3,6,9-trioxaundecamethylene bis(2-ethylhexanoate)

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:

May 5, 1993 to May 7, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

The test was conducted by recognized guidelines acceptable at the time of the study.

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

U.S. EPA-TSCA, 40 CFR, Part 797

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test material sample (batch #010241) was received from The C P . Hall Company on November 30, 1992, in good condition and was issued ABC reference #TS-6174. The sample upon receipt was observed to be an amber liquid and was stored at room temperature. Sample purity was not specified. This sample was used for the preliminary study started on December 15, 1992, and the definitive test attempts started on December 28, 1992, January 12, 1993, January 19, 1993, and January 26, 1993.

A second sample (batch #010241) was received from The C P. Hall Company on February 16, 1993, in good condition and was issued ABC reference #TS-6321. The sample upon receipt was observed to be an amber liquid and was stored at room temperature. Sample purity was not specified. All test concentrations were based on the total compound, i.e., not corrected for sample purity. This sample was used for the preliminary study started on March 9, 1993, and the definitive attempts started February 22, 1993, February 25, 1993, March 9, 1993, March 31, 1993, and April 27, 1993, and the for the definitive study performed on May 5, 1993.

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

The definitive study was conducted at the following nominal concentrations of the test material at: the water-soluble fractions (WSFs) of 10, 18, 32, 56, 100, 180, and 320 mg/L. The WSF of each nominal test concentration was reached by adding the appropriate weights of compound to 1.0 L of dilution water in 1.0-L beakers. Records of all standard weights and dilution values were maintained. Before addition to the beakers, 0.1 mL of dimethylfonnamide (DMF) was added to each sample to increase dispersion of the compound in the dilution water. The vehicle blank: chamber received a 0.1-mL aliquot of DMF, which was equivalent to the highest amount used in any test solution.

The solutions were stirred overnight. The solutions were stopped from stirring to allow the undissolved compound to collect on the surface of the water. At this time, the solutions were placed in a waterbath to adjust to test temperature. After approximately 4.5 hours, 200 mL of each of the test solutions was siphoned into each of two 250-mL test beakers, taking care to leave all undissolved compound in the vessel in which the solutions were stirred.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test specimens of Daphnia magna were obtained from an in-house daphnid culture that ABC Laboratories has maintained since 1977. The primary culture was obtained from the Columbia National Fisheries Research Laboratory (CNFRL), Columbia, Missouri, in 1977. A trace of the daphnid strain indicated that CNFRL acquired their culture from the U.S. Fish & Wildlife Service Fish Control Laboratory, LaCrosse, Wisconsin, in 1960 and they obtained their culture from Pennsylvania State University in 1954 (2).

All daphnids were cultured and tested in a temperature-controlled area at 20 (±2)°C. The lighting was maintained at approximately 60 footcandles on a 16-hour daylight photoperiod. Light reading measurements are kept on file at ABC Laboratories. The daphnid cultures were maintained in 1.5-L glass containers and were cultured in hard blended water. Hard blended water is a combination of well water and reverse osmosis water blended to a hardness of 130 to 160 mg/L as CaC03 (Table I). The hard blended water was biologically aged prior to use. That is, the water was held in a tank containing
aquatic organisms. The water was filtered with a sediment filter prior to use. During the holding period, the daphnids were fed a suspension of at least one algae species cultured at ABC: Selenastrum capiicomutum and/or Ankistrodesmus falcatus at least every 3 days. Along with the algae, the daphnids were fed a supplement consisting of fish food (Zeigler Bros., Inc., Gardners, PA) and Fleischmann's active dry yeast. Results of the analysis of the fish food are kept on file at ABC Laboratories.

First-instar daphnids (< 24 hours old) were selected for the test. To provide test daphnids that were <24 hours old, the adult daphnids were isolated by transferring to a fresh culture water/food suspension the previous day.

All test daphnids were provided by ABC culture #93-I3 on May 5, 1993. The adults from this culture had been cultured for 35 days and were considered acceptable with no signs of stress, disease, or physical damage. Since the culturing and testing parameters of temperature, dilution water, and lighting were the same, no acclimation period was necessary. Test daphnids were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

138 mg/L as CaC03 (hardness)

Test temperature:

see table below

pH:

see table below

Dissolved oxygen:

see table below

Salinity:

not available

Conductivity:

340 umhos/cm (conductivity)

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56, 100, 180 and 320 mg/L

Details on test conditions:

The static Daphnia magna bioassay was conducted in 250-mL glass beakers that had a 200-mL test volume. The mean depth and mean surface diameter for the test solution in the test vessels were 6.3 cm and 6.3 cm, respectively. All test vessels were covered with loose-fitting petri dish covers to minimize evaporation and prevent contamination during the study. These vessels were kept at 20 (± 2)°C in a temperature-controlled waterbath. The lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods. Light intensity measured over the waterbath in April and May 1993 ranged from 59 to 61 footcandles (634 to 692 lux). Light intensity data are kept on file at ABC Laboratories.

