Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

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:

31 October 2011 to 16 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

-

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Stability in water: Not indicated
Solubility in water: ≤24.5 ng/L at 20 °C (determined in NOTOX project 497887)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter was kept for possible analysis of undissolved residue.

Frequency: at t=0 h and t=96 h
Volume: 1 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 1 ml were taken for possible analysis.

Vehicle:

no

Details on test solutions:

The batch of FAT 41043/A tested was a red powder, solid with a purity of 99.8 % and consisted of two isomeric forms. The substance was not completely soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 15-minute period of magnetic stirring combined with a treatment with ultrasonic waves. The obtained aqueous mixture was subsequently magnetically stirred for a period of two days to ensure maximum dissolution in test medium and subsequently filtered through a 0.45 µm membrane filter (rc55, Whatman).The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation (length definition, mean, range and SD):
Limit test: 2.7 ± 0.2 cm

- Weight at study initiation (mean and range, SD):
Limit test: 0.46 ± 0.07 g

- Method of breeding: F1 from a single parent-pair bred in UV-treated water.
- Feeding during test: no

HOLDING

Quarantine/Acclimatisation: At least 12 days after delivery

Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO3: 46.7 mg/l
KCl: 4.2 mg/l

Measurements: pH, nitrate and nitrite concentration and ammonia concentration: once a week. Temperature: every day.
In addition, pH and temperature were measured before transferring the fish to the test system.

Water quality parameters: Were kept within the optimum limits for the respective fish species.

Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

180 mg CaCO3/l

Test temperature:

21.0-22.2 °C

pH:

7.4-7.8

Dissolved oxygen:

5.9-9.2 mg O2/l

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations: A 0.45 µm filtered solution prepared at a loading rate of 100 mg/l (limit concentration)

Measured concentrations: Analysis of the sample taken from the limit concentration (i.e. the undiluted filtrate) at the start of the test showed actual concentration of 220 µg/l. The measured concentration dropped to 14 µg/l, i.e. 6.5 % of initial at the end of the test. Based on these results, the average exposure concentration was calculated to be 56 µg/l.

Note that the measured concentration at the end of the test was above the water solubility of the test substance which was determined to be ≤24.5 ng/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 9.8 litres of test solution
- Type (delete if not applicable): open
- Aeration: no
- Type : static
- No. of organisms per vessel: 7 (limit test)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: 0.33 g/litre

TEST MEDIUM / WATER PARAMETERS
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO3: 46.7 mg/l
KCl: 4.2 mg/l

Measurements pH, nitrate and nitrite concentration and ammonia concentration: once a week. Temperature: every day.
In addition, pH and temperature were measured before transferring the fish to the test system.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): NOEC and 24, 48, 72 and 96 h EC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

56 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: -

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 56 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: -

Details on results:

- Other biological observations: No effects
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses:no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
The 96h-LC50 for carp exposed to PCP was estimated to be 0.15 mg/l (95 % confidence interval between 0.10 and 0.22 mg/l). This effect was already reached within 24 hours of exposure.

Reported statistics and error estimates:

The average exposure concentrations were calculated as, being the geometric means of the concentrations of FAT 41043/A measured in the samples taken at the start (Ct=0) and the end of the test (Ct=96).

No LC50 could be calculated because the test substance proved to be non-toxic (LC50 > maximum soluble concentration).

Sublethal observations / clinical signs:

 Incidence of mortality and total mortality during the limit test

FAT 41043/A % of filtrate prep. at 100 mg/l	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3h	24h	48h	72h	96h
control	7	0	0	0	0	0	0
100 (56 µg/l)	7	0	0	0	0	0	0

() – between brackets: average exposure concentration

Validity criteria fulfilled:

yes

Conclusions:

The 96 h-LC50 was beyond the range tested, i.e. exceeded an average exposure concentration of 56 µm/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l.

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for carp could not be reached.

Description of key information

The short term toxicity to fish of FAT 41043/A was investigated according to OECD Guideline Nr. 203, EU Method C.1 and GLP principles. The 96h-LC50 was beyond the range tested, i.e. exceeded an average exposure concentration of 56 µg/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l.

Additional information

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for carp could not be reached.