Test; adopted: Oct. 02, 2012). It was performed to assess the effects of Macrolex Orange R on the reproductive output of parthenogenetic Daphnia magna STRAUS within a 21 day exposure period under semi-static conditions.

An acute Daphnia toxicity test preceded the test of this report. It provided information about the range of concentrations to be used in the reproduction test. The following nominal concentrations were tested: 1, 10 and 100 mg/L. No toxic effects were observed in the acute test up to the highest tested concentration of 100 mg/L (nominal).

Young female Daphnia, aged less than 24 hours at the start of the test, were exposed to a range of concentrations, nominally 0.1, 0.316, 1.0, 3.16 and 10 mg/L of Macrolex Orange R dissolved in water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and folded filters.

During the test a temperature range of 18 - 22 °C was maintained in the test vessels, with a maximum temperature fluctuation of +/- 2 °C in each individual test. Test vessels were not aerated during the test. A photoperiod of 8 hours darkness and 16 hours light was maintained, the light intensity not exceeding 15 – 20 μE x m 2 x s-1. Feeding of the Daphnia with living cells of Desmodesmus subspicatus was done daily. The ration was 0.1 – 0.2 mg C / Daphnia / day which was regarded sufficient for achieving the required number of offspring in the controls.

After a 21d-exposure period, the total number of living offspring produced per parent animal which did not die accidentally or inadvertently during the test was assessed. The reproductive output of the Daphnia exposed to the test item was compared to that of the control in order to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) according to Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment. Additionally, the survival of the parent Daphnia and the time to production of first brood was determined. The following valus were determined after 21 d [mg/L]: NOEC (reproduction): 0.255, LOEC (reproduction): 0.642, NOEC (mortality): ≥ 0.642, LOEC (mortality): > 0.642.

The results are expressed in time-weighted mean measured concentrations.

Effective concentrations ranged from 7.2 % to 15.8 % of the nominal values in the freshly prepared media and from 2.6 % to 11.0 % in the media after 48 hours, or 72 hours of exposure.

All values were calculated using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

Description of key information

A study was performed to assess the effects of Macrolex Orange R on the reproductive output of parthenogenetic Daphnia magna STRAUS within a 21 day exposure period under semi-static conditions according to EC Method C.20, which equals in all relevant parts the OECD 211.

An acute Daphnia toxicity test preceded the test of this report. It provided information about the range of concentrations to be used in the reproduction test. The following nominal concentrations were tested: 1, 10 and 100 mg/L. No toxic effects were observed in the acute test up to the highest tested concentration of 100 mg/L (nominal).

Young female Daphnia, aged less than 24 hours at the start of the test, were exposed to a range of concentrations, nominally 0.1, 0.316, 1.0, 3.16 and 10 mg/L, of Macrolex Orange R dissolved in water. After a 21d-exposure period, the total number of living offspring produced per parent animal which did not die accidentally or inadvertently during the test was assessed.

A 21d-NOECreproduction of 0.255 mg/L was determined for Macrolex Orange R towards Daphnia and a 21d-LOECreproduction of >0.642 mg/L.

The results are expressed in time-weighted mean measured concentrations.

Effective concentrations ranged from 7.2 % to 15.8 % of the nominal values in the freshly prepared media and from 2.6 % to 11.0 % in the media after 48 hours, or 72 hours of exposure.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.255 mg/L