1,2,3-Propanetricarboxylic acid, 2-(1-oxobutoxy)-, 1,2,3-trihexyl ester

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Test Guideline 211

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD GLP; UK GLP; EC Commission Directive 2004/10/EC

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-890-4

EC Name:

-

Cas Number:

82469-79-2

Molecular formula:

C28H50O8

IUPAC Name:

1,2,3-trihexyl 2-(butanoyloxy)propane-1,2,3-tricarboxylate

Constituent 2

Reference substance name:

1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er

IUPAC Name:

1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er

Details on test material:

Citroflex® B-6; n-Butyryltri-n-hexyl citrate (BTHC); CAS # 82469-79-2; Lot/Batch No. 0000085635; Expiration Date, 6 February 2009; Purity, 99.3%; Clear oily liquid; Storage Conditions, room temperature in the dark.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Test concentrations were not corrected for the purity of the test substance.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples (100 ml) of the freshly prepared control and test media were taken from the media remaining in the preparation flasks on Days 0, 7, 14 and 20. Following the transfer of the Daphnia to fresh media, duplicate samples (100 ml) of expired (ca. 24-hour-old) media were collected from the pooled contents of the replicate control and test vessels on Days 1, 8, 15 and 21. All samples of test media were placed into polypropylene bottles containing ethyl acetate (20 ml) to stabilise samples. On each occasion, one of the samples was analysed initially and the second (reserve) sample was stored in a freezer for further analysis, if required. Reserve samples were analysed when the results obtained for the original samples were considered anomalous (all samples from Day 7 fresh and Day 15 expired; at 0.0320 mg/l on Day 20; and at 0.0128 and 0.2 mg/l on Day 21).

Test solutions

Vehicle:

yes

Details on test solutions:

Test media were prepared daily during the definitive test to maintain measurable levels of the test substance between renewals. Each week, a series of concentrated stock solutions were prepared as follows. The test substance (50 mg) was dissolved in tetrahydrofuran (THF;10 ml) to provide a solution with a nominal concentration of 5 mg/ml. This solution was serially diluted with THF to provide intermediate solvent stock solutions at 0.128, 0.32, 0.8 and 2.0 mg/ml. Each day an aliquot (100 µl) of the appropriate solvent stock solution was dispersed in the dilution medium, Elendt M4, (800 ml) in a volumetric flask (1-litre). The contents of each flask were then vigorously shaken before being made up to volume (1-litre) to provide the test media at nominal concentrations of 0.0128, 0.032, 0.08, 0.2 and 0.5 mg/l. The highest concentration was limited by the solubility of the test substance in the media.
The solvent control medium (0.1 ml THF/L was prepared using the same formulation as for the test media but without the test substance.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Appliqué (IRChA), France. Stock cultures of the Daphnia were maintained as parthenogenic cultures in covered one litre capacity glass vessels containing Elendt M4 culture medium (approximately 500 to 800 ml).
Cultures were held for 25 days prior to the definitive test in a temperature-controlled laboratory at nominally 20 ± 2ºC (within 20 ± 1ºC at least 24 hours prior to the definitive test). A photoperiod of 16 hours light : 8 hours dark was maintained, with periods (1 hour) of subdued lighting at the beginning and end of each light phase. The light intensity of the culture area is checked at three-month intervals and the last measurement conducted prior to this test was 618 lux (11 June 2008). A maximum of fifteen adult Daphnia were maintained in each culture vessel.
Cultures were fed daily with a suspension of the unicellular green algae Pseudokirchneriella subcapitata. Algal cultures were grown in synthetic mineral salts medium. Concentrated algal cell suspensions were prepared by removing and centrifuging aliquots of algal culture and resuspending the algal pellets in small volumes of dilution medium. Appropriate volumes of these concentrated suspensions were added to each Daphnia culture to provide nominally 0.1 to 0.2 mg carbon per daphnid per day, except during the initial three days of culture when a slightly lower ration was given.
The day before the start of the test, all neonate daphnids were removed from the laboratory cultures. The following day, any neonates produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessel and held in a separate vessel. These animals, which were less than 24 hours old, were used in the test. The temperature of the holding water immediately prior to the addition of the neonates to the test vessels for the definitive test was 19.8°C.
The test organisms were maintained and the test conducted in Elendt M4 medium. The medium used in the study was prepared in de-ionised water.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None

Test conditions

Hardness:

