Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(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

Study period:

19 Feb 2017 to 24 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-145-3

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature without exposure to light in a tightly sealed container

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Total organic carbon (TOC)

Details on sampling:

The total organic carbon was determined in samples at exposure initiation, at renewal, and at exposure termination. The samples were collected from all test item loading rates and the control at exposure initiation (fresh) and at renewal (24-h old). Moreover, the samples were also collected from the test item loading rates: filtrate of a loading of 6.25 and 100 mg/L and the control at renewal (fresh) and at exposure termination (24-h old)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Appropriate amounts of the test item were weighed separately in glass flasks with ISO test water and mixed with appropriate volumes of the ISO test water. The mixtures were visually heterogeneous (with visible undissolved particles). The mixtures and the control (800 mL ISO test water) were left for mechanical shaking for 48 hours (30ºC, 90 rpm, darkness). After 48 hours of mechanical shaking, the mixtures (visually heterogeneous) and the control were left at room temperature for a settling phase (no shaking). After 24 hours of settling, the mixtures were still visually heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (HAWG filter, 0.22 µm, Millipore). Filtration was chosen as the separation method since centrifugation was not possible with the large volumes of test solution. Each filtrate was visually homogeneous (without any visible undissolved particles). From each filtrate and the control, two samples were collected for chemical analyses (100 mL each). One was transferred to chemical analyses at exposure initiation, the second one was stored under test conditions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Originated from the standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology.
- Age: Only organisms up to 24 hours old (not first brood progeny) and in good physiological condition were used in the tests.

CULTURING
Daphnia magna was cultured in glass beakers with a capacity of 150 mL (one parent per vessel) containing 100 mL of ISO test water at room temperature. The daily light cycle was 16 h light : 8 h dark (KANLUX electronic time programmer, Poland). The culture was maintained in the ISO test water. The Daphnia magna were fed with a suspension of algae; mixture of two species Pseudokirchneriella subcapitata : Desmodesmus subspicatus (in 2:1 ratio) originating from separate cultures in the Laboratory of Aquatic Toxicology. Group B vitamins and micronutrients necessary for proper growth were supplied with the lyophilized suspension of Spirulina sp.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

233.6 mg/L as CaCO3

Test temperature:

19.4 - 21.4 ºC

pH:

7.26 - 7.67

Dissolved oxygen:

8.0 - 8.9 mg/L

Conductivity:

563 μS/cm

Nominal and measured concentrations:

- Nominal concentrations (loading rates): 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L..

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers of 150 mL capacity, containing 100 mL of a given test item concentration or the control.
- Type: The beakers were covered with transparent lids in order to minimise evaporation and to prevent accidental contamination
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Age: less than 24 h old
- Feeding during test: no

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water recommended by the OECD Guideline No. 202
- Temperature: temperature was continuously recorded using a sensor submerged in an additional test vessel containing 100 mL of the test medium
- Intervals of water quality measurement: The pH values and the dissolved oxygen concentrations were measured at exposure initiation, i.e. before splitting up into replicates, at renewal and at exposure termination in pooled replicates

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark (KANLUX electronic time programmer, Poland)
- Light type: Fluorescent

EFFECT PARAMETERS MEASURED:
The effect of the test item on immobilisation of Daphnia magna was estimated after 24 h and 48 h of exposure. The Daphnia magna was considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.

RANGE-FINDING STUDY
- Test concentrations: 100-fold diluted filtrate of a loading of 10 mg/L, 10-fold diluted filtrate of a loading of 10 mg/L, filtrate of a loading of 10 mg/L, filtrate of a loading of 100 mg/L plus the control were used for exposure.
- Results used to determine the conditions for the definitive study: At exposure termination, the immobilisation of Daphnia magna was 100% in the test item concentration: filtrate of a loading of 100 mg/L and 25% in the test item concentration: filtrate of a loading of 10 mg/L. In the remaining test item concentrations and the control, no immobilisation of Daphnia magna was observed during exposure.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

27.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% C.I. 20.32 - 37.29

Details on results:

During exposure, no immobilisation was observed in the control. At exposure termination, in the control no immobilisation was observed. In the filtrate of a loading of 6.25, 12.5, 25, 50 and 100 mg/L, the immobilisation was 5, 15, 60, 75 and 85%, respectively. See 'Any other information on results incl. tables'.