Reference substance (positive control):

no

Key result

Duration:

24 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: > the water soluble fraction of 100 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: > the water soluble fraction of 18 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Results not from a definitive test result and to be treated as supplemental data

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: EC50 value calculated with 95 % confidence limits of the WSF of 84 to 250 mg/L.

Details on results:

RESULTS AND DISCUSSION - DEFINITIVE TEST

A 48-hour static Daphnia magna test with TegMeR® 804 was completed on May 7, 1993. All results were based on the water-soluble fractions of the nominal concentrations of 10, 18, 32, 56, 100, 180, and 320 mg/L. Table II presents the individual immobility and behavioral observations for daphnids exposed to TegMeR® 804. All test solutions were clear with no visible surface film or precipitate throughout the study. No immobility or abnonnal effects were observed in the control or vehicle blank beakers during the study. At 24 hours, one daphnid was found stuck to the side of the 100 mg/L A chamber above the solution surface. Since this was not a compound-related immobility, this daphnid was not included in percent mortality data or in calculating the EC 50 values. At 24 hours, immobility was as follows: 0% at 10 mg/L, 25% at 18 mg/L, 25% at 32 mg/L, 10% at 56 mg/L, 11% at 100 mg/L, 35% at 180 mg/L, and 20% at 320 mg/L. At 48 hours, immobility was as follows: 5% at 10 mg/L, 25% at 18 mg/L, 30% at 32 mg/L, 20% at 56 mg/L, 37% at 100 mg/L, 50% at 180 mg/L, and 35% at 320 mg/L. The 48-hour NOEC was 10 mg/L based on the lack of >5% effects at this concentration.

The 24- and 48-hour EC50 values were > 100 and > 18 mg/L, respectively (see table below)).

The table below presents the water quality parameters measured during the definitive test. Temperature was 20°C when measured at 0 and 48 hours. The pH values ranged from 8.0 to 8.5. Dissolved oxygen concentrations ranged from 7.4 to 8.5 mg/L during the test. These values represented 85 to 98% saturation at 20°C, respectively. The dissolved oxygen was considered adequate for testing (5); therefore, the test chambers were not aerated during the test. The continuous temperature graph from the data logger showed that the waterbath temperature was within protocol limits during the study.The study was conducted following the Good Laboratory Practice regulations (1), and the final report was reviewed by ABC Laboratories' Quality Assurance Unit. All original raw data were provided to The C P . Hall Company with the final report. A copy of the report was retained at ABC Laboratories, Inc.

METHODS. RESULTS. AND DISCUSSION - SUPPLEMENTAL DATA

The initial range-finding test (12/15/92 to 12/17/92) was performed at the nominal concentrations of 0.10, 1.0, 10, and 50 mg/L with a concurrent control. Each control or test concentration received five daphnids. There were oily droplets on the surface of the 1.0-, 10-, and 50-mg/L test concentrations. The control and 0.10-mg/L test concentration were clear during the test. There were no immobilities observed in the control, 0.10-, 1.0-, and 10-mg/L test concentrations during the test. The 50-mg/L test concentration elicited 100% immobility during the 48-hour exposure.

All subsequent definitive test attempts had duplicate replicates of each control, vehicle blank (if used), and test concentration, with 10 daphnids per replicate for a total of 20 daphnids per control or test concentration. The second, third, and fourth attempts of the definitive test also had a third replicate to which no daphnids were admitted for performing 0-hour water chemistry. All changes of test concentrations were discussed with the sponsor.

Based on the preliminary data, the first attempt of the definitive test (12/28/92 to 12/30/92) was peifonned at the nominal concentrations of 5.0, 10, 20, 40, and 80 mg/L with concurrent vehicle blank (acetone) and control chambers. The test solutions were sonicated at the time of preparation to help the test material go into solution. During the test, the controls were clear and all test solutions had oily droplets on the surface of the test solutions. During the test, the 5.0-, 10-, 20-, 40-, and 80-mg/L test chambers elicited 0, 45, 5, 20, and 20% cumulative immobility at 48 hours. This attempt of the definitive test was not used due to the lack of the anticipated dose response. Insufficient immobility occurred and a dose response could not be demonstrated over a reasonable range (<37 to >63%); therefore, an EC50 could not be calculated.

