Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2009 - 1 June 2009

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 211 (Daphnia magna Reproduction Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials a modification of the standard method for the preparation of aqueous materal was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test material with dechlorinated tap water for 24 hours and then removing the undissolved test material by centrifugation at 40000 g for 30 minutes to give a saturated solution with a nominal concentration of approximately 1.9 mg/L (concentration based on chemical analysis of a saturated solution prepared during a solubility trial).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 3, 7, 10, 15 and 17 and of the expired test preparations on Days 1, 4, 8, 11, 16, and 18, Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Test Water
The test water used for the definitive test was the same as that used to maintain the stock animals.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener) giving water with a total hardness of approximately by 140 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.

Solubility Trial
The method of preparation for the test material was based on the results of an acute toxicity test to 1st instar Daphnia magna. However, this was conducted using reconstituted water as the diluent, therefore it was considered appropriate to conduct a solubility trial to determine the concentration of the dissolved test material in a saturated solution using dechlorinated tap water as the diluent. The saturated solution was prepared by stirring an excess (50 mg/L) of test material.
An amount of test material (1125 mg) was dispersed, in 22.5 litres of dechlorinated tap 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 the stirring periods samples were taken for chemical analysis after the following pre-treatment:
• Centrifugation at 40000 g for 30 minutes

Definitive Test
Based on the results of an acute toxicity test to 1st instar Daphnia magna the following test concentrations were assigned to the definitive test: 0.00071, 0.0023, 0.0071, 0.023 and 0,071 mg/L. However, chemical analysis conducted during the test showed significantly lower measured concentrations. Therefore the results will be expressed in terms of time-weighted mean measured concentrations of 0.00023, 0.00058, 0.0022, 0.0059 and 0.021 mg/l.

Experimental preparation
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 initial test material dispersions at a concentration of 50 mg/L.
An amount of test material (550 mg) was dispersed in 11 litres of dechlorinated tap water and stirred using a propeller stirrer at approximately 1600 rpm at approximately 21°C for a period of 24 hours. After 24 hours the stirring was stopped and the undissolved test material removed by centrifugation at 40000 g for 30 minutes to give the saturated solution. An aliquot (200 mL) of the saturated solution was added to dechlorinated tap water and the volume adjusted to 2 litres to give a stack solution with a nominal concentration of 0.19 mg/L. Aliquots (7.5, 24, 75, 242 and 747 mL) of this stock solution were each added separately to a final volume of 2 litres of dechlorinated tap water to give the time-weighted mean measured test concentrations of 0.00023, 0.0058, 0.0022, 0.0059 and 0.021 mg/L respectively.
On days when sampling of the fresh test media was not required aliquots (3,8, 12, 38, 121 and 374 mL) of the stock solution were each added separately to a final volume of 1 litre of dechlorinated tap water to give the time-weighted mean measured test concentrations of 0.00023, 0.0058, 0.0022, 0.0069 and 0.021 mg/L respectively.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
On Day 4 of the test it was noted that some undissolved test material remained in the supernatant after centrifugation, therefore for the remainder of the test it was considered appropriate to filter the supernatant through Postlip filter paper (10 µm retention size) in order to obtain a testable solution.
Additional filtration was not performed for the solubility trial 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 samples were verified by chemical analysis on Days 0, 3, 7, 10, 15 and 17 (fresh media) and on Days 1, 4, 8, 11, 16 and 18 (old media).
The concentration of the saturated solution was taken on Days 0, 3, 7, 10, 15 and 17 for chemical analysis.

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 dechlorinated tap water at a temperature of 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 at a light intensity preferably not exceeding 1000 lux. Each culture was fed daily with a suspension of algae (Chloralla 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None.

Hardness:

108 to 132 mg/l as CaCO3 in the control and highest surviving test group throught the test.

