Trioctyl benzene-1,2,4-tricarboxylate

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 to 31 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Version / remarks:

2007

Deviations:

no

Principles of method if other than guideline:

Study was conducted according to a standard guideline

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: 1190
Expiry date: 23 May 2020
Analysed purity content: 100%
Substance type: Technical product
Appearance: Slightly yellowish, transparent liquid
Storage conditions: Ambient temperature (room temperature, dark and dry, GLP-storage room)

Analytical monitoring:

yes

Details on sampling:

To verify exposure concentrations, sediment, pore water and overlying water samples were taken at test start and test end.

Vehicle:

yes

Remarks:

Acetone

Details on sediment and application:

Wet sediment preparation:
A peat suspension was prepared two days before mixing it into the other constituents of the artificial sediment. Deionised water (1.85 L) was added to peat and mixed using a homogenising device. The pH of this suspension was adjusted to 6.0 using CaCO3. This suspension was incubated for two days under gentle stirring and maintained under conditions identical to those of the test, and the pH then measured again. The peat suspension was then mixed with the other constituents (75% sand and 20% kaolin). The pH of the resulting artificial sediment was measured to be 6.10, indicating that no further adjustment was necessary. The sediment was incubated for four days under test conditions. Two days before test start, the test item was introduced into the wet sediment and allowed to equilibrate.

Artificial sediment spiking:
The test item was applied using spiked sediment. The test item was dissolved in acetone to form a primary stock solution, followed by serial dilutions of this stock solution (also prepared in acetone) to obtain Individual stock solutions for each treatment group. Defined volumes of these stock solutions were mixed thoroughly into 60 g sand (per treatment group) and the acetone was allowed to fully evaporate over a 2.5-hour period. Spiked sand was subsequently mixed into wet sediment using a homogenising device. Spiked sediment was incubated under test conditions with gentle aeration for two days. Untreated controls and solvent controls which did not contain test item were also included in the study design.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test organism origin: ECT Oekotoxikologie GmbH, Böttgerstraße 2 – 14, 65439 Flörsheim am Main, Germany
Origin of animals used in testing: Holding and breeding in the test facility under standardised laboratory conditions
Breeding: Performed in glass beakers (250 mL) under the same conditions as in the test
Breeding medium: Reconstituted water (according to OECD Guideline 203)
Food and feeding (breeding): Twice a week with commercial fish food (Tetra Min)
All test organisms were derived from a synchronised culture setup 19 days prior to test start.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

Reconstituted water was used as overlying water. Sediment contained powdered leafs of stinging nettle (Folia urticae) as a food source.

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

N/A

Hardness:

Control: 274 – 277 mg/L CaCO3;
Solvent control: 275 – 277 mg/L CaCO3;
Test item treatment (1000.1 mg/L): 275 – 276 mg/L CaCO3

Test temperature:

20.4 – 20.8°C

pH:

Control: 7.46– 8.21;
Solvent control: 7.47– 8.35;
Test item treatments: 7.46– 8.32

Dissolved oxygen:

Control: 7.84– 8.92 mg/L;
Solvent control: 8.25– 8.91 mg/L;
Test item treatments: 7.69– 8.84 mg/L

Salinity:

N/A

Ammonia:

Control: 0 – 8 mg/L NH4+;
Solvent control: 0 – 8 mg/L NH4+;
Test item treatments: 0 – 8 mg/L NH4+

Conductivity:

≤10 µS/cm (according to OECD Guideline 203)

Nominal and measured concentrations:

Nominal test item concentrations: 0 (untreated control), 0 (solvent control), 42.6, 93.8, 206.5, 454.5, 1000.1 mg/kg dry weight

Details on test conditions:

Tests were conducted using glass beakers (250 mL) as test vessels, with 10 worms test vessel and a sediment loading of 1:4 (sediment depth to the depth of overlying water). Freshly prepared test vessels including spiked sediment were equilibrated for two days under identical test conditions prior to test start. Stinging nettle (Urtica dioica) leaf powder was incorporated into the sediment to provide a food source. Tests included six replicates for control groups (untreated and solvent controls) and three replicates for treatment groups. Tests were started upon the introduction of synchronised worms to the spiked sediment. Test vessels were gently and constantly aerated throughout the test.

Tests were conducted under a 16:8 h (light:dark) photoperiod, with light intensity of 406 lux at 400 – 700 nm. Dissolved oxygen concentrations and pH were monitored in one test vessel of each treatment group at test start, once a week and at test end. Total water hardness (CaCO3) was assessed in one control vessel and in one vessel of the highest treatment group at test start, once weekly and at test end. Ammonium concentrations (mg/L NH4 + ) were monitored in one test vessel of each treatment group 3 times per week.

