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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 with GLP certificate. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

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:

no

Vehicle:

no

Details on test solutions:

The test item solution was prepared by dissolution of 100.4 mg of the test item in 1000 mL of the test medium to obtain a nominal concentration at 100 mg/L (experimental concentration = 100.0 mg/L). The solution was prepared in 2 L glass bottle and shaken for 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata
- Source: Culture at SGS Multilab Rouen, strain from SAG Gottingen collection (ID n° 61.81).
- Method of cultivation: Temperature: 23°C ± 2°C. Light: continuous, with plant growth specific spectra fluorescent bulb, between about 4440 and 8880 Lux. The algae are maintained at exponential growth rate by adding aliquots of algal culture to fresh reconstituted water samples weekly.
- Culture medium: Freshwater (reconstituted from salts)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

22.1-22.5 °C

pH:

7.9-9.4

Dissolved oxygen:

Not specified

Salinity:

Not specified

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 250 mL capacity
- Test volume: 100 mL
- Aeration: No
- Initial cells density: 5000 to 10000 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Application: The WSF for 100 mg/L loading rate was prepared and divided into 7 replicates. 6 replicates were inoculated in the range of 5000 to 10000 cells per ml. The 7th replicate was used as a colour control.

TEST MEDIUM / WATER PARAMETERS:
- Source/ preparation of dilution water: According to OECD Guideline 201

OTHER TEST CONDITIONS:
- Agitation: Yes
- Photoperiod: continuous light
- Light intensity: In a range of 5600-5700 lux

EFFECT PARAMETERS MEASURED:
- Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence at 683 nm.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Results with reference substance (positive control):

Results with reference substance (potassium dichromate) valid
- The Erc50 value based on growth rate inhibition: 1.05 mg/L

Reported statistics and error estimates:

Not specified

Cell densities

Loading rate (mg/L)	28 Mar 2018 04 : 08 pm	29 Mar 2018 04 : 22 pm	30 Mar 2018 03 : 38 pm
Control	37678	327235	2037643
	31879	304400	2028467
	32307	301288	1358463
	27539	269034	1442467
	26014	249053	1584433
	26845	302483	2174782
100	36174	322612	1868896
	29131	315251	2380580
	34976	292499	1577444
	33286	330624	2108192
	34944	315484	1844968
	27747	295463	1748289

Growth rate and yield inhibition

Loading rate (mg/L)	Growth rate (day^-1)	Growth inhibition (%)	Yield  (cells/mL)	Yield inhibition (%)
Control	1.9892		2032483
	1.9877		2023307
	1.8543		1353303
	1.8743		1437307
	1.9055		1579273
	2.0109		2169622
100	1.9604	-1.2	1863736	-5.5
	2.0409	-5.4	2375420	-34.5
	1.9040	1.7	1572284	11.0
	2.0005	-3.3	2103032	-19.1
	1.9561	-1.0	1839808	-4.2
	1.9382	-0.1	1743129	1.3

Growth rate and yield inhibition average

Nominal concentrations (mg/L)	Growth rate (day^-1)	Growth inhibition (%)	Yield (cells/mL)	Yield inhibition (%)
Control	1.9370	0.0	1765883	0.0
100	1.9668	-1. 5	1916235	-8.5

Validity criteria fulfilled:

yes

Conclusions:

In this study, test item was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values : The EC50 value with for inhibition of growth rate (ErC50) after 72 hours was > 100 mg/L. The EC10-value with for inhibition of growth rate (ErC10) after 72 hours was >100 mg/L. The NOEC-value for inhibition of growth rate after 72 hours was >= 100 mg/L.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species, Pseudokirchneriella subcapitata, was exposed to test material at a loading rate of 100 mg/L for 72 h using the WSF method (test substance shaken for 24 hours in aqueous medium then filtered through a 0.2µm Whatman cellulose nitrate membrane filter)

The study was conducted under static conditions with an initial cell density of 5000 to 10000 cells/mL. Six replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates).

The test item was measured but not detected by chemical analysis at any time during the range-finding 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. As such analysis was not performed during the definitive study.

 

In this study, test item was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values : The EC50 values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were > 100 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were >= 100 mg/L, respectively.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) > 100 mg/L based on loading rate of 100 mg/L

72h-ErC10 (Pseudokirchneriella subcapitata) > 100 mg/L based on loading rate of 100 mg/L

72h-NOECr (Pseudokirchneriella subcapitata) >= 100 mg/L based on loading rate of 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

One key study is available (SGS, 2018) to assess the influence of the registered substance on the growth of the freshwater green algae species, pseudokirchneriella subcapitata, under 72h static conditions, according to OECD guideline 201. The study carries a GLP statement.

The algae were exposed to the test substance at a loading rate of 100 mg/L for 72 h. Six replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

All validity criteria of the test guideline were fulfilled (increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates).

 

In this study, test item was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata with the following effect values : The EC50 values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were > 100 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were >= 100 mg/L, respectively.