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 aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 December 2014 to 26 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and from the bulk test preparation of each test group at 0 hours.
- Samples were also taken from the pooled replicates (replicates R1-R4) at 48 hours.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Appendix 4 (attached).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM
- 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 21 °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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Theoretical hardness of 250 mg/L as CaCO3 (see Appendix 4, attached)

Test temperature:

20 to 22 °C (see Table 3, attached)

pH:

7.8 to 8.0 (see Table 3, attached)

Dissolved oxygen:

8.7 to 8.9 mg O2/L (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

PRELIMINARY TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

PROCEDURE
- Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work (see Appendix 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE-FINDING TEST
- The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
- In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of
1.0, 10 and 100 mg/L.
- Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2.0 and 2.0 L of test water to give the 1.0, 10 and 100 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.
- The stirring was stopped after 23 hours and the mixtures allowed to stand for approximately 1 hour.
- A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down,
to a depth of approximately 5 cm from the bottom of the vessel.
- A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
- In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 to 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.
- Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

EXPERIMENTAL PREPARATION
- Nominal amounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2.0, 2.0 and 2.0 liters of test water to give the 1.0, 3.2, 10, 32 and 100 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.
- The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.
- A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel.
- A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

EXPOSURE CONDITIONS
- As in the range-finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations.
- Four replicate test and control vessels were prepared.
- The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 to 22 °C with a photoperiod of 16 hours light (509 to 524 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
- The control group was maintained under identical conditions but not exposed to the test item.
- Test preparations were not renewed during the exposure period.

EVALUATIONS
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.
- The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
- Water temperature was recorded daily throughout the test.
- Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.
- The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter.
- Temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period.

VALIDATION CRITERIA
- Results of the test are considered valid if the following performance criteria are met:
(a) No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
(b) The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate conducted between 21 July 2014 and 23 July 2014 (see Appendix 3, attached)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Appendix 5) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- The the WAF was therefore prepared using a stirring period of 23 hours followed by a one Hour settlement period.

RANGE-FINDING TEST
- Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No significant immobilisation was observed at 1.0 mg/L loading rate WAF. However, 40 %
immobilisation was observed at 10 mg/L loading rate W AF and 100 % immobilization was
observed at 10 mg/L loading rate WAF.
- In the control, 20 % immobilization was observed, the cause of which is considered to be attributable to natural causes.
- Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the
definitive test.
- Chemical analysis of the fresh test preparations at 0 hours (see Appendix 6) showed measured concentrations of less than the limit of quantification (LOQ), which was determined to be 0.15 mg/L, to 0.714 mg/L.
- Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from less than the LOQ to 0.748 mg/L indicating that the test item was stable under test conditions.

DEFINITIVE TEST
- Chemical analysis of the fresh test preparations at 0 hours (see Appendix 6) 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 LOQ (determined to be 0.15 mg/L) to 1.20 mg/L.
- The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

IMOBILISATION DATA
- Cumulative immobiliSation data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (attached).
- Analysis of the immobilisation data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the results shown in the table below.
- The No Observed Effect Loading rate after 24 and 48 hours exposure was 1.0 mg/L loading rate
WAF. Correspondingly the Lowest Effect Loading rate was considered to be 3.2 mg/L loading rate WAF.

VALIDATION CRITERIA
- The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 20 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the W AFs.
At the start of the mixing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to
be clear colourless water columns with powdered test item at the water surface and throughout the water column.
- After 23 hours stirring and a I-Hour standing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear colourless water columns with powdered test item at the water surface and settled at the bottom of the vessel.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear, colourless solutions.

Results with reference substance (positive control):

- The results from the positive control with potassium dichromate were within the normal range for the reference item.

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- The EL50 values and associated 95 % confidence limits at 24 and 48 hours were calculated by
Probit analysis using Linear Maximum-Likelihood regression.
- The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate at 24 and 48 hours were calculated using the Fisher's Exact Binomial Test with Bonferroni correction.
- All results were calculated using the ToxRat Professional computer software package (ToxRat).

RESULTS FROM PROBIT ANALYSIS OF IMOBILISATION DATA

Time (h)	EL50 (mg/L loading rate WAF)	95 % confidence limits (mg/L loading rate WAF)
24	5.2	3.7 to 7.3
48	2.0	1.5 to 2.7

Validity criteria fulfilled:

yes

Conclusions:

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.

Executive summary:

GUIDELINE

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

 

METHODS

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.

 

RESULTS

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.

 

CONCLUSION

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.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 November 2008 - 18 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, without deviations from standard test guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

A range was tested between 2.2 and 100 mg/L while a range between 1 and 100 mg/L was mentioned in amendment 3 to the protocol. A spacing factor of 2.2 is the maximum allowed according to the guideline. The study integrity was not adversely affected.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.2, 4.6, 10, 22, 46, and 100 mg/l

- Sampling method: Water samples for analysis were taken from all test concentrations and the control, with a frequency of time=0 and time=48 hours

- Sample storage conditions before analysis: Samples were analyzed on the day of the sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: Dispersion. Preparation of test solutions with individually prepared loading rates. These were treated with ultrasonic waves for 10 minutes followed by stirring for 2 days to ensure reaching maximum dissolution. The resulting dispersions were filtered through a 0.4 micron membrane filter to remove undissolved test material. This resulted in clear and colorless test solutions up to and including loading rates of 10 mg/L and slightly yellow solutions at loading rates between 22 and 100 mg/L.

