1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation: 20-03-2015 Study Completion: 27-07-2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Substance code: ZZYD20140123-0866
Batch: 04503/MA
Purity: >98%
Expiry Date: 2016.2
Conditions of Storage: Avoid direct heating, dirt, chemical contamination, sunlight, UV or ionising radiation
Stability under storage conditions: Stable at normal ambient temperature and pressure.
Oxidization not expected based on structure and functional groups.
logPow=13.4
Vapour Pressure: < 0.001 mm Hg (100°F); 0.1333Pa (37.7C)
Water Solubility: <1.0mg/L (test data, 25C);
2.644e-010mg/L (EPI Estimated)

Analytical monitoring:

not specified

Details on sampling:

N/A

Details on test solutions:

Synthetic sewage
A synthetic sewage was made by dissolving the following amounts of substances in 1L of ultra pure water produced by Millipore MilliQ A 10:
1) Peptone - 16g
2) Meat extract - 11g
3) Urea - 3g
4) NaCl - 0.7g
5) CaCl2.2H20 - 0.4g
6) MgSO4.7H2O - 0.2g
7) K2HPO4 - 2.8g

Solubility of test substance was <1.0mg/L as described by the sponsor. The test substance was weighed and added directly into the test vessels.

Reference substance stock solution
A solution of 0.2501g of 3,5-dichlorophenol in 250mL water was prepared. Warm water was used to accelerate the dissolution and the solution was made up to volume after cooling to room temperature. The pH of the solution was checked and adjusted with NaOH and H2SO4 to pH 7-8.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge, from a domestic sewage treatment plant is normally used as the microbial inoculums based on "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic System, 2013, 209 Activated Sludge, Respiration Inhibition Test" and "OECD Guidelines for the Testing of Chemicals, 209, Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation, 2010)". Characteristics which make activated sludge suitable for acute respiration inhibition test to activated sludge are their higher cell densities, no pre-adaption to chemicals, and their ease of giving lower scattering of results.

Sludge collection: a fresh sample of activated sludge for the range-finding was collected from the aeration stage of Longhua Wastewater Treatment Plant in Shanghai, which treated predominantly domestic sewage, on Mar 23. The sludge was aerated until use.

Sludge treatment: On return to the laboratory the sludge was washed, with an isotonic solution containing 0.85% NaCl(w/v). After centrifuging the supernatant was decanted. This procedure was repeated three times (4000rpm, 4°C, centrifuge 20min). A small amount of the washed sludge was weighed and dried by moisture meter at 105 °C for 1h to calculate the dry weight. The dry weight of sludge used in range-finding test count up to 7.78% of the gross weight, respectively.

Sludge suspension: The amount of wet sludge needed to be suspended in isotonic solution was calculated based on the dry weight. The wet sludge was then placed into solution to obtain the required sludge solids concentration of 3 g/L. Sludge was then suspended with a mixer and aerated until use. The final concentration of the activated sludge in the test medium was 1.5g/L.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Hardness:

N/A

Test temperature:

20.1 °C

pH:

pH 7-8

Dissolved oxygen:

See attached results tables 19.2 - 19.3

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

(Nominal)
Test group -
10mg/L (1 replicate)
100mg/L (1 replicate),
1000mg/L (3 replicates)

Reference substance
7.80mg/L
12.50mg/L
20.00 mg/L
32.00mg/L

Details on test conditions:

Test substance group (Fr):
The test substance was added directly with 16mL synthetic sewage feed, well mixed, ultra-pure water added till 250mL, then mixed with 250mL activated sludge suspended solution.
Final concentrations of 10mg/L (1 replicate),100mg/L (1 replicate), and 1000mg/L (3 replicates) were obtained.

Blank control group(Fs):
16ml synthetic sewage feed with ultra-pure water added up to 250mL, then mixed with 250mL activated sludge suspended solution(2 replicates).

Abiotic control(FA):
The test substance was added directly with 16mL synthetic sewage feed, well mixed, then ultra-pure water added till 500mL, The final concentration was 1000mg/L.

Reference substance group( Fa):
Reference substance stock solution was added together with 16ml synthetic sewage feed, well mixed, ultra-pure water added till 250mL, and then mixed with 250mL activated sludge suspended solution. Final concentrations of 7.80mg/L, 12.50mg/L, 20.00 mg/L, and 32.00mg/L were obtained.

Procedure
The range-finding test was performed,
FT = test substance group;
FB = blank controls;
FA = abiotic control;
FR = reference substance group (3,5-dichlorophenol).

Test systems were incubated in 1000mL glass vessels at the test temperature under forced aeration (ca. 1L air/minute) by glass tube, to maintain the sludge flocs in suspension.

After a 3 h exposure period, sample were transferred from the aeration vessel to BOD flask and self-stirred immediately, the concentration of dissolved oxygen was then measured. The electrode was removed, and the mixture returned to the aeration vessel and aerated and stirred.

The pH of the control, reference substance and test substance were measured at the beginning and end of contact time.

