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

Endpoint summary

Description of key information

Short-term toxicity to fish: LC50 (96h) > 100 mg/L based on nominal loading rate (OECD 203)

Long-term toxicity to fish: Key study, NOEL (28d) 1.2 mg/L loading rate WAF (OECD 215); Supporting study, NOEC (28d) 0.357 mg/L (OECD 215)

Short-term toxicity to Daphnia: Key study, EL50 (48h) 2.0 mg/L loading rate WAF (OECD 202); Supporting study, EC50 (48h) 0.35 mg/L (OECD 202)

Long-term toxicity to Daphnia: NOEL (28d) 1.0 mg/L loading rate WAF

Algae: EC50 (72h) > 100 mg/L based on nominal loading rates; NOEC 3.2 mg/L based on nominal loading rates (OECD 201)

Microorganisms: EC50 (3h) 250 mg/L (OECD 209)

Additional information

Fish

Short-term toxicity

The acute toxicity of the test item to Gobiocypris rarus was investigated in a key study under static conditions in accordance with the Guidelines for the testing of chemicals (HJ/T 153-2004) and the Guidelines for the Testing of Chemical Effects on Biotic Systems (2nd edition, Beijing: China Environment Press, 2013) with reference to Procedure 203 of the Guidelines for Testing of Chemicals “Fish Acute Toxicity Test” (OECD; 1992).

 

A range-finding test was conducted with five fish per treatment (no replicates) at nominal concentrations of 1.00, 10.0 and 100 mg/L. No adverse effect was observed up to 100 mg/L and, in the main test, fish (three replicates of seven fish) were exposed to the saturated solution obtained by dissolving 0.3 g of test material in 3 L of water (a Limit Test). Water samples taken from the control and the treatments in the Limit Test (0 h, 24 h, 48 h, 72 h and 96 h) were analysed. The mean measured concentration of the saturated solution was 0.284 mg/L. The analytical results showed that the concentration of the test substance was consistent in the test medium throughout the 96-hour test period (deviation within 20%). Thus a static procedure was reasonable.

During the limit test period, the pH values of the control mediums and test mediums were between 7.85 and 8.02, the Dissolved Oxygen (DO) values varied from 72% and 92% of the air saturation at the test temperature, and the total hardness was in the range of 136 mg (CaC03)/L to 142 mg (CaC03)/L. During the test, the temperature of the test mediums were maintained in the range of 23.1 to 23.5°C, and all fish in the control group were normal. With the same conditions, potassium dichromate was used as the positive control substance, and the resulting 24 h-LC50 was 298 mg/L. The study met the acceptability criteria prescribed by the protocol and Test Guidelines (The mortality of control < 10%; pH: 6.0 ~ 8.5; dissolved oxygen concentration: > 60% of the air saturation value: total hardness: 10 - 250 mg (CaC03)/L; temperature: 23± 2 °C; 24 h-LC50 in the range of 200 mg/L to 400 mg/L). Therefore the test was considered valid. During the definitive test period, all fish in the control groups and treated groups were alive and appeared normal. The results showed that under valid static test conditions, the 96 h-LC50 of test item to fish (Rare minnow, Gobiocypris rarus) was greater than the nominal concentration of saturated solution (measured concentration was 0.284 mg/L), while the maximum concentration causing no mortality (96 h-LC0) was equal to the saturated solution (prepared by dissolving 0.3 g test material in 3 L of water, measured concentration was 0.284 mg/L).

The LC0 (96 h) and LC50 (96h) values for a nominal concentration of 100 mg/L (measured concentration 0.284 mg/L were determined to be > 100 mg/L.

Long-term toxicity

A key study was performed to assess the effect on growth of juvenile rainbow trout (Oncorhynchus mykiss) of the test material. The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (2000) No 215, "Fish, Juvenile Growth Test” referenced as Method C.14 of Commission Directive 2001/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, fish were exposed, in groups of 16 to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L for a period of 28 days at a temperature of 14 to 16 °C under semi-static test conditions. Any mortalities or adverse reactions to exposure were recorded daily throughout the exposure period. The fish were fed daily at a rate of 4% bodyweight with trout pellets and the weight of each fish determined at 0, 14 and 28 days.

 

With the exception of the 4.8 mg/L loading rate WAF fresh test preparations on Days 0 and 13 which gave measured concentration of 0.165 and 0.134 mg/L respectively, chemical analysis of the 2.4 and 4.8 mg/L loading rate WAF test preparations at all sampling occasions showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.13 mg/L). This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis.

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis. The No Observed Effect Loading Rate (NOEL) was 1.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) was 2.4 mg/L loading rate WAF.

In a supporting study,under semi-static conditions of 72 h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004), “The Guidelines for the Testing of Chemicals, Effects on Biotic Systems” (the 2nd edition) (2013); Procedure 215 of the “Guidelines for Testing of Chemicals” of the OECD “Fish, Juvenile Growth Test” (2000) etc.