Test temperature:

20 to 22 °C

pH:

7.5 to 8.1

Dissolved oxygen:

88 to 113 % ASV (Dissolved oxygen concentration expressed as a percentage of Air Saturation Value)
8.7 to 9.8 mg/l O2

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentration (mg/l) Time weighted mean measured concentration (mg/L) % of nominal
0.00071 0.00023 32
0.0023 0.00058 25
0.0071 0.0022 31
0.023 0.0059 26
0.071 0.021 30

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass flask
- Type (delete if not applicable): open / closed covered with a plastic lid
- Aeration: covered with plastic lid to reduce evaporation
- Renewal rate of test solution (frequency/flow rate): renewed daily. Adult daphnia transferred to fresh media by wide-bore pipeette
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): for each test and control group, ten replicates
- No. of vessels per control (replicates): ten
- No. of vessels per vehicle control (replicates): ten

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water
- Total organic carbon: 1.041 (mean of 8 samples)
- Particulate matter: not measured
- Metals: copper (<0.008 mg/L); iron (<13.481 µg/L); lead (<1.138 µg/L); mercury (<0.012 µ/L); nickel (<1.788 µg/L);
- Pesticides: (total) 0.000 µg/L
- Chlorine: free chlorine (0.231 mg/L); Total chlorine (0.297 mg/L)
- Alkalinity: not measured
- Ca/mg ratio: not measured
- Conductivity: not measured
- Culture medium different from test medium: no
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: 16 hours light, 8 hours of dark with 20 minutes of dawn and dusk transition periouds at a light intensity not exceeding 1000 lux.
- Light intensity: 493 to 549 lux

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Acute toxicity test to 1st instar Daphnia magna test concentrations were assigned to the definitive test. Chemical analysis conducted during the test showed significantly lower measured concentrations, so the results were expressed in time weighted means.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.021 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Parental Daphnia generation (P1)

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.021 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: 1 mortality in the treatment group, zero mortalities in the control group
- No. of offspring produced per day per female: average by day 21 in control group = 82; range for treatment groups = 70 to 95
- Body length and weight of parent animals: 3.9 to 4.8 mm

Solubility Trial
The results of the solubility trial indicated that a test concentration of 2.0 mg/L could be attained.

Definitive Test
Based on results of an Acute Toxicity to Daphnia magna study, nominal test concentrations of 0.00071, 0,0023, 0.0071, 0,023 and 0.0071 mg/L were selected for the definitive test. However, chemical analysis conducted during the test showed significantly lower measured concentrations. Therefore the results will be expressed in terms of time-weighted mean measured concentrations of 0.00023, 0,00058, 0.0022, 0.0059 and 0.021 mg/L.

Physico-chemical Measurements
Temperature was maintained at 20°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The temperatures in some of the test vessels in the majority of freshly prepared media was measured to be above the 20 ± 1°C range given in the protocol during the definitive test. This deviation was considered not to affect the outcome or validity of the test given that no adverse effects were observed in the daphnids throughout the test at any concentration employed.
Throughout the test the light intensity was observed to be in the range 493 to 549 lux.
The water hardness was observed to be in the range 108 to 132 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.

Observations on Test Material Solubility
The freshly prepared test media were observed to be clear, colourless solutions whereas the old test media were observed to be pale green dispersions due to the presence of algal cells used as feed for the daphnids.

Validation Criteria
The following validation criteria were achieved during the test:
Required Actual
a Control mortality ≤20% 0%
b Dissolved oxygen >3 mg O2/L >7.9 mg O2/L
c pH (control group) deviation ≤1.5 0.9
d Mean number of live young per surviving adult (control group) ≥60 after 21 days 82
e Coefficient of variation for control group ≤25% 22%

Lethal Effects on the Parental Generation (P1)
Mortality was observed at the time-weighted mean measured test concentrations of 0.0022, 0.0058 and 0.021 mg/L. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 0.0022, 0.0069 and 0.021 mg/L test groups were not significantly different (P≥0.05) when compared to the control group.
EC50 (immobilisation) values based on time weighted mean measured test concentrations were estimated throughout the test to be >0.021 mg/L.

Sub-lethal Effects on the Parental Generation (P1)
After 21 days there were no statistically significant differences between the control and all the test groups in terms of the number of live young produced per adult.
The EC50 (reproduction) value based on the time-weighted mean measured test concentration was estimated to be greater than 0.021 mg/L.
Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P≥0.05) from the control and each test group.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥0.05) between the control and each test group in terms of length of the daphnicis after 21 days exposure to the test material.

