Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

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:

18 November 2008 - 18 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with agreed protocols and standard test guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Light intensity was not measured in the limit test. Normal growth was observed during the test period, hence, intensity was close to/within the required range. The study integrity was not adversely affected by the deviation.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 23, Guidance document on aquatic toxicity testing of difficult substances and mixtures (December 2000)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling and Recording of Cell Densities

At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe.

Concentrations. Final test concentrations of the test item were prepared as 0.45 filtered dispersions of loading rates of 1.0, 3.2, 10, 32, and 100 mg/L

Sampling for Analysis of Test Concentrations

During the final test samples for analysis were taken from all test concentrations and the control at time = 0 hours, time = 24 hours, and time = 72 hours. The volume sampled was 2 ml taken from the approximate center of the test vessels. Samples were analysed on the day of sampling. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling

- Sample storage conditions before analysis: Samples were analyzed on the day of the sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:

The test substance being a reaction mixture consisted of black lumps and was not soluble in test medium at the concentrations tested. Preparation of the test solutions started with individually prepared loading rates. These were treated with ultrasonic waves for 10 minutes followed by magnetic stirring for 2 days to ensure reaching maximum dissolution in the test medium. The resulting dispersions were subsequently filtered through a 0.45 micron membrane filter to remove the larger undissolved test material. This resulted in a clear and colorless test solution at a loading rate of 1.0 mg/L and slightly yellow solutions (increasingly colored) at loading rates between 3.2 and 100 mg/L. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

- Eluate: NA.
- Differential loading: NA.
- Controls: Negative controls and positive control/reference toxicant
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): NA
- Concentration of vehicle in test medium (stock solution and final test solution): NA
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Solutions prepared from loading rates between 3.2 and 100 mg/L appeared as slightly yellow colored solutions.

Test organisms (species):

other: Pseudokirchneriella subcapitata, strain NIVA CHL 1

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: Pseudokirchneriella subcapitata, strain NIVA CHL
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation): NDA

- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24C. The light intensity was 60 to 120 uE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Stock culture medium was M1(according to the NPR 6505, formulated using MilliRO water).

ACCLIMATION
- Acclimation period: NDA
- Culturing media and conditions (same as test or not): M1 medium was used for stock culture medium; M2 medium was used for preculture medium

-Any deformed or abnormal cells observed: At the end of the final test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae. Microscopic observations at the end of the test revealed a normal appearance of the exposed cells when compared to the control.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

the project started with a limit test. Six replicates of exponentially growing algae were exposed to a control and a 0.45 micron filtered solution prepared at a loading rate of 100 mg/L.

Hardness:

no data available

Test temperature:

The temperature of the test medium was 22.3C at the start of the test. During the exposure period the temperature in the incubator was maintained between 22 and 23.7C. Temperature remained within the limits prescribed by the protocol

pH:

7.7 to 8.1

Dissolved oxygen:

no data available

Salinity:

na/freshwater

Nominal and measured concentrations:

Nominal loading rates were 0, 1.0, 3.2, 10, 32, and 100 mg/l

Initial measured test concentrations were used because the levels analyzed at 24 hours were all below the limit of detection of the analytical method. Corresponding initial measured concentrations were n.d., 0.008, 0.03, 0.07, 0.108, and 0.112 mg/L. Maximum solubility appeared to be in the order of 0.1 mg/L.

Details on test conditions:

TEST SYSTEM
Test vessel: 100 ml, all glass, containing 50 mL of the test solution
- Material, size, headspace, fill volume: 100 ml, all glass, containing 50 mL of test solution
- Aeration: During incubation the algal cells were kept in suspension by continuous shaking
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): NA
- Initial cells density: 104cells per ml.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, M1 medium for stock culture and M2 for pre-culture

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: M2 Formulated with Milli-Q tap water subsequently passed over activated carbon and ion exchange cartridges.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination using TLD lames of the type “cool white” of 30 Watt, with a light intensity within the range of 85 to 96 uE.m-2s-1.
- Light intensity and quality: continuous illumination using TLD lames of the type “cool white” of 30 Watt, with a light intensity within the range of 85 to 96 uE.m-2s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometic measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength = 20 mm).

TEST CONCENTRATIONS
-Results used to determine the conditions for the definitive study: The project started with a limit test. Six replicates of exponentially growing algae were exposed to a control and a 0.45 micron filtered solution prepared at a loading rate of 100 mg/L. The mean cell densities and the percentage reduction of growth rate and inhibition of yield during the limit test are presented in the attached Tables 1 and 2.

