Bis(2-ethylhexyl) terephthalate

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-24 to 2004-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

bis (2-ethylhexyl) terephthalate

IUPAC Name:

bis (2-ethylhexyl) terephthalate

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) terephthalate

EC Number:

229-176-9

EC Name:

Bis(2-ethylhexyl) terephthalate

Cas Number:

6422-86-2

Molecular formula:

C24H38O4

IUPAC Name:

1,4-bis(2-ethylhexyl) benzene-1,4-dicarboxylate

Constituent 3

Reference substance name:

1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester

IUPAC Name:

1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester

Constituent 4

Reference substance name:

Di (2-ethylhexyl) terephthalate, DEHT; Dioctyl terephthalate, DOTP

IUPAC Name:

Di (2-ethylhexyl) terephthalate, DEHT; Dioctyl terephthalate, DOTP

Details on test material:

Storage conditions of test material: Room temperature in the dark.
Lot/Batch No.: TD-3033023
Physical appearance: clear, colorless liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analysis for the test material was performed on Days -2 (preparation of the sediment), 0, and 28. Samples on Day -2 were analyzed for the concentration of the test material in the sediment only in order to confirm correct dosing of the test system. Samples of sediment from the control groups were also analyzed at Day -2. Samples (pooled replicates) were taken for analysis of concentration of DOTP in the sediment, overlying water and interstitial water on Days 0 and 28.

Test substrate

Vehicle:

no

Details on sediment and application:

A formulated sediment was used with the following composition:
76% w/w industrial quartz sand
20% w/w kaolinite clay
4% sphagnum moss peat

The peat was air dried and homogenized to give a particle size of < 1 mm. Calcium carbonate was added to bring the pH range to 7.0 ± 0.5. Amounts of test material (150, 270, 480, 840, and 1500 mg) were each separately weighed into vials and then washed into 1.50 kg (dry weight) of artificial soil with an aliquot of deionised reverse osmosis water and then further deionised reverse osmosis water added (total of 579 mL) to give test concentrations of 100, 180, 320, 560, and 1000 mg/kg with a nominal moisture content of 40% of dry weight. The control was prepared using 1.50 kg (dry weight) of artificial soil and 579 mL of deionised reverse osmosis water without addition of test material.

The prepared sediment was dispensed to glass beakers (600-mL) to give a 2-cm layer and then covered with an 8-cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. The test vessels were then aerated (approximately 1 bubble/sec) via narrow bore glass tubes approximately 2-3 cm above the sediment layer. The vessels were left for 2 days prior to addition of the organisms. After the 2-Day equilibration period, aeration was stopped and larvae were placed in each test and control vessel.

TEST WATER
Reconstituted water (Elendt M4) was used to maintain stock animals and in the toxicity test.

Test organisms

Test organisms (species):

Chironomus riparius

Details on test organisms:

CULTURE CONDITIONS
Common name: midge
Supplier: The test organisms were cultured within the testing facility
Age at study initiation: 2-3 days old
Substrate: maintained in glass beakers with 5-10 mm layer of fine quartz sand covered by reconstituted water (Elendt M4)
Temperature: room temperature approximately 21 °C
Photoperiod: 16 h light / 8 h darkness cycle with 20-min dawn and dusk transition
Aeration: gently through narrow bore glass tubes to avoid disturbing substrate
Feeding: larvae were fed Tetramin® flake food (250 mg/vessel/day) as a suspension in water.

Study design

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Exposure duration

Duration:

28 d

Exposure phase:

total exposure duration

Remarks:

On day 10, 2 extra replicates prepared for control and test concentrations were sacrificed for determination of larval survival and weight.

Test conditions

Hardness:

Controls: 248 and 340 mg/L as CaCO3 for days 0 and 28, respectively.
Test (1000 mg/kg): 282 and 300 mg/L as CaCO3 for days 0 and 28, respectively. Difference was considered to be due to the addition of calcium carbonate to sediment during preparation to adjust pH to 7.0.

Test temperature:

21 °C

pH:

No treatment related differences

Dissolved oxygen:

No treatment related differences

Salinity:

No data

Ammonia:

Controls: 0.39 and 0.05 mg/L as NH3 for days 0 and 28, respectively.
Test (1000 mg/kg): 0.93 and 0.05 mg/L as NH3 for days 0 and 28, respectively

Nominal and measured concentrations:

Nominal concentrations: 100, 180, 320, 560, and 1000 mg/kg
Measured concentrations: Analysis of sediment on Day -2 (the day sediment was prepared) showed measured concentrations to range from 91% to 113% of nominal. Analysis of sediment on Day 0 showed the measured concentrations to range from 90% to 121% of nominal (with the exception of the 1000 mg/kg test concentration which was 55% of nominal). Analysis of a duplicate frozen 1000 mg/kg sample gave a measured concentration of 92% of nominal. The low value obtained for the initial analysis of the 1000 mg/kg sample was considered to be possibly due to sampling error. Analysis of the sediment on Day 28 showed the measured concentrations to range from 77% to 97% of nominal. The mean measured concentrations ranged from 88% to 100% of nominal. Therefore, the results were based on nominal concentrations.

Water Analysis: Analysis of the interstitial water on Days 0 and 28 showed measured concentrations to be less than the limit of detection for all concentrations < 1000 mg/kg. On Day 28, a concentration of 2.33 mg/L was detected in interstitial water containing organisms exposed to 1000 mg/kg. Analysis of the overlying water on Days 0 and 28 showed measured concentrations to be less than the limit of detection for all concentrations.