Dilution medium = 254 to 270 mg/L as CaCO3
Control medium = 254 to 266 mg/L as CaCO3
Test medium at 0.416 mg/L (measured) = 252 to 260 mg/L as CaCO3

Test temperature:

19.5 to 22.0 °C

pH:

7.48 to 8.13

Dissolved oxygen:

95% to 110% air saturation (ASV)

Salinity:

Not applicable

Nominal and measured concentrations:

0 (medium control and solvent control), 0.0128, 0.032, 0.08, 0.2 and 0.5 mg/L (nominal)
0 (medium control and solvent control), 0.0114, 0.0294, 0.0742, 0.196, 0.416 (arithmetic mean measured)

Details on test conditions:

Two formulation trials were performed to identify a suitable solvent for use in the study and to examine the potential for losses by adsorption of the test substance under test conditions. The solvent selected for use in the study was tetrahydrofuran (THF). The results of analysis during the first trial indicated that Citroflex® B-6 was unstable under the conditions of the test therefore an exposure regime with daily renewal of media was selected for use in the study. The results of the second trial demonstrated that the losses of Citroflex® B-6 were not due to adsorption onto glassware.

A range finding test was conducted at nominal concentrations of 0.005, 0.01, 0.05, 0.1 and 0.5 mg/l. The range finding test employed exposure conditions and test media formulation techniques comparable to those subsequently used in the definitive test. Based on the results of the range finding test, the definitive test employed nominal concentrations of 0.0128, 0.032, 0.08, 0.2 and 0.5 mg/l. The highest concentration was considered to be the maximum practicable to employ given the physical effects (floating) observed in the range finding test that were attributed to the insolubility of the test substance. Control animals were exposed to dilution medium alone and to dilution medium containing tetrahydrofuran. In the definitive test, ten, individually-housed, Daphnia were exposed for 21 days to the dilution medium control and to each test level and twenty individuals to the solvent control. Daphnia were added to the test vessels (glass jars, ca. 60 ml capacity) containing control or test medium, according to a random group order.

The test media were renewed daily. On each occasion of renewal, any dead animals were discarded and the surviving parental Daphnia (mobile and immobile) were transferred to fresh control or test media in a second set of vessels. Any neonates, unhatched eggs or carapaces present in the expired media were counted and then discarded. After completion of the analytical and environmental measurements, the vessels containing the expired media were emptied, rinsed thoroughly with purified water and left to drain until used for the next renewal.

A concentrated suspension of Pseudokirchneriella subcapitata was added directly to the medium in each control and test vessel at the start and subsequently on each day during the definitive test. The volume of algal ration given during the definitive test was estimated to be in the range of 0.1 to 0.2 mg carbon per daphnid per day, except during the initial two days of the test when a slightly lower ration was given (nominally 0.075 mg carbon per daphnid per day).

The temperature of the test area was continuously monitored in an additional vessel containing the same volume of dilution medium as the test vessels.

A photoperiod of 16 hours light : 8 hours dark was maintained, with periods (one hour) of subdued lighting at the beginning and end of each light phase. No supplementary aeration was employed and the pH of the test media was neither adjusted nor controlled during the study.

Temperature measurements were taken daily, while pH and DO were measured on three occasions each week.

The total hardness of the control and test medium at 0.5 mg/l (nominal) were measured on Days 3, 10 and 17.

The light intensity of the test area is checked at approximately three-month intervals; the measurements conducted prior to and after this test were made on 11 June and 9 October 2008).

Immobility was defined as the inability to free-swim in the test medium and death was defined as the cessation of all movement. The numbers of mobile, floating (swimming at the media surface), immobile, dead and gravid (animals with eggs in the brood pouch) parental Daphnia were recorded daily, together with any general observations of their size and general appearance (if different from the controls). At the end of the definitive test, after observations of the Daphnia were made, the body length (taken as the distance from the apex of the helmet to the base of the spine) of each surviving adult was measured using a microscope and stage mounted graticule. From Day 8 (the first occasion when juveniles were observed), the numbers of live, dead or floating neonates and the presence of any aborted (unhatched) eggs or dead juveniles in each vessel were recorded daily.

