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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2015 to 28 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotoc Systems [M], 2nd edition. Beijing: China Environment Press. 2013: 30-36.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 27861-2011, Chemicals-Fish acute toxicity test. Beijing: SAC, 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 29763-2013, Chemicals-Rare Minnow (Gobiocypris rams) acute toxicity test Beijing: SAC, 2013.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples (20.0 mL) were taken (in duplicate) from each concentration during the limit test at 0, 24, 48, 72 and 96 h.
- On each occasion, one sample was analysed after certain pre-treatments (see below) and the remaining samples were retained in case further analysis was required.

Vehicle:

no

Details on test solutions:

DILUTION MEDIUM
- Good quality tap water which had been dechlorinated for at least 24 hours was used.
- The total hardness of the dilution water was 145 mg/L (CaCO3).
- Oxygen concentration was about 92% of the air saturation value.
- The pH was 7.82 at room temperature.
- Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The latest measuring result is showed in Table 10 (attached).

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- The Rare minnow, Gobiocypris rarus, was used in the test on the basis of such important practical criteria as, for example, their ready availability throughout the year, ease of maintenance, convenience for testing and any relevant economic, biological or ecological factors.
- The test species (Batch No.: F20141215), were obtained as fry from a commercial fish supplier of Institute of Hydrobiology, Chinese Academy of Sciences.
- Fish were held at least for 12 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. In the present test, the test fish were held 23 d for the range-finding test and 41 d for limit test. Fish to be used in the test were held for 7 days in water of the quality and temperature to be used in the test.
- A photoperiod of 16 hours light (light intensity: 1000 lux - 1500 lux), provided by overhead fluorescent tubes, and 8 hours dark was maintained.
- The oxygen concentration was more than 80 % of the air saturation value.
- Temperature was controlled between 23.0 and 23.5 °C.
- Fish were fed daily during the holding period on proprietary fish food. They were held without food for approximately 24 hours before being placed into the test vessels. The ingredients of the fish food were crude protein (> 36.0 %); crude fat (> 2.0 %); crude fibre (< 3.0 %); crude ash (< 12.0 %); moisture (< 10.0 %). Characteristics of the fish food are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The latest measuring result is showed in Table 11 (attached).
- After the 48 hour settling-in period, no mortality was observed in the following 7 days and the batch of fish (Batch: F20141215) was accepted.
- The average wet-weight and the length of the fish used in the limit test was 0.289 g and 2.61 cm, with relative standard deviation (RSD) of 7.63 % and 8.12 %, respectively (see Table 1, attached).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

136 to 142 mg/L as CaCO3

Test temperature:

23.1 to 23.5 °C

pH:

7.85 to 8.02

Dissolved oxygen:

72 to 92 %

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations of 1.00, 10.0 and 100 mg/L

Details on test conditions:

APPARATUS
- Oxygen meter, thermometer and pH meter (HACH HQ 40 d. The Dissolved Oxygen, temperature and pH value can be directly read by immersing the probe in the water sample when the reading is stable).
- Equipment for determination of hardness of water (Model: 16900, made by HACH company (the hardness can be directly read by immersing the probe in the water sample when the reading is stable).
- Analytic Balance (AG135, Accuracy 0.1 mg, METTLER TOLEDO, Switzerland).
- Tanks made of glass material, with a sealable inert lid, and with a capacity of approximately 5 L (Haimen Sanhe Zuping Glass Instrument Factory. Jiangsu).
- Thermostatic water bath (Chang Yuan Medical Instrument Factory, Jiangsu).

REFERENCE SUBSTANCE
- The reference substance was potassium dichromate, K2Cr2O7 (CAS: 7778-50-9; Purity: > 99.8%; Lot number: NH20131115; Nanjing Chemical Reagent Co., Ltd).
- The test with the reference substance is performed at least once with each batch of fish as a means of assuring that the laboratory test conditions are adequate and have not changed significantly. The resulting 24 h-LC50 should be in the range of 200 to 400 mg/L. Otherwise, the test is regarded as invalid.
- Recent results of this study are shown in Table 5 (attached).