At the end of testing each replicate was examined, and worms were sieved from sediment, and the total number of living and dead individuals per replicate recorded. Worms were considered dead if (1) they did not react after gently stimulus; (2) they showed signs of decomposition and (3) if worms are missing. Additionally, the general condition of single individuals, any abnormal behaviour of worms and/or abnormal appearance of sediment/overlying water were recorded. The dry weight of 30 representative worms was determined at test start. At test end, worm weight was assessed as well. The total organic carbon (TOC) content of the sediment was determined prior to the test start.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (nominal concentrations: 3.13, 6.25, 12.5, 25.0, 50.0 mg/kg dry weight)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

655.3 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Worm dry weight

Remarks on result:

other: 95% confidence intervals: 184.0 – 2179.7 mg/kg dw

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

2 206.4 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Worm number (living)

Remarks on result:

other: 95% confidence intervals: 1161.0 – 7117.0 mg/kg dw

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

318.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Worm dry weight

Remarks on result:

other: 95% confidence intervals: 130.7 – 774.7 mg/kg dw

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

407.9 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Worm number (living)

Remarks on result:

other: 95% confidence intervals: 284.6 – 617.1 mg/kg dw

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

218.4 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Worm dry weight

Remarks on result:

other: 95% confidence intervals: 90.5 – 527.0 mg/kg dw

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

168.8 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Worm number (living)

Remarks on result:

other: 95% confidence intervals: 93.2 – 269.5 mg/kg dw

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

454.4 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Worm dry weight

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

454.4 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Worm number (living)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

206.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Worm dry weight

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

206.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Worm number (living)

Details on results:

Up to the 206.5 mg/kg dw test item concentration exposure group, most parent worms were found at test end. The lowest survival rate up to this treatment was 86.7 %. At concentrations 454.5 and 1000.1 mg/kg dw test item, the survival rate was 76.7 and 73.3%, respectively. It can be concluded that the effect of the test item was predominantly on the reproduction and not mortality. Dead worms were not found during the course of the test and at test end. Exclusively at the highest treatment (1000.1 mg/kg dw test item), adult worms retrieved at test end exhibited copious mucus production. Attempts to wash the worms using sieves were unsuccessful in rinsing-off these mucus spots and fine peat debris that adhered to them. This might have affected the dry weight determination in this treatment group at the end of the test to an uncertain extent and may have contributed to the relatively high coefficient of variation recorded for this treatment group. No abnormal behaviour was observed in any treatment groups. Recoveries of Trioctyl benzene-1,2,4-tricarboxylate in sediment samples were within 81 to 96 % of nominal concentrations at test start and test end and nominal values were confirmed analytically, therefore toxicity data were based on nominal concentrations.

Results with reference substance (positive control):

An EC50 value of 17 mg/kg dry weight (nominal) for ‘worm number (living)’ (95% confidence intervals: 14.22 – 20.54 mg/kg dry weight) was shown with the reference substance (pentachlorophenol). The result is consistent with those reported in Annex 6 of the OECD Guideline 225 (EC50worm number (living) = 19.9 mg/kg (geometric mean), range 4.0 to 37.9 mg/kg). It is therefore concluded that the Lumbriculus breeding culture used in the study with Trioctyl benzene-1,2,4-tricarboxylate was not abnormally (in)sensitive.

Reported statistics and error estimates:

Comparison of untreated controls and solvent controls:
Untreated controls and solvent controls were compared via STUDENT`s t-test (p ≤ 0.05, two-sided). No significant difference between these two groups was found. Subsequently, both control groups were pooled to strengthen the statistical power.

Pre-testing:
The data set was tested for normal distribution (Shapiro-Wilk´s test, p ≤ 0.01); variance homogeneity (Levene´s test, p ≤ 0.01) and monotonicity (trend analysis by contrasts, p ≤ 0.05). The data set was found to be normally distributed, variances were found to be homogeneous and a monotone trend was determined.

Determination of LOEC`s and estimation of effect concentration ECx:
The Williams t-test (p ≤ 0.05, one-sided smaller) was used to determine LOECs for all addressed endpoints. Effect concentrations (ECx) were determined using concentration-response modelling. For the endpoint worm number, probit analysis (maximum likelihood regression) with adjustment to normal distribution and fitting over replicates was performed. Confidence intervals were determined by Bootstrapping. The data fit was justified by the p(Chi2 ) statistics for scattering of data and p(F) statistics for the slope. As a result, the scattering was low and the slope was significantly different from zero. The ECx based for worm dry weight were determined by non-linear regression (logistic 3 parameter; weighting, 1/var(y)). The EC10 was set at parameter b0 for the modelling. The fit was justified by the significance of modelling parameters b0, b1 and b2 and the lack of fit criteria. As a result, the parameters were significant and a lack of fit was not found.

