2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA TSCA, 40 CFR, Part 797.1600 as modified in Consent Agreement 40 CFR, Part 799, FR Vol 54 No. 223, 21 Nov 1989

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Concentrations of each replicate for each concentration were determined on study day 0, 1, 7 and every 7 days thereafter, including study termination day by means of spectrophotometric analysis. In addition control, carrier blank and fortified water sanples of the test substance were analysed on each sample day.

Details on test solutions:

Mean measured concentrations of the purified presscake solutions: 0.36, 0.58, 1.2, 2.5 and 4.8 µg/l (corresponding to nominal 0.31, 0.63, 1.3, 2.5 and 5 µg/l).
5 µg/l was considered to be the solubility limit of the test substance.
Two replicates for each concentration

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Fertilised eggs < 4 hours before study initiation.
Unfertilised rainbow trout eggs and sperm were obtained from Mt. Lassen Trout Farm. They were collected from adult brood fish, placed into plastic bags and placed with ice in a cooler, shipped overnight to the ABC Lab. The eggs and sperm were tempered from 3.8 to 7.3 °C during a 2hours equilibration period. Once the temperature was reached, the eggs were combined with the sperm in a dry plastic bowl resting in a ca. 7 °C water bath and gently mixed. Enough isotermal water was added to cover the eggs and the mixture was gently stirred to ensure maximum fertilisation. Approx. one minute ater the water addition, the eggs were rinsed several times then covered again with water and allowed to wter harden for ca. 2 hours before ditribution to the test system incubation cups.
test solutions had been flowing through the exposure aquaria for 39 hours prior to the addition of the eggs at the initiation of the main study.
Five newly fertilised rainbow trout eggs were introduced for each replicate up to a total of 40 eggs for each inubator cup ( 80 eggs for each concentration, total of 560 eggs for control, blank and test solutions). In addition, 50 eggs were placed in separate incubator cups in each of the control replicates for determining viability (fertilisation success). Egg mortality was recorded daily and dead eggs were removed to prevent fungal growth.

Viability (fertilisation success): After 11-days of exposure the viability eggs were removed from the control chambers and placed in a 10% glacial acetic acid solution. After several minutes in the solution he embryos became clear. Fertilisation and embryo development were indicated by the presence of a neural keel, which was visible as a white line.
The final number of viable eggs used in the study for each replicate was 37.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

122 d

Remarks on exposure duration:

post hatch

Hardness:

Alkalinity: control, low and high test concentration 178-202 total as CaCO3
Hardness: 158-184 mg/l (total, as CaCO3)

Test temperature:

Test temperature was determined for control, blank and the five concentrations through the whole study period in alternate replicate until day 105; from day 112 , then in each chamber.
Temperature during incubation: 10+/-11.5 °C
Temperature during fry growth: 12+/-1.5 °C

pH:

7.9 - 8.7 for control, blank and the five concentrations through the whole study period in alternate replicate beginning on day 0.

Dissolved oxygen:

Dissolved oxygen was determined for control, blank and the five concentrations through the whole study period in alternate replicate until day 105; from day 112 in each chamber due to increasing szie and oxygen demand.
Prior to day 60 post-hatch (study day 96) D.O. ranged from 8.2 mg/l (at 11.5 °C) to 10.6 mg/l (at 11.2 °C) which is 80 and 100 % D.O. saturations respectively. Following day 60 post-hatch the saturation ranged from 54 to 83 %.

Salinity:

Conductivity: 280 to 428 µS/cm for control, blank and the five concentrations through the whole study period

Nominal and measured concentrations:

concentrations were decided after a DRF study of 30 days at the following doses 0, 0.31, 0.63, 1.3, 2.5 and 5 µg/l
concentrations of the main test: 0, 0.31, 0.63, 1.3, 2.5 and 5 µg/l based on no effects observed at any of the doses of the DRF study
The concentrations of the test solutions were measured and they ranged bewteen 92 to 113 % of the nominal value

Details on test conditions:

Un-ionised ammonia was also measured 3.2 - 19.8 µg/l for control and one tested sample

Key result

Duration:

122 d

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatchability, fry survival and fry growth (lenght and weight)

Details on results:

No statistically significant reductions in hatchability were detected at any concentration.
No statistically significant survival reductions were indicated at any test level for either the 20 or 60 days post hatch intervals. Marginally significatn reductions in sruvival were detected at 2.5 µg/l for both the 90 and 122 day post-hatch intervals. However, these reductions were not considered to be concentration related or biologically significant. The 2.5 µg/l concentration was therefore not considered as an effect level with regard to survival.
Lenght, which was measued at 6, 90 and 122 days post-hatch was not significantly reduced at any test lvel. Weight which was measured at day 122 post-hatch (study termination) was not significantly reduced at any test level.