The vessels were also checked daily for carapaces moulted by the parental generation and, where present, these were removed from the vessels and counted.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: floating, parental

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: floating, parental

Effect concentrations 5

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

parental length

Effect concentrations 6

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

parental length

Effect concentrations 7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Effect concentrations 8

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Effect concentrations 9

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Effect concentrations 10

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Effect concentrations 11

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

0.247 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Remarks on result:

other: 0.134 to 0.391

Effect concentrations 12

Duration:

14 d

Dose descriptor:

other: EC20

Effect conc.:

0.354 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Remarks on result:

other: 0.268 to 0.460

Effect concentrations 13

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Effect concentrations 14

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

0.218 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Remarks on result:

other: 0.153 to 0.353

Effect concentrations 15

Duration:

14 d

Dose descriptor:

other: EC20

Effect conc.:

0.275 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Remarks on result:

other: 0.219 to 0.377

Effect concentrations 16

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Remarks on result:

other: 0.369 to 0.468

Effect concentrations 17

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.303 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Remarks on result:

other: 0.165 to 0.410

Effect concentrations 18

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

> 0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Effect concentrations 19

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.416 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

all live neonates

Effect concentrations 20

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.204 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

excluding floating neonates

Remarks on result:

other: 0.150 to 0.281

Details on results:

Additional effect concentrations:
Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
21 d EC20 0.266 mg/L meas. (arithm. mean) test mat. reproduction excluding floating neonates 0.291 to 0.326
21 d EC50 0.419 mg/L meas. (arithm. mean) test mat. reproduction excluding floating neonates 0.381 to 0.474
21 d NOEC 0.196 mg/L meas. (arithm. mean) test mat. time to first brood
21 d LOEC 0.416 mg/L meas. (arithm. mean) test mat. time to first brood

In the range finding test, one parental animal died on Day 2; no other mortality occurred during the test although all of the adults at 0.5 mg/l were observed swimming at the surface of the medium (floating). Compared to the solvent control group, neonate production was reduced at 0.5 mg/l, with a mean number of neonates per adult of 10 compared with 21 for the control. The results of analysis confirmed that the solvent stock solutions were stable for at least 8 days after preparation (ranging between 80 and 110% of nominal) and that the aqueous test media could be adequately achieved (between 74 and 143% of nominal in fresh media) but not maintained between renewals (decreasing to between 56 and 80% of initial).

In the definitive test, measured concentrations in samples of freshly prepared media ranged from 74 to 171% of nominal concentrations, with the majority of values ranging from 82 to 122% of nominal. In samples of expired (ca. 24-hour-old) media, the measured concentrations ranged from 47 to 188% of their nominal values (with the majority of values between 47 and 116%) and on most occasions, the measured concentrations had decreased from their starting values. The overall geometric mean measured concentrations of Citroflex® B-6 were 0.0114, 0.0294, 0.0742, 0.196 and 0.416 mg/l and these values were used in test calculations.

An EC50 value for parental generation mortality could not be calculated because insufficient mortality occurred. The sub-lethal effect of “floating” (swimming at the media surface) was exhibited by two to eight adults at 0.416 mg/l throughout the test; at 0.196 mg/l, at most, two adults were observed floating on occasions during the test. This sub-lethal effect was a physical effect attributed to the presence of the test substance in the
media.

Statistical analysis of the body lengths of surviving adults after 21 days of exposure to Citroflex® B-6 indicated that growth was adversely affected at 0.416 mg/l.

Statistical analysis of the total number of live neonates produced by each surviving adult in the test groups compared to the solvent control group indicated that reproduction was significantly reduced at a Citroflex® B-6 concentration of 0.416 mg/l (-19.5%; p<0.001). At the highest test level, substantial numbers of the neonates observed in the vessels were floating (38%). Floating neonates were also observed at 0.196 mg/l, but the numbers affected were minimal (5%). Floating was a physical effect attributed to the presence of test substance in the media and represents a worst-case estimate.

The time taken to produce the first brood of neonates was significantly delayed at a Citroflex® B-6 concentration of 0.416 mg.

Reported statistics and error estimates:

Insufficient parental mortality precluded the calculation of an EC50 value.

The body lengths of all surviving parental Daphnia in each group at the end of the test were compared to those in the solvent control group using a multiple comparisons Dunnett’s test. The dilution medium and solvent control groups were compared using a t-test.

The cumulative number of live neonates produced per adult in the solvent control group was compared with those in each test group using a multiple comparisons Williams’ test (Williams; 1971, 1972). Where suitable data was generated, the 14- and 21-day EC10, EC20 and EC50 values were estimated by non-linear regression (logistic curve) and the 95% confidence limits were derived by the likelihood ratio method. The dilution medium and solvent control groups were compared using a t-test. The statistical analysis was then repeated after subtracting the number of floating neonates from the total number of live neonates produced in each vessel to evaluate the impact of this sub-lethal physical effect. The time to the appearance of the first brood of neonates per adult Daphnia surviving until Day 21 of the test was analysed for each replicate. The control groups and all treated groups were compared using two-tailed Linear-by-linear Association test for a trend in time correlated with increasing dose (Agresti et al 1990).