The second attempt at the definitive test (1/12/93 to 1/13/93) was perfonned at the same test nominal concentrations without sonication because it was thought that perhaps the sonication may have volatilized some of the test material. The solutions appeared the same as the previous test. The daphnids were observed to be surfacing in the control solutions at 24 hours; therefore, the study was terminated at 24 hours. The toxicity at 24 hours was 10, 10, 60, 75, and 65% immobility in the 5.0-, 10-, 20-, 40-, and 80-mg/L test concentrations, respectively. The third attempt of the definitive test (1/19/93 to 1/21/93) was peifonned at the nominal concentrations of 1.5, 3.0, 6.0, 12, 24, and 48 mg/L. The control, vehicle blank (acetone), and 1.5-mg/L test concentrations were clear during the test. All higher concentrations had oily droplets on the surface or a surface film observed during the test. The 1.5-, 3.0-, 6.0-, 12-, 24-, and 48-mg/Ltest concentrations elicited 15, 0, 0, 5, 35, and 35% immobility, respectively. This study was not used as the definitive test because insufficient immobility occurred and a'dose response could not be demonstrated over a reasonable range (< 37 to > 63 %); therefore, an EC50 could not be calculated.

The fourth attempt of the defmitive test (1/26/93 to 1/27/93) was peifonned at the same nominal concentrations as the previous test, but with the addition of a 96-mg/L test concentration. At 24 hours the 1.5-, 3.0-, 6.0-, 12-, 24-, 48-, and 96-mg/L test concentrations elicited 0, 30, 0, 5, 0, 0, and 0% immobility, respectively. This study was terminated at 24 hours due to the lack of a dose response. After reviewing all the previous data with the sponsor, it was decided that it would be appropriate to perfonn the study as WSFs of the nominal test concentrations. Based on this decision and all previous toxicity, the test concentrations for the fifth attempt of the definitive test (2/22/93 to 2/23/93) were set at the WSFs of 100, 180, 320, 560, and 1000 mg/L.

The test solutions for the fifth and all subsequent attempts of the definitive test were prepared in the same maimer. The WSF of each nominal test concentration was reached by adding the appropriate weights of compound to 1.0 L of dilution water. Records of all standard weights and dilution values were maintained. Before addition to the water, 0.1 mL of vehicle (if used) was added to each sample to increase dispersion of the compound in the dilution water. The vehicle blank chamber received a 0.1-mL aliquot of vehicle, which was equivalent to the highest amount used in any test solution.

The solutions were stirred overnight. The solutions were stopped from stirring to allow the undissolved compound to collect on the surface of the water. At this time, the solutions were placed in a waterbath to adjust to test temperature. After several hours, 200 mL of the solutions were siphoned into each of two 250-mL test beakers, taking care to leave all undissolved compound in the vessel in which the solutions were stirred.

The test solutions for the fifth attempt of the definitive test were prepared on February 22, 1993, and were allowed to stir overnight. On February 23, 1993, the solutions were place in a waterbath to stand for several hours. The stirring was too vigorous, and the test substance could be seen as numerous fine droplets suspended in the test solution. Since the droplets did not separate from the water as expected, the solutions were discarded.

The nominal test concentrations for the sixth attempt of the definitive test (2/25/93 to 2/26/93) were the same as the previous attempt the WSFs of 100, 180, 320, 560, and 1000 mg/L). A control and vehicle blank (acetone) were included. All control solutions were clear during the test. All solutions containing TegMeR® 804 were cloudy during the test. At 24 hours the 100, 180, 320, 560, and 1000 mg/L WSFs elicited 75, 65, 55, 75, and 60% immobility, respectively. This attempt of the definitive test was terminated at 24 hours due to excessive immobility. It was also noted that the dissolved oxygen was low in all chambers but the controls. Therefore, the vehicle for all subsequent tests was changed to DMF.

A second range-finding experiment (3/2/93 to 3/4/93) was conducted with five Daphnia magna in nominal exposure concentrations of 1.0, 10, 100, and 1000 mg/L with concurrent control and vehicle blank chambers. No immobility or abnormal effects were observed in the control, vehicle blank, or 1.0-mg/L beakers during the test. At 24 hours, one immobile daphnid was noted in the 10-mg/L test beaker, and no immobile daphnids were noted in the 100- and 1000-mg/L test beakers. At 48 hours, 60% immobility was seen in the 10 mg/L test solution, 40% immobility was seen in the 100 mg/L test solution, and 60% immobility was seen in the 1000 mg/L test solution. All test solutions were clear with no visible precipitate or surface film throughout the 48-hour exposure period.

