Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

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:

Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

EC Number:

916-868-7

Molecular formula:

C32H22CoN10O8.C10-14H21-29NH2 and C32H22CoN10O8.Na

IUPAC Name:

Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Test material form:

solid

Details on test material:

- Physical state / Appearance: Solid / 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. The test solutionsfor groups 1 and 2.2 mg/L were prepared separately (differential loading) by directly adding test substance to test medium according to the loading rate described in the table below and stirring for about 2 days. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). Undissolved test substance was observed, then filtration with a membrane filter (pore width 0.2 µm) was done. The first 50-100 mL of filtered solution was discarded (used to condition the filter). The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media. The exposure was started after separation of the undissolved material. Fresh test solutions were prepared and renewed daily to maximize the exposure to all soluble components of the test substance.
Test substance mass less then 1 mg/L could not be accurately weighed. Therefore the lower concentrations were prepared by diluting the 1 mg/L WSF according to the following scheme: See "Any other information on materials and metods incl. tables"

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Justification for species other than prescribed by test guideline:
- 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.20 – 3.20 mmol/L

Test temperature:

19.8 – 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), 0.046, 0.1, 0.22, 0.46, 1, 2.2 mg/L as loading rate based on test substance mass without a 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
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 animals / test vessel (0.1 animals/mL)
- Diet: No feeding during the exposure period.

TEST MEDIUM / WATER PARAMETERS
A synthetic fresh water (Elendt M4) is used as media for culture and test purposes
- Ca/mg ratio: about 4 : 1

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: About 20 – 850 lux at a wave length of 400-750 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.
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.
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 pH and dissolved oxygen content of the test solutions was measured after 0h and 24h (new test solutions) 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 highest test concentration and the control were analyzed at the start and the end of one renewal interval as an additional water quality parameter.

RANGE-FINDING STUDY
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤2.2 should be used, preferably encompassing the range from no effect to 100% immobilization.
In the first experiment the 48 hour EC50 was <0.46 mg/L

Reference substance (positive control):

yes

Remarks:

sodium chloride

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.119 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 since the analytical detection limit was above the water solubility of the test substance because a reliable method for analyses in the required concentration range could not be developed. However, all reasonable efforts were taken to produce a water saturated fraction (WSF) of the test substance in test media, following the guidance in OECD 23. The test organisms were exposed to a water saturated fraction (WSF) of the test substance at the given loading rate and the effect concentrations should be expressed based on the loading rate as recommended in OECD 23.

Results with reference substance (positive control):

In order to verify that the Daphnia magna culture is responding normally to toxic stress, tests with a reference substance, sodium chloride, are conducted monthly. Reference substance tests are conducted according to OECD 202 guidelines and in accordance with GLP, but without a GLP status.
The EC50(48h) of the reference substance sodium chloride was 4.18 g/L (experiment date: 09 May 2017, project number: 50E0789/12E060).
This result is within the range of 3.88 – 7.22 g/L (Ref. 12) and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Any other information on results incl. tables

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
7.9	571	2.40	0.86	1.0	24 May 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	pH value after 24 h	O2value [mg/L] at 0 h	O2value [mg/L] after 24 h
0 (control)	7.9	8.0	8.6	9.0
0.046	7.9	8.0	8.7	9.1
0.1	7.9	8.0	8.7	9.0
0.22	7.9	8.0	8.6	8.9
0.46	7.9	8.0	8.7	9.0
1.0	8.0	8.0	8.7	9.1
2.2	8.0	8.0	8.7	9.1
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	pH value after 48 h	O2value [mg/L] at 24 h	O2value [mg/L] after 48 h
0 (control)	8.0	8.0	8.7	8.8
0.046	7.9	8.0	8.6	8.7
0.1	7.9	8.0	8.6	8.7
0.22	7.9	8.0	8.7	8.8
0.46	7.9	7.9	8.7	8.7
1.0	8.0	8.0	8.6	8.7
2.2	8.0	7.9	8.6	8.7
measured in the replicate 1 of each concentration

Total organic carbon measured in the test goups and the control at the start and the end of the exposure

Test Group [mg/L]	TOC [mg/L]
	Day 0	Day 3
0 (control)	0.9	1.0
2.2	1.1	1.7

 

Dissolution behavior

The following observations were recorded for the dissolution behavior of the test substance in the test water:

 

Test Group [mg/L]	0 hour	24 hours		48 hours
		Old test solution	Fresh test solution
0 (control)	0	0	0	0
0.046	0	0	0	0
0.1	0	0	0	0
0.22	0	0	0	0
0.46	1	1	1	1
1.0	1	1	1	1
2.2	1	1	1	1
Explanation of abbreviations: 0:          no remarkable observations, clear test medium 1:          coloration caused by the test substance (light yellowish)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is acute very toxic to aquatic invertebrates.