Effects on the Filial Generation (F1)
Information on the effects of the test material on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 8 of the test.
Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation,

Lowest Observed Effect Concentration
The Lowest Observed Effect Concentration (LOEC) based on the time-weighted mean measured test concentrations of the test media was considered to be greater than 0.021 mg/l on the basis that of this loading rate no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the control and the 0.021 mg/l test group in terms of numbers of live young produced per adult by Day 21.

No Observed Effect Concentration
The "No Observed Effect Concentration" (NOEC) based on the time-weighted mean measured test concentrations of the test media was 0.021 mg/l as there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Verification of Test Concentrations
Analysis of the saturated solution showed measured concentrations ranging from 0.0628 to 0.450 mg/l with the exception of Day 3 which showed a measured concentration of 5.3 mg/l. This high measured value was considered to be due to differences in stirring and/or possible undissolved test material present in the test medium although every effort was made to ensure that all the test material was removed.
Analysis of the freshly prepared media showed measured values to range from less than the limit of quantitation (LOQ) of the analytical method to 0.0314 mg/l. Analysis of the old or expired test media showed measured values to range from less than the limit of quantitation (LOQ) of the analytical method to 0.0180 mg/l.
Measured concentrations from Day 3 fresh media and subsequent old or expired media on Day 4 showed measured values to range from 0.000343 to 0.0901 mg/l. This was due to the high measured concentration of the saturated solution.
The measured concentrations were significantly lower than those obtained from the solubility trial with the exception of Day 3 media. This was considered to be due to the additional use of filtration through Postlip BW/S filter paper (10 µm retention size) after centrifugation which was necessary to remove undissolved test material not removed by centrifugation. Additional filtration was not performed prior to this as undissolved test material was not observed in the supernatant after centrifugation.
A similar effect was observed during the Acute Toxicity to Daphnia magna test which showed measured results to be greater than an order of magnitude less in the definitive test than during the pre-study media preparation trial.
It was therefore considered justifiable to base the results on the time-weighted mean measured test concentrations of the test media to give a "worst case" analysis of the data. Where analytical results were given as less than the limit of quantitation of the analytical method (LOQ) a value of half the LOQ was used for calculation of the time-weighted mean measured concentrations.

Nominal Test Concentration (mg/l) Time Weighted Mean Measured Concentration (mg/l) % of nominal
0.00071 0.00023 32
0.0023 0.00058 25
0.0071 0.0022 31
0.023 0.0059 26
0.071 0.021 30

The 21-Day EC50 (immobilisation) value based on the time-weighted mean measured test concentrations of the test media was estimated to be greater than 0.021 mg/l.
The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations of the test media was estimated to be greater than 0.021 mg/l.
The "Lowest Observed Effect Concentration" (LOEC) and "No Observed Effect Concentration" (NOEC) based on the time-weighted mean measured test concentrations was 0.021 mg/l.
The use of time-weighted mean measured test concentration to calculate the results did not significantly affect the results of the test.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Analysis of numbers of live young produced per adult
Mean and standard deviation values derived from the number of live young produced per surviving adult in the control and all the test groups were used for the analysis. No significant differences were found between the control and each test group in terms of the number of live young produced per adult by Day 21.

A corrected chi-squared statistic (Breslow and Day 1980) was performed to show whether mortalities in the 0.0022, 0.0059 and 0.021 mg/L test groups observed during the test, were statistically significant when compared to the control group. The chi-squared table value for one degree of freedom is 3.85 at the 0.05 probability level. There was no significant difference (p<0.05) between the observed mortalities in the control and the 0.0022 mg/l test group.

There was no significant difference (p<0.05) between the observed mortalities in the control and the 0.0059 and 0.021 mg/L test groups.

Results from the control and each test groups Daphnia length data, determined for the surviving daphnids on termination of the test were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparision procedure for comparing several treatments with a control. Mean and standard deviation values derived from the lenght of the surviving daphnids of the parental generation in the control and all of the test groups were used for the analysis. No statistically significant differences (p>0.05) were found between the control and all test groups in terms of length of the surviving parental daphnids on Day 21 of the test.