Viability (fertilisation success) : 97 %

Hatch was 95% complete in all chambers by day 37. Percent hatch in the 0.36, 0.58, 1.2, 2.5 and 4.8 µg/l test concentrations was 99, 99, 100, 99 and 100 % respectively. Moreover, there was NO observed concentration realted delay with regard to time to hatch

Swim up: Newly hatched fry began swimming up fromthe bottom of the test chambers at 12 days post-hatch. Swim up was 75 % complete in all chambers by day 19 post-hatch. There was NO observed concentration related dealy in time to swim up.

Morphological and behavioural abnormalities: Abnormalities (light discolouration, quiescence, distended abdomen, curved body posture, resting on chamber bottom on both ventral and lateral surfaces, erratic swimming pattern, irregular respiration, surfacing, and outer marging of operculum reduced by 2-4 mm) were noted in only a few fish at any given time and were NOT considered to be concentration related effects

Fry survival: no test concetration was considered to have an effect level on fry survival

Lenght and weight: no statistically significant reductions in lenght was observed at day 60, 90 and 122 post-hatch, and in weight were indicated at day 122 post-hatc for any test level.

Validity criteria fulfilled:

yes

Conclusions:

The substance was tested for long term toxicity to fish following OECD 210. Under the experimental condition the nominal NOEC was greater than 5 µg/L.

Executive summary:

A study was conducted to determine the long-term toxicity of the test substance (99.61% purity) to Rainbow trout (Onchorhyncus mykiss) according to the method of EPA TSCA, 40 CFR, Part 797.1600 as modified in Consent Agreement 40 CFR, Part 799, FR Vol 54 No. 223, 21 Nov 1989, comparable to OECD Guideline 210 (Fish early stage toxicity test). Eighty fertilised eggs were exposed during incubation up to 122 d post-hatch (Day 156 of the study) in a flow-through system at nominal concentrations of 0, 0.31, 0.63, 1.3, 2.5 and 5 µg/L. Hatchability (time and percentage), fry survival, length and weight, together with abnormalities and behaviour (swim up), were measured at different time points post-hatch. Test substance concentrations were measured in each replicate of each dose group on Days 0, 1, 7 and every 7^th day thereafter, including study termination day, by spectrophotometric analysis. No statistically significant reductions in hatchability were noted at any concentration. No statistically significant survival reductions occurred at any test level at the 20 and 60 days post hatch intervals. Marginally significant reductions in survival were recorded at 2.5 µg/L for the 90 and 122 -d post-hatch intervals. However, these were not considered dose-related or biologically significant. The 2.5 µg/L concentration was therefore not regarded as an effect level with regard to survival. Length, measured at 6, 90 and 122 d post-hatch, was not significantly reduced at any dose level. Weight, measured at Day 122 post-hatch (study termination), was also not significantly reduced in any group. Based on these results, the nominal NOEC was determined to be ≥5 µg/L. A LOEC could not be identified since there were no dose-related effects (ETAD, 1991).

Additional information

A study was conducted to determine the long-term toxicity of the test substance (99.61% purity) to Rainbow trout (Onchorhyncus mykiss) according to the method of EPA TSCA, 40 CFR, Part 797.1600 as modified in Consent Agreement 40 CFR, Part 799, FR Vol 54 No. 223, 21 Nov 1989, comparable to OECD Guideline 210 (Fish early stage toxicity test).Eighty fertilised eggs were exposed during incubation up to 122 days post-hatch (Day 156 of the study) in a flow-through system at nominal concentrations of 0, 0.31, 0.63, 1.3, 2.5 and 5 µg a.i./L. Hatchability (time and percentage), fry survival, length and weight, together with abnormalities and behaviour (swim up), were measured at different time points post-hatch. Test substance concentrations were measured in each replicate of each dose group on Days 0, 1, 7 and every 7^th day thereafter, including study termination day, by spectrophotometric analysis. No statistically significant reductions in hatchability were noted at any concentration. No statistically significant survival reductions occurred at any test level at the 20 and 60 days post hatch intervals. Marginally significant reductions in survival were recorded at 2.5 µg/L for the 90 and 122 day post-hatch intervals. However, these were not considered dose-related or biologically significant. The 2.5 µg/L concentration was therefore not regarded as an effect level with regard to survival. Length, measured at 6, 90 and 122 days post-hatch, was not significantly reduced at any dose level. Weight, measured at Day 122 post-hatch (study termination), was also not significantly reduced in any group. Based on these results, the nominal NOEC was determined to be ≥ 5 µg a.i./L. A LOEC could not be identified since there were no dose-related effects (ETAD, 1991).