PREPARATION OF THE TEST SOLUTION
- In the range-finding test, the test solution was prepared by directly adding appropriate amounts of test item to dilution water (see table, below) and then facilitating its dispersion by stirring for 30 min.
- The test substance (black powder) was deposited at the bottom of solution.
- In the limit test, the saturated solution was prepared by adding 0.3000 g test substance to 3L of test medium in glass aspirator bottles.
- The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 24 hrs on a magnetic stirplate with a telfon stirbar at room temperature (about 23 to 25°C).
- The vortex height was set at least 10% of the static liquid height.
- At the end of the 24 hours, stirring was stopped and the test substance was allowed to settle.
- The saturated solution was allowed to stand for 1 hour at room temperature prior to the removal of any undissolved test item by filtration through 0.45 µm millipore membrane (first approximate 100 mL discarded) to produce a 100 % v/v saturated solution of the test item.

OBSERVATIONS AND EVALUATIONS
- Any abnormal responses of the fish observed were recorded, such as mortality, inactivity, abnormal swimming pattern, other abnormal behaviour, etc.
- Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produced no reaction.
RANGE-FINDING TEST
- The range-finding test, carried out under static conditions, was conducted to determine the range
of concentrations for the subsequent test.
- Groups of fish (5 per group) were exposed to the test solutions with nominal concentration of 1.00, 10.0 and 100 mg/L.
- One control group was also included in the study using test water without the test substance.
- Each test tank contained 3 L of test solution.
- No replicates were used.
- The test fish were randomly chosen and put in appropriate test solutions after the temperature had been adjusted to the required value. This was done within 30 min.
- During the test, the following conditions were maintained: Light: 16 hours photoperiod daily (light intensity: 1000 lux to 1500 lux); temperature: 23.0 to 23.5 °C; oxygen concentration: 70 % to 90 % of the air saturation; no aeration; no feeding.
- Test duration was 96 hours, and the test animals were observed daily. If dead fish was observed, they were removed and discarded. Fish mortality was recorded at 24, 48, 72 and 96 h and the maximum concentration causing no mortality was determined.

LIMIT TEST
- A Static method was adopted in the limit test.
- The stability of the test solution was confirmed by results indicated in Table 3 (attached) and deviation was within 20%.
- In the range-finding test one fish was dead in the 100 mg/L treatment group. The death may have been due to physical factors and unrelated to the test item, so a limit test at the concentration of saturated solution was assigned in the main test.
- One control group was also included in the test.
- Three replicates, each containing 3L of test solution, were assigned for each treatment group and control group, and the initial number of testing fish was 7 for each replicate.
- The test fish were randomly chosen and put in appropriate test solutions after the temperature had been adjusted to the required value. The group assignment was completed within 30 min.
- During the test, the following conditions were maintained: Light: 16 hours photoperiod daily (light intensity: 1000 lux to 1500 lux); temperature: 23.1 to 23.5 °C; oxygen concentration 72 to 92% of the air saturation; no aeration; no feeding.
- At 24, 48, 72 and 96 h, fish mortality was recorded, and observations on individual behaviour were performed.
- Measurements of pH, dissolved oxygen and temperature were carried out and recorded daily.

VALIDITY OF THE TEST
- Control group: A control group, comprising the same number of fish as that exposed at the test concentration, was placed into test water alone.
- Reference substance test: With the conditions maintained as designed for this test, potassium dichromate was used as the test substance and the resulting 24 h-LC50 was determined to be 298 mg/L. Results of this study are shown in Table 5 (attached).
- Fish loading rate: 0.5 g to 0.6 g fish (wet weight) per litre of test medium.
- Validity of test result: During the test period no mortality was observed in the controls and all fish in the control group were normal. The pH values of the control mediums and test mediums were between 7.85 to 8.02 and the Dissolved Oxygen (DO) values varied from 72 to 92 % of the air saturation at the test temperature. Total hardness was in the range of 136 to 142 mg/L (CaC03). During the test, the temperature of the test media was maintained in the range of 23.1 to 23.5 °C. With the same conditions, potassium dichromate was used as the positive control substance, and the resulting 24 h-LC50 was determined to be 298 mg/L. The study met the acceptability criteria prescribed by the protocol (mortality of control < 10%; pH: 6.0 - 8.5; dissolved oxygen concentration: > 60% of the air saturation value; total hardness: 10 ~ 250 mg/L (CaC03); temperature: 23 ± 2 °C; 24 h-LC50 of potassium dichromate in the range of 200 to 400 mg/L). Therefore the test was considered valid.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: saturated solution

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.284 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: saturated solution

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: saturated solution

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 0.284 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: saturated solution

