Benzene-1,3,5-tricarboxylic acid

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:

2017-05-12 to 2017-08-23

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified 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 test vesels.

Vehicle:

no

Details on test solutions:

The stock solution (100 mg/L of the test item were weighed out) was prepared with dilution water (see Table 1) one day prior to the start of the exposure (at -24hours). The stock solution was strirred with a magnetic stirrer appproximately 1100 rpm for 24 hours at romm temperature. 5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution of 100 mg/L with dilution water (see Table 1), were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100 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: 7.59
Test solutions: 7.20 to 7.54

Dissolved oxygen:

Control: 9.07 mg/L
Test solutions: 8.48 to 8.96 mg/L

Nominal and measured concentrations:

nomimal : 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
measured: 6.56- 13.0- 25.3- 52.1- 104 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): loosely covered with watch glasses
- Material, size, headspace, fill volume: Glass beakers (4 (10) x 7 (H) cm), 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: 448 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: yes in the highest concentration of 100 mg/L by addition of 1 M NaOH prior to start of the exposure.
- 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] IMMOBILIZATION [%]
24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
100 100 100 100 100% mortality after 24 hours
10 0 0 0 0 0 0
1 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.:

70.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: pH not adjusted

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: pH adjusted for neutralisation

Details on results:

Other effects were not observed

Results with reference substance (positive control):

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Current Study Valid Range
EC50 1.60 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 1.43- 1.79mg/L

Test duration: 2017-05-04 to 2017-05-05

Measured Concentrations and Percent of Nominal Concentration of the Test ltembenzene-1,3,5-tricarboxylicacidduring the DefinitiveTest

 

Samplingdate	2017-06-07 Start of the exposure, 0hours		2017-06-09 End of the exposure, 48hours
Start ofanalysis	2017-06-07		2017-06-09
Nominal test itemconcentration [mg/L]	benzene-1,3,5-tricarboxylic acid
	Meas. conc.rmCl/Ll	%	Meas. conc.[mC1/Ll	%
100*	107	107	104	104
100	106	106	105	105
50.0	51.8	104	52.1	104
25.0	26.0	104	25.3	101
12.5	13.2	105	13.0	104
6.25	6.57	105	6.56	105
Control	<LOQ		<LOQ

 Meas.conc.=measuredconcentrationofthetestitem,meanvalueof2injections,dilutionfactorstakenintoaccount

% = percent of the nominal concentration of the test ite

LOQ = limit of quantification of the analytical method (0.300 mg/L of the test   item)

* =pH-neutralised

Absolute Numbers of immobile Daphnids after 24 and 48h of Exposure in the DefinitiveTest (n=20,divided into 4 replicates with 5daphnids each)

benzene-1,3,5-

NUMBER

OF IMMOBILEDAPHNIDS

ITOTAL NUMBEROF

DAPHNIDS

tricarboxylicacid

24h

48h

Nominal

Replicates

Replicates

testitem

1

2

3

4

MV

1

2

3

4

MV

concentration

rmg/LJ

100*

0/

5

0 /5

0 /5

0 /5

0 /20

0 /5

0 /5

0 /5

0 /5

0 /20

100

5/

5

5 /5

5 /5

5 /5

20 /20

5 /5

5 /5

5 /5

5 /5

20 /20

50.0

0/

5

0 /5

0 /5

0 /5

0 /20

0 /5

0 /5

0 /5

0 /5

0 120

25.0

0/

5

0 /5

0 /5

0 /5

0 /20

0 /5

0 /5

0 /5

0 /5

0 /20

12.5

0/

5

0 /5

0 /5

0 /5

0 /20

0 /5

0 /5

0 /5

0 /5

0 /20

6.25

0/

5

0 /5

0 /5

0 /5

0/20

0 /5

0 /5

0 /5

0 /5

0 /20

Control

0/

5

0 /5

0 /5

0 /5

0120

0 /5

0 /5

0 /5

0 /5

0 /20

Validity criteria fulfilled:

yes

Conclusions:

In this study, no biological effects occurred in the tested concentration range of 6.25 mg/L -100 mg/L. Thus, the test item is not toxic.
The validity criteria of the test guidelines were fulfilled.

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 of 2, prepared by dilution of the stock solution of 100.0 mg/L with dilution water, were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100 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 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 104 to 107% of the nominal values at the start of the exposure (0 hours) and 101 to 105% of the nominal values at the end of the exposure (48 hours).

Based on the nominal concentrations of the test item the 48 hour-EC₅₀ for Daphnia magna > 100 mg/L.

Description of key information

In the acute immobilization test withDaphnia 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 of2, prepared by dilution of the stock solution of100.0 mg/Lwith dilution water, were tested as follows:6.25 - 12.5 - 25.0 - 50.0 - 100 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 itemwereanalytically verifiedvia 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 104 to 107% of the nominal values at the start of the exposure (0 hours) and 101 to 105% of the nominal values at the end of the exposure (48 hours).

Based on the nominal concentrations of the test item the 48 hour-EC₅₀for Daphnia magna >100 mg/L for adjusted pH and 70.7 mg/L for non-adjusted pH

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Based on the nominal concentrations of the test item the 48 hour-EC₅₀for Daphnia magna was >100 mg/L for adjusted pH and 70.7 mg/L for non-adjusted pH