Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

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:

4 January 2005 - 21 January 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control (replicates R1 — R4 pooled) and the 2.2 mg/L test group (replicates R1— R2 and R3 - R4 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility of the test material the test concentration used in the definitive test was prepared as a saturated solution prepared from an initial test material dispersion at a concentration of 50 mg/l.
An amount of test material (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for a period of 24 hours. After 24 hours the stirring was stopped and the undissolved test material removed by centrifugation (40000 g for 30 minutes) followed by filtration through Postlip filter paper (10 µm retention size) to give the saturated solution with nominal test concentration of 2.2 mg/I*.
* Concentration based on results obtained from the media preparation trials.
Media preparation trials using a saturated solution method of preparation indicated that the test material adsorbed to 0.2 µm filters. Therefore, filtration (0.2 µm) was considered to be an inappropriate method for the removal of undissolved test material in the preparation of a saturated solution. Centrifugation at either 10000 g or 40000 g for 30 minutes showed measured concentrations of 3.59 and 2.17 mg/L respectively following a stirring period of 24 hours and 3.74 and 2.32 mg/l respectively following a 48-Hour stirring period. It was therefore considered appropriate to use a stirring period of 24 hours and to use centrifugation at 40000 g for 30 minutes to remove the undissolved test material. During the definitive test it was noted that some undissolved test material remained in the supernatant after centrifugation it was therefore considered appropriate to filter the supernatant through Postlip filter paper (10 µm retention size).
Additional filtration was not performed for the range-finding test or media preparation trials as undissolved test material was not observed in the supernatant after centrifugation, this was considered to be due to differences in stirring although every effort was made to ensure stirring period were identical
The concentration and stability of the test material in the test preparations were verified by chemical analysis 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 approximately 20°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 the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used 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:

None

Hardness:

250 mg/L as CaCO3

Test temperature:

20.7 - 20.8 °C

pH:

8.0

Dissolved oxygen:

94 to 96% (expressed as a percent of air saturation value) for both control and treatment for all replicates
8.3 - 8.5 mg O2/L

Salinity:

no information

Nominal and measured concentrations:

Nominal Concentration: 2.2 mg/L

Details on test conditions:

Test Water
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Procedure

Media preparation trials
Preliminary solubility work was conducted as no solubility data was available for the test material. This work showed that the highest attainable test concentration (by visual inspection of the test media) which could be prepared was 0.20 mg/l by spiking the test medium with an aliquot (100 µl/l) of a solvent stock solution in dimethylformamide. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were undertaken in order to determine the most appropriate method of test media preparation.

Solvent spike trial
An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution from which a serial dilution was prepared in dimethylformamide to give a further solvent stock solution of 20 mg/20 ml. An aliquot (500 µl) of the 20 mg/10 ml solvent stock solution was dispersed in 5 litres of reconstituted water to give a nominal test concentration of 0.20 mg/l. Samples were taken for chemical analysis as follows:
• Filtration through 0.2 µm Gelman Acrocap filter (approximately first 100 ml discarded to pre-condition the filter)
• Filtration through 0.2 µm Gelman Acrocap filter (approximately first 500 ml discarded to pre-condition the filter)
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes

Saturated solution trial
A saturated solution was prepared by stirring an amount of test material (550 mg) in 11 litres of reconstituted water at 1500 rpm for 24 or 48 hours at approximately 21°C. After stirring samples were taken for chemical analysis as follows:
• Filtration through 0.2 µm Gelman Acrocap filter (approximately first 100 ml discarded to pre-condition the filter)
• Filtration through 0.2 µm Gelman Acrocap filter (approximately first 500 ml discarded to pre-condition the filter)
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes

Range-finding test

The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
Based on the results for the media preparation trials it was considered that the most appropriate method of preparation of test solutions was the use of a saturated solution.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution.
An amount of test material (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for a period of 24 hours. After 24 hours the stirring was stopped and the undissolved test material removed by centrifugation (40000 g for 30 minutes) to give the 100% v/v saturated solution from which serial dilutions were prepared in reconstituted water to give the 10 and 1.0% v/v saturated solution test concentrations.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20°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 group was maintained under identical conditions but not exposed to the test material.

Definitive test

Based on the results of the range-finding test a "Limit test" was conducted. The test material solution for the definitive test was prepared by stirring an excess (50 mg/l) of test material in reconstituted water at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by centrifugation (4000 g for 30 minutes) to give a 100% v/v saturated solution of the test material with nominal test concentration of 2.2 mg/l*.
* Concentration based on results obtained from the media preparation trials.

