Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2005 - 4 February 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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 2.2 mg/l test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored (approximately -20°C) for further analysis. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and high purity of the test material the test concentration used in the definitive test was a saturated solution prepared from an initial test material dispersion at a concentration of 50 mg/I.
An amount of test material (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of 21°C for a period of 24 hours. After 24 hours the stirring was stopped and any large particles of undissolved test material removed by filtration through Postlip BW/S filter paper (10 µm retention size) prior to centrifugation (40000 g for 30 minutes) to give the saturated solution with nominal test concentration of 2.2 mg/l*. An aliquot (2 litres) of the 2.2 mg/1 saturated solution was inoculated with 10 ml of algal suspension to give the required test concentration of 2.2 mg/I*.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.
Media preparation trials conducted for the Acute Toxicity to Daphnia magna test 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/I 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 range-finding 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) prior to centrifugation in the definitive test.
Additional filtration was not performed for the media preparation trial as it was not considered necessary at the time.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

24 ± 1°C

pH:

7.3 - 8.6 (test vessels)
7.5 - 8.4 (control vessels)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal : 2.2 mg/L (Concentration determined from the media preparation trials conducted)
Measured: Geometric Mean Measured Test Concentrations: 0.027 mg/l (R1 - R3) and 0.028 mg/l (R4 - R6)

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Procedure

Media preparation trials
Preliminary solubility work was conducted for the Acute Toxicity to Daphnia magna study 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) that 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.

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 results of the media preparation trial conducted indicated that a dissolved test material concentration of 2.2 mg/l was obtained following centrifugation (40000 g for 30 minutes) of an initial test material dispersion of 50 mg/l.
Scenedesmus subspicatus cells were exposed to a series of nominal test concentrations of 0.022, 0.22 and 2.2 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
An amount of test material (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of 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 a saturated solution of the test material with a nominal test concentration of 2.2 mg/l*. Given that a significant amount of undissolved test material was visible in the supernatant it was considered appropriate to centrifuge the saturated solution again to remove as much undissolved test material as possible. A series of dilutions was made from the 2.2 mg/l saturated solution to give further stock solutions of 0.22 and 0.022 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (2.5 ml) to give the required test concentrations of 0.022, 0.22 and 2.2 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
* Concentration determined from the media preparation trials conducted.

Definitive test
Based on the result 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 the test material in culture medium for a period of time and then removing any undissolved test material by centrifugation (40000 g for 30 minutes) to give a saturated solution.

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
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution were prepared for the treatment group and three flasks for the control group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.40 x 10^6 cells per ml. Inoculation of 2 litres of test medium with 10 ml of this algal suspension gave an initial cell density of 10^4 cells per ml and had no significant dilution effect.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Physico-chemical measurements
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Verification of test concentrations
Water samples were taken from the control and the 2.2 mg/l test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored (approximately -20°C) for further analysis. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Media Preparation Trials
Preliminary solubility work was conducted for the Acute Toxicity to Daphnia magna study 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) that 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.

Solvent spike trial
The solvent spike media preparation trial showed measured concentrations of 0.0526 mg/l and less than the limit of quantitation (LOQ) following centrifugation at 10000 g and 40000 g respectively and 0.0237 mg/I and less than the LOQ following filtration using a pre-conditioning volume of 100 ml and 500 ml respectively. These results indicated that using a solvent spike method of preparation was not suitable for this test material due to the variability of the results obtained.

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
The results showed no effect on growth at the nominal test concentrations of 0.022, 0.22 and 2.2 mg/l.
Based on this information a "Limit test" was conducted for the definitive test. The test material solution for the definitive test was prepared by stirring an excess (50 mg/l) of test material in culture medium for a period of time and then removing any undissolved test material by filtration through Postlip BW/S filter paper (10 µm retention size) followed by centrifugation to give a saturated solution.

Definitive Study

Growth data
From the data, it is clear that neither the growth (r) or the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.
The EC50 value with respect to the area under the growth curve (biomass) EbC50 (72 h) was determined by inspection of the area under the growth curve data after 72hours.
The EC50 value with respect to growth rate, ErC50 (0 - 72 h), was determined by inspection of the growth rates for the period 0 - 72 hours.
Accordingly the following results were determined from the data:
EbC50 (72 h): > 2.2 mg/l
ErC50 (0 - 72 h): > 2.2 mg/l

where EbC50 is the test concentration that reduced biomass by 50% and ErC50 is the test concentration that reduced specific growth rate by 50%.

Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using a Students t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences between the control and 2.2 mg/l test concentration (P≥0.05) and, therefore the "No Observed Effect Concentration" (NOEC) was 2.2 mg/l.