Based on this preliminary test, the seventh attempt of the definitive test (3/9/93 to 3/11/93) was performed at the same nominal concentrations as the previous test (the WSFs of 100, 180, 320, 560, and 1000 mg/L). A control and vehicle blank (DMF) were included. All solutions were clear during the test. The 100, 180, 320, 560, and 1000 mg/L WSFs elicited 60, 100, 80, 70, and 60% immobility at 48 hours. Except for one surfacing daphnid in the control B replicate at 24 hours, there were no abnonnal effects in the control or vehicle blank chambers. Since excessive immobility occurred and a dose response could not be demonstrated over a reasonable range (<37 to >63%), the EC50 and/or its 95% confidence limits could not be calculated. Therefore this test was not used as the definitive test.

The eighth attempt of the definitive test (3/31/93 to 4/1/93) was performed with nominal test concentrations set at the WSFs of 1.8, 3.2, 5.6, 10, 18, 32, and 56 mg/L, based on the excessive immobility observed in the sixth and seventh tests. A control and vehicle blank (DMF) were included. All solutions were clear during the test. At 24 hours, surfacing daphnids were noted in the control and vehicle blank test chambers; therefore, the study was terminated at 24 hours.

The ninth attempt of the definitive test (4/27/93 to 4/29/93) was performed with test concentrations set at the WSFs of 3.2, 5.6, 10, 18, 32, 56, and 100 mg/L, based on the excessive immobility observed in the sixth and seventh definitive test attempts and the lack of immobility in the eighth definitive test attempt. A control and vehicle blank (DMF) were included. All solutions were clear during the test, except for the 100 mg/L WSF test concentration, which had oily droplets on the surface observed at 24 and 48 hours. The 3.2-, 5.6-, 10-, 18-, 32-, 56-, and 100-mg/L WSF concentrations elicited 5, 10, 15, 5, 35, 20, and 50% immobility at 48 hours. This study was not used as the definitive test because insufficient immobility occurred and a dose response could not be demonstrated over a reasonable range (<37 to >63%); therefore, an EC50 could not be calculated.

The tenth and final attempt of the definitive test was performed from May 5, 1993, to May 7, 1993. The nominal test concentrations were chosen to include concentrations from the WSF tests that had excessive immobility and concentrations from the WSF tests that had insufficient immobility. The nominal concentrations were the WSFs of 10, 18, 32, 56, 100, 180, and 320 mg/L with concurrent control and vehicle blank (DMF) chambers. All test solutions were clear with no visible surface film or precipitate throughout the study. No immobility or abnonnal effects were observed in the control or vehicle blank beakers during the study. At 24 hours, one daphnid was found stuck to the side of the 100-mg/L A chamber above the solution surface. Since this was not a compound-related immobility, this daphnid was not included in percent mortality data. At 24 hours, immobility was as follows: 0% at 10 mg/L, 25% at 18 mg/L, 25% at 32 mg/L, 10% at 56 mg/L, 11% at 100 mg/L, 35% at 180 mg/L, and 20% at 320 mg/L. At 48 hours, immobility was as follows: 5% at 10 mg/L, 25% at 18 mg/L, 30% at 32 mg/L, 20% at 56 mg/L, 37% at 100 mg/L, 50% at 180 mg/L, and 35% at 320 mg/L.

The tenth attempt of the definitive test was used as the definitive test. The results are discussed in the "Results and Discussion — Definitive Test" section above.

Reported statistics and error estimates:

The data were analyzed using the computerized EC50 program developed by Stephan et al..

Stephan, C E . , K . A . Busch, R. Smith, J. Burke and R.W. Andrews. 1978. A Computer Program for Calculating an LC5 0 . U.S. Environmental Protection Agency, Duluth, Minnesota, pre-publication manuscript, August 1978.

Stephan, C E . 1977. Methods for Calculating an LC5 0 , p. 65-84. In F.L. Mayer and J.L. Hamelink (Eds.). Aquatic Toxicology and Hazard Evaluation. ASTM Special Technical Riblication 634. ASTM. Philadelphia.