Juvenile fish in exponential growth phase were placed, after being weighed, in test chambers and were exposed to a range of sub-lethal concentrations of the test substance dissolved in water under appropriate semi-static conditions. The test duration was 28 days. At the end of the test, the fish were weighed again. Effects on growth rates were analysed using a regression model in order to estimate the concentration that would cause a 50% variation in growth rate (EC50).

During the whole test period, the pH values of the control and test media were between 7.73 and 7.99, and the Dissolved Oxygen (DO) values varied from 69% to 96% of the air saturation at the test temperature. The temperature of the test media was maintained at 23.1 to 23.4°C. All fish in the control group were normal. The mean weight of fish in the control group increased more than 50%. The study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration no less than 60% of the air saturation value; temperature 23 ± 2 °C and no more than ± 1 °C between test chambers; increasing rate of the mean wet-weight of fish no less than 50% of the initial weight). The test was valid.

In order to confirm the stability of the test substance in the test medium, concentrations of the test samples during the test period were analysed by UPLC-PDA. Concentrations of the test substance were quantified by UPLC-PDA. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of test item (0.50, 1.00, 2.00, 5.00 and 10.0 mg/L): A=139059c-55817, with good linearityof r2=0.9988. The result showed that linearity for the test item with the concentration range of 0.50 mg/L to 10.00 mg/L was good. The analytical results showed that the concentration of the test substance of the test solution was stable throughout a period of 72 h (deviation within 20%). Thus a semi-static test of 72 h-renewal was acceptable. The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatment groups. Fish in all treatment and control groups were alive and appeared normal.

 

The LOEC (lowest observed effect concentration) to juvenile rare minnow (Gobiocyprisrarus) was determined to be > 0.357 mg/L and the NOEC (No observed effect concentration) of the test substance was reported as 0.357 mg/L.

Daphnia magna

Short-term toxicity

A key study was performed to assess the acute toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (W AF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 20 to 21 °C under static test conditions. The number of immobilizedDaphniaand any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.221 to 3.6 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) determined to be 0.15 mg/L, to 1.20 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Following exposure of the freshwater invertebrate, Daphnia magna to the test item, the EL50 (48h) value was reported as 2.0 mg/L loading rate WAF (confidence limits 1.5 to 2.7 mg/L loading rate WAF). The No Observed Effect Loading Rate (NOELR) was reported as 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOELR) was reported as 3.2 mg/L loading rate WAF.

 

In a supporting study, the acute toxicity of the test material to Daphnia magna followed the methods described in the OECD 202. Following a preliminary limit test, twenty daphnids (4 replicates of 5 animals) were exposed to individually prepared dispersions of the test material over a range of nominal loading rates of 2.2, 4.6, 10, 22, 46, and 100 mg/L for 48 hours at a temperature of 20 to 21 °C under static test conditions. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours. A positive control conducted concurrently used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.1, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L for 48 hours at a temperature of 21°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

The 48-Hour EL50 for the test material to Daphnia magna based on nominal loading rates was 6.8 mg/L with 95% confidence intervals of 4.6 - 10 mg/L. The No Observed Effect Loading rate was 4.6 mg/L nominal loading rate. Chemical analysis of the test preparations at time =0 and after 48 hours showed measured test concentrations ranging from 0.022 to 0.17 mg/L. No concentration dependent general trend was consistently observed with increasing loading rate (this was true for time =0 hour and 48 hour test durations as well as average exposure concentrations).  Samples taken at the start of the final test showed measured concentrations that corresponded to 1% or less relative to the individual loading rates. The measured concentrations decreased significantly during the test period.  The dissolved test material may have been one or several components of the test material. Extra peaks with unknown nature were observed in the chromatograms. These were expected to derive from unknown start and or reaction or 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 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.35 mg/L with 95% confidence intervals of 0.31 – 0.40 mg/L.

 

Long-term toxicity

A key study was performed to assess the chronic toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211,"Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 "Daphnid Chronic Toxicity Test".

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Based on the results of an acute toxicity test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to aWater Accommodated Fraction (WAF) of the test item over a range of nominal loading rates 0.10, 0.22, 0.45, 1.0 and 2.2 mg/L for a period of 21 days. The test solutions were renewed dailythroughout the test.The numbers of live and dead adult daphnia and young daphnids (live and dead) weredetermined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin flake food suspension.

Chemical analysis of the test preparations at all sampling occasions showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.019 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave EL10 values of 0.67 mg/L loading rate WAF (immobilisation) and 0.8 mg/L loading rate WAF (reproduction). The EL50 values reported were 1.2 mg/L loading rate WAF (immobilisation) and 1.3 mg/L loading rate WAF (reproduction). The No Observed Effect Loading Rate (NOELR) was determined to be 1.0 mg/L loading rate WAF, the Lowest Observed Effect Loading Rate (LOELR) was 2.2 mg/L loading rate WAF and the Maximum Acceptable Toxicant Loading Rate (MATL) was reported as 1.5 mg/L loading rate WAF.

Algae

A key 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”.

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.

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.

Inhibition of sewage sludge micro-organisms

A key study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3-Hour EC50 of 250 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 94 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3-Hour EC50 value of 7.1 mg/L (95% confidence limits 4.6 - 11 mg/L).