2,4,6-trimethylbenzene-1,3-diamine

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:

20-09-2016 to 28-04-2017

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:

2004

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all concentration levels and in the control. At the start of the exposure (0 hours), sampling was carried out after preparation of the test item concentrations. At the end of the exposure (48 hours), samples of the old media were taken from additional replicates, which were prepared with test media, but without daphnids and incubated under test conditions until sampling.

Vehicle:

no

Details on test solutions:

A stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with dilution water (see Table 2) prior to the start of the exposure (at 0 hours). The stock solution was mixed thoroughly by manual agitation. 5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution of 10.0 mg/L with dilution water, were tested as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L.
The test concentrations were selected based on the results of a non-GLP preliminary range finding test.
Control: Dilution water without test item incubated under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test. Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

18 - 22 °C, constant within ± 1 °C

pH:

Control: 7.55
Test solutions: 7.46 to 7.52

Dissolved oxygen:

Control: 9.49 mg/L
Test solutions: 9.27 to 9.34 mg/L

Nominal and measured concentrations:

0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: PE beakers, 110 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 8.5 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start:602 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was determined in all groups after 24 h and 48 h.

TEST CONCENTRATIONS
- Range finding study:
Immobilization Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 0 0 0 0 0 0
10.0 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

Test concentrations 0.500 – 1.00 – 2.00 – 4.00 mg/L

Current Study Valid Range
EC50 0.964 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 0.551 - 1.00 mg/L



Test duration: 2016 12-13 to 2016-12-14.

Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2016-12-20 Start of the exposure, 0 hours		2016-12-22 End of the exposure, 48 hours
Start of analysis	2016-12-21		2016-12-22
Nominal test itemconcentration [mg/L]
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
10.0	11.2	112	11.3	113
5.00	5.56	111	5.69	114
2.50	2.67	107	2.77	111
1.25	1.33	106	1.37	110
0.625	0.728	116	0.687	110
Control	< LOQ		< LOQ

Validity criteria fulfilled:

yes

Conclusions:

Based onthe nominal concentrations of the test item the 48 hour-EC50 for Daphnia magna was 2.52 mg/L (95% confidence limits: 2.28 - 2.80 mg/L) and thus the test item is considered to be toxic to aquatic invertebrates.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004), which is equivalent to the Council Regulation (EC) No.440/2008 Method C.2 (2008). The study was conducted under static conditions over a period of 48 hours. Five test item concentrations in a geometric series with a separation factor o 2, prepared by dilution of the stock solution of 10.0 mg/L with dilution water, were tested as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L.Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the test item were analytically verified via via HPLC-DAD at the start (0 hours) and at the end of the exposure (48 hours) in all concentration levels and in the control.

The measured concentrations of the test item were in the range of 106 to 116% of the nominal values at the start of the exposure (0 hours) and 110 to 114% of the nominal values at the end of the exposure (48 hours).

Based onthe nominal concentrations of the test item the 48 hour-EC₅₀ for Daphnia magna was 2.52 mg/L (95% confidence limits: 2.28 - 2.80 mg/L).

Description of key information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004), which is equivalent to the Council Regulation (EC) No.440/2008 Method C.2 (2008). The study was conducted under static conditions over a period of 48 hours.Five test item concentrations in a geometric series with a separation factor o2, prepared by dilution of the stock solution of 10.0 mg/L with dilution water, were tested in the concetration range 0.625 to 10.0 mg/L

Based onthe nominal concentrationsof the test item the 48 hour-EC50 for Daphnia magnawas 2.52 mg/L (95% confidence limits: 2.28 - 2.80 mg/L) indicating that the text item is toxic to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.52 mg/L