2,4,6-trinitrobenzene-1,3,5-triamine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP certificate. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate of compliance with good laboratory practice according to directives 2004/9/CE and 2004/10/CE

Specific details on test material used for the study:

- Storage condition of test material: Room temperature, away from light

Analytical monitoring:

yes

Remarks:

All samples found to be

Vehicle:

no

Details on test solutions:

The test item solution was prepared by dissolution of 50.5 mg of the test item in 500 mL of the test medium to obtain a nominal concentration at 100 mg/L (experimental concentration = 100.8 mg/L). The solution was prepared in 1 L glass bottle and shaken during about 24h. Then this preparation was filtered through 0.2 µm filter (Whatman cellulose nitrate membrane filters). This solution was used for exposure and identified as 100mg/L loading rate (WSF).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS
- Breeder: Culture at SGS France Laboratoire de Rouen
- Age at the beginning of the test: Daphnids less than 24 hours old and produced from parthenogenically reproducing brood female population.
- Culture condition: Temperature: at 20°C ± 2°C, Light: light/dark cycle 16/8 hours, Freshwater (reconstituted from salts).

Every working day, daphnids were filtered on the following sieves (200 µm, 560 µm and 800 µm). Daphnids which pass through the 800 µm sieve and remain on the 560 µm sieve were used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

19.6-20.0 °C

pH:

7.8-7.9

Dissolved oxygen:

7.3-7.4 mg/L

Salinity:

None

Conductivity:

Not specified

Nominal and measured concentrations:

Loading rate of 100 mg/L

Details on test conditions:

TEST METHOD:
Static

TEST SYSTEM:
- Test vessels: glass flask 125 mL capacity
- Test volume: 50 mL
- Aeration: No
- Type of flow-through (e.g. peristatic or proportional diluter): Not applicable
- Renewal of test solutions: None
- No. of organisms per vessel: 5 (control and test material groups)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Application: Limit test at approximately 100 mg/L loading rate (WSF weighed exactly) was prepared and was divided in 4 replicates. A control (4 replicates) was performed at the same time with only test medium. The test was conducted in 125mL glass flasks with 50 mL solution per flask. Five daphnids were introduced into each replicate.

TEST MEDIUM / WATER PARAMETERS:
- Dilution water: ISO Test water, according to OECD 202
- Culture medium: Freshwater (reconstituted from salts).
- Conductivity: Not specified

OTHER TEST CONDITIONS:
- Photoperiod: light/dark cycle 16/8 hours.
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED:
Biological parameters:
- Immobilisation and other observations: Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made

Water Quality Parameters:
Dilution water: Prior to the start of the exposure (0 h) the water quality parameters (i.e, pH value, dissolved oxygen concentration, temperature) of the dilution water were measured.
Test media: the water quality parameters (i.e, pH value, dissolved oxygen concentration, temperature) of the dilution water were measured the day of preparation.
Temperature: The incubator temperature was recorded throughout the period of the test.

TEST CONCENTRATIONS
loading rate of 100 mg/L (Water Soluble Fraction)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- BIological data: Immobilization of daphnids was 0% at 24 and 48h at a loading rate of 100 mg/L

- Measured Exposure Concentrations during the definitive test: The concentration of the test item was measured during the test item solution preparation: Nominal concentration of 100 mg/L (experimental concentration = 100.8 mg/L).

- Water Quality Parameters: The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.43 mg/L

Reported statistics and error estimates:

Not specified

Ecotoxicity test results

 

 

Data at 24 h

 

Loading rate (mg/L)	Daphnids immobilised per glass flask*				Total of immobilised daphnids	Percentage of immobilisation	pH	Dissolved O2 (mg/L)
	Replicate A	Replicate B	Replicate C	Replicate D
Control	0	0	0	0	0	0%	7.8	8.3
100	0	0	0	0	0	0%	7.8	7.3

* 5 daphnids per glass flask

 

Data at 48 h

 

Loading rate (mg/L)	Daphnids immobilised per glass flask *				Total of immobilised daphnids	Percentage of immobilisation	pH	Dissolved O2 (mg/L)
	Replicate A	Replicate B	Replicate C	Replicate D
Control	0	0	0	0	0	0%	7.9	8.7
100	0	0	0	0	0	0%	7.9	7.4

* 5 daphnids per glass flask

 

The daphnids immobilisation was determined at 0% for the 100 mg/L loading rate after 48 hours.

EC50-48H > 100 mg/L (loading rate)

Validity criteria fulfilled:

yes

Conclusions:

Based on the loading rate concentration of the test item, the 48 h-EC50 to Daphnia magna was > 100mg/L.

Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test substance to Daphnia magna, under static conditions.

The daphnids were exposed to the test item at the loading rate of 100 mg/L for 48 hours using the WSF method (test susbtance shaken in aqueous medium for 24 hours then filtered through a 0.2µm Whatman cellulose nitrate membrane filter).

This method was used as the solubility of the test substance was unknown but was found to be inferior to 0.5 µg/L.

The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 19.6 -20.0 °C. The tested solutions were observed to be clear throughout the exposure period.

The concentration of the test item was measured at the start of the study. The test item was not detected during chemical analysis at any time during the test but this was expected due to the extremely low solubility of the test item (determined as < 0.5 µg/L) which is below the limit of quantification of the method.

 

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

Immobilization of daphnids for the definitive test was 0% (24 h); 0% (48 h) at the loading rate of 100 mg/L

The EC50 -value of the reference item, potassium dichromate at 1.43 mg/L after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

 

Based on the loading rate concentrations of the test item, the 48 h-EL50 to Daphnia magna was > 100 mg/L.

Description of key information

OECD Guideline 202, EU Method C.2, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) > 100 mg/L based on loading rate of 100 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

One key study is available (SGS, 2018) to assess the 48h acute toxicity of the registered substance to Daphnia magna, under static conditions, according to OECD Guideline 202 with GLP statement.

The daphnids were exposed to the test item at the loading rate of 100 mg/L for 48 hours. This method was used as the solubility of the test substance was unknown but was found to be inferior to 0.5 µg/L. The control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 19.6 -20.0 °C. The tested solutions were observed as clear throughout the exposure period.

The concentration of the test item was measured at the start of the study. The test item concentrations in this relevant range were successfully maintained for the duration of the test. 

 

The water quality parameters (i.e. pH-values and dissolved oxygen concentrations), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

Immobilization of daphnids for the definitive test was 0% (24 h); 0% (48 h) at the loading rate of 100 mg/L

The EC50 -value of the reference item, potassium dichromate at 1.43 mg/L after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

 

Based on the loading rate concentrations of the test item, the 48 h-EL50 to Daphnia magna was > 100 mg/L.