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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 April 2015 to 21 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

measurement of room temperature instead of vessel temperature and water hardness measurements not taken from highest surviving concentration; no impact on outcome of study (see below)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

measurement of room temperature instead of vessel temperature and water hardness measurements not taken from highest surviving concentration; no impact on outcome of study (see below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

yes

Remarks:

measurement of room temperature instead of vessel temperature and water hardness measurements not taken from highest surviving concentration; no impact on outcome of study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

VERIFICATION OF TEST CONCENTRATIONS
- Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis.
- Samples of the fresh test preparations were taken on Days 0, 6, 13 and 20.
- Samples of the expired test preparations were taken on Days 1, 7, 14, and 21.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
- Only samples at the No Observed Effect Loading Rate and above were analysed.

Vehicle:

no

Details on test solutions:

TEST WATER
- The reconstituted water (Elendt M7 medium) used for the definitive test was the same as that used to maintain the stock animals and is defined in Appendix 2 (attached).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SPECIES
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 2, attached) in a temperature controlled room at approximately 20 °C.
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis.
- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable

Hardness:

236 to 274 mg/L as CaC03

Test temperature:

20.1 to 22.1 °C (see Table 10, attached)

pH:

- Refer to Table 10 (attached)

Dissolved oxygen:

≥ 8.3 mg O2/L (see Table 10, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Water Accomodated Fractions of nominal loading rate 0.10, 0.22, 0.45, 1.0 and 2.2 mg/L

Details on test conditions:

VALIDATION OF MIXING PERIOD
- Preliminary work (see Appendix 3, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

DEFINITIVE TEST
- Based on the results of an acute toxicity test Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (W AF) of the test item over a range of test concentrations of 0.10, 0.22, 0.45, 1.0 and 2.2 mg/L for a period of 21 days.
- The test solutions were renewed daily throughout the test.

EXPERIMENTAL PREPARATION
- Prior to addition of the test item a glass siphon tube was placed in the test media.
- Nominal amounts of test item (10 and 22 mg) were each separately added to the surface of 10 L of test water to give the 1.0 and 2.2 mg/L loading rates respectively.
- After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- During the stirring phase the vessels were sealed with a minimal headspace.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- A length of Tygon tubing was attached to the top of the glass siphon tube. A glass wool plug was inserted into the opposite end of the tubing and the WAF was removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0 and 2.2 mg/L loading rate WAFs.
- Microscopic observations of the W AFs were performed after filtering and showed no micro dispersions or undissolved test item.
- Dilutions were performed from the 1.0 mg/L loading rate WAF to create 0.10, 0.22 and 0.45 mg/L loading rates.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 1, 6, 7, 13, 14, 20 and 21 (see Appendix 4, attached).

EXPOSURE CONDITIONS
- For each concentration, a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation.
- For each test and control group, ten replicate test vessels were prepared.
- Test vessels were maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light (not exceeding 1500 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for
21 days.
- Each vessel was randomly assigned to a position in the laboratory.
- The test vessels were not aerated.
-Diluent water only was aerated prior to use.
- The control group was maintained under identical conditions but not exposed to the test item.
- Test preparations were renewed daily throughout the test.
- The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
- Each daphnid received approximately 5 to 10 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 μL of Tetramin flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

TEST ORGANISM OBSERVATIONS
- On a daily basis, the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (Fi) daphnia and the number of discarded unhatched eggs were counted.
- An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.
- The number of daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobiliSation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
- At the end of the test, the length of each surviving parent animal was determined.

WATER QUALITY CRITERIA
- Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal.
- The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter.
- Temperature was measured using a Hanna Instruments HI 93 510 digital thermometer.
- Measurements were made on one replicate for each test concentration.
- The room temperature was also monitored hourly using the Delta Building Management System.
- Water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week. On Days 14 and 21 the aged media from the 1.0 mg/L loading rate W AF group was taken in place of the highest test concentration as there was not enough test media available in the 2.2 mg/L loading rate WAF. Additionally, on Day 21 the aged media from the control group was not taken in error.

VALIDATION CRITERIA
- For the test to be valid, the following criteria should be fulfilled:
(i) Mortality of parent test animals in the controls should not exceed 20 % at the end of the test.
(ii) The mean number of living offspring in the control should be at least 60 per surviving adult daphnia at the end of the test.
(iii) The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be equal to or less than 25 %.
(iv) No ephippia are produced in the controls.
(v) Dissolved oxygen concentration should remain greater than 3 mg O2/L throughout the test.
(vi) pH of the controls should be within the range 6 to 9 pH units and not vary by greater than 1.5 units throughout the test.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation and reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation and reproduction

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % confidence limits 0.26 to 0.91 mg/L

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % confidence limits 0.84 to 1.7 mg/L

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits 0.60 to 0.90 mg/L

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits 1.2 to 1.5 mg/L

Details on results:

VERIFICATION OF TEST CONCENTRATIONS
- 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.
- The LOQ was determined to be 0.019 mg/L.
- These results do 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.

TEST ORGANISM OBSERVATIONS
- Observations for each test and control group are summarized in Tables 1 to 7 (attached).
- The total cumulative production of live young is given in Table 8 (attached).
- Total number of offspring per parent daphnia (for each replicate) alive at the end of the test is shown in Figure 1 (attached).
- Data for each individual replicate vessel are given in Appendices 6 to 11 (attached).

