1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthyl)ethan-1-one

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP requirements and OECD test method. Study is well documented and the data complete.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see overall remarks

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

Appropriate amounts of the test substance were weighed out and dissolved in triethylene glycol, followed by 20 hours of mechanically stirring. During stirring, the stock solutions were protected from light. New stock solutions were prepared weekly and subsequently placed in the flow-through system.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Eggs were obtained from the commercial fish hatchery Atlanta in Hellevoetsluis (Netherlands). At the start of the test the eggs were verified to be in the young blastula stage.
20 fertilized were used after the first 24 hours of exposure.

Test type:

other: Intermittent flow through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Hardness:

Ca. 200 mg/l.

Test temperature:

variation of temperature (measured in one of the control vessels) : 24.0 - 25.8°C

pH:

variation of pH value: 7.8 - 8.3

Dissolved oxygen:

lowest measured oxygen concentration : 7.1 mg/l

Nominal and measured concentrations:

Nominal: 0, 10, 20, 35, 50 and 75µg/l.
See also table 1

Details on test conditions:

An intermittent flow-through system was used to dose the test solutions. The test was carried Out under a 16h light/8h dark regime in a temperature controlled room. The water supply was also temperature controlled.

Reference substance (positive control):

no

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

missing tail fin

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

50 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

missing tail fin

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

>= 75 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

>= 75 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

malformation induced in the egg stage

Details on results:

The continuous monitoring of the temperature showed that there were no disturbances in the temperature of the culture room.
The measured actual concentrations confirmed the correct dosing of the test substance, being between 104% and 110% of the nominal values.

Reported statistics and error estimates:

Statistical significance for growth was determined with the two-tailed Dunnett-test with a 95% and 99% significance level. In both cases the observations at each concentration were compared with those of the control. In case of significance at the 99% level only that significance is given. The NOEC for condition was not determined statistically.

The actual concentration as determined by chemical analysis varied between 104% and 110% of the nominal values, which confirmed the correct dosing of the test substance. Because this is above 80% of the nominal concentrations it is common practice according to EU regulations, to express the results of the test in nominal concentrations.

Table 1 Results of the chemical analysis of the test solutions as carried out during the early life stage test with AHTN

	Nominal Concentration in µg/l						Column
Days	0	10	20	35	50	75	75^1)
	Measured Concentration in µg/l
1	<5	12.6	22.2	37.9	54.8	76.4	66.6
9	<5	11.2	21.4	38.4	52.9	83.3	64
16	<5	10.5	23.3	38	58.1	82.2	67.8
23	<5	10.8	22	36.8	47.4	76.6	69.2
30	<5	9.7	19.6	34	45.8	74.8	59
Average(std dev)	<5	11.0(1.1)	21.7(1.4)	37.0(1.8)	51.8(5.1)	78.7(3.8)	65.3(4.0)
% of nominal		110	109	106	104	105	87

¹⁾ Intended concentration by column technique

Validity criteria fulfilled:

yes

Conclusions:

The solvent triethylene glycol does not contribute to the observed effects.
The NOEC and LOEC values for the induction of missing tail fins are 35µg/l and 50µg/l respectively.
Hatching of the eggs and the survival of the fish larvae at the end of the test were not affected by AHTN at a concentration ≤75µg/l.
At concentrations up to 75µg/l there were no malformations induced in the egg stage, as was observed in the range-finding test at concentrations of 200µg/l and higher.
Concentrations ≥50µg/l induced lack of the tail fin and curling or curving of the body and AHTN causes disturbed swimming behaviour from 65µg/l.

Executive summary:

The influence of AHTN on the induction of egg and larval malformations (in particular missing tail fins) in Zebra fish species was determined in the Fish Early Life Stage Toxicity Test. An intermittent-flow-through system was used to expose eggs/larvae for 34 days to AHTN. The test substance was dosed by diluting concentrated solutions in the solvent triethylene glycol. Five test substance concentrations were tested together with a control and a solvent control. The nominal concentrations of AHTN tested were 10, 20, 35, 50 and 75 µg/l. The actual concentration as determined by chemical analysis.

A number of 4 replicates, each with 20 fish eggs/larvae, were exposed at each concentration or control. Mortalities were recorded. The mortality in the controls and the AHTN concentrations was between 2.5% and 5%; this indicates that the NOEC and LOEC values are ≥75µg/l and> 75µg/l respectively.

To investigate the possible contribution of the solvent triethylene glycol to the induction of malformations, fish were also exposed to an AHTN solution prepared without the use of a solvent, i.e. dosing of the test substance using a generator column. The concentration dosed by the column was intended to be µg/l the actual concentration as determined by chemical analysis appeared to vary between 59.l and 69µg/l (average 65µg/l).

In the controls and at 35µg/l, the exposed eggs/larvae showed a normal development and the phenomenon of the missing tail fins was never observed, but missing tail fins were observed at 50 and 75µg/l and after dosing via the column generator at 65µg/l. At 75µg/l, all fish had missing fins tail and most of them showed curving and/or curling of the body, together with a disturbed swimming behaviour. At 50µg/l and after column dosing the adverse effects were also observed, but to a lesser extent than at 75µg/l.

Description of key information

The long-term toxicity of the test item on fish was determined in 35d-Early Life Stage tests with Fathead Minnow and Zebrafish as well as in a 21d-growth test with Bluegill Sunfish. With 0.089 mg/L, the NOEC in the growth test was the higher value. Larval development and growth was the more sensitive endpoint with NOEC 0.035 mg/L in both e.l.s. tests.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

35 µg/L

Additional information

The test item is classified with respect to aquatic life. In the environment it is most concentrated immediately below the sewage treatment plant (STP) discharge point. Although the test item has a short half-life in river water, its high adsorption to sediment in combination with an expected steady state concentration in river water due to a continuous release from the STP, justifies the need for highly reliable long-term toxicity data. One study on the effect on fish growth and two early life stage development studies are included to reduce uncertainty for the PNEC derivation. All tests were performed according to OECD guidelines and recorded and reported under GLP conditions.

The 21d-growth study on Blue gill sunfish (Lepomis macrochrius) with the NOEC of 0.089 mg/l included mortality, growth and clinical signs such as loss of equilibrium, irregular respiration and cessation of food intake. However, this was considered to be insufficient to cover all relevant effects of prolonged exposure.

Next, an early life-stage study was performed on Fathead Minnow according to OECD TG 210 under GLP. The major recorded effect was a reduction in length caused by the absence of the caudal fin. The NOEC was 0.035 mg/L, LC50 was 0.100 mg/L. The test was repeated with Zebrafish to confirm the observations. Also in this species the test substance caused the absence of the caudal fin and curling or curving of the body. In this study the NOEC was also 0.035 mg/L. These studies are selected as the key studies for the risk assessment.