- Eluate: Not applicable.

- Differential loading: Preparation of test solutions with individually prepared loading rates

- Controls: Untreated negative control; and concurrent positive control/reference toxicant

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water

- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Loading rates between 22 and 100 mg/L had a slightly yellow color.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain: Daphnia magna (Straus 1820, at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions)
- Source: In house laboratory cultures with a known history
- Age at study initiation (mean and range, SD): less than 24 h old, from parental daphnids of more than two weeks old
- Weight at study initiation (mean and range, SD): no data available.
- Length at study initiation (length definition, mean, range and SD): no data available.
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data available.
- Peripheral shell growth removed prior to test initiation: no data available.
- Method of breeding: parthenogenises
- Feeding during test : The daphnids received no food during exposure
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: from parental daphnids of more than two weeks old
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Each culture was fed daily with a suspension of freshwater algae
- Feeding frequency: daily
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress, presence of males, ephippia or discolored animals and there was no delay in the production of the first brood

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

19.6 ºC to 21 ºC

pH:

There were no treatment related differences in pH. pH was at 8 at the start of the test in controls and all test concentrations, and the pH was between 8 and 8.1 at the end of the 48 duration.

Dissolved oxygen:

There were no treatment related differences in oxygen concentration (range from 8.7 to 8.8 at start of test and 9.1 to 9.3 at end of test)

Salinity:

not applicable/freshwater

Nominal and measured concentrations:

Nominal loading rates of 2.2, 4.6, 10, 22, 46, and 100 mg/l.
Corresponding to average measured concentrations of 0.009, 0.026, 0.049, 0.058, 0.026, and 0.045 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass vessels
- Type (delete if not applicable): closed

- Material, size, headspace, fill volume: 100 mL glass vessels containing 80 mL of test solution.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): static.

- No. of organisms per vessel: 5 per vessel; 20 per concentration
- No. of vessels per concentration (replicates): 20 per concentration; 4 replicates
- No. of vessels per control (replicates): 4 vessels in control; 5 organisms per vessel
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Test organisms were introduced to the test solutions within 20 minutes after their preparation.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted ISO medium from purified tap water; including M7 medium
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No difference
- Intervals of water quality measurement: N/A

OTHER TEST CONDITIONS
- Adjustment of pH: NA
- Photoperiod: 16 hours light and 8 hours darkness cycle
- Light intensity: NDA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At 24 hours and 48 hours, the criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim within approximately 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: NA
- Range finding study. A limit test was used
- Test concentrations: nominal loading rate of 100 mg/l for limit test
- Results used to determine the conditions for the definitive study: A total of 95% of the daphnia exposed to the filtered 100 mg/L loading rate became immobilized during the test period. Based on this information loading rates of 2.2, 4.6, 10, 22, 46, and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval was 4.6 - 10 mg/L loading rate

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: the 95% confidence interval was 0.026 - 0.049 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: NDA
- Observations on body length and weight: NDA
- Other biological observations: NDA
- Mortality of control: 0
- Other adverse effects control: None noted
- Abnormal responses: NDA
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Loading rates between 22 and 100 mg/L had a slightly yellow color whereas loading rates up to 10 mg/L were clear and colorless
- Effect concentrations exceeding solubility of substance in test medium: N/A

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Mortality: immobility was observed at concentration ≥ 0.32 mg/L
- EC50/LC50:
Time (h) 24
EC50 (mg/l) 0.57 mg/L
95% Confidence interval (mg/l) = 0.52 to 0.66

Time (h) 48
EC50 (mg/l) = 0.35
95% Confidence interval (mg/l) = 0.31 and 0.40

- Other: The responses are within the ranges of the expected concentrations at the different concentrations and are in agreement with historical data

Reported statistics and error estimates:

No data available

Cumulative Immobilisation data in the Limit Test

Please see Table 1 JPEG file attached

 

Cumulative Immobilisation Data in the Definitive Test

Please see Table 3 JPEG file attached

 

Chemical analysis of test loading rates (Please see Table 2 JPEG file attached for Measured Exposure Concentrations)

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. A concentration dependant general trend was not 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. 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of 6.8 mg/L nominal loading rate with 95% confidence intervals of 4.6 - 10 mg/L loading rate. The No Observed Effect Loading rate at 48 hours was 4.6 mg/L nominal loading rate.

Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test material to daphnia magna followed that described in the OECD 202.

METHODS

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 ºC to 21 ºC 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.

RESULTS

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.

CONCLUSION

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.

Description of key information

Following exposure of the freshwater invertebrate, Daphnia magna to the test item in the key study, 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 (OECD 202 and EU Method C.2). An earlier supporting study based on nominal loading rates reported an EL50 value of 6.8 mg/L (OECD 202).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2 mg/L

Additional information

Key study

GUIDELINE

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

METHODS

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. 

RESULTS

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.

CONCLUSION

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.

 

Supporting study

GUIDELINE

A study was performed to assess the acute toxicity of the test material to daphnia magna followed that described in the OECD 202.

METHODS

Following a preliminary limit test, twenty daphnids (4 replicates of5 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 ºC to 21 ºC 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.

RESULTS

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.

CONCLUSION

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.