N-(dodecylphenyl)naphthalen-1-amine

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:

5 October 2000 to 17 November 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material were verified by chemical analysis of the un-centrifuged and centrifuged samples at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media).

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

For the purpose of the definitive study the test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (250 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 250 mg/25 ml solvent stock solution. An aliquot (2.0 ml) of this solvent stock solution was dispersed in dechlorinated tap water and the volume adjusted to 20 litres to give the 1.0 mg/l test concentration.
The stock solution was inverted several times and the test concentration was mixed using a flat bladed stirrer to ensure adequate mixing and homogeneity.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, Nr Skipton, Yorkshire, UK and maintained in-house since 24 October 2000. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 1 November 2000 to 13 November 2000. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14°C with a dissolved oxygen content of greater than or equal to 9.5 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive study. There was less than 1% mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.2 cm (sd = 0.2) and a mean weight of 0.96 g (sd = 0.11) at the end of the definitive study. Based on the mean weight value this gave a loading rate of 0.48 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100 mg/l as CaCO3.

Test temperature:

14 ± 1 °C.

pH:

7.6 and 8.0.

Dissolved oxygen:

9.2 - 10.3 mg O2/l

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

In the range-finding study fish were exposed to a series of nominal test concentrations of 0.010, 0.10 and 1.0 mg/l.
Based on the results of the range-finding study a "Limit test" was conducted for the definitive study at a test concentration of 1.0 mg/l to confirm that at the highest attainable test concentration of 1.0 mg/l.
The time-weighted mean measured concentration of the centrifuged samples using the mean of the measured test concentrations of replicates R1 and R2 were calculated (see Any other information for full details).

Details on test conditions:

Test Water
The test water used for both the range-finding and definitive studies was the same as that used to maintain the stock fish.
Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Procedure
Range-finding study
During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. However, at a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 4451283) showed a water solubility value of 1.96 mg/l.
Based on this information it was considered appropriate to conduct the range-finding test using a homogenous dispersion of the test material to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration.
Therefore, in the range-finding study fish were exposed to a series of nominal test concentrations of 0.010, 0.10 and 1.0 mg/l. The test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 100 mg/10 ml solvent stock solution. Serial dilutions were prepared from this to give further solvent stock solutions of 10 and 1.0 mg/10 ml. Aliquots (2.0 ml) of each solvent stock solution were separately dispersed into a final volume of 20 litres of dechlorinated tap water to give the 0.0 10, 0.10 and 1.0 mg/l test concentrations.
In the range-finding study 3 fish were added to each 20 litre test and control vessel and maintained at 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions.
The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl/l of dimethylformamide.
Each vessel was covered to reduce evaporation. After 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

Definitive study
Based on the results of the range-finding study a "Limit test" was conducted for the definitive study at a test concentration of 1.0 mg/l to confirm that at the highest attainable test concentration of 1.0 mg/l, no mortalities or sub-lethal effects of exposure were observed.

Exposure conditions
As in the range-finding study 20 litre glass exposure vessels were used for each test concentration.
At the start of the study 10 fish were placed in each test vessel at random in the prepared test solutions. The test vessels were then covered to reduce evaporation and maintained at 14 ± 1 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control and solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 μl/l of dimethylformamide.
A semi-static test regime was employed in the study involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Physico-chemical measurements
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the study. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the study after 96 hours, represent those of the used or 24-hour old test preparations.

Verification of test concentrations
Two samples of each of the solvent control and replicate test groups were taken at 0 (fresh test media), 24, 48, 72 (old and fresh test media) and 96 hours (old test media). One sample was analysed un-centrifuged and one sample after centrifugation. A further set of samples (as above) were taken and stored frozen (approximately -20 °C) for further analysis if necessary.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.34 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding Study
During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. However, at a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.
Based on this information it was considered appropriate to conduct the range-finding test using a homogenous dispersion of the test material to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration. Therefore, the range-finding study was conducted using a series of nominal test concentrations of 0.010, 0.10 and 1.0 mg/l.
There were no sub-lethal effects of exposure during the range-finding study.
The results showed no mortalities at the test concentrations of 0.010, 0.10 and 1.0 mg/l.
Based on this information, a single test concentration, in duplicate, of 1.0 mg/l was selected for the definitive study. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration of 1.0 mg/l, no mortalities or sub-lethal effects of exposure were observed.

Definitive Study
Mortality data
There were no mortalities in 20 fish exposed to a test concentration of 1.0 mg/l for a period of 96 hours.
The results of the definitive study showed the highest test concentration resulting in 0% mortality to be greater than or equal to 1.0 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 1.0 mg/l and the No Observed Effect Concentration (NOEC) to be 1.0 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.
The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guidelines. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water.
At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

Sub-lethal effects
There were no sub-lethal effects of exposure observed in 20 fish exposed to a test concentration of 1.0 mg/l for a period of 96 hours.

Physico-chemical measurements
Temperature was maintained at 14 ± 1 °C throughout the study, while there were no treatment related differences for oxygen concentration or pH.
The pH of the solvent control group was observed to vary between 7.6 and 8.0. This variation was considered not to affect the validity or integrity of the study given that no mortalities or adverse reactions to exposure were observed in the solvent control group and the Test Guideline states that the pH should not vary by more than 1 unit.