Table 1. Analysis of Numbers of Live Young Produced per Adult

Time-weighted Mean Measured Concentration (mg/L)		Number of Live Young Produced per Adult by Day 21
Control	Mean	82
	Standard Deviation	18
0.00023	Mean	70
	Standard Deviation	24
0.00058	Mean	77
	Standard Deviation	13
0.0022	Mean	81
	Standard Deviation	15
0.0059	Mean	90
	Standard Deviation	14
0.021	Mean	95
	Standard Deviation	3.1

Analysis of Mortality in the Parental Generation

Table 2a. Results of the 0.0022 mg/L test group

	Treated	Control	Total
Mortalities	1	0	1
No Mortalities	9	10	19
Total	10	10	20

Table 2b. Results for the 0.0059 and 0.021 mg/l test groups:

	Treated	Control	Total
Mortalities	2	0	2
No Mortalities	8	10	18
Total	10	10	20

Table 3. Analysis of the Daphnia Length Data

Time-weighted Mean Measured Concentration (mg/l)		Daphnia Lengths at Day 21 (mm)
Control	Mean	4.5
	Standard Deviation	0.2
0.00023	Mean	4.3
	Standard Deviation	0.3
0.00058	Mean	4.5
	Standard Deviation	0.2
0.0022	Mean	4.3
	Standard Deviation	0.2
0.0059	Mean	4.5
	Standard Deviation	0.3
0.021	Mean	4.5
	Standard Deviation	0.2

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test material resulted in no significant mortalities at all of the test concentrations employed during the test.
The 21-Day EC50 (immobilisation) values for the parental Daphnia generation (P1) were estimated to be greater than 0.021 mg/L based on the time-weighted mean measured test concentrations of the test media.
The 21-Day EC50 (reproduction) was greater than 0.021 mg/L based on the time-weighted mean measured test concentrations test media.
No significant impairment of reproduction was observed at the test concentrations employed in the test.

Executive summary:

A 21-day toxicity test was conducted on Nigrosine, with the Daphnid Daphnia magna Straus. This test determines the adverse effects of the test substance on survival, reproduction, and growth of the daphnid. The life-cycle test was conducted at 21 °C with time-weighted mean measured test concentrations of 0.00023, 0.00058, 0.0022, 0.0059 and 0.021 mg/L. Each exposure concentration consisted of ten replicates: to evaluate the effects on reproduction, growth and mortality. A toxic response was not achieved through the highest exposure concentration of 0.021 mg/L; therefore, the LOEC and NOEC values were 0.021 mg/L. In addition the 21 -Day EC50 values (immobilisation and reproduction) were estimated to be greater than 0.021 mg/L.

Description of key information

Exposure of Daphnia magna to the test material resulted in no significant mortalities at all of the test concentrations employed during the test.

The 21-Day EC50 (immobilisation) values for the parental Daphnia generation (P1) were estimated to be greater than 0.021 mg/L based on the time-weighted mean measured test concentrations of the test media.

The 21-Day EC50 (reproduction) was greater than 0.021 mg/L based on the time-weighted mean measured test concentrations test media.

No significant impairment of reproduction was observed at the test concentrations employed in the test.

Additional information

A 21-day toxicity test was conducted on Nigrosine, with the Daphnid Daphnia magna Straus. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). This test determines the adverse effects of the test substance on survival, reproduction, and growth of the daphnid. The life-cycle test was conducted at 21 °C with time-weighted mean measured test concentrations of 0.00023, 0.00058, 0.0022, 0.0059 and 0.021 mg/L. Each exposure concentration consisted of ten replicates: to evaluate the effects on reproduction, growth and mortality. A toxic response was not achieved through the highest exposure concentration of 0.021 mg/L; therefore, the LOEC and NOEC values were 0.021 mg/L. In addition the 21 -Day EC50 values (immobilisation and reproduction) were estimated to be greater than 0.021 mg/L.