2-methyl-p-phenylenediamine

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:

From February 22, 2010 to March 11, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed by Japanese competent authorities according to GLP and equivalent OECD standardized guidelines.

Reason / purpose for cross-reference:

other: reference to report on algae tests performed on fresh and aged solutions to support that parent compound is more toxic than degradation products.

Qualifier:

according to guideline

Guideline:

other: Testing Method for New Chemical Substances Alga growth inhibition test, Acute daphnia immobilization test and Acute fish toxicity test (21/11/2003; No.1121002, Pharmaceutical and Food Safety Bureau, Japanese Ministry of Health, Labour and Welfare)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration of test substance in all test groups in test solutions and test cultures at the beginning of the exposure, 24, 48 hours and 72 hours after the beginning of the exposure was analyzed.- Sampling method and Sample storage conditions before analysis:At T0: test solutions were mixed after sampling 1.0 mL from each of the middle layer of the all test vessels, in each test group. Then, diluted arbitrarily with purified water (for those which are expected to be beyond the range of calibration curve), 0.75 mL sampled and 0.75 mL of Acetonitrile added, mixing and then LC/MS measurement.At T 24, 48 and 72 after the beginning of the exposure: samples were mixed after sampling 1.0 mL from each of the middle layer of the all test vessels, in each test group, then were centrifuged and the supernatants diluted arbitrarily with purified water (for those which were expected to be beyond the range of calibration curve), 0.75 mL sampled and 0.75 mL of Acetonitrile added, mixing and then LC/MS measurement.

Vehicle:

no

Details on test solutions:

Storage method and confirmation of stability:During the test period, the test substance was stored in a refrigerating chamber for the use of storing test substances in this facility (storage conditions: cold storage, dark place, capped tightly). After the completion of the experiment, Infrared Absorption Spectrum of the stored test substance was measured. Since the obtained spectrum coincided with the one measured before the experiment, it was judged that the test substance was stable during the storage period. PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: through dilution of a 100 mg/L stock solution (direct addition of 50 mg/L in a 500 ML volumetric flask and stirred by stirred 3 min, capped tightly). Test solutions were prepared in 300 mL glass conical flasks (air-permeable silicon plug), stirred by hand shaking.- Eluate: no- Differential loading: no- Controls: dilution water- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not used- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM- Common name: Pseudokirchneriella subcapitata- Strain: ATCC22662- Source (laboratory, culture collection): American Type Culture Collection- Method of cultivation: Subcultured under sterile condition with using Gorham medium. The recommended medium shown in the test guideline was used for both pre-culture and the test after adjustment, filtration sterilization (0.22µm). ACCLIMATION- Acclimation period: Duration of pre-culture; February 19, 2010 - February 23, 2010Algae were pre-cultured under the same conditions as the test to ensure that they are in the exponential growth phase when beginning exposure. - Culturing media and conditions (same as test or not): yes- Any deformed or abnormal cells observed: no cell with the change of shape or abnormality was observed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

21.6-22.0°C

pH:

7.9-8.2

Dissolved oxygen:

Not reported

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0.2, 0.38, 0.72, 1.4, 2.6 and 5.0 mg/LMeasured concentrations: T0: 0.212, 0.387, 0.784, 1.55, 2.60 4.93 mg/lT24H: 0.0698, 0.0599, 0.08, 0.114, 0.160, 0.208 mg/lT48H: 0.0218, 0.00848, 0.00942, 0.0180, 0.0267, 0.0453 mg/lT72H: 0.00739, <0.001, <0.001, <0.001, 0.00638, 0.0129 mg/l

Details on test conditions:

TEST SYSTEM- Test vessel: 300 mL glass conical flask (IWAKI) (with air-permeable silicon plug) - Type (delete if not applicable): open - Material, size, headspace, fill volume: volume of test solution: 100 mL/vessel- Aeration: shake culture (100 rpm)- Type of flow-through (e.g. peristaltic or proportional diluter): /- Renewal rate of test solution (frequency/flow rate): No, static- Initial cells density: 5000 cells/mL- Control end cells density: 2040000 cells/mL- No. of organisms per vessel: /- No. of vessels per concentration (replicates): 3- No. of vessels per control (replicates): 6- No. of vessels per vehicle control (replicates): /GROWTH MEDIUM- Standard medium used: yes, the one recommended in the guideline - Detailed composition if non-standard medium was used: /TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: the same as for culture medium.- Total organic carbon: not reported- Particulate matter: not reported- Metals: not reported- Pesticides: not reported- Chlorine: not reported- Alkalinity: not reported- Ca/mg ratio: not reported- Conductivity: not reported- Culture medium different from test medium: no- Intervals of water quality measurement: temperatures, light intensities, and number of revolutions in incubation chamber were measured once a day. pH was measured at T0 and T72H (in the backup vessels, that is without algae)OTHER TEST CONDITIONS- Sterile test conditions: Yes. Subcultured under sterile condition with using Gorham medium. Germfree status has been confirmed by conducting periodical bacterium examinations (at around every 6 months). Where appropriate, sterile test vessels and other equipment were used. Besides, inoculation of Algae was performed under sterile condition. test medium sterilized by filtration (0.22µm).- Adjustment of pH: No- Photoperiod: continuously illuminated - Light intensity and quality: 65 to 75 µE/m2/s with white fluorescent lamp (near liquid surface)- Salinity (for marine algae): /EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: 1.0 mL (0.5 mL at 72 hours) of test cultures were sampled from each test vessel at the beginning of the exposure, 24, 48 and 72 hours after the beginning of the exposure, and they were mixed with 9.0 mL (9.5 mL at 72 hours) of electrolyte for the use of electronic particle counter and then biomasses of them were measured by the particle counter. The test substance changes in its structure in dilution water, and particles which are thought to be due to the degraded product produced over time. Since these particles are included in the results of particle counting, biomasses were calculated by subtracting background values from the counting results of test cultures, where the counting results of the test solutions in backup vessels at each time were used as the background values in each test group.TEST CONCENTRATIONS- Spacing factor for test concentrations: 1.9- Justification for using less concentrations than requested by guideline:/- Range finding study: Yes- Test concentrations in the range finding study: 0.2, 0.5 and 5 mg/L:- Results used to determine the conditions for the definitive study: Preliminary test was conducted, with the aim of confirming the toxicity of degraded product, with using test solutions immediately after the preparation and at 24 hours after the preparation:Test solution immediately after the preparation: 0.2 mg/L: -1.9% inhibition; 0.5 mg/L: 17.2% inhibition; 5.0 mg/L: 125% inhibitionTest solution at 24 hours after the preparation: 0.2 mg/L: 2% inhibition; 0.5 mg/L: 4.9% inhibition; 5.0 mg/L: 120% inhibition

