N-(dodecylphenyl)naphthalen-1-amine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 October 2000 to 15 November 2000.

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test"

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Method C.2 of Commission Directive 92/69/EEC

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:

Two samples of each of the solvent control (replicates R1 - R2 pooled) and the 1.0 mg/l test group (replicates R1 - R2 and R3 - R4 pooled) were taken at 0 and 48 hours. One sample was analysed uncentrifuged and one sample after centrifugation. Further samples (in duplicate) were taken at 0 and 48 hours and stored frozen (approximately -20°C) for further analysis if necessary.

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/2.5 ml solvent stock solution. An aliquot (200 μl) was dispersed in reconstituted water and the volume adjusted to 2 litres to give the 1.0 mg/l test concentration.
The solvent stock solution and the prepared concentration were inverted several times to ensure adequate dissolution, mixing and homogeneity.
The concentration and stability of the test material in the test concentration were verified by chemical analysis of the un-centrifuged and centrifuged samples at 0 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced overnight were then removed for testing.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

21°C

pH:

7.8 - 7.9

Dissolved oxygen:

8.4 mg O2/l

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

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 immobilisation or adverse reactions to exposure were observed.

Details on test conditions:

Range-finding study
The test concentration to be used in the definitive study was determined by a preliminary 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 Daphnia magna 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 (50 μl) of each solvent stock solution were separately dispersed into a final volume of 500 ml of reconstituted water to give the 0.010, 0.10 and 1.0 mg/l test concentrations.
Each of the prepared solvent stock solutions and test concentrations were inverted several times to ensure adequate mixing and homogeneity.
In the range-finding study 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation.
After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
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.

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 immobilisation or adverse reactions to exposure were observed.

Preparation of the test material
For the purpose of the definitive study the required amount of test material was added to each test vessel using the method described above.

Exposure conditions
As in the range-finding study 250 ml glass jars containing approximately 200 ml of test solution were used. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the prepared test solutions. Four replicate test vessels were prepared with duplicate control and solvent control vessels. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
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.
The test solutions were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Physico-chemical measurements
Water temperature was recorded daily throughout the study. Dissolved oxygen concentrations and pH were recorded at the start and termination of the study.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.38 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

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. At a test concentration of 1.0 mg/l it was possible to obtain a homogenous dispersion at 0 hours. However, a slick of test material formed after 24 hours. Furthermore, a study, The Determination of the General Physico-Chemical Propel-ties 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.
No immobilisation was observed at the test concentrations of 0.010, 0.10 and 1.0 mg/l.
Based on this information, a single test concentration of four replicates 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 immobilisation or adverse reactions to exposure were observed.

Definitive Study
Immobilisation data
There was no immobilisation in 40 daphnids exposed to a test concentration of 1.0 mg/l for a period of 48 hours.
The No Observed Effect Concentration after 24 and 48 hours exposure was 1.0 mg/l. The No Observed Effect Concentration is based upon zero immobilisation 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.

Physico-chemical measurements
Temperature was maintained at 21°C throughout the study, while there were no treatment related differences for oxygen concentration or pH.

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Not specified

Cumulative Immobilisation Data in the Range-finding Study

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
Solvent Control	0	0
0.010	0	0
0.10	0	0
1.0	0	0

 

Cumulative Immobilisation Data in the Definitive Study

Nominal Concentration (mg/l)		Cumulative Immobilised Daphnia (Initial Population: 10 per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1 R2	0 0	0	0	0 0	0	0
Solvent Control	R1 R2	0 0	0	0	0 0	0	0
1.0	R1 R2 R3 R4	0 0 0 0	0	0	0 0 0 0	0	0

R₁– R₄= Replicate 1 to 4

 

Physico-Chemical Measurements

Nominal Concentration (mg/l)		0 Hours			24 Hours	48 Hours
		pH	mg O2/l	T°C	T°C	pH	mg O2/l	T°C
Control	R1 R2	7.8 7.8	8.4 8.4	21.0 21.0	21.0 21.0	7.9 7.9	8.4 8.4	21.0 21.0
Solvent Control	R1 R2	7.8 7.8	8.4 8.4	21.0 21.0	21.0 21.0	7.9 7.9	8.4 8.4	21.0 21.0
1.0	R1 R2 R3 R4	7.8 7.8 7.8 7.8	8.4 8.4 8.4 8.4	21.0 21.0 21.0 21.0	21.0 21.0 21.0 21.0	7.9 7.9 7.9 7.9	8.4 8.4 8.4 8.4	21.0 21.0 21.0 21.0

R₁– R₄= Replicate 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 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 concentration of the centrifuged samples the 48-Hour EC50 was estimated to be greater than 0.38 mg/l. Correspondingly the No Observed Effect Concentration was 0.38 mg/l.

Executive summary:

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

 

Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at a concentration of 1.0 mg/l for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

The 48-Hour EC50 for the test material to Daphnia magna 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 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 2.96 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 (17000 6). The results indicated that the test material adsorbed to the filter matrix of the 0.2 μm filters and that a proportion of the test material was present as a dispersion at a 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 (17000 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 hours showed measured concentrations ranging from 87% to 89% of nominal for the un-centrifuged samples and from 29% to 39% of nominal for the centrifuged samples. Analysis of the old or expired test media at 48 hours showed measured concentrations ranging from 77% to 86% of nominal for the un-centrifuged samples and from 40% to 46% of nominal for the centrifuged samples.

 

The variability shown in the measured test 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 only partially dispersed in a complex biological testing system.

 

Given the variability in the results from chemical analysis and the low measured values shown for the centrifuged test media, it was considered justifiable to base the results on the time-weighted mean measured test concentration 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 48-Hour EC50 based on the time-weighted mean measured test concentration of the centrifuged test media was greater than 0.38 mg/l and correspondingly the No Observed Effect Concentration was 0.38 mg/l.

Description of key information

Acute toxicity of the test material to Daphnia magna. Limit Test performed in a GLP accredited laboratory in accordance with OECD Guideline 202 and EU Method C2.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.38 mg/L

Additional information

A study was performed to assess the acute toxicity of the test material to Daphnia magna.

 

Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at a concentration of 1.0 mg/l for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

The 48-Hour EC50 for the test material to Daphnia magna 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 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 2.96 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 (17000 6). The results indicated that the test material adsorbed to the filter matrix of the 0.2 μm filters and that a proportion of the test material was present as a dispersion at a 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 (17000 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 hours showed measured concentrations ranging from 87% to 89% of nominal for the un-centrifuged samples and from 29% to 39% of nominal for the centrifuged samples. Analysis of the old or expired test media at 48 hours showed measured concentrations ranging from 77% to 86% of nominal for the un-centrifuged samples and from 40% to 46% of nominal for the centrifuged samples.

 

The variability shown in the measured test 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 only partially dispersed in a complex biological testing system.

 

Given the variability in the results from chemical analysis and the low measured values shown for the centrifuged test media, it was considered justifiable to base the results on the time-weighted mean measured test concentration 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 48-Hour EC50 based on the time-weighted mean measured test concentration of the centrifuged test media was greater than 0.38 mg/l and correspondingly the No Observed Effect Concentration was 0.38 mg/l.