2-methyl-p-phenylenediamine

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:

From 2016-06-22 to 2016-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: 6-MWC-149-1- Expiration date of the lot/batch:2018-02-08- Purity test date: 2015-02-10STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material:Hygroscopic, refrigerator, under inert atmosphere- Stability under test conditions:Concentrated stock solutions were prepared once for the test with DMF- Solubility and stability of the test substance in the solvent/vehicle:the measured test item concentrations did not remain stable within the of ± 20% of the nominal concentrations- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:/TREATMENT OF TEST MATERIAL PRIOR TO TESTING no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels, the solvent control and the control were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and thereafter every 24 hours (0, 24 and 48 hours) in all test replicates. If an effect of 100% mortality is observed during the study, the respective replicate was not be analysed anymore in the following sampling interval(s). - Sampling method, Sample storage conditions before analysis:Samples of the stock solutions were taken directly after preparation (-216 hours) and at the end of the exposure (48 hours). Samples of the aqueous test solutions was taken directly from each test vessel at -192, -168, -144, -48, -24, 0, 24 and 48 hours. These samples were stabilised by addition of HPLC water : acetonitrile (10 : 90) containing 0.2% formic acid and 0.2% ascorbic acid.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method:A stock solution with a nominal concentration of 22000 mg/L of the test item was freshly prepared with DMF before the start of the equilibration period (at -216 hours), mixed thoroughly by manual agitation and was used as the highest test concentration and as a stock solution for the preparation of the further concentration levels. The concentrated stock solutions were prepared once for the test with DMF and filled into syringes, which were covered with aluminium foil. The stock solutions were introduced into the mixing chambers by a precision syringe pump.- Controls: Two replicates of dilution water without test item incubated under the same conditions as the test groups. Additionally a solvent control was tested under the same conditions as the test groups. For this purpose, dilution water with 0.1 mL DMF/L in a continuous flow.- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 mL/L- Evidence of undissolved material (e.g. precipitate, surface film, etc.):All test item concentrations were visually clear throughout the exposure period. The stock solution and the test solution of ≥ 1.00 mg/L showed a clear, brown color throughout the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM - Common name: Daphnia magna - Strain/clone: STRAUS (Clone 5) - Source: Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany; Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany - Age of parental stock (mean and range, SD): no data - Feeding during test: no - Food type/Amount/Frequency: The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility. ACCLIMATION: Acclimatisation is not necessary, because the dilution water was equivalent to the culture medium. METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids will be removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was for the test. The daphnids were inserted with a small amount of dilution water at the start of the exposure by a pipette.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

/

Hardness:

178 mg CaCO3/L (Dilution Water at the Start of the Exposure Intervals (0 and 24 hours))

Test temperature:

18 - 22 °C, constant within ± 1 °C (actual 17.5 – 21 °C)

pH:

From 6.92 to 7.19 at 0 hours; from 7.14 to 7.24 at 24 hours and from 7.36 to 8.23 at 48 hours

Dissolved oxygen:

From 4.1 to 6.66 mg/L at 0 hours; from 5.32 to 6.88 mg/L at 24 hours and from 5.00 to 7.63 mg/L at 48 hours

Salinity:

/

Conductivity:

488-495 µS/cm

Nominal and measured concentrations:

Nominal: 0.0939 - 0.207 - 0.455 - 1.00 - 2.20 mg/LGeometric mean measured: 0.0725 - 0.152 - 0.311 - 0.605 - 1.26 mg/L

Details on test conditions:

TEST SYSTEM - Test vessel: Glass beakers (8.0 (ID) x 9.5 (H) cm, 250 mL capacity) with an outlet covered with mesh (495 m) allowing flow-through of test and control solutions and avoiding an escape of the young daphnids. The test vessels will be loosely covered with a pierced petri dish. Mixing chambers: Brown glass flasks (6.4 (ID) x 14.0 (H) cm, 250 mL capacity) with an outlet as overfill protection covered with a pierced screw cap. - Material, size, headspace, fill volume: Test volume: Approximately 200 mL per test vessel - Type of flow-through (e.g. peristaltic or proportional diluter): Multi channel tubing pumps provided the water flow-through. Each replicate of the concentration levels, the solvent control and the control were supplied by an own channel. The dilution water was pumped into mixing chambers. For each concentration level, the solvent control and the control one mixing chamber was provided. The stock solutions of the test item with different concentrations, prepared with DMF, and pure DMF for the solvent control were introduced into the mixing chambers by a precision syringe pump. The test solutions in the mixing chambers were gently stirred for homogenous mixing. The test solutions were pumped from the mixing chambers into the test vessels. The delivery system was calibrated prior to experimental starting including determination of the flow-through rate through each test vessel. - Renewal rate of test solution (frequency/flow rate): The flow rate of a test chamber was 41.7 mL/h +/- 10%, corresponding to a minimum of 37.5 mL/h and a maximum of 45.9 mL/h. (1000 mL/d, equivalent to a medium renewal: 5 times the volume of the whole test vessel per day). The general operation of the test item delivery system was daily. The flow-through rates should not vary more than 10% among the test vessels.Equilibration period: Test solutions flows through the test vessels at least 48 hours prior to exposure. - No. of organisms per vessel:20 daphnids, divided into 2 replicates, each with 10 daphnids - No. of vessels per concentration (replicates):2 - No. of vessels per control (replicates):2 - No. of vessels per vehicle control (replicates):2 - Biomass loading rate:/ TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water:Same as culture medium : Elendt M4 - Intervals of water quality measurement: pH and dissolved oxygen were measured in al the tests concentrations and controls at T0, T24 and T48H. Temperature, conductivity, total hardness, pH and dissolved oxygen were also measured in the dilution water at T0 and T24H OTHER TEST CONDITIONS - Adjustment of pH:no - Photoperiod/Light intensity:The test was carried out under diffuse light (ambient daylight conditions) and a natural light/dark cycle. EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation was determined in replicates after 24 and 48 hours. An animal was considered immobile, if it is not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Relevant additional observations were recorded, if necessary. VEHICLE CONTROL PERFORMED: yes RANGE-FINDING STUDY: 2 were performed - Test concentrations: 1) 1 mg/L (nominal) = 0.675 mg/L geometric mean measured (55% immobilisation at 48H). 2) 0.455 mg/L (nominal) = 0.225 mg/L geometric mean measured (20% immobilisation at 48H). - Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.491 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 0.385-0.658