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.02 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.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.03 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2.01-2.06

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.212 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): yes- Observation of abnormalities (for algal test): Yes: neither cell morphological change (contraction, expansion, rupture, etc.) nor cell agglutination was observed in concentration group 4 or the lower groups, and the algae looked normal compared with those in the controls. In concentration groups 5 and 6, sufficient observation could not be done because the number of cells was too low.- Unusual cell shape: No- Flocculation: No- Adherence to test vessels: No- Aggregation of algal cells: No- Any stimulation of growth found in any treatment: 1.1% stimulation in one replicate from conc. 1 (0.2 mg/L) at 72H.- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance changes in its structure in dilution water, and particles which are thought to be due to the degraded product produced over time.- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? YesThe latest 72-hour Median Effect Concentration, which was calculated by growth rates method, is shown below.ErC50: 0.826 mg/L (95% confidence interval: 0.773 - 0.884 mg/L, Exposure duration: December 15, 2009 – December 18, 2009) Average ErC50 since June, 2000 is ErC50 ± S.D = 0.821 ± 0.0828 mg/L, n=20 (Minimum – maximum: 0.687 – 0.965 mg/L)

Reported statistics and error estimates:

ErC50 and associate 95% confidence limits were determined by least squares linear regression analysis of the logarithm of test concentrations against the growth inhibition (%) relative to the control. NOEC: Bartlett test for equal variance (α=0.01) was performed with using all test groups’ data; however, homogeneity of variance was not confirmed. Therefore, Bartlett’s equal variance test was performed with excluding the data of concentration group 6 for which clear inhibition was confirmed when compared with the data of controls, and homogeneity of variance was confirmed: hence, one-way analysis of variance (1-way ANOVA, α=0.05) and Williams test for multiple comparison (α=0.05, two tailed) were performed to determine the NOEC.

Structure of the test substance changed in water, and the test substance had become almost undetected in the test solution at 24 hours after the preparation. Although the identification of the degraded product has not been achieved, toxicity of the product has been confirmed by prior examination. Therefore, the toxicities of test substance and degraded product were comprehensively assessed by using the measured concentrations at the time of preparing test solution for calculating the test results.

Other tests performed by the lead registrant on fresh and aged test solutions allowed to demonstrate that degradation/transformation processes continue to take place after 24H and lead to the formation of less toxic products. A re-calculation was therefore performed by the assessor based on the geometric mean measured concentrations at the beginning and the end of exposure to consider the worst case situation and the higher toxicity of the parent compound.

Validity criteria fulfilled:

yes

Remarks:

Controls: the biomass increased by a factor of at least 16 within 72H. The mean CV for section-by-section specific growth rate didust not exceed 35%. The CV of average specific growth rate did not exceed 7%

Conclusions:

ECr50= 1.02 mg/l and NOEC=0.11 mg/l based on geometric mean measured concentrations.

Executive summary:

The test substance 2,5 -diaminotoluene was tested on Pseudokirchneriella subcapitata during 72H exposure in static conditions. The test followed Japanese guidelines, equivalent to OECD guidelines, and GLP. Nominal concentrations were 0.2, 0.38, 0.72, 1.4, 2.6 and 5.0 mg/L. Initial measured concentrations were almost the same as nominal values, but concentrations rapidly fell into solution to reach range of less than the detection limit to 4% of the nominal values at the end of exposure. Identification of the degraded product was not achieved, but tests on 24 hour-aged solutions indicated that they presented equivalent toxicities. ECr50 and NOEC reported in the study therefore related to initial measured concentrations and were determined to be 2.03 mg/l and 0.212 mg/l respectively.

In agreement with the sponsor, these were re-calculated considering the geometric mean measured concentration of the test substance and were determined to be 1.02 mg/l and 0.11 mg/l respectively. This study met all the validity criteria and was thus considered as reliable.

Description of key information

The test substance was tested on Pseudokirchneriella subcapitata during 72H exposure in static conditions. The test followed Japanese guidelines, equivalent to OECD guidelines, and GLP. Nominal concentrations were 0.2, 0.38, 0.72, 1.4, 2.6 and 5.0 mg/L. Initial measured concentrations were almost the same as nominal values, but concentrations rapidly fell into solution to reach range of less than the detection limit to 4% of the nominal values at the end of exposure. In agreement with the sponsor, and because tests performed on fresh and aged solutions revealed a higher toxicity of the parent compound, the concentrations were re-calculated. The geometric mean measured concentrations were determined to be 1.02 mg/l and 0.11 mg/l respectively. This study met all the validity criteria and was thus considered as reliable.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.02 mg/L

EC10 or NOEC for freshwater algae:

0.11 mg/L