Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

287-007-4

EC Name:

Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Cas Number:

85408-46-4

Molecular formula:

C17H12N4O3 . C17H12N4O3 . Cr. C12H28N to C14H32N

IUPAC Name:

Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance
- color: yellow to orange

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at ambient temperature.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. Each test solution was prepared separately (differential loading) by directly adding test substance to test medium according to the loading rate described and stirring for about 2 days. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). The aqueous fraction was inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect). After centrifugation, a positive Tyndall effect was observed. Therefore, the solutions were filtered with a membrane filter (Whatman OE66, pore width 0.2 µm). The first 50-100 mL of filtered solution were discarded (used to condition the filter). After filtration the solutions were stirred for approx. 10 minutes to achieve adequate dissolved oxygen levels. The control was treated in the same way.
The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media.
Fresh test solutions were prepared and renewed daily.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 parthenogenetically reproducing females for a maximum of 4 weeks and fed live unicellular algae (Desmodesmus subspicatus), cultured continuously at the test facility. All individuals in each mass culture originate from a single female and are thus genetic clones. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start of the exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.

ACCLIMATION
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, and temperature (20 ±1°C).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

2.20 – 3.20 mmol/L

Test temperature:

19.5-20.5 °C (continuous monitoring)

pH:

7.5 – 8.5

Dissolved oxygen:

Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 hours prior to use.

Conductivity:

550 - 650 µS/cm

Nominal and measured concentrations:

0 (control), 100 mg/L as loading rate based on test substance mass without correction for purity or composition

Details on test conditions:

TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL)
- Test volume: 50 mL
- Aeration: none
- Diet: No feeding during the exposure period.
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal period was 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.1 animals/mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 202.
- Ca/mg ratio: about 4 : 1
- Culture medium different from test medium:

EXPERIMENTAL PROCEDURE
The exposure was started by impartially distributing 20 neonate (< 24 hours old) Daphnia magna evenly among the 4 test vessels per test group. The neonate daphnids all originated from the same mass culture and were thus genetic clones. The Daphnia were transferred into the test vessels with minimal culture water by capturing each daphnid in a pipette then gently expelling it onto a Teflon mesh (70 µm) held by forceps. Each daphnid was then immediately transferred from the mesh into the corresponding test vessel from lowest to highest concentration.
Each test vessel was visually checked for immobilized daphnids after 0, 24 and 48 hours. In addition any abnormal behavior or appearance was documented.
For test renewal, daphnids in each replicate were captured with a pipette and transferred into test vessels with fresh test solution.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily in old and new test solutions.
The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, TOC and conductivity) were determined after aeration and prior to use in the test.
The pH and dissolved oxygen content of the test solutions was measured after 0h and 24h (new test solutions) without daphnids and after 24 h and 48 h (old test solutions) in replicate 1 of each tested concentration.
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.
The TOC (Total organic carbon) values of the test concentration and the control were analyzed at the start and the end of one renewal interval as an additional water quality parameter.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 111-664 lux at a wave length of 400 -750 nm

RANGE-FINDING STUDY
In a preliminary test (experimental conduct in accordance with GLP, without a GLP status) a concentration of 100 mg/L caused no immobilisation or symptoms after 48 hours.
According to the OECD-guideline, the highest suggested test concentration is 100 mg/L for a limit test.
The raw data of the range finding test are archived together with the raw data of this study.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mobility

Details on results:

ANALYTICAL CONCENTRATION CONTROL
Concentration control analysis was not performed because a sufficiently sensitive method for analyses in the required concentration range was not available. However all reasonable efforts were taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a multi-constituent material, the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD 23, for tests with chemicals that can not be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based loading rate (for mixtures).

Any other information on results incl. tables

Acute toxicity test

Number of mobile Daphnids 

Loading rate [mg/L]	Number of mobileDaphniaat each observation period
	Replicate 1 [h]			Replicate 2 [h]			Replicate 3 [h]			Replicate 4 [h]			∑	∑
	0	24	48	0	24	48	0	24	48	0	24	48	24 h	48 h
0 (control)	5	5	5	5	5	5	5	5	5	5	5	5	20	20
100	5	5	5	5	5	5	5	5	5	5	5	5	20	20
Number of test animals per replicate: 5 Number of test animals per test group: 20

WATER quality

Culture medium

Physical and chemical characteristics of the M4-medium used. 

pH value	Conductivity [µS/cm]	Total hardness [mmol/L]	Acid capacity up to pH 4.3 [mmol/L]	TOC[mg/L]	Date of measurement
8.2	581	2.44	0.83	1.1	22 Feb 2017

 

ph and dissolved oxygen content

pH- and oxygen values in the test solutions after 0 h and 24 h (interval 1) 

Test Group [mg/L]	pH value at 0 h^a	pH value after 24 h^a	O2value [mg/L] at 0 h^a	O2value [mg/L] after 24 h^a
0 (control)	7.9	7.8	8.3	9.3
100	7.9	7.8	8.2	9.2
^a measured in the replicate 1 of each concentration

 

 

pH- and oxygen values in the test solutions after 24 h and 48 h (interval 2) 

Test Group  [mg/L]	pH value at 24 h^a	pH value after 48 h^a	O2value [mg/L] at 24 h^a	O2value [mg/L] after 48 h^a
0 (control)	7.9	8.1	7.9	9.3
100	7.9	8.1	8.0	9.1
^a measured in the replicate 1 of each concentration

 

Total organic carbon (TOC) content

TOC values in the test solutions after 0 h and 24 h in interval 1 

Loading rate [mg/L]		after 0 h [mg/L] fresh test solution		after 24 h [mg/L] aged test solution
0 (control)		0.9		1.0
100		2.1		2.4

The TOC in the treatment group was higher than in the control test medium. The higher TOC is likely due to soluble organic components from the test substance. The TOC value confirms qualitatively that the daphnids in the WAF were exposed to soluble organic components from the test substance.

 

Dissolution behavior

All of the test solutions were visibly clear and colorless after preparation and remained so after 24 and 48 hours of exposure in the test. No undissolved test substance was visible and there were no other remarkable observations.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is with high probability not acutely harmful to aquatic invertebrates.