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

Long-term toxicity to fish

Administrative data

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Test Guideline 210 OPPTS 850.1400

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity 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:

Samples (100 ml) were taken from each replicate control and test vessel at the start of the test (freshly-prepared), on Day 1 pre-hatch and on Days 0, 2 (at 0.16 mg/l), 7, 9, 14, 15 (at 0.4 mg/l), 21 and 28 post-hatch. Single or duplicate samples (100 ml) of fresh or expired media were also taken from the preparation vessels containing the aqueous media on Days 0 and 1 pre-hatch and Days 8, 9, 27 and 28 post-hatch to verify the correct preparation of the media and its stability during dosing. Additional samples were also taken on Days 13 and 14 post-hatch at 0.0256 and 0.16 mg/l, and on Days 21 and 22 post-hatch at 0.0256 and 1 mg/l because anomalous values were obtained on previous sampling occasions.

All samples of media were placed into polypropylene bottles containing ethyl acetate (20 ml) to stabilise the samples. On each occasion, one of the samples was analysed initially and the second (reserve) sample (were taken) was stored in a freezer in case further analysis was required. These reserve samples were analysed when the results obtained for the original samples were considered anomalous.

Test solutions

Vehicle:

yes

Details on test solutions:

Test solutions were prepared on a daily basis to maintain measurable levels of the test substance in the test vessels. Each week, a series of concentrated solvent stock solutions were prepared as follows. The test substance (500 mg) was dissolved in tetrahydrofuran (THF; 50 ml) to provide a solution with a nominal concentration of 10 mg/ml. This solution was serially diluted with THF to provide intermediate solvent stock solutions at 0.256, 0.64, 1.6 and 4.0 mg/ml. Each day, an aliquot (100 μl/litre) of the appropriate solvent stock solution was dispersed in the diluent water (dechlorinated and softened tap water) and these aqueous stock preparations were mixed for approximately 10 minutes. On cessation of stirring, these aqueous preparations were continuously delivered, via a peristaltic pump, to replicate test vessels at each of the following nominal concentrations: 0.0256, 0.064, 0.16, 0.4 and 1.0 mg/l

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

Fertilised eggs, between 22 and 48 hours old at the start of the definitive test, were obtained from in-house laboratory breeding stocks of mature fathead minnow, which were originally supplied by Aquatic Research Organisms, Hampton, New Hampshire, USA.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Remarks on exposure duration:

5-day hatch; 28-day post-hatch

Post exposure observation period:

None

Test conditions

Hardness:

156 to 180 mg/L as CaCO3

Test temperature:

24.6 to 26.6°C

pH:

7.67 to 8.30

Dissolved oxygen:

62 to 98% air saturation (ASV)

Salinity:

Not applicable

Nominal and measured concentrations:

0 (water control and solvent control), 0.0256, 0.064, 0.16, 0.4 and 1.0 mg/L (nominal)
0 (water control and solvent control), 0.0221, 0.0542, 0.135, 0.252, and 0.539 mg/L (geometric mean measured)

Details on test conditions:

A formulation trial was performed to identify a suitable solvent for use in the study; tetrahydrofuran was selected. The results of analysis during this trial also indicated that Citroflex® B-6 was unstable under the conditions of the test; therefore an exposure regime with continual renewal of the media was necessary for use in the study.

A range finding test was conducted at nominal concentrations of 0.01, 0.1 and 1 mg/l. Based on the results of the range finding test, the definitive test employed nominal concentrations of 0.0256, 0.064, 0.16, 0.4 and 1 mg/l. The highest concentration intentionally exceeded the stated limit of aqueous solubility (0.6 mg/l) to ensure that the maximum attainable concentration that was practicable to prepare was employed. Control groups comprised diluent water alone and diluent water containing 100 μl THF/l. Two vessels were employed for each control and test group. Test vessels were glass aquaria, with approximate dimensions of 17 x 10 x 15 cm fitted with a surface overflow (12 cm above the base), to hold an approximate volume of two litres of medium. At the start of the definitive test, each test vessel contained two glass dishes (6 cm diameter) located on the base of the vessel. Fifteen fertilised eggs were placed into each glass dish, giving a total of 30 eggs per vessel and 60 eggs per treatment group. A photoperiod of 16 hours light : 8 hours dark, with a 1-hour transition period of subdued lighting at the beginning and end of each light phase, was maintained during the study, and measurements of temperature, pH and dissolved oxygen were made in each control and test vessel twice per week. The total hardness of the control and test media in one replicate vessel per group was measured on Day 4 pre-hatch (protocol deviation) and Day 28 post-hatch. In addition, the total hardness and chlorine content of the diluent water supplied to the test area was measured each week during the definitive test. The light intensity of the test area was 347 lux (the light intensity of the test area was checked at three-month intervals and the last measurement conducted prior to this test was made on 11 June 2008). Fry were fed with a suspension of Liquifry No.1 and newly-hatched (24-hour-old) Artemia (brine shrimp) nauplii (Artemia International LLC, Texas, USA) that were prepared in diluent water.

