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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Mar 2017 to 31 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

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

Details on sampling:

At exposure initiation and at the first renewal analyses were performed in samples of every test item loading rate and the control. At exposure termination analyses were performed in 96-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 a total volume was filled up to 4 L. 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 pre-conditioned nitrocellulose filter of 0.22 µm pores (Millipore 0.22 µm GSTF, Merck). The filtrates were collected and used for exposure. Glass test vessels of 4 L were pre-conditioned by filling up with the respective filtrate, discarding the contents and refilling to be used for exposure.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Hamilton-Buchanan
- Source: Embryos originated from the laboratory zebrafish breeder the European Zebrafish Resource Center, Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Germany
- Age at study initiation: approximately 3 months old
- Codition: fish were healthy and in good physiological condition. The health of fish is monitored daily on regular basis and hygiene in the animal facility is monitored on regular basis, according to the animal monitoring and veterinary care programme.
- Average length at study termination (SD): 1.96 cm (± 0.18)
- Average weight at study termination (SD): 0.11 g (± 0.02)

ACCLIMATION
Before exposure initiation in the definitive test, the fish adaptation to the test conditions lasted for 12 days. The wild-type AB line zebrafish originated from the embryos were acquired from the laboratory culture and they were adapted to the test conditions before exposure initiation. The test organisms were kept in the reconstituted water at a temperature stabilised with a heater and with continuous aeration. Once the larvae were hatched, they were fed with various feeds to learn how to chase and catch their prey. Nutritional enrichment was supplied by providing alive Daphnia magna and Artemia salina in addition to a regular Tetra Min® flake fish food (Tetra, Germany). Later, the juvenile zebrafish were kept in a larger volume aquarium with environmental enrichment supplied: colored bottom, bubbling aeration system, shaded area, stones of various dimensions and alive submerged macrophyte plants for shelter. The aim of the adaptation was to confirm the health status of the fish and to acclimatise to the test conditions. Fish mortality during the 12-day period before definitive testing was 0.7% (i.e. 1 dead fish out of 142 fish in the batch for testing). The adaptation was performed in an aquarium of 300 L in reconstituted water with constant filtration and aeration system. Reconstituted water was filtered through layers of sponge (mechanic filter), CarboMax™ (activated carbon), ZeoWonder™ and BioCeraMax™ (biofilters) aerated with fresh air and disinfected by UV lamp. The fish were fed with flake food and live feed (i.e. Daphnia magna and Artemia salina). Feeding the fish to be used in the definitive test was stopped for 24 h before exposure initiation. The fish were healthy, free from external parasites and visible deformations, which was confirmed by the veterinary control.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

216 mg/L as CaCO3

Test temperature:

22.3 - 23.2 °C

pH:

7.28 - 7.79

Dissolved oxygen:

81 - 98% of air saturation value

Conductivity:

545 µS/cm

Nominal and measured concentrations:

- Nominal loading rates: 0 (control), 37.0, 66.7, 120, 216 and 388.8 mg/L (based on a range-finding study

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels
- Type: Closed; vessels were covered with glass lids in order to minimise evaporation and to prevent accidental loss of fish (due to jumping out) and accidental contamination. The bottom of glass vessels with fish was coloured but the walls were transparent to facilitate observations during exposure.
- Fill volume: 4 L
- Aeration: Continuous; the gentle bubbling aeration was supplied with a glass tube to the bottom and served also as the orientation reference for the fish.
- Renewal rate of test solution: Daily; on every renewal all alive fish were transferred from the spent solution to the respective freshly prepared solution using a fish-net catcher.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Fish loading: 0.28 g/L
- Feeding during test: no

PREPARATION OF THE TEST MEDIA
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 a total volume was filled up to 4 L. 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 pre-conditioned nitrocellulose filter of 0.22 Um pores (Millipore 0.22 Um GSTF, Merck). The filtrates were collected and used for exposure. Glass test vessels of 4 L were pre-conditioned by filling up with the respective filtrate, discarding the contents and refilling to be used for exposure.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituent water (ISO 6341 : 1982) was prepared based on deionized water by adding appropriate amounts of stock solutions of reagent grade chemicals. See 'Details test organism' for the preparation method. The composition is tabulated in 'Any other information on materials and methods incl. tables'.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Twice daily (before and after renewal)

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h darkness
- Light type: fluorescent

EFFECT PARAMETERS MEASURED
During the test observations for mortality and for symptoms of intoxication were conducted and recorded after 3, 6, 24, 48, 72 and 96 h of exposure. These were observations for loss of equilibrium, changes in swimming behaviour, respiration or pigmentation. The fish are considered dead if there is no reaction to external stimuli (touching of the caudal peduncle).