Verification of test concentrations
During preliminary solubility work the highest test concentration that was achieved without the presence of visible undissolved test material was 0.20 mg/l. At a test concentration of 1.0 mg/l a homogenous dispersion was observed with a slick of test material forming after 24 hours. Therefore, it was considered appropriate to conduct the test using a test concentration of 1.0 mg/l to ensure that the test organisms were exposed to the maximum possible dissolved test material concentration. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.
During the range-finding study, samples were taken for chemical analysis from the 1.0 mg/l test concentrations to assess the amount of test material bioavailable to the test organisms. Samples were taken and analysed after both single and double 0.2 μm filtration and following centrifugation (17,000 G). The results showed that the test material adsorbed to the filters and centrifugation of these samples confirmed that a proportion of the test material was present as a dispersion.
Based on these results it was considered appropriate to analyse samples taken from the definitive study with no pre-treatment and after 30 minutes centrifugation (17,000 G). The results of the centrifuged samples indicate the concentration of test material in solution and hence bioavailable to the test organisms.
Chemical analysis of the fresh test media at 0, 24, 48 and 72 hours showed measured concentrations ranging from 55% to 92% of nominal for the un-centrifuged samples and from 37% to 74% of nominal for the centrifuged samples. Analysis of the old or expired test media at 24, 48, 72 and 96 hours showed measured concentrations ranging from 16% to 45% of nominal for the un-centrifuged samples and from 11% to 30% of nominal for the centrifuged samples.
The variability shown in the measured concentrations from analysis was considered to be due to variations in sampling and/or analysis of a test material at a low nominal test concentration which was partially dispersed in a complex biological testing system.
Post-study work was conducted where a test concentration of 1.0 mg/l was stirred using a propeller stirrer for approximately 48 hours to determine whether a higher level of dissolved test material could be achieved. This work confirmed the results of the definitive study in that variable results were shown from analysis of the resultant test media and higher measured concentrations of dissolved test material were not achieved.
Given the variability in the results from chemical analysis and the decline in the measured test concentrations over each 24 hour media renewal period it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media in order to express the results in terms of the bioavailable test material and to give a "worst case" analysis of the data.
The 96-Hour LC50 based on the time-weighted mean measured test concentration of the centrifuged media was greater than 0.34 mg/l and correspondingly the No Observed Effect Concentration was 0.34 mg/l.
The use of the time-weighted mean measured test concentration of centrifuged test media was shown to significantly affect the results of the study.

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Not specified

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Range-finding Study

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population = 3)
	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0
Solvent Control	0	0	0	0
0.010	0	0	0	0
0.10	0	0	0	0
1.0	0	0	0	0

 

Cumulative Mortality Data in the Definitive Study

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population = 10)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0
1.0 R1	0	0	0	0	0	0	0
1.0 R2	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 to 2

 

Physico-Chemical Measurements

Nominal Concentration (mg/l)	Time (Hours)
	0 Hours (Fresh Media)			24 Hours (Old Media)			24 Hours (Fresh Media)
	pH	mg O2/l	T°C	pH	mg O2/l	T°C	pH	mg O2/l	T°C
Control	7.6	9.8	14.0	7.9	9.9	13.5	7.7	10.1	14.0
Solvent Control	7.6	9.8	14.0	7.9	9.9	13.5	7.7	10.2	14.0
1.0 R1	7.6	9.7	14.0	8.0	9.9	13.5	7.7	10.2	14.0
1.0 R2	7.6	9.7	14.0	7.9	10.0	13.5	7.7	10.2	14.0
Nominal Concentration (mg/l)	Time (Hours)
	48 Hours (Old Media)			48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/l	T°C	pH	mg O2/l	T°C	pH	mg O2/l	T°C
Control	7.9	10.2	13.5	7.7	10.1	14.0	7.9	9.3	14.0
Solvent Control	8.0	10.2	13.5	7.7	10.1	14.0	8.0	9.2	14.0
1.0 R1	8.0	10.3	13.5	7.7	10.1	14.0	8.0	9.3	14.0
1.0 R2	8.0	10.3	13.5	7.7	10.2	14.0	8.0	9.4	14.0
Nominal Concentration (mg/l)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/l	T°C	pH	mg O2/l	T°C
Control	7.7	9.8	14.0	7.9	9.3	13.0
Solvent Control	7.7	9.8	14.0	7.9	9.3	13.0
1.0 R1	7.7	9.8	14.0	8.0	9.4	13.0
1.0 R2	7.7	9.8	14.0	8.0	9.3	13.0

R₁– R₂= Replicates 1 to 2

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 1.0 mg/l. Correspondingly the No Observed Effect Concentration was 1.0 mg/l.
Based on the time-weighted mean measured test concentrations of the centrifuged test media, the acute toxicity of the test material to rainbow trout gave a 96-Hour LC50 value of greater than 0.34 mg/l. The No Observed Effect Concentration was 0.34 mg/l.