Reference substance (positive control):

yes

Remarks:

A solution of 0.2501g 3,5-dichlorophenol in 250mL water was prepared. Warm water was used to accelerate the dissolution and the solution made up to volume after cooling to room temperature. pH was checked and adjusted with NaOH and H2SO, to pH 7-8.

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: There was no respiration inhibition effect of the test substance on activated sewage sludge. 3h IC50 was greater than 1000mg/L. The definitive test was unnecessary.

Details on results:

The respiration rate of FA (Abiotic Control) was -0 .18mg/L, no abiotic respiration was observed.

Respiration inhibition percentage of the test substance on the respiration of activated sewage sludge were 18.66%, 4.14% and 9.34% under the concentration of 1000mg/L in the range-finding test. t-Test indicated no significant differences between range-finding test group and blank control (p=0.16, p>0.05). There was no respiration inhibition effect of the test substance on the activated sewage sludge.
3h IC50 was greater than 1000mg/L. (Table 19.3).

The definitive test was unnecessary.

Results with reference substance (positive control):

The respiration inhibition of 3h IC50 of reference substance was 11.64mg/Lh, 95%
confidence limit was 10.87~12.41 .

Reported statistics and error estimates:

N/A

N/A

Validity criteria fulfilled:

yes

Remarks:

Validation described in further detail in method section.

Conclusions:

3h IC50 of 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DODECYL AND OCTYL TRIESTERS inhibiting respiration of activated sludge was >1000mg/L.

Executive summary:

The study was conducted according to OECD Guidelines for the Testing of Chemicals, 209, Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation, 2010)".

No respiration inhibition effect of the test substance on the respiration of activated sewage sludge was observed in the range-finding test of 1000mg/L, 3h IC50 was greater than 1000mg/L. The definitive test was therefore not necessary. 

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: O2 consumption test (Huels method)

Principles of method if other than guideline:

The "Oxygen Consumption Test" offers the possibility of measuring chronic toxicity effects in bacteria with difficult substances e.g. those which are sparingly soluble, coloured, emulsifying or dispersive, even slightly volatile products can be tested.
Principle of the test: Glass vessels (e.g. 100 mL BOD-flasks) with known volume will be completely filled with culture solution, bacterial suspension and test substance in different concentrations (emulsified if insoluble in water), bubble-free sealed with glass stoppers and incubated for 5 to 6 hours at 25 ± 2°C on a shaking machine. Test vessels without bacterial suspension and vessels without test substance serve as controls. The differences between the oxygen content in the vessels at the beginning and the end of the incubation time is a measurement for the bacterial oxygen consumption.

GLP compliance:

yes

Specific details on test material used for the study:

N/A

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2.0g of the test substance was weighed in 20 mL neutralised emulsifier and emulsified in a shaking incubator at 25 °C for 19h at 200 rpm. 1 mL of this test solution was given into the test vessel (100 mL) for the highest test concentration (1000 mg/L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).
- Controls: yes
- Chemical name of vehicle (emulsifier): Nonylphenol plus 10 EO plus 5 PO
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1mL/100mL
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not mentioned

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: not described
- Preparation of inoculum for exposure: approx. 19h incubation of liquid culture of test organism
- Pretreatment: 200mL stock solution I and II each were filled in an sterile 5 L-flask, containing 3520mL deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80mL pre-culture were added and additionally 5 min stirred and areated.
- Initial biomass concentration: approx. 10 x 10^5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.306)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4.75 h

Remarks on exposure duration:

The incubation time is slightly below the scheduled value, but this had no influence on the oxygen consumption (4.5 mg O2/L)

Post exposure observation period:

none

Hardness:

not mentioned

Test temperature:

Preculture: 24.8 - 24.9 °C
Incubation: 25.4 - 26.5 °C

pH:

not mentioned

Dissolved oxygen:

see "Remarks on results"

Salinity:

see "Details on test conditions"

Conductivity:

N/A

Nominal and measured concentrations:

1, 10, 100 and 1000 mg/L (nominal concentrations)

Details on test conditions:

INOCULUM/TEST ORGANISM
- Species/strain: Pseudomonas putida migula
- Source: Dr. Reinhard Kanne, Bayer AG, Leverkusen, Germany
- Method of cultivation: not described
- Preparation of inoculum: approx. 19h incubation of liquid culture of test organism
- Pretreatment: 200 mL stock solution I and II each were filled in an sterile 5 L-flask, containing 3520 mL deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80 mL pre-culture were added and additionally 5 min stirred and areated.
- Cell concentration of the inoculum: approx. 10 x 10^5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.306)

TEST SYSTEM
- Number of culture flasks:
a) 4 flasks per concentration with test substance, 2 of this flasks were used for determination of autooxidation of the test substance by adding of 1 mL HgCl2 solution, final concentration in the flasks approx. 3 mg/L

b) 4 flasks without test substance but with HgCl2 addition for determination of the oxygen content at the beginning of the test

c) 5 flasks without test substance, without HgCl2 for determination of the uninfluenced bacterial oxygen consumption. 1 flask of these was used to measure the oxygen contents after 4.5 h to determine the end of incubation. This value was not used for evaluation.

- Measuring equipment: oxygen electrode
- Termination of test: 1 mL HgCl2 solution (final concentration approx. 3 mg HgCl2/L) were added to each test vessel to stop the reaction.