Subjective assessments were made on the viability of the embryos (e.g. clouded eggs were recorded as being dead). Each day, the number of healthy fry was recorded, together with the number of fry found dead in the egg dishes and the number of dead eggs. The type and incidence of post-hatch sub-lethal effects were recorded and compared to controls. At study termination, all surviving fry were sacrificed by means of an anaesthetic overdose. The standard length of individual fry was measured with a digital caliper, to the nearest 0.01 mm. Subsequently, fry were placed in an oven set to a temperature of approximately 60°C and dried for two days. The fry were then weighed to obtain the dry weight of each fry.

Hatching success was defined as the percentage of embryos that produced live fry, and was based on the number of eggs initially introduced to the egg dishes. Fry that hatched but were found dead in the egg dish or vessel were counted as a successful hatch and were recorded as post-hatch mortalities. Post-hatch survival was defined as the percentage of fry surviving to the termination of the study and was based on the number of successfully hatched eggs. Overall survival was defined as the percentage of fry surviving to the termination of the study and was based on the number of eggs initially introduced to the egg dishes, corrected for unaccounted and accidental mortalities that occurred. The lowest-observed effect concentration (LOEC) was defined as the lowest concentration tested at which the test substance was observed to have a statistically significant specified adverse effect on hatching success, survival, length and/or weight (at p<0.05), when compared with the solvent control and where all concentrations above the LOEC had a harmful effect equal to or greater than those observed at the LOEC. The no-observed effect concentration was defined as the concentration immediately below the LOEC.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Effect concentrations 2

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Effect concentrations 3

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 6

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 7

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 8

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

dry

Effect concentrations 9

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

dry

Effect concentrations 10

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: overall study result, based on worst-case estimate

Effect concentrations 11

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.539 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: overall study result based on worst-case estimate

Details on results:

In the rangefinding test, after 15 days post-hatch exposure, Citroflex® B-6 had no effects on either the hatching success (93%) or survival (86 to 100%) of fathead minnow at any of the concentrations employed. Therefore, the NOEC for survival during the rangefinder was considered to be 1 mg/l (nominal).

In the definitive test, measured concentrations ranged from 70 to 111% of nominal concentrations in samples taken prior to the addition of fertilized eggs. Thereafter, measured concentrations typically ranged from 56 to 133% of their nominal values at concentrations of 0.0256 to 0.16 mg/l and from 35 to 96% at 0.4 and 0.1 mg/l, although on occasions, values fell outside these ranges. The variability in the individual values reflects the low solubility and poor dispersability of the test substance in the water. Based on a geometric mean, the overall measured concentrations of Citroflex® B-6 in the test vessels were 0.0221, 0.0542, 0.135, 0.252 and 0.539 mg/l.

Hatching commenced four days after the eggs were introduced into the test system and the majority of eggs had hatched by the fifth day (Day 0 post-hatch), although hatching was not complete until the seventh day (Day 2 post-hatch). Hatching success was not affected by exposure to Citroflex® B-6, ranging from 98 to 100%.

Compared to the solvent control, survival was not significantly affected at any of the concentrations employed in the test and no treatment-related sub-lethal effects were observed. After 28 days post-hatch exposure, mortality ranged between 2 and 7% within the test groups and there was no significant difference between the control groups, where mortality was ≤ 2% (solvent control).

At the end of the definitive test, the average dry weight of the fry at 0.539 mg/l was found to be statistically significantly lower (p<0.05) than those in the solvent control when a parametric test (Williams’ test) was used. However, using a non parametric test (Shirley’s test), no statistical differences were identified between the solvent control and any of the test groups. No statistical differences were observed in fish lengths between the solvent control and any of the test groups, and there was no significant difference between the control groups.

Reported statistics and error estimates:

Hatching success and cumulative mortality at Day 28 (post-hatch) for each group was compared to the solvent control group using one-tailed Fisher’s exact tests (Fisher 1973). The LC50 could not be estimated for either hatching success or cumulative mortality because sufficient dose-related effects were not observed.

The lengths and dry weights of the surviving fish on Day 28 (post-hatch) were analysed using an F1 test for monotonicity. The treated groups were then compared to the solvent control group using the parametric two-tailed Williams’ test (1971, 1972) and the solvent control group was compared to the control group using a t-test.