VALIDITY CRITERIA
In the definitive test, the validity criteria were met according to OECD Guideline No. 202 (2004):
- The percentage of immobilisation of Daphnia magna in the control was 0% (validity criterion: not more than 10%),
- The dissolved oxygen concentrations in the test vessels were within the range of 7.9 - 8.9 mg/L (validity criterion: not less than 3 mg/L).

RESULTS CHEMICAL ANALYSIS
In the definitive test, TOC was analysed. However, since the test substance is a UVCB, the TOC analysis was only conducted to assess the stability of the test substance. The results were corrected by the value determined for the control sample in a series and presented as the concentrations of TOC. At exposure initiation and at renewal analyses were performed in samples of each test item loading rate and the control. On the renewal and at exposure termination analyses were performed in samples of the filtrate of a loading of 6.25 and 100 mg/L and the control. At exposure initiation, in fresh samples the analysed TOC concentration was below the LoQ value (<1.0 mg C/L) in the control and in the range of 1.68 - 3.86 mg C/L in the test item loading rates. At renewal, in old samples the analysed total organic carbon concentration was below the LoQ value (<1.0 mg C/L) in the control and in the range of 1.68 - 3.61 mg C/L in the test item loading rates. At renewal, in fresh samples the analysed total organic carbon concentration were: below the LoQ value (<1.0 mg C/L) in the control, 1.77 mg C/L in the test item loading rates filtrate of a loading of 6.25 mg C/L and 3.71 mg C/L in the test item loading rates filtrate of a loading of 100 mg/L. At exposure termination, in old samples the analysed total organic carbon concentration were: 1.85 mg C/L in the control, 2.11 mg C/L in the test item loading rates filtrate of a loading of 6.25 mg/L and 4.25 mg C/L in the test item loading rates filtrate of a loading of 100 mg/L.

Results with reference substance (positive control):

The test with the reference material potassium dichromate was performed from February 08, 2017 to February 10, 2017. The 48-h EC50 of the reference substance was determined to be 0.85 mg/L.

Any other information on results incl. tables

Table: Immobilisation, definitive test

Test item loading rate	Number of Daphnia magna	Number of immobilised Daphnia magna								Total of immobilised Daphnia magna [%]
		24 h				48 h
		Replicates
		A	B	C	D	A	B	C	D	24 h	48 h
Control	20	0	0	0	0	0	0	0	0	0	0
6.25 mg/L	20	0	0	0	0	0	1	0	0	0	5
12.5 mg/L	20	0	0	0	0	0	2	1	0	0	15
25 mg/L	20	0	0	0	0	3	3	4	2	0	60
50 mg/L	20	0	0	0	0	4	3	4	4	0	75
100 mg/L	20	1	0	1	0	5	3	4	5	10	85

ADDITIONAL EFFECT VALUES

- EL10 (48h): 7.73 mg/L (95% C.I.: 3.80 - 11.51)

- EL20 (48h): 11.93 mg/L (95% C.I.: 7.04 - 16.53)

Table: Results of total organic carbon analyses, definitive test

Test item loading rate	Mean concentration of TOC (n=3) determined in samples [mg C/L]
	At exposure innitiation (fresh)	After 24 h of exposure		at exposure termination
		24-h old	fresh
Control	< LoQ	< LoQ	< LoQ	1.85
6.25 mg/L	1.68	1.68	1.77	2.11
12.5 mg/L	2.33	2.29	--	--
25 mg/L	3.03	2.7	--	--
50 mg/L	3.49	3.44	--	--
100 mg/L	3.86	3.61	3.71	4.25

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl tables'.