Details on test conditions:

Replicates: 6 (2 extra replicates prepared for sacrifice at day 10)
Organisms per Replicate: 20
Exposure vessels: 600-mL glass beakers, 8 cm in diameter
Photoperiod: 16 h light followed / 8 h dark; fluorescent lighting with 20-minute dawn/dusk transition period
Light intensity: 496 to 541 lux
Test Parameters: Survival
Dilution water: Reconstituted water (Elendt M4)
Aeration: yes, approximately 24 h after having allowed the larvae to settle in the sediment

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

180 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: All differences were considered significant at the α=0.05 (95% confidence) level.

Effect concentrations 2

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: All differences were considered significant at the α=0.05 (95% confidence) level.

Effect concentrations 3

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: All differences were considered significant at the α=0.05 (95% confidence) level.

Details on results:

Day 10 Larval Survival and Growth:
Two extra replicates were included in the test to determine larval survival and growth after 10 days of exposure to DOTP. After ten days of exposure there was no significant effect (p < 0.05) on larval growth (weight) at any concentration. There was a dose-dependent, toxic effect on the numbers of surviving larvae on day 10. However, the numbers of larvae surviving on Day 10 were similar to the number observed emerging to adult flies on Day 28. Summary data for larval survival and growth are provided in the Results section below.

Day 28 Emergence:
The numbers of adult midges emerging after 28 days in the definitive test are provided in the summary table located in the Results section below. Numbers in excess of twenty emerging as adults in some of the control and 100 mg/kg vessels were likely due to a counting error during introduction of the larvae at the start of the test. It is assumed that the error rate in the other test vessels was similar to that of the control and 100 mg/kg vessels. The sediment was inspected at the end of the study. No larvae were observed in the sediments. The statistical analyses performed on the data at the end of the test took into account the variation in numbers obtained. The result of which there was no overall impact on the results of the test as a clear concentration-dependent effect was observed. Statistical analysis of emergence ratio data, transformed by the square root arcsin function, showed no significant differences (p < 0.05) between the control, 100 and 180 mg/kg groups. There were significant differences between the control, 320, 560 and 1000 mg/kg groups. There was no effect of treatment on the numbers of males and females that emerged. Based on emergence, the no observed effect concentration after 28 days was 180 mg/kg. The lowest concentration causing 100% mortality was > 1000 mg/kg. The EC50 value for emergence was > 1000 mg/kg (which resulted in a 68% emergence rate). The mean emergence in the controls was 100%, satisfying the validation criterion that the control rate should be > 70%. The mean development rate per vessel was calculated for each test concentration. The overall means for the control and each concentration were calculated. An EC50 value was calculated following normalization of the data against the control mean. The EC50 value for development rate was > 1000 mg/kg.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Emergence ratio data were analyzed using a Dunnett's multiple comparison test. The emergence ratio was the number of midges emerged per vessel/number of larvae introduced per vessel. The emergence ratio values were transformed by the square root arcsin function prior to statistical analysis. The 28-Day EC50 value (reduction in emergence) and associated confidence limits were calculated by the maximum-likelihood probit method (if applicable). The 28-Day EC50 (development rate) and associated confidence limits (if applicable) of normalized mean development rates also were calculated.

Any other information on results incl. tables

	10-Day Survival and Growth Data
	Number of Larvae			Dry Weight of Surviving Larvae
Nominal Conc. (mg/kg)	Live Larvae	Dead Larvae	Larvae Unaccounted For	Total Dry Weight (mg)	Mean Individual Larvae Dry Weight (mg)
Control R5	18	0	2	13.9	0.8
Control R6	20	0	0	12.4	0.6
100 R5	18	0	2	17.5	1.0
100 R6	18	0	2	16.0	0.9
180 R5	17	0	3	13.7	0.8
180 R6	20	0	0	15.4	0.8
320 R5	17	0	3	10.2	0.6
320 R6	16	0	4	9.0	0.6
560 R5	15	0	5	7.9	0.5
560 R6	17	0	3	12.5	0.7
1000 R5	12	1	7	8.7	0.7
1000 R6	11	0	9	7.1	0.6
R5-R6 = replicates 5 and 6 prepared at the start of study for sacrifice on Day 10
	28-Day Adult Emergence and Development Rate
	Total Emergence at Day 28
Nominal Conc. (mg/kg)	Replicate 1	Replicate 2	Replicate 3	Replicate 4	Mean Development Rate
Control	20	20	21	21	0.0562
100	21	23	26	22	0.0568
180	20	13	19	16	0.0559
320	6	16	16	18	0.0564
560	12	13	17	13	0.0558
1000	14	14	15	11	0.0536

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The mean emergence in the controls was 100% thereby satisfying the validation criteria that greater than 70% emergence should be attained.

Conclusions:

Exposure of sediment dwelling larvae to DOTP for 10 days may result in decrease in survival. Larvae exposed to concentrations up to 1000 mg/kg are not expected to exhibit delayed growth. Exposure of sediment dwelling larve to DOTP for up to 28 days is not expected to adversely affect the rate of development, however emergence of the adults may be decreased at concentrations greater than 180 mg/kg.

Executive summary:

In a 28-d sediment toxicity study, Chironomus riparius were exposed to DOTP at nominal concentrations of 100, 180, 320, 560, and 1000 mg/kg under static conditions. At day 10, larval survival and growth was evaluated. There was no difference in larval growth among organisms exposed to up to 1000 mg/kg. Data indicated a toxic effect on the early larval stages of the test organism. Analysis of emergence data at day 28 resulted in an EC50 value of >1000 mg/kg. The NOEC after 28 days was 180 mg/kg. The NOEC was based on no significant reduction in emergence. The highest test concentration resulting in 0% mortality was 100 mg/kg and the lowest test concentration resulting in 100% mortality was >1000 mg/kg.