Data were analysed using ToxRat Professional (version 3.3.0; RATTE, 2018).

Reductions of worm number (living) and worm dry weight following 28-day exposure to Trioctyl benzene-1,2,4-tricarboxylate in artificial sediment

	Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	% reduction*
worm number (living)	control	-
	solvent control	-
	42.6	-2.2
	93.8	-3.5
	206.5	8.0
	454.5	29.7
	1000.1	34.8
worm dry weight	control	-
	solvent control	-
	42.6	2.8
	93.8	-0.2
	206.5	1.8
	454.5	39.2
	1000.1	58.6

^* control and solvent control were pooled as significant differences between these groups were not found. Negative values signify increases compared to the combined controls

 

 

pH, O2 content, water hardness and ammonia content during the course of the test

Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	pH in overlying water
	1^st week	2^nd week	3^rd week	4^th week	test end
control	7.61	7.46	8.15	8.12	8.21
solvent control	7.47	8.10	7.94	8.35	8.34
42.6	7.57	7.99	8.10	7.98	7.94
93.8	7.53	7.95	7.64	8.15	7.99
206.5	7.50	7.46	7.97	8.10	8.13
454.5	7.46	8.01	8.18	7.73	7.98
1000.1	7.46	7.89	8.32	7.84	8.15

 

Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	O2 (mg/L) concentration in overlying water
	1^st week	2^nd week	3^rd week	4^th week	test end
control	8.92	8.75	8.81	7.84	8.12
solvent control	8.91	8.64	8.43	8.25	8.45
42.6	8.28	8.76	8.13	8.13	8.31
93.8	8.46	8.13	8.12	8.10	8.05
206.5	7.69	8.64	8.12	7.99	7.98
454.5	8.43	8.35	8.58	8.10	8.13
1000.1	8.84	8.13	7.99	7.85	7.95

 

Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	water hardness (mg/L CaCO3) in overlying water
	1^st week	2^nd week	3^rd week	4^th week	test end
control	275	276	274	274	277
solvent control	275	275	275	275	277
1000.0	275	275	275	275	276

 

Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	ammonia content (mg/L NH4^+)
	test start	day 4	day 6	day 8	day 11	day 13	day 15
control	1.6	8	6	8	8	8	8
solvent control	1.6	8	6	6	6	6	8
42.6	1.6	8	4	6	6	6	6
93.8	1.6	8	4	6	6	6	6
206.5	1.6	8	8	8	6	6	6
454.5	1.6	8	6	6	6	6	6
1000.1	1.6	8	3.2	1	1	1	1
Trioctyl benzene-1,2,4-tricarboxylate mg/kg dw nominal	ammonia content (mg/L NH4^+)
	day 18	day 20	day 23	day 25	day 27	day 28	-
control	0	0	0	0	0	0
solvent control	0	0	0	0	0	0
42.6	0	0	0	0	0	0
93.8	0	0	0	0	0	0
206.5	8	6	0	0	0	0
454.5	5	6	0	0	0	0
1000.1	5	6	8	8	8	8

LOQ = 0.3818 mg/L NH₄⁺

Validity criteria fulfilled:

yes

Remarks:

Average worm number (living) in controls increased by a factor of 2.6, overlying water pH ranged from 7.46 – 8.35, oxygen concentration was >30% of the max. air saturation (lowest value measured: 7.69 mg/L O2 = 87.9% max. oxygen concentration)

Conclusions:

The 28-day EC10 values for Lumbriculus variegatus exposed to Trioctyl benzene-1,2,4-tricarboxylate in artificial sediment were 168.8 mg/kg dw (95% confidence intervals: 93.2 – 269.5 mg/kg dw) and 218.4 mg/kg dw (95% confidence intervals: 90.5 – 527.0 mg/kg dw), based on worm number (living) and worm dry weight, respectively. The 28-day NOEC values were 206.5 mg/kg dw for both worm number (living) and worm dry weight.

Executive summary:

The chronic toxicity of Trioctyl benzene-1,2,4-tricarboxylate to the freshwater sediment dwelling organism, Lumbriculus variegatus, was assessed according to OECD Guideline 225 (‘Sediment-water Lumbriculus toxicity test using spiked sediment’).

