Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Mar 2017 to 05 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Analytical monitoring:

yes

Remarks:

Total organic carbon (TOC)

Details on sampling:

Since the test item is a multiconstituent, in the definitive test, total organic carbon (TOC) was analysed to verify the stability of the test item. At exposure initiation and at the renewal analyses were performed in samples of every test item loading rate and the control to determine the total oranic carbon. At exposure termination analyses were performed in 48-hours old samples (i.e. not renewed) of every test item loading rate and the control.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Filtrates of five different loading rates and a control were prepared. The separate portions of the test item were weighed directly into the test medium in a glass flask. Every portion was quantitatively transferred into the respective glass vessel by multiple washing with test medium and filling up to the respective total volume. The resulting mixtures and the control (i.e. test medium without test item) was incubated at temperature approx. 30 °C for 48 h with continuous mechanical shaking at 90 rpm in darkness. Next, every mixture and the control was kept at room temperature for another 24 h without shaking and with continuous aeration. Every mixture and the control were separately filtered through a pre-conditioned cellulose filter and next through a preconditioned nitrocellulose filter of 0.22 Wm pores (Millipore 0.22 Wm GSTF, Merck). The filtrates were collected and used for exposure. Each filtrate was visually homogeneous and without any visibly non-dissolved particles. Glass beakers of 150 mL were pre-conditioned by filling up with the respective filtrate, discarding the contents and refilling to be used for exposure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Originated from the standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology

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 with daily cycle 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 lyophilised suspension of Spirulina sp.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water is of defined composition and therefore the total hardness is expected 250 mg/L ± 25 mg/L as CaCO3.

Test temperature:

20.2 - 21.9 ºC

pH:

- pH measured in fresh samples: 7.72 - 7.89
- pH measured in spent samples: 7.79 - 7.95

Dissolved oxygen:

- Dissolved oxygen measured in fresh samples: 7.8 - 8.1 mg/L
- Dissolved oxygen measured in spent samples: 7.8 - 8.0 mg/L

Conductivity:

565 µS/cm

Nominal and measured concentrations:

- Nominal concentrations (loading rates): 0 (control), 11.4, 20.6, 37.0, 66.7 and 120 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: Closed, the beakers were covered with transparent lids in order to minimise evaporation and to prevent accidental contamination.
- Aeration: No
- Renewal rate of test solution: Once after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Feeding during test: No
- Age: Only organisms up to 24 hours old (not first brood progeny) and in good physiological condition were used in the tests.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Based on information on the test item’s properties it contains 4.7% chromium. The test media containing known chelating agents (such as EDTA) should be avoided for testing substances containing metals. Therefore, the reconstituted ISO medium was to be used in the definitive test instead of the Elendt M7 medium, which was used in the preliminary non-GLP test. The composition of the reconstituted water (ISO 6341 : 1982) is tabulated in 'Any other information on materials and methods incl. tables'.
- Alkalinity: 67.7 mg/L as NaHCO3
- Culture medium different from test medium: No
- 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. The temperature was measured continuously.

OTHER TEST CONDITIONS
- 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. The Daphnia magna should be considered immobile if they showed no ability to swim within 15 seconds after swirling the test vessel.

RANGE-FINDING STUDY
- Nominal concentrations (loading rates): 0 (control), 5, 10, 50 and 100 mg/L.
- Results used to determine the conditions for the definitive study: In the control, in the filtrate of a loading of 5 mg/L, in the filtrate of a loading of 10 mg/L, in the filtrate of a loading of 50 mg/L and in the filtrate of a loading of 100 mg/L no Daphnia magna immobilisation was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mobility

Details on results:

During exposure, no immobilisation was observed in the control. At exposure termination no immobilisation was observed in any treatment.
- Test item solubility: Each filtrate was visually homogeneous and without any visibly non-dissolved particles.

Results with reference substance (positive control):

The test with the reference material potassium dichromate was performed from March 01, 2017 to March 03, 2017. The 48-h EC50 of the reference substance was determined to be 0.66 mg/L. The 48-h EC10 and EC20 were determined to be 0.47 and 0.53 mg/L, respectively, and the 48-h NOEC and LOEC were determined to be 0.56 and 1.00 mg/L.

Reported statistics and error estimates:

Probit method calculations and analysis by Step-down Cochran-Armitage Test Procedure.

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

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

Description of key information

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

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria (IPO, 2017). In this study daphnids (D. magna, 20 per concentration) were introduced in test vessels containing filtrates of the test substance with the following loading rates: 0 (control), 11.4, 20.6, 37.0, 67.7 and 120 mg/L for 48 hours under semi-static conditions (test medium renewal after 24 hours). As organisms were exposed to a nominal loading rate of the test substance, effect concentrations are expressed as nominal. Analytical quantification of the test substance was performed by measuring the total organic carbon (TOC) using a validated method. Immobilisation was recorded after 24 and 48 hours exposure. No immobilisation was observed up to the highest loading rate. Based on the findings, the 48-h EL50 value was determined at > 120 mg/L.