Due to concerns over the homogeneity of variance of the data, further analysis of the lengths and dry weights of the fish was undertaken. One of the assumptions of parametric analysis such as Williams’ test is that the variability in the data is the same across all groups. This was tested using Bartlett’s test, and for dry weights, a p value of 0.0191 was obtained. While this result was significant at the 5% level, it was not significant at the 1% level, indicating that there may be a problem with the validity of the assumption of equal variance. To examine the validity of the results of the Williams’ test, the non-parametric equivalent (Shirley’s test) was also performed on lengths and dry weight data. The results of the non-parametric test were not found to be significant for either lengths or dry weights. The contradictory conclusions of these two analyses could be caused by the growth data not meeting the assumptions of the parametric test and/or the relative power of the statistical tests.

Any other information on results incl. tables

Hatching Success:

Nominal Citroflex® B-6 concentration (mg/l)	Mean measured Citroflex® B-6 concentration (mg/l)	Total number of eggs*	Total number of dead eggs**	Hatching success (%)
Control	ND	60 (59^#)	-	100
Solvent control	ND	60 (59^#)	-	100
0.0256	0.0221	60	-	100
0.064	0.0542	60	-	100
0.16	0.135	60	1	98
0.4	0.252	60 (59^#)	-	100
1.0	0.539	60	-	100
ND = None detected ( < 0.0025 mg/l; limit of detection *  All eggs were examined microscopically to confirm viability at time of exposure initiation ** Number of dead eggs removed from test vessel at time of detection on Day 1 (#)  Only 59 eggs were used in test calculations because one fry was accidentally killed during cleaning

Survival - Day 0 to 28 Post-Hatch:

Nominal Citroflex® B-6 concentration (mg/l)	Mean measured Citroflex® B-6 concentration (mg/l)	Total number of hatched fry [A]	Total number of dead (unaccounted) fry ***	Total number of surviving fry on Day 28 [B]	Post-hatch survival (%) *	Overall survival (%) **
Control	ND	60 (59^#)	0	59	100	100
Solvent control	ND	60 (59^#)	1	58	98	98
0.0256	0.0221	60	4	56	93	93
0.064	0.0542	60	1	59	98	98
0.16	0.135	59	1 (1)	57	97	95
0.4	0.252	60 (59^#)	0 (1)	58	98	98
1.0	0.539	60	1	59	98	98
ND = None detected (< 0.0025 mg/l; limit of detection)      * = Based on total number of successfully hatched eggs (=B/A)     ** = Based on total number of eggs at initiation (see Table 2)    *** = Number of live larvae back-calculated from the numbers of fry surviving on Day 28 by adding the sum of observed larval mortalities throughout the exposure period    ( )  =Number of animals that could not be accounted for on Day 28 based on the number of dead eggs and larvae             removed during the study.  These animals were allocated as having died on Day 5 post-hatch without being identified (^#) = Only 59 eggs were used in test calculations because one fry was accidentally killed during cleaning

Mean Length and Dry Weight Parameters for Surviving Fry on Day 28 Post-Hatch:

Nominal Citroflex® B-6 concentration (mg/l)	Mean measured Citroflex® B-6 concentration (mg/l)	Length (mm)				Dry weight (mg)
		mean	SD	p Par.	p N-par.	mean	SD	p Par.	p N-par.
Control	ND	17.3	2.0	0.942	0.996	13.7	4.9	0.503	0.868
Solvent control	ND	17.3	2.9	-	-	14.5	7.8	-	-
0.0256	0.0221	17.9	2.2	1.000	1.000	14.7	6.0	1.000	1.000
0.064	0.0542	17.2	2.2	1.000	1.000	13.4	5.8	0.652	1.000
0.16	0.135	17.5	2.6	1.000	1.000	14.1	6.7	0.652	1.000
0.4	0.252	17.4	2.4	1.000	1.000	13.5	5.9	0.551	0.972
1.0	0.539	16.8	2.2	0.319	0.282	12.0	5.5	0.037*	0.157
ND = None detected ( < 0.0025 mg/l; limit of detection) SD = Standard deviation Par. = Parametric tests (Williams' test for Citroflex® B-6; t test for control) N-par. = Non-parametric test (Shirley's test for Citroflex® B-6; Wilcoxon rank sum for control) * = Statistically significant (p < 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Mean control hatching success > 60%, and mean control post-hatch survival >= 70%.

Conclusions:

Hatching success: Citroflex® B-6 had no effect on the hatching success of fathead minnow eggs exposed at concentrations up to and including 0.539 mg/l.

Post-hatch survival: The LC50 value for post-hatch survival could not be calculated because insufficient mortality occurred.

Sub-lethal effects: No effects on appearance or behaviour were observed.

Growth: Based on the dry weights and lengths of surviving fry on Day 28 post-hatch, the following NOEC and LOEC values were derived from the data. Although the results of the original statistical analysis (parametric Williams’ test) of the dry weight data were not corroborated by a subsequent non-parametric analysis (Shirley’s test), the results from the parametric analysis for dry weight are considered to represent the worst case (although based on the non-parametric statistical analysis performed, it is not a realistic case) and, therefore, were used to determine the LOEC and NOEC for dry weight.