1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-13 to 2018-05-29

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)

Version / remarks:

updated 13th April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

April 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Date of Production: June 16, 2015
Expiry date: June 16, 2020

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE SAMPLES

Sampling and analysis date:
September 13, 2017 Start of experiment,
Start of 1st renewal period
September 14, 2017 End of the 1st renewal period
Start of 2nd renewal period
September 15, 2017 End of the 2nd renewal period
End of experiment

Four replicate samples were analysed from the test solutions at the start and at the end of the renewal periods.

Four replicate samples were analysed from the control as well.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the water-accommodated fraction (WAF) method as described in OECD Guidance Document No. 23. A supersaturated solution (100 mg test material/L nominal loading) was prepared by adding the appropriate amount of the test item to the test medium (0.0512 g test item in 512 mL ISO medium on day -1 and 0.0501 g test item in 500 mL ISO medium on day 0). The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred rigorously for a period of 24 hours to achieve an equilibrated concentration on each occasion. The test solution was then filtrated through a membrane filter (0.45 µm; Thermo Nalgene® membrane) to separate the possible non-dissolved test material on both occasion. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period).
- Controls: Positive control, negative control
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: The daphnids were bred in the Laboratory. Originally they were obtained from Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Health: Apparently healthy animals were used in this test with a known history
- Feeding during test: No. The daphnids were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

ACCLIMATION
Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Animals were exposed to the test concentration or included as control over a period of 48 hours. The test animals were not fed during the test. Immobility or mortality was observed by visually after 24 and 48 hours.

Post exposure observation period:

Not preformed.

Hardness:

The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3). The measured hardness of the ISO medium used in the study was 213.6 mg/L (as CaCO3).

Test temperature:

20.0 - 20.4°C

pH:

7.95 - 8.18

Dissolved oxygen:

7.27 - 8.06 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.193 mg/L (geometric mean; saturation concentration in the test medium, based on water-accommodated fractions of the test item)

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type: open
- Material, size, fill volume: Glass; nominal volume: 50 mL; fill volume: 40 mL
- Aeration: No
- Renewal rate of test solution:
September 13, 2017 Start of experiment,
Start of 1st renewal period
September 14, 2017 End of the 1st renewal period
Start of 2nd renewal period
September 15, 2017 End of the 2nd renewal period
End of experiment

- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: According to guideline (ISO Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness.
- Light intensity: Artificial illumination

EFFECT PARAMETERS MEASURED: Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY
- Test concentrations: 0.1 , 1, 10, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the non-GLP Preliminary Range-Finding Test (see above), the main test was a limit test performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including only one test concentrationperformed at saturation limit (equivalent to 100 mg/L nominal concentration) and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

> 0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

BIOLOGICAL RESULTS AND PERFORMANCE OF THE TEST
Testing units were kept in a climate chamber at a temperature of 19.9 – 20.8 °C, while temperature in the test glasses ranged between 20.0 - 20.4 °C. The pH was measured as 7.95 - 8.18 and the dissolved oxygen concentration between 7.27 - 8.06 mg/L during the study. The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness. There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected (see Table 7 of Appendix I).

VALIDITY OF THE STUDY
Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 7.27 - 8.06 (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Results with reference substance (positive control):

The date of the latest study with reference item Potassium dichromate was: 05 – 06 September 2017. The 24h EC50 value was determined to be 1.53 mg/L (95% conf. limits: 1.30 – 1.78 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Reported statistics and error estimates:

A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary. The NOEC, LOEC and ECx values of the test item were determined directly from the raw data.

Table 1: Immobilization of the test animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
Saturated test concentration (0.193 mg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

 

Table 2: Brown 3 concentrations measured during the study

Mean of the measured concentrations (mg/L) with the 95% confidence intervals (n=4)	Mean of the measured concentrations (mg/L) with the 95% confidence intervals (n=4)		Measured concentration in % of the start
First renewal period
Start (September 13, 2017)		End (September 14, 2017)
0.287±0.053		0.113±0.014		39
Second renewal period
Start (September 14, 2017)		End (September 15, 2017)
0.314±0.040		0.136±0.037			43

The ISO control samples show a small absorbance. The amount of any interfering reagent component was less than 20 % of the lowest concentration of the calibration curve.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than the solubility limit of the test item in the test medium, which corresponds to a measured concentration of 0.193 mg/L and a nominal loading rate of 100 mg/L.

Executive summary:

The acute toxicity of the test item was assessed on Daphnia magna in a GLP-compliant Acute Immobilisation Test according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010. Young daphnids were exposed in a semi-static regime for 48 hours to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)). In addition, a postive control with the reference substance Potassium dichromate and a negative control (only ISO Medium) was conducted. The quantification of the test item was performed by UV-VIS spectrophotometry. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment. The mean determined exposure concentration was 0.193 mg/L which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods. Healthy, young daphnids less than 24 hours of age were used. Twenty daphnids (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the controls. The immobilisation of the test animals was recorded 24 and 48 hours after test start. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Based on the results of this study, the test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than the solubility limit of the test item in the test medium, which corresponds to a measured concentration of 0.193 mg/L and a nominal loading rate of 100 mg/L.  All validity criteria were met and therefore the study can be considered as valid.

Description of key information

In a Daphnia sp. Acute Immobilisation Test according to OECD Guideline 202, the 48-h EC50 was higher than the solubility limit of the test item in the test medium, which corresponds to a geometric mean measured concentration of 0.193 mg/L and a nominal loading rate of 100 mg/L.

Additional information

The acute toxicity of the test item was assessed on Daphnia magna in a GLP-compliant Acute Immobilisation Test according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010. Young daphnids were exposed in a semi-static regime for 48 hours to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)). In addition, a postive control with the reference substance Potassium dichromate and a negative control (only ISO Medium) was conducted. The quantification of the test item was performed by UV-VIS spectrophotometry. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment. The mean determined exposure concentration was 0.193 mg/L which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods. Healthy, young daphnids less than 24 hours of age were used. Twenty daphnids (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the controls. The immobilisation of the test animals was recorded 24 and 48 hours after test start. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Based on the results of this study, the test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than the solubility limit of the test item in the test medium, which corresponds to a geometric mean measured concentration of 0.193 mg/L and a nominal loading rate of 100 mg/L.  All validity criteria were met and therefore the study can be considered as valid.