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

Exposure conditions
In the definitive test 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the study 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20°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 group was maintained under identical conditions but not exposed to the test material.
The test preparations 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-cheniical measurements

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter and the temperature was measured using a Hama Instruments HI 93510 digital thermometer.

Verification of test concentrations

Water samples were taken from the control (replicates R1 - R4 pooled) and the 2.2 mg/l test group (replicates R1- R2 and R3 - R4 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Evaluation of data

An estimate of the EC50 values was given by inspection of the immobilisation data.

Positive Control
A positive control using potassium dichromate as the reference material was conducted using test concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l (Safepharm Laboratories Project No: 39/731).
An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (100 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 1 litre to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.

Evaluation of data

An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 values and associated confidence limits at 24 and 48 hours was calculated using the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.071 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.071 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Media Preparation Trials
Preliminary solubility work was conducted as no solubility data was available for the test material. This work showed that the highest attainable test concentration (by visual inspection of the test media) which could be prepared was 0.20 mg/L by spiking the test medium with an aliquot (100 µl/L) of a solvent stock solution prepared in dimethylfolinamide. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were undertaken in order to determine the most appropriate method of test media preparation.

Solvent spike trial
The solvent spike media preparation trial showed measured concentrations of 0.0526 mg/L and less than the limit of quantitation (
Saturated solution trial
The saturated solution media preparation trial showed measured concentrations of less than the limit of quantitation of the analytical method following filtration using a pre-conditioning volume of either 100 mL or 500 mL. Centrifugation at either 10000 g or 40000 g showed measured test concentrations of 3.59 and 2.17 mg/L respectively following a stirring period of 24 hours and 3.74 and 2.32 mg/L respectively following a 48-Hour stirring period.
The results of the media preparation trials showed that higher measured test concentrations were obtained using a saturated solution method of media preparation when compared to the use of a solvent spike method, indicating that the saturated solution method maximised the amount of dissolved and hence bioavailable test material present. Increasing the stirring period of the saturated solution did not result in a significantly increased level of test material in solution. Therefore, it was considered appropriate to prepare the test media for the definitive test as a saturated solution using a 24-Hour stirring period followed by centrifugation at 40000 g for 30 minutes to remove the undissolved test material.

Range-finding Test

No immobilisation was observed at the test concentrations of 1.0, 10 and 100% v/v saturated solution.
Based on this information, the test material solution for the definitive test was prepared by stirring an excess (50 mg/l) of test material in reconstituted water for a period of time and then removing any undissolved test material by filtration to give the 100% v/v saturated solution with nominal test concentration of 2.2 mg/L*. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.
* Concentration based on results obtained from the media preparation trials.

Definitive Test

Immobilisation data
There was no immobilisation in 20 daphnids exposed to a nominal test concentration of 2.2 mg/l for a period of 48 hours.
The No Observed Effect Concentration after 24 and 48 hours exposure was 2.2 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

Observations on test material solubility
The test material preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements
Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Verification of test concentrations
Preliminary solubility work conducted, due to the lack of solubility data for the test material, showed that the highest attainable test concentration (by visual inspection of the test media) which could be prepared was 0.20 mg/l by spiking the test medium with an aliquot (100 µl/l) of a solvent stock solution prepared in dimethylformamide. Based on this information the test material fell into the category of a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were undertaken in order to determine the most appropriate method of test media preparation.
Based on the results of the media preparation trials conducted it was considered that the solvent spike method of media preparation would not be suitable for ecotoxicology studies due to the low results which were obtained when using this method. However, it was considered that the preparation of a saturated solution was the most suitable method of preparation. Further, filtration was considered to be an inappropriate method for the removal of undissolved test material in preparation of a saturated solution due to possible adsorbtion to the filters. Therefore, centrifugation at 40000 g for 30 minutes was used to remove the undissolved test material and therefore maximise the dissolved test material concentration.
Analysis of the test preparations at 0 hours showed measured test concentrations of 0.060 and 0.053 mg/l. Analysis of the test preparations at 48 hours showed measured test concentrations of 0.098 and 0.073 mg/l. The measured concentrations were lower than those obtained from the media preparation trials. This was considered to be due to the additional use of filtration through Postlip BW/S filter paper (10 µm retention size) after centrifugation for the definitive test which was considered necessary to remove undissolved test material not removed by centrifugation. Additional filtration was not performed for the range-finding test or media preparation trials as undissolved test material was not observed in the supernatant after centrifugation, this was considered to be due to differences in stirring although every effort was made to ensure stirring period were identical.
The measured test concentration gave an estimate of the dissolved concentration of the test material and hence bioavailable to the test organisms.
Given that no significant decline in measured test concentrations was observed it was considered justifiable to base the results on the mean measured test concentrations in order to give a "worst case" analysis of the data. The 48-Hour EC50 based on the mean measured test concentrations was greater than 0.071 mg/l and correspondingly the No Observed Effect Concentration was 0.071 mg/l.
The use of mean measured test concentrations was considered to have significantly affected the results of the test.
This test showed that there were no toxic effects of saturation.