Any other information on results incl. tables

Parental Mortality During the Study

Observation time (day)	% mortality / measured Citroflex® B-6 concentration (mg/l)
	Control	Solvent control	0.0114	0.0294	0.0742	0.196	0.416
2	0	0	0	0	0	0	10
4	0	0	0	0	0	0	10
7	0	0	0	0	0	0	10
14	0	0	0	0	0	0	10
21	0	5	10	0	0	0	20
The mortality observed at 0.114 and 0.416 mg/L was not considered significant or treatment related

Summary of Statistical Analysis - Mean Number of Live Young per Adult and Lengths of Surviving Adults

Parameter	Concentration	Sample size	Mean	% Inhibition	p
Mean live young per adult after 21 days	Control	10	77.10	1.9	0.607^T
	Solvent control	19	78.58	0.0	-
	0.0114 mg/l	9	75.22	4.3	0.638^W
	0.0294 mg/l	10	78.20	0.5	0.638^W
	0.0742 mg/l	10	76.10	3.2	0.498^W
	0.196 mg/l	10	74.60	5.1	0.218^W
	0.416 mg/l	8	63.25	19.5	< 0.001***^W
Parameter	Concentration	Sample size	Mean	% Inhibition	p
Mean live young per adult after 21 days excluding floaters	Control	10	77.10	1.9	0.635^T
	Solvent control	19	78.58	0.0	-
	0.0114 mg/l	9	75.22	4.3	0.675^W
	0.0294 mg/l	10	78.20	0.5	0.675^W
	0.0742 mg/l	10	75.80	3.5	0.477^W
	0.196 mg/l	10	70.60	10.2	0.013*^W
	0.416 mg/l	8	39.13	50.2	< 0.001***^W
Parameter	Concentration	Sample size	Mean	% Inhibition	p
Length of surviving adults	Control	10	3.66	1.9	0.053^T
	Solvent control	19	3.73	0.0	-
	0.0114 mg/l	9	3.63	2.6	0.051^D
	0.0294 mg/l	10	3.73	0.0	>0.999^D
	0.0742 mg/l	10	3.74	-0.2	>0.999^D
	0.196 mg/l	10	3.73	0.0	>0.999^D
	0.416 mg/l	8	3.59	3.9	0.002**^D
p values are for the comparison with Solvent control using Williams' test (W), Dunnett's test (D)  and the t-test (T)  * p < 0.05, ** p < 0.01, *** p < 0.001

Summary of Statistical Analysis - Distribution of First Broods by Study Day

Group	Day		p
	8	9
Control	10	0
Solvent control	19	0
0.0114 mg/l	9	0
0.0294 mg/l	10	0
0.0742 mg/l	10	0
0.196 mg/l	10	0
0.416 mg/l	6	2	0.0196*
P values are asymptotic linear by linear test for all groups up to and including the current row except control and solvent control  * p < 0.05

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Less than 20% parental mortality in control groups. Mean number of live neonates per surviving parent in the control groups greater than or equal to 60.

Conclusions:

For the parental generation, a sub-lethal physical effect of floating at the surface of the media was observed at 0.196 and 0.416 mg/l. However, chemical toxicity of Citroflex® B-6 (i.e. decreased body length) only occurred at 0.416 mg/l. A conservative parental generation NOEC of 0.0742 mg/l is established by the physical effect of floating. For reproduction endpoints, the number of live neonates floating at the surface because of the physical effect of Citroflex® B-6 resulted in a conservative NOEC and LOEC of 0.196 and 0.416 mg/l, respectively. The chemical toxicity of Citroflex® B-6 (i.e. significantly delayed time to production of the first brood) also had a NOEC and LOEC of 0.196 and 0.416 mg/l, respectively.
Therefore, the lowest NOEC in this Daphnia magna reproduction test for Citroflex® B-6 is derived from a physical effect (i.e. floating) on the parental generation, which is considered to be a consequence of its low water solubility rather than chemical toxicity.
Environmental concentrations above the solubility limit of Citroflex® B-6 are very unlikely to occur. Therefore, it seems most appropriate to base risk assessment for invertebrates on chemical toxicity.