Reference substance (positive control):

yes

Remarks:

potassium dichromate run proximal in time to definitive test

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): In the controls, cell density increased by an average factor of >16 within 2 days

See attached Table 3 of Mean cell densities and Figure 1 of growth curves

- Observation of abnormalities (for algal test):
- Unusual cell shape: There were no abnormalities detected in any of the control or test cultures
- Colour differences: The loading rate of 1 mg/L was clear and colorless but the loading rates between 3.2 and 100 mg/L were slightly yellow solutions that were increasingly colored with increased loading.

Results with reference substance (positive control):

Algae were exposed for a period of 72 hours to potassium dichromate concentrations of 0.18, 0.32, 0.56, 1.0, 1.8, and 3.2 mg/L and to a blank control. The initial cell density was 1x104 cells/mL. Under conditions of the reference study with Pseudokirchneriella subcaptitata, potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 1.0 mg/L and higher. The 72 hour EC50 for growth rate reduction was 1.7 mg/L with a 95% confidence interval ranging from 1.2 to 2.5 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. The 72 hour EC50 for yield inhibition was 0.58 mg/L with a 95% confidence interval ranging from 0.36 to 0.92 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Psedokirchneriella subcapitata to the test material gave 72 hour EC50 values for growth rate reduction of greater than 100 mg/L based on nominal loading rates, and correspondingly the No Observed Effect Concentration was 3.2 mg/L.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the freshwater algaPseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals, guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”.

Methods. The batch of test item tested consisted of black lumps with a purity of approximately 95.5%. The test item was not soluble in test medium at the concentrations tested. All test solutions were individually prepared based on the fact that the test item is considered a reaction mixture. Due to the poor solubilityi n water, preparation further included a 2-day magnetic stirring period followed by filtration to ensure reaching maximum dissolution at the various test groups.

A final test was performed based on the result of the preceding limit test. Exponentially growing algal cultures were exposed to a control and 0.45 micron filtered test item loading rates prepared at 1.0, 3.2, 10, 32, and 100 mg/L. The total test period was 72 hours and the initial algal cell density was 10⁴cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start and after 24 hours of exposure.

Results. Analysis showed that the measured test concentrations at the start of the test were 0.008, 0.03, 0.07, 0.11, and 0.11 mg/L at 0.45 micron filtered solutions prepared at respectively 1.0, 3.2, 10, 32, and 100 mg/L loading. Hence maximum solubility appeared to be in the order of 0.1 mg/L. Analysis after 24 hours showed that measured concentrations had all decreased below the limit of detection of the analytical method. 

Exposure ofPsedokirchneriella subcapitatato the test material gave 72 hour EC₅₀ values for growth rate reduction of greater than 100 mg/L based on nominal loading rates, and correspondingly the No Observed Effect Concentration was 3.2 mg/L.

Extra peaks with a varying degrees of response and with unknown nature were observed in the chromatograms over time. The origin of these extra peaks is unknown but they may be caused by degradation products. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Description of key information

Exposure of Psedokirchneriella subcapitata to the test material gave 72 hour EC50 values for growth rate reduction of greater than 100 mg/L based on nominal loading rates, and correspondingly the No Observed Effect Concentration was 3.2 mg/L (OECD 201).

Additional information

Introduction. A study was performed to assess the effect of the test material on the growth of the freshwater alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals, guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”.

Methods. The batch of test item tested consisted of black lumps with a purity of approximately 95.5%. The test item was not soluble in test medium at the concentrations tested. All test solutions were individually prepared based on the fact that the test item is considered a reaction mixture. Due to the poor solubilityi n water, preparation further included a 2-day magnetic stirring period followed by filtration to ensure reaching maximum dissolution at the various test groups.

A final test was performed based on the result of the preceding limit test. Exponentially growing algal cultures were exposed to a control and 0.45 micron filtered test item loading rates prepared at 1.0, 3.2, 10, 32, and 100 mg/L. The total test period was 72 hours and the initial algal cell density was 104cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start and after 24 hours of exposure.

Results. Analysis showed that the measured test concentrations at the start of the test were 0.008, 0.03, 0.07, 0.11, and 0.11 mg/L at 0.45 micron filtered solutions prepared at respectively 1.0, 3.2, 10, 32, and 100 mg/L loading. Hence maximum solubility appeared to be in the order of 0.1 mg/L. Analysis after 24 hours showed that measured concentrations had all decreased below the limit of detection of the analytical method. 

Exposure of Psedokirchneriella subcapitata to the test material gave 72 hour EC50 values for growth rate reduction of greater than 100 mg/L based on nominal loading rates, and correspondingly the No Observed Effect Concentration was 3.2 mg/L.

Extra peaks with a varying degrees of response and with unknown nature were observed in the chromatograms over time. The origin of these extra peaks is unknown but they may be caused by degradation products. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

The study met the acceptability criteria prescribed by the protocol and was considered valid.