Test organisms of synchronised age were exposed to artificial sediment spiked with the test item in a sediment-water system, at nominal test concentrations of 42.6, 93.8, 206.5, 454.5, 1000.1 mg/kg dry weight over a 28-day period. Test item stock solutions were prepared using acetone as a solvent, then mixed into portions of dry sand carrier and the acetone subsequently allowed to evaporate completely, following which the sand portions were mixed into the respective batches of wet sediment. Untreated and solvent controls were also employed. Stinging nettle (Urtica dioica) leaf powder was incorporated into the sediment to provide a food source during the tests. Effect concentrations (ECx, LOEC, NOEC) were calculated for reproduction (total number of worms (living)) and worm biomass (dry weight). Mortality was recorded qualitatively. Sediment samples taken for chemical analysis on days 0 and 28 of tests showed that measured sediment concentrations remained >80% of nominal concentrations, thereby demonstrating that the test item remained intact and predominantly in the sediment phase in all treatment levels throughout the test. Reference substance (pentachlorophenol) results (EC50 value of 17 mg/kg dw (nominal) for worm number (living)) were consistent with those reported in Annex 6 of OECD Guideline 225, thereby concluding that the Lumbriculus breeding culture used in the study with Trioctyl benzene-1,2,4-tricarboxylate was not abnormally (in)sensitive.

All validity criteria of the OECD Guideline 225 were met, the average worm number (living) in the controls increased by a factor of 2.6 (required: at least 1.8), the overlying water pH ranged from 7.46 – 8.35 (required: 6 – 9), and oxygen concentration in overlying water was >30% of the maximum air saturation (lowest value measured was 7.69 mg/L O₂ corresponding to 87.9% maximum oxygen concentration (8.75 mg/L O₂ = 100 % at 20.6°C)).

The 28-day EC10 values for L. variegatus exposed to Trioctyl benzene-1,2,4-tricarboxylate in artificial sediment were 168.8 mg/kg dw (95% confidence intervals: 93.2 – 269.5 mg/kg dw) and 218.4 mg/kg dw (95% confidence intervals: 90.5 – 527.0 mg/kg dw), based on worm number (living) and worm dry weight, respectively. The 28 -day NOEC values were 206.5 mg/kg dw for both worm number (living) and worm dry weight.

Description of key information

The chronic toxicity of Trioctyl benzene-1,2,4-tricarboxylate to the freshwater sediment dwelling organism, Lumbriculus variegatus, was assessed according to OECD Guideline 225 (‘Sediment-water Lumbriculus toxicity test using spiked sediment’).

Test organisms of synchronised age were exposed to artificial sediment spiked with the test item in a sediment-water system, at nominal test concentrations of 42.6, 93.8, 206.5, 454.5, 1000.1 mg/kg dry weight over a 28-day period. Test item stock solutions were prepared using acetone as a solvent, then mixed into portions of dry sand carrier and the acetone subsequently allowed to evaporate completely, following which the sand portions were mixed into the respective batches of wet sediment. Untreated and solvent controls were also employed. Stinging nettle (Urtica dioica) leaf powder was incorporated into the sediment to provide a food source during the tests. Effect concentrations (ECx, LOEC, NOEC) were calculated for reproduction (total number of worms (living)) and worm biomass (dry weight). Mortality was recorded qualitatively. Sediment samples taken for chemical analysis on days 0 and 28 of tests showed that measured sediment concentrations remained >80% of nominal concentrations, thereby demonstrating that the test item remained intact and predominantly in the sediment phase in all treatment levels throughout the test. Reference substance (pentachlorophenol) results (EC50 value of 17 mg/kg dw (nominal) for worm number (living)) were consistent with those reported in Annex 6 of OECD Guideline 225, thereby concluding that the Lumbriculus breeding culture used in the study with Trioctyl benzene-1,2,4-tricarboxylate was not abnormally (in)sensitive.

All validity criteria of the OECD Guideline 225 were met, the average worm number (living) in the controls increased by a factor of 2.6 (required: at least 1.8), the overlying water pH ranged from 7.46 – 8.35 (required: 6 – 9), and oxygen concentration in overlying water was >30% of the maximum air saturation (lowest value measured was 7.69 mg/L O₂corresponding to 87.9% maximum oxygen concentration (8.75 mg/L O₂= 100 % at 20.6°C)).

The 28-day EC10 values for L. variegatus exposed to Trioctyl benzene-1,2,4-tricarboxylate in artificial sediment were 168.8 mg/kg dw (95% confidence intervals: 93.2 – 269.5 mg/kg dw) and 218.4 mg/kg dw (95% confidence intervals: 90.5 – 527.0 mg/kg dw), based on worm number (living) and worm dry weight, respectively. The 28 -day NOEC values were 206.5 mg/kg dw for both worm number (living) and worm dry weight.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

168.8 mg/kg sediment dw