Results with reference substance (positive control):

Positive Control
Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al (1977)) at 24 and 48 hours based on the nominal test concentrations gave the following results:

3 h EC50: >3.2 mg/L*
24h EC50: 1.5 mg/L (95% CL: 1.4 - 1.7)
48h EC50: 1.2 mg/L (95% CL: 1.0 - 1.3)

The No Observed Effect Concentrations after 24 and 48 hours were 1.0 mg/L and 0.56 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
* A 3-Hour EC50 value was not calculated as less than 50% immobilisation occurred at this time point.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/L (sd = 0.24).

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours was calculated using the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Table 1. Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/L)	cumulative immobilised Daphnia (initial population: 5 per replicate)
	24h			48h
	No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control R1	0	0	0	0	0	0
R2	0			0
R3	0			0
R4	0			0
2.2 R1	0	0	0	0	0	0
R2	0			0
R3	0			0
R4	0			0

Table 2. Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/L)	cumulative immobilised Daphnia (initial population: 10 per replicate)
	3 hours				24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	0	0	2	3	5	25
1.8	0	0	0	0	8	8	16	80	10	10	20	100
3.2	1	2	3	15	10	10	20	100	10	10	20	100

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 2.2 mg/L. Correspondingly the No Observed Effect Concentration was 2.2 mg/L.
Based on the mean measured test concentrations the 48-Hour EC50 was estimated to be greater than 0.071 mg/L. Correspondingly the No Observed Effect Concentration was 0.071 mg/L.
This study showed that there were no toxic effects at saturation.

Executive summary:

A study was performed to assess the acute toxicity of Nigrosine Base to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test". A preliminary solubility trial was conducted due to lack of data available. The results of a solvent spike media preparation trial indicated that using a solvent spike method of preparation was not suitable for this test material due to the variability of the results obtained. A saturated solution media preparation trial showed that higher measured test concentrations were obtained using a saturated solution method of media preparation when compared to the use of a solvent spike method, indicating that the saturated solution method maximised the amount of dissolved and hence bioavailable test material present. The definitive test solution was prepared by stirring an excess (50 mg/L) of test material in reconstituted water for a period of time and then removing any undissolved test material by filtration to give the 100% v/v saturated solution with nominal test concentration of 2.2 mg/L. This experimental design confirms to the Limit test design. During the definititve test there was no immobilisation in 20 daphnids exposed to a nominal test concentration of 2.2 mg/L for a period of 48 hours at approximately 20 °C. The No Observed Effect Concentration after 24 and 48 hours exposure was 2.2 mg/L. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The concentration verification test showed that there were no toxic effects of saturation. The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/L (sd = 0.24).

The acute toxicity of the Nigrosine Base to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 2.2 mg/L. Correspondingly the No Observed Effect Concentration was 2.2 mg/L. Based on the mean measured test concentrations, the 48-Hour EC50 was estimated to be greater than 0.071 mg/L. Correspondingly the No Observed Effect Concentration was 0.071 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

The acute toxicity of Nigrosine BASE to Daphnia magna was measured using a limit test and nominal test concentrations of 2.2 mg/L resulting in 48-Hour LC50 of greater than 2.2 mg/L.  Based on the mean measured test concentrations the 48-Hour EC50 was estimated to be greater than 0.071 mg/L. Correspondingly the No Observed Effect Concentration was 0.071 mg/L. This study showed that there were no toxic effects at saturation.

Additional information

One key study was chosen for this endpoint, of Klimisch 1 reliability and performed to the OECD Guidelines for Testing of Chemicals (1992) No. 202 "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC and gave a 48-Hour EC50 of greater than 2.2 mg/L based on nominal concentrations. Correspondingly the No Observed Effect Concentration was 2.2 mg/L. Based on the mean measured test concentrations the 48-Hour EC50 was estimated to be greater than 0.071 mg/L. Correspondingly the No Observed Effect Concentration was 0.071 mg/L. This study showed that there were no toxic effects at saturation.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).