The following data show that the cell concentration of the control cultures increased by a factor of 57 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:
Mean cell density of control at 0 hours: 9.77 x 10^3 cells per ml
Mean cell density of control at 72 hours: 5.52 x 10^5 cells per ml

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Physico-chemical measurements
Temperature was maintained at 24± 1°C throughout the test.
The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on test material solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

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) that 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 adsorption to the filter matrix. Therefore, centrifugation at 40000 g for 30 minutes was used to remove the undissolved test material and therefore maximise the dissolved test material concentration.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.0501 and 0.0515 mg/l (replicates R1-R3 and R4-R6 pooled respectively). These measured concentrations were lower than the predicted value (2.2 mg/l) obtained from the media preparations trials conducted. This was considered to be due to the additional use of filtration through Postlip BW.S filter paper (10 µm retention size) to remove as much undissolved test material as possible prior to centrifugation. Additional filtration was not performed for the media preparation trial as it was not considered necessary at the time.
Analysis of the test preparations at 72 hours showed a marked decline in measured test concentrations to below the limit of quantitation (LOQ) of the analytical method employed. These results were inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test period.
Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. Following current regulatory advice, in cases where the measured test concentration was less than the LOQ of the analytical method a concentration of half the LOQ was used for calculation purposes.
The geometric mean measured test concentrations were determined to be:

Nominal Concentration (mg/l) Geometric Mean Measured Test Concentration (mg/l)
2.2 (R1-R3) 0.027
2.2 (R4-R6) 0.028

The following results were determined from the data based on the geometric mean measured test concentrations:
EbC50 (72 h):> 0.028 mg/l
ErC50 (0 - 72 h): > 0.028 mg/l
No Observed Effect Concentration (NOEC) 0.028 mg/l

The use of mean measured test concentrations in the calculation of the results of the test had a significant effect on the conclusions of the test.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using a Students t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences between the control
and 2.2 mg/I test concentration (P≥0.05) and, therefore the "No Observed Effect Concentration" (NOEC) was 2.2 mg/l.

The geometric mean measured test concentrations of the samples using the geometric mean of the measured test concentrations of replicates RI - R3 and R4 - R6 pooled were calculated as follows:
geometric mean measured test concentration (mg/I) =square root of the (measured concentration at the start of the test (mg/I) x measured concentration at the end of the test (mg/I))

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 values of greater than 2.2 mg/l. Correspondingly the No Observed Effect Concentration was 2.2 mg/l.
The effect of the test material on the growth of Scenedesmus subspicatus based on the geometric mean measured test concentration gave EC50 values of greater than 0.028 mg/l. Correspondingly the No Observed Effect Concentration was 0.028 mg/l.
This study showed that there were no toxic effects at saturation.

Executive summary:

Nigrosine BASE was tested in an Algal Inhibition Limit Test. The objective was to determine the affect of Nigrosine BASE on the growth and biomass of Scenedesmus subspicatus (CCAP 276/20) over a 72-Hour exposure period at 24 ± 1 °C. The pH values of the control cultures increased from pH 7.5 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. The algae were exposed over a 72-hour period with a concentration of 2.2 mg/L nigrosine. Samples were taken on a daily basis at 0, 24, 48 and 72 hours. The mean cell densities versus time were estimated for the definitive test. The EC50 value with respect to the area under the growth curve (biomass) EC50 (72 h) was determined by inspection of the area under the growth curve data after 72hours. The EC50 value with respect to growth rate, EC50 (0 - 72 h), was also determined by inspection of the growth rates for the period 0 - 72 hours. The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 values of greater than 2.2 mg/l. Correspondingly the NOEC was 2.2 mg/l.

The effect of the test material on the growth of Scenedesmus subspicatus based on the geometric mean measured test concentration gave EC50 values of greater than 0.028 mg/l. Correspondingly the NOEC was 0.028 mg/l.

Description of key information

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 values of greater than 2.2 mg/L. Correspondingly the No Observed Effect Concentration was 2.2 mg/L.

The effect of the test material on the growth of Scenedesmus subspicatus based on the geometric mean measured test concentration gave EC50 values of greater than 0.028 mg/L. Correspondingly the No Observed Effect Concentration was 0.028 mg/L.

This study showed that there were no toxic effects at saturation.

Additional information

Nigrosine BASE was tested in an Algal Inhibition Limit Test.  The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The objective was to determine the effect of Nigrosine BASE on the growth and biomass of Scenedesmus subspicatus (CCAP 276/20) over a 72-Hour exposure period at 24 ± 1 °C. The pH values of the control cultures increased from pH 7.5 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. The algae were exposed over a 72-hour period with a concentration of 2.2 mg/L nigrosine. Samples were taken on a daily basis at 0, 24, 48 and 72 hours. The mean cell densities versus time were estimated for the definitive test. The EC50 value with respect to the area under the growth curve (biomass) EC50 (72 h) was determined by inspection of the area under the growth curve data after 72 hours. The EC50 value with respect to growth rate, EC50 (0 - 72 h), was also determined by inspection of the growth rates for the period 0 - 72 hours. The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 values of greater than 2.2 mg/L. Correspondingly the NOEC was 2.2 mg/L.

The effect of the test material on the growth of Scenedesmus subspicatus based on the geometric mean measured test concentration gave EC50 values of greater than 0.028 mg/L. Correspondingly the NOEC was 0.028 mg/L.