OTHER MEASUREMENTS
In both tests temperature was continuously recorded using an electronic device with a sensor submerged in the glass vessel containing the control (HI 141 thermologger, Hanna Instruments). The pH values and dissolved oxygen concentrations were measured in every freshly prepared test item loading rate (i.e. filtrate) and the control at exposure initiation and on every renewal as well as in every spent test item loading rate and the control on every renewal and at exposure termination in each glass vessel with fish (inoLAB Level 3 pH-Oxi-meter, WTW, Germany). In the test at exposure initiation conductivity was measured in the control (Multi 3430, WTW, Germany and the total hardness was determined by complexometric titration with EDTANa2 (ethylenediamine-tetraacetic acid disodium salt) (digital titration apparatus Solarus, Hirschmann Laborgeräte GmbH&Co., Germany.

PRELIMINARY NON-GLP TEST
- Test loading rates: 0 (control), 5 (diluted), 10 and 100 mg/L.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Results used to determine the conditions for the definitive study: 40% mortality was observed after 96 exposure in 100 mg/L loading rate treatment. The test item loading rate used in the definitive test was determined on the basis of the preliminary test results.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 388.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mortality (fish)

Details on results:

- Observations: In the control and in the filtrate of a loading of 37.0 mg/L as well as the filtrate of a loading of 66.7 mg/L no mortality of fish nor any symptoms of intoxication were observed during exposure. In the filtrate of a loading of 120 mg/L at exposure termination one fish was found dead. In the filtrate of a loading of 216 mg/L after 72 h of exposure one fish was found dead and no other symptoms nor mortality was observed till exposure termination. In the filtrate of a loading of 388.8 mg/L after 72 h of exposure one fish was found dead and at exposure termination two fish were found dead. Therefore, the fish mortality increased up to 20% at the highest loading rate. An overview of the results on mortality are provided in 'Any other information on results incl. tables'.
- Test item solubility: Mixtures were heterogeneous with visibly non-dissolved particles.

Reported statistics and error estimates:

The Fisher’s Exact Binomial Test with Bonferroni Correction was used to indicate possible significant results.

Sublethal observations / clinical signs:

Table: Mortality - definitive test

Treatment	Number of fish tested	Number of dead fish in each observation term						Total of dead [%]
		3 h	6 h	24 h	48 h	72 h	96 h
Filtrate of a loading of 388.8 mg/L	10	0	0	0	0	1	2	20
Filtrate of a loading of 216 mg/L	10	0	0	0	0	1	1	10
Filtrate of a loading of 120 mg/L	10	0	0	0	0	0	1	10
Filtrate of a loading of 66.7 mg/L	10	0	0	0	0	0	0	0
Filtrate of a loading of 37 mg/L	10	0	0	0	0	0	0	0
Fitlrate of the control	10	0	0	0	0	0	0	0

ADDITIONAL EFFECT VALUES

96-h LL10: 199.0 mg/L

96-h LL20: 359.9 mg/L

96-h LOELR: 388.8 mg/L

96-h NOELR: 216.0 mg/L

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The test item is with high probability not acutely harmful to fish.

Description of key information

The test item is with high probability not acutely harmful to fish.

Additional information

The short-term toxicity to freshwater fish was determined in a study according to OECD TG 203 and in compliance with GLP criteria (IPO, 2017). In this study, groups of 10 zebrafish (Danio rerio) were exposed for 96 hours to filtrates of the test substance with the following loading rates: 0 (control), 37.0, 66.7, 120, 216, and 388.8 mg/L. The study was semi-static with a 24-hour renewal of test media. Analytical quantification of the test substance was performed by measuring the total organic carbon (TOC) using a validated method. Incidences of mortality and clinical effects were recorded after 3, 6, 24, 48, 72 and 96 hours exposure. All OECD validity criteria were met. In the filtrate of a loading of 120 mg/L at exposure termination one fish was found dead. In the filtrate of a loading of 216 mg/L after 72 h of exposure one fish was found dead and no other symptoms nor mortality was observed till exposure termination. In the filtrate of a loading of 388.8 mg/L after 72 h of exposure one fish was found dead and at exposure termination two fish were found dead. Based on these findings the 96-h LL50 is determined to be > 388.8 mg/L.