VALIDATION CRITERIA
- The validation criteria shown below were achieved during the test.
- Control mortality: 0 % (requirement ≤ 20 %)
- Mean number of live young per surviving adult (control group): 109 (requirement ≥ 60 after 21 days)
- Coefficient of variation for control group: 9.1 % (requirement ≤ 25 %)
- Ephippia produced: 0 (requirement = 0)
- Dissolved oxygen: ≥ 8.3 mg O2/L (requirement > 3 mg O2/L)
- pH (control group): 0.7 (requirement = deviation ≤ 1.5)

LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- Mortality (immobilisation) occurred predominantly at the highest test loading rate of 2.2 mg/L resulting in 90% mortality by Day 13.
- Significant mortality (immobilisation) also occurred throughout the test in the 1.0 mg/L loading rate WAF group resulting in 40% mortality by day 21 indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test item.

SUB-LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- There was a significant effect on the colour of the daphnids in that 100 % of the surviving daphnids on Day 5 at the loading rate of 2.2 mg/L loading rate W AF were markedly paler in colour than the control animals. Observations of pale daphnids were also made at a loading rate of 1.0 mg/L.
- After 21 days the length of each surviving adult was determined, the results of which are given in Table 9 (attached). The results showed that there were no statistically significant differences (P ≥ 0.05) between the control and the 0.10, 0.22, 0.45, 1.0 and 2.2 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 5, attached).

EFFECTS ON REPRODUCTION
- After 21 days there were no statistically significant differences between the control and the 0.10, 0.22, 0.45 and 1.0 mg/L loading rate WAF groups. The 2.2 mg/L loading rate WAF group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult (see Appendix 5, attached).

EFFECTS ON THE FILIAL GENERATION (F1)
- Information on the effects of the test item on the F 1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
- Young were first produced in the control test group on Day 8 of the test. Two dead young were observed in the 0.22 mg/L loading rate WAF group on day 15. There were no unhatched eggs observed throughout the test.

LOWEST OBSERVED EFFECT LOADING RATE
- The "Lowest Observed Effect Loading Rate" (LOEL) was 2.2 mg/Las this test group produced significantly fewer live young per adult (P < 0.05) than the control group.

NO OBSERVED EFFECT LOADING RATE
- The "No Observed Effect Loading Rate" (NOEL) was 1.0 mg/L as there were no significant mortalities (immobilisation) observed in the parental generation (Pl) and there were no significant differences (P ≥ 0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

MAXIMUM ACCEPTABLE TOXICANT LOADING RATE
- The "Maximum Acceptable Toxicant Loading Rate" (MATL) was calculated to be 1.5 mg/L loading rate WAF.

WATER QUALITY CRITERIA
- results of the water quality measurements are given in Table 10 (attached).
- Temperature was maintained at 20 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.
- Room temperature which was recorded approximately every hour using the Delta Building Management System ranged from 20.1 to 22.1 °C (see Figure 1, attached).
- The water hardness was observed to be in the range 236 to 274 mg/L as CaCO3 in the control and the highest surviving test group (or 1.0 mg/L loading rate WAF group on days 14 and 21) throughout the test. The results of the water hardness measurements are given in Table 11 (attached).
- Throughout the test the light intensity was observed to be in the range 573 to 662 lux (see Table 12, attached).

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.
OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- At the start of the mixing period the 1.0 and 2.2 mg/L loading rates were observed to be clear colourless water columns with test item dispersed throughout.
- After 23 hours stirring and a one hour standing period, the 1.0 and 2.2 mg/L loading rate was observed to remain as at the start of stirring.
- Microscopic inspection of the W AF after filtration showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, all loading rates were observed to be a clear, colourless solutions.

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- The 21-Day ELx (immobilization) values and associated confidence limits were calculated by Probit analysis using Linear Maximum-Likelihood regression.
- The ELx (reproduction) values and associated confidence limits after 21 days were calculated by Probit analysis using Linear Maximum-Likelihood regression.
- All results were calculated using the ToxRat Professional computer software package (ToxRat).
- For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate " (NOEL) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using Shapiro-Wilk's Test, Levene's Test and Multiple Sequentially-rejective U-test after Bonferroni-Holm (see Appendix 5, attached).
- Results from the control and each test groups daphnia length data, determined for the surviving daphnids on termination of the test, were compared using using Shapiro-Wilk' s Test, Levene's Test and Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment (see Appendix 5, attached).
- All statistical analyses were performed using the ToxRat Professional computer software package (ToxRat).
- The Maximum Acceptable Toxicant Loading Rate (MATL) was calculated using the geometric mean method and the equation MATL value = square root of (NOEL * LOEL) where NOEL = No Observed Effect Loading Rate (mg/L) and LOEL = Lowest Observed Effect Loading Rate (mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Exposure ofDaphnia magnato 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.

Executive summary:

GUIDELINE

A 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".

 

METHODS

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 a Water 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.

 

RESULTS

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.

 

CONCLUSION

Exposure ofDaphnia magnato 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.

Description of key information

Exposure of Daphnia magnato 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 (OECD 211, EU Method C.20 and OPPTS 850.1300).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

GUIDELINE

A study was performed to assess the chronic toxicity of the test item to Daphnia 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".

 

METHODS

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 magnawere 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.

 

RESULTS

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.

 

CONCLUSION

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.