Executive summary:

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Following a preliminary range-finding study fish were exposed, in two groups of ten, to an aqueous solution of the test material, at a single concentration of 1.0 mg/l for a period of 96 hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours.

 

The 96-Hour LC50 based on nominal test concentrations was greater than 1.0 mg/l and correspondingly the No Observed Effect Concentration was 1.0 mg/l.

 

The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Furthermore, a study, The Determination of the General Physico-Chemical Properties of the test material (Safepharm Laboratories Project Number: 445/283) showed a water solubility value of 1.96 mg/l.

 

Pre-study analyses were performed on test samples prepared at a nominal test concentration of 1.0 mg/l. Samples were taken and analysed after both single and double 0.2 μm filtration and following centrifugation (17,000 G). The results indicated that the test material adsorbed to the filter matrix of the 0.2 pm filters and that a proportion of the test material was present as a dispersion at the concentration of 1.0 mg/l.

 

Based on these results it was considered appropriate to analyse samples taken from the definitive study with no pre-treatment and after 30 minutes centrifugation (17,000 G). The results of the centrifuged samples indicated the concentration of test material in solution and hence bioavailable to the test organisms.

 

Chemical analysis of the fresh test media at 0, 24, 48 and 72 hours showed measured concentrations ranging from 55% to 92% of nominal for the un-centrifuged samples and from 37% to 74% of nominal for the centrifuged samples. Analysis of the old or expired test media at 24, 48, 72 and 96 hours showed measured concentrations ranging from 16% to 45% of nominal for the un-centrifuged samples and from 11% to 30% of nominal for the centrifuged samples.

 

The variability shown in the measured concentrations from analysis was considered to be due to variations in sampling and/or analysis of a test material at a low nominal test concentration which was partially dispersed in a complex biological testing system.

 

Post-study work was conducted where a test concentration of 1.0 mg/l was stirred using a propeller stirrer for approximately 48 hours to determine whether a higher level of dissolved test material could be achieved. This work confirmed the results of the definitive study in that variable results were shown from analysis of the resultant test media and higher measured concentrations of dissolved test material were not achieved.

 

Given the variability in the results from chemical analysis and the decline in the measured test concentrations over each 24 hour media renewal period it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media in order to express the results in terms of the bioavailable test material and to give a "worst case" analysis of the data.

 

The 96-Hour LC50 based on the time-weighted mean measured test concentration of the centrifuged media was greater than 0.34 mg/l and correspondingly the No Observed Effect Concentration was 0.34 mg/l.

Description of key information

A GLP accredited laboratory study performed in accordance with OECD Guideline 203 and EU Method C.1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.34 mg/L

Additional information

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss).

Following a preliminary range-finding study fish were exposed, in two groups of ten, to an aqueous solution of the test material, at a single concentration of 1.0 mg/l for a period of 96 hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours.

 

The 96-Hour LC50 based on nominal test concentrations was greater than 1.0 mg/l and correspondingly the No Observed Effect Concentration was 1.0 mg/l.

 

The test concentration of 1.0 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under the OECD Guidelines. Furthermore, a study, showed a water solubility value of 1.96 mg/l.

 

Pre-study analyses were performed on test samples prepared at a nominal test concentration of 1.0 mg/l. Samples were taken and analysed after both single and double 0.2 μm filtration and following centrifugation (17,000 G). The results indicated that the test material adsorbed to the filter matrix of the 0.2 pm filters and that a proportion of the test material was present as a dispersion at the concentration of 1.0 mg/l.

Based on these results it was considered appropriate to analyse samples taken from the definitive study with no pre-treatment and after 30 minutes centrifugation (17,000 G). The results of the centrifuged samples indicated the concentration of test material in solution and hence bioavailable to the test organisms.

 

Chemical analysis of the fresh test media at 0, 24, 48 and 72 hours showed measured concentrations ranging from 55% to 92% of nominal for the un-centrifuged samples and from 37% to 74% of nominal for the centrifuged samples. Analysis of the old or expired test media at 24, 48, 72 and 96 hours showed measured concentrations ranging from 16% to 45% of nominal for the un-centrifuged samples and from 11% to 30% of nominal for the centrifuged samples.

The variability shown in the measured concentrations from analysis was considered to be due to variations in sampling and/or analysis of a test material at a low nominal test concentration which was partially dispersed in a complex biological testing system.

 

Post-study work was conducted where a test concentration of 1.0 mg/l was stirred using a propeller stirrer for approximately 48 hours to determine whether a higher level of dissolved test material could be achieved. This work confirmed the results of the definitive study in that variable results were shown from analysis of the resultant test media and higher measured concentrations of dissolved test material were not achieved.

 

Given the variability in the results from chemical analysis and the decline in the measured test concentrations over each 24 hour media renewal period it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged test media in order to express the results in terms of the bioavailable test material and to give a "worst case" analysis of the data.

 

The 96-Hour LC50 based on the time-weighted mean measured test concentration of the centrifuged media was greater than 0.34 mg/l and correspondingly the No Observed Effect Concentration was 0.34 mg/l.