Water Quality Measurements During the Definitive Static Acute Toxicity Test of the Water-Soluble Fraction of The Test Material to Daphnia magna

	0 Hour			48 Hours
Nominal Concentration (mg/L)	Temp(a) °C	DO(b)	pH©	Temp(a) °C	DO(b)	pH©
Control A	20	7.4	8.1	20	8.1	8.4
Control B	20	7.5	8.1	20	8.3	8.4
Vehicle Blank A	20	8.2	8.3	20	8.4	8.5
Vehicle Blank B	20	8.3	8.3	20	8.4	8.5
10 A	20	8.2	8.4	20	8.5	8.5
10 B	20	8.2	8.4	20	8.5	8.5
18 A	20	8.3	8.4	20	8.5	8.5
18 B	20	8.4	8.4	20	8.5	8.5
32 A	20	8.4	8.4	20	8.4	8.5
32 B	20	8.4	8.4	20	8.4	8.5
56 A	20	8.4	8.4	20	8.3	8.0
56 B	20	8.4	8.4	20	8.3	8.5
100 A	20	8.4	8.4	20	8.4	8.5
100 B	20	8.5	8.4	20	8.4	8.5
180 A	20	8.5	8.4	20	8.3	8.5
180 B	20	8.5	8.4	20	8.3	8.5
320 A	20	8.4	8.4	20	8.3	8.5
320 B	20	8.4	8.4	20	8.3	8.5

a Temperature measured with a mercury thermometer

b Dissolved oxygen concentration - YSI Model 54 ARC dissolved oxygen meter

c pH - Beckman Model 39841 probe with a Beckman 34 pH meter

Note: Dissolved oxygen saturation corrected for altitude at the test temperature of 20°C is 8.7 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION - SUPPLEMENTAL DATA

The 48-hour EC50 calculated from these combined data was the WSF of 140 mg/L, with 95 % confidence limits of the WSF of 84 to 250 mg/L. This value is ABC Laboratories' best estimate of the EC50 for Daphnia magna exposed to the test material, but it is not a definitive test result and should be treated as supplemental data.

CONCLUSIONS - DEFINITIVE TEST

The nominal duplicate test concentrations for this study were the water-soluble fractions of 10, 18, 32, 56, 100, 180, and 320 mg/L. The 48-hour EC50 for Daphnia magna exposed to the test material was > 18 mg/L. The 48-hour NOEC was 10 mg/L.

Description of key information

In a key study with the test substance, the nominal duplicate test concentrations for this study were the water-soluble fractions of 10, 18, 32, 56, 100, 180, and 320 mg/L. The 48-hour EC50 for Daphnia magna exposed to the test material was > 18 mg/L. The 48-hour NOEC was 10 mg/L. In supplemental data from this same study, the 48-hour EC50 calculated from these combined data was the WSF of 140 mg/L, with 95 % confidence limits of the WSF of 84 to 250 mg/L. This value is the testing laboratories' best estimate of the EC50 for Daphnia magna exposed to the test material, but it is not a definitive test result and should be treated as supplemental data.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

140 mg/L

Additional information

The test substance is a member of the Glycol Esters Category submitted by The American Chemistry Council's (ACC) Aliphatic Esters Panel (Panel) under the High Production Volume (HPV) Chemical Challenge Program (ACC, 2003). Information from a Screening Information Data Set (SIDS) developed in that program serves as supplementary or weight-of-evidence in the current dossier. The glycol esters chosen to supply additional information are very closely related in molecular structure They exhibit similar behavior with respect to physicochemical, environmental fate Category ester compounds with similar structures and functionalities were generally of minimal or low acute toxicity to Dapnia magna at or above the effective water solubility limits of the substances. The structurally similar material, decanoic acid, mixed diester with octanoic acid and triethylene glycol (CAS 68583 -52 -8), had a 48-hour EC50 of 440 mg/L The test material 2,2'-ethylenedioxydiethyl bis(2 -ethylhexanoate) (CAS 94 -28 -0) caused no immobilization of daphnids in 96 h at the nomial test concentration of 97 mg/L. Ethane-1,2 -diylbis(oxyethane-2,1 -diyl) bisheptanoate (CAS 7434 -40 -4) had a stated 48-h EC50 value in excess of 9.1 mg/L, displaying some minimal toxicity. Heptanoic acid, oxybis(2,1 -ethanediyloxy-2,1 -ethanediyl)ester (CAS 70729 -68 -9) had an estimated 24-h EC50 of 3800 mg/L, well above the solubility limit of the substance. Heptanoic acid, ester with 2,2,4 -trimethyl-1,3 -pentanediol (CAS 71839 -38 -8) had a 48 -h EC50 in excess of 2000 mg/L, well above the water solubility limit of the test substance..