Details on results:

ANALYTICAL METHOD FOR DETERMINING TEST ITEM IN WATER
- Specificity: Under the UPLC condition and at retention time 1.0 min, the chromatographic peak emerged for the test substance sample (see Figure 2, attached) and there was no chromatographic peak for the blank sample (see Figure 4, attached). Thus the UPLC method was specific for the test item.
- Calibration curve: A series of standard solutions with concentration at 0.50, 1.00, 5.000, 10.0 and 20.0 were measured under the UPLC-PDA conditions described above. Based on the test result, a linear regression equation was obtained between the concentration and the UPLC-PDA response: A = 141703c-56787, with good linearity of r = 0.9997, where A represents peak area (µV*s); and c is the concentration of the test substance (mg/L) (see Figure 1, attached). The results show that linearity for concentration range of 0.50 mg/L to 20.0 mg/L was good.
- Precision: Under the above conditions, 10 mg/L solution of the test substance was analysed six times and the results are shown in Table 4 (attached). The relative standard deviation was 0.86%, which was below the limit of 5%.
- Recovery test: The recovery samples with concentrations of 0.10 mg/L and 1.00 mg/L were prepared by adding 0.50 mL and 5.00 mL of standard solution (10.0 mg/L) to a total volume of 50.0 mL test water
with three replications. Recover samples (20.0 mL)were extracted twice with 20.0 mL dichloromethane. The combined upper organic phases were dried with rotary evaporator, and then re-dissolved with 2.00 mL mobile phase (10-fold concentrated). The final solutions were analysed by UPLC-PDA. Measurements obtained from the recovery test are shown in Table 3 (attached) whilst Figure 3 (attached) shows the chromatograph of the recovery sample from the recovery test. The mean recovery rate was 99.2 % (0.10 mg/L) and 105% for the concentration (1.00 mg/L). The relative standard deviation was 4.01 % and 3.24% respectively.
- Detection limit: The detection limit was 0.06 mg/L (S/N > 3), and the quantitation limit was 0.20 mg/L (S/N > 10).

ANALYSIS OF TEST ITEM IN TEST SOLUTIONS
- Analytical results for the test samples from the Limit Test are given in Table 4 (attached).
- Figure 4 and Figure 5 (attached) show the chromatographs of the control sample and the treated sample from the limit test.
- The mean measured concentration of samples was 0.284 mg/L.
- Results indicated that concentration of test item was stable (within 80 % of the initial concentration) in the water during the test period. Thus the static method used in the Limit Test was reasonable.

TEST CONDITIONS
- Values of pH, dissolved oxygen concentration, total hardness and temperature of the control and treatment groups during the limit test are shown in Table 5 (attached).
- During the test period, the pH values of the control mediums and test media were between 7.85 and 8.02, Dissolved Oxygen (DO) values varied from 72 to 92% of the air saturation at the test temperature, and total hardness was in the range of 136 to 142 mg/L (CaCO3).
- Temperature was maintained in the range of 23.1 to 23.5 °C in the test media.

MORTALITY AND EFFECTS
- Table 7 and Table 8 (attached) show the mortality data during the range-finding test and limit test
respectively.
- Table 9 shows the summary of the visual observations (for behaviour or abnormalities) during the limit test.
- All fish in the control and treated groups were alive and appeared normal.

Results with reference substance (positive control):

- Results with the refererence substance, potassium dichromate, were as expected (see Table 5, attached).

Reported statistics and error estimates:

DATA PROCESSING
- Trimmed Speannan-Karber Method (Version 1.5, USEPA) can be used to calculate the LC50 and
95% confidence limits.
- Due to its low toxicity to fish in this study, only LC50 and 95% confidence limits of the reference substance were calculated by this method.

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

GUIDELINE

The acute toxicity of the test item to Gobiocypris rarus was investigated 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).

 

METHODS

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.

 

RESULTS

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).

 

CONCLUSION

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

Description of key information

The LC0 (96 h) and LC50 values for a nominal concentration of 100 mg/L (measured concentration 0.284 mg/L were determined to be > 100 mg/L (OECD 203, OPPTS 850.1075 and relevant Chinese guidelines).

Additional information

GUIDELINE

The acute toxicity of the test item to Gobiocypris rarus was investigated 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).

 

METHODS

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.

 

RESULTS

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 raruswas 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).

 

CONCLUSION

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