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: / - Observations on body length and weight:/ - Other biological observations:/ - Mortality of control: 0% - Other adverse effects control: no - Abnormal responses:/ - Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All test item concentrations were visually clear throughout the exposure period. The stock solution and the test solution of ≥ 1.00 mg/L showed a clear, brown color throughout the exposure period. This did not interfere with determination of daphnid immobility. - Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes- Limit test: noThe EC50-value with 95% confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:EC50: 2.00mg/L (CI 1.22 - 3.29 mg/L)The EC50-value of the reference item potassium dichromate after 24 hours is within the required concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

Estimation of the EC10- and EC50-Value after 48 hours:Equation:Sigmoidal dose-response, variable slope: Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))Log concentration [mg/L]Immobilisation after 48 hours [%]-1.1400-0.81800-0.5074020-0.21870500.10090100

2,5‑Diaminotoluene	IMMOBILIZATION [%]
Test item concentration [mg/L]	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2
2.20	90	100	95	90	-^1)	95
1.00	0	10	5	70	50	60
0.455	0	0	0	40	20	30
0.207	0	0	0	0	0	0
0.0939	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0
Control	0	0	0	0	0	0

¹⁾                      = after 24 hours a mortality of 100 percent was observed

Validity criteria fulfilled:

yes

Remarks:

In the control group, no daphnids were immobilised or showed any signs of disease or stress (discoloration or unusual behaviour such as trapping on the surface of the water, during the 48H test. The dissolved O2 at the end of the exposure was ≥ 4.10 mg/L

Conclusions:

Based on the geometric mean measured concentrations of the test item 2,5 Diaminotoluene, the 48 hour EC50 for Daphnia magna was 0.491 mg/L (95% confidence limits: 0.385 – 0.658 mg/L).

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item 2,5‑Diaminotoluene were determined according to OECD 202 (2004) and GLP. The study was carried out as a flow-through exposure design over a period of 48 hours with five concentrations of the test item 2,5‑Diaminotoluene ranging from 0.939 to 2.20 mg/L in a geometric series with a separation factor of 2.2. The test item2,5‑Diaminotoluene is a tan to brown solid, which is soluble in water (20°C).A stock solution with a nominal concentration of 22000 mg/L of the test item was freshly prepared with DMF before the start of the equilibration period (at -216 hours), mixed thoroughly by manual agitation and was used as the highest test concentration and as a stock solution for the preparation of the further concentration levels.Twenty daphnids (divided into 2 replicates with 10 daphnids each) were exposed to each concentration level, the solvent control and the control. No immobility was observed in solvent control and control. The concentrations of the test item were analytically verified via LC-MS/MS. The measured concentrations of the test item were in the range of 0.0645 to 1.30 mg/L during the exposure period. Since the measured test item concentrations did not remain stable within the of ± 20% of the nominal concentrations, the effect levels are based on the geometric mean measured concentrations.Based on the geometric mean measured concentrations of the test item2,5‑Diaminotoluene, the 48 hour‑EC₅₀for Daphnia magna was 0.491 mg/L (95% confidence limits: 0.385 – 0.658 mg/L). The validity criteria were fulfilled.

Description of key information

This study was carried out as a flow-through exposure design over a period of 48 hours to maintain as far as possible the test item concentrations. Indeed, the previous daphnia test, performed by the Japanese authorities in semi-static regime showed that at 24H, the concentrations fell to below the detection limit, inducing an artificially low EC50 measurement. In this new test, even if measured concentrations did not remain stable within the of ± 20% of the nominal concentrations, they were still detectable and were in the range of 0.0645 to 1.30 mg/L during the exposure period. Based on the geometric mean measured concentrations of the test item, the 48 hour‑EC50 for Daphnia magna was 0.491 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.491 mg/L