TEST SUBSTANCE CONCENTRATIONS: 1, 10, 100 and 1000 mg/L
- Preparation of test substance solutions: 2.0 g of the test substance was weighed in 20 mL neutralised emulsifying solution (Nonylphenol, 10 EO, 5 PO) and emulsified in a shaking incubator at 25 °C for 19 h at 200 rpm. 1 mL of this test solution was given into the test vessel (100 mL) for the highest test concentration (1000 mg/L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).

DURATION OF THE TEST: 4.75 h

ANALYTICAL PARAMETER: O2 consumption

TEST CONDITIONS
- Composition of solutions:
a) Trace element solution (autoclaved), per 500 mL distilled water:
0.0275 g Al2(SO4)3 x n H2O
0.014 g KJ
0.014 g KBr
0.0275 g TiO2
0.014 g SnCl2 x 2 H2O
0.014 g LiCl
0.1945 g MnCl2 x 4 H2O
0.307 g H3BO3
0.0275 g ZnSO4 x 7 H2O
0.0275 g CuSO4 x 5 H2O
0.0295 g NiSO4 x 6 H2O
0.0275 g Co(NO3)2 x 6 H2O
b) Stock solution I (sterilized):
20 g D(+)Glucose
4.24 g Na NO3
2.40 g K2HPO4
1.20 g KH2PO4
30 mL Trace element solution (a)
1000 mL distilled water
c) Stock solution II (steril filtrated):
0.200 g FeSO3 x 7 H2O
3.71 g MgSO4 x 6 H2O
made up to 1 L with distilled water
d) NaCl solution (autoclaved):
0.500 g NaCl made up to 1 L distilled water

Reference substance (positive control):

no

Key result

Duration:

4.75 h

Dose descriptor:

EC10

Effect conc.:

> 0.98 g/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The test substance did not affect the bacterial respiration in the tested concentration range:
EC10 > 0.98 g act. ingr./L
EC50 > 0.98 g act. ingr./L

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: not mentioned

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

No statitistics performed

Oxygen concentration

	control	1 mg/L	10 mg/L	100 mg/L	1000 mg/L
Initial average	8.24	8.32	8.08	8.24	8.30
Average after incubation	3.74	3.71	3.77	3.66	3.53
O2 consumption during incubation	4.50	4.61	4.31	4.58	4.78
Absolute difference		-0.11	0.19	-0.08	-0.28
Relative difference [%]		-2.4	4.3	-1.7	-6.2

All oxygen concentrations are given in mg O₂ / L

Validity criteria fulfilled:

yes

Conclusions:

The test substance did not affect the bacterial respiration in the tested concentration range.

Executive summary:

The study of the bacteriotoxicity of the structurally related substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl esters was carried out by a method developed by the testing laboratory. Principle of the method: 100 mL BOD bottles closable by means of ground stoppers are to be charged with the culture solution, the suspension of bacteria (Pseudomonas putida Migula) and the test substance in stepped concentrations, closed so as to leave no air bubbles and incubated for 5 -6 hours at 25 ± 2 °C in a shaking incubator.

Batches free of test substance serve as control. The difference between the oxygen content of the solutions in the individual containers at time zero and after the incubation time gives the bacterial oxygen consumption. Comparison of the amounts of oxygen consumed in the control and test batches gives information about the concentration-dependent influence of the test substance on the oxygen consumption.

For the test 2.0 g of the test substance was weighed in 20 mL neutralised emulsifier (nonylphenol 10 EO, 5 PO) and emulsified in a shaking incubator at 25 °C for 19 h at 200 rpm. 1 mL of this test solution was given into the test vessel (100 mL) for the highest test concentration (1000 mg/L, corresponding to 980 mg a.i./L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).

The structurally related substance did not affect the bacterial respiration in the tested concentration range (1, 10, 100 and 1000 mg/L; corresponding to 1, 9.8, 98 and 980 mg a.i./L),: EC10 > 0.98 g a.i./L, EC50 > 0.98 g a.i./L.

Description of key information

The toxicity of 1,2,4 -benzenetricarboxylic acid, mixed dodecyl and octyl triesters was conducted according to OECD Guidelines for the Testing of Chemicals, 209, Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation, 2010)". No respiration inhibition effect of the test substance on the respiration of activated sewage sludge was observed in the range-finding test of 1000mg/L, 3h IC50 was greater than 1000mg/L. The definitive test was therefore not necessary. 

Furthermore, data are available for a key study on the read across material 1,2,4-Benzenetricarboxylic acid, decyl octyl ester. The study from Diefenbach R (1994)of the bacteriotoxicity of the structurally related substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl esters was carried out by a method developed by the testing laboratory. The structurally related substance did not affect the bacterial respiration in the tested concentration range (1, 10, 100 and 1000 mg/L; corresponding to 1, 9.8, 98 and 980 mg a.i./L),: EC10 > 0.98 g a.i./L, EC50 > 0.98 g a.i./L

The available information demonstrates a lack of toxic effects in the range of the water solubility.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

0.98 g/L