Tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-18 - 2018-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

yes

Remarks:

The pH of the first control and two reference item concentrations were not measured at the start of the test. The validity criteria were met and therefore it can be concluded that this deviations has no effect on the outcome of the study.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts on an hourglass were added to the dark brown test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Remarks:

Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 19 and 21°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

The pH in all test vessels, before addition of sludge was between 7.4 and 7.9. After the 3 hour exposure period the pH was between 7.2 and 8.2.

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

Salinity:

not applicable

Nominal and measured concentrations:

Combined limit/range-finding test
Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the six controls.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The combined limit/range-finding test showed no inhibition of the respiration rate at a concentration of 10 and 100 mg/L and an average of 2% inhibition of the respiration rate at a concentration of 1000 mg/L.

Results with reference substance (positive control):

3,5-dichlorophenol
EC50 (3h) = 6.3 mg/L
95% CI: 5.1 - 7-5 mg/L

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

Results - Respiration Rate/Inhibition, pH Values

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	^2	8.0	30.66	20.44
C 2	0	7.4	7.9	33.80	22.53
C 3	0	7.4	7.7	39.16	26.11
C 4	0	7.4	7.7	40.56	27.04
C 5	0	7.4	7.7	42.20	28.13
C 6	0	7.4	7.7	36.46	24.31
C Mean				37.14	24.76 (RC)
SD				4.36	2.91
CV (%)				12	12
R 1	1.0	^2	7.9	33.91	22.61	8.70
R 2	3.2	^2	8.1	26.04	17.36	29.89
R 3	10	7.4	8.1	12.82	8.55	65.48
R 4	32	7.5	8.2	5.91	3.94	84.09
T 1	10	7.4	7.8	38.49	25.66	-3.63
T 2	100	7.4	7.7	39.37	26.25	-6.00
T 3a	1000	7.4	7.7	39.74	26.49	-7.00
T 3b	1000	7.4	7.8	34.13	22.75	8.10
T 3c	1000	7.4	7.8	34.97	23.31	5.84
T3 Mean				36.28	24.19 (RT)	2.32 (IT)
TA	1000	7.4	7.2	0.13	0.09	99.65

Glossary

C: Control

R: Reference item, 3,5-dichlorophenol

T: Test item, Lexolube1 - Pyromellitate

TA: Abiotic control of Lexolube1 - Pyromellitate

SD: Standard deviation

CV: Coefficient of variation

¹  The amount of suspended solids in the final test mixture was 1.5 g/L.

² The pH was not measured

R_C: Total respiration in the control

R_T: Total respiration with Lexolube1 - Pyromellitate

I_T: % inhibition of total respiration relative to R_C

Interpretation of results

EC_x

For the reference item, calculation of the EC₅₀value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.

For Lexolube1 - Pyromellitate, no EC₅₀value could be calculated because the test item proved to be non-toxic (EC₅₀> 1000 mg/L)

NOEC determination

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions^®GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the test item was not toxic to waste water bacteria (activated sludge) at 1000 mg/L (NOEC).
The EC50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate the test items for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no-observed effect concentration (NOEC). The influence of the test item on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study was performed according to OECD Guideline 209 (2010) in compliance with GLP.

The batch of test item tested was a clear colourless liquid with a purity of 99.8%. No correction was made for the purity/composition of the test item. The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

There was no significant oxygen uptake from abiotic processes. The batch of activated sludge was also tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid. No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg/L. Under the conditions of this present test, the test item was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.

Description of key information

The objective of the study (Buitenweg, 2018) was to evaluate the test items for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no-observed effect concentration (NOEC).The influence of the test item on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study was performed according to OECD Guideline 209 (2010) in compliance with GLP.

The batch of test item tested was a clear colourless liquid with a purity of 99.8%. No correction was made for the purity/composition of the test item. The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses were compared to the controls.

There was no significant oxygen uptake from abiotic processes.The batch of activated sludge was also tested for sensitivity with the reference item3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg/L.Under the conditions of this present test, the test item was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC). The EC50 was above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L