Dihydro-3-(tripropenyl)furan-2,5-dione

Long-term toxicity to fish

Administrative data

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-13 to 2013-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tripropenyl succinic acid

Cas Number:

147384-84-7

Molecular formula:

R-C4H6O4 , whereas R=C8-C10-alkyl-(branched, unsaturated)

IUPAC Name:

Tripropenyl succinic acid

Details on test material:

- Name of test material (as cited in study report): Tripropenyl succinic acid
- Physical state: Viscous liquid, pale brown
- Analytical purity: 99.3 %
- Purity test date: 2013-03-04
- Lot/batch No.: 12-SK002
- Expiration date of the lot/batch: January 2014
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L (factor 3.16)
- Sampling method: Samples of test media including control were taken from alternating test replicates in 3 sampling intervals from freshly prepared and corresponding 2 – 3 d old test solutions.
- Sample storage conditions before analysis: All samples were stored at room temperature before preparation and until start of analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate amounts of the test item were weighed out on an object slide and placed in glass aquaria (dimensions: 11.5/13/20 cm), filled with 2.5 L of dilution water. The test item dispersions were mixed with a magnetic stirrer for up to 24 hours at test temperature.
- Eluate: Dilution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L (factor 3.16)
- Controls: 30 eggs in dilution water (without test item, 3 replicates with 10 eggs each) will be tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbuundesamt, Schichauweg 58, 12307 Berlin).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning rectangular dishes (26 cm x 14 cm x 6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. After approx. 3 h the glass dishes were gently removed. About 300 eggs were taken and immediately distributed to the test solution and dilution water (for control).
- Subsequent handling of eggs: After 4 hours eggs were checked for fertilization. Under a stereo microscope every embryo was checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were discarded. Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Test conditions

Hardness:

Low hardness to avoid precipitation of Ca-Salts.

Test temperature:

please refer to "Any other information on materials"

pH:

please refer to "Any other information on materials"

Dissolved oxygen:

please refer to "Any other information on materials"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dishes (inner diameter 13.5 cm, water height about
5 cm) were used. The volume of the test media in the dishes was about 500 mL.
- Aeration: Gentle aeration was provided. Semi-static conditions with daily renewal of the test media was performed.
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5


OTHER TEST CONDITIONS
- pH: 6 – 8.5 within a range of 0.5 °C
- Photoperiod: 16 h photoperiod daily
- Light intensity: 0.1 - 10 µmol photons • m-2 • s-1on water surface (diffuse light)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biological parameters Observations were made daily.

Hatched eggs The number of hatched eggs was determined daily until 5 days post-hatch.

Post hatch period Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the control group have hatched (day 4 of the study).

Mortality Criteria for mortality vary according to life stage:

For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, was checked daily. Dead eggs were discarded.

For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.

For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/or lack of reaction to mechanical stimulus.

Further effects
Abnormal appearance and behaviour were also recorded.The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour will also be recorded by visually inspecting each replicate.

Measurement of fish size Fish size was determined at the end of the exposure (post-hatch day 5) of the control and of two test groups where no significant mortality (0.316 mg/L) and mortality close to 50 % (1.00 mg/L) were observed, respectively.
The fish were euthanized in a Benzocaine solution and the total length of each fish was measured to the nearest 0.001 mm with a microscope camera and corresponding software (ImageFocus, EUROMEX BV).


Measurement of
dry body weight At the end of exposure (post-hatch day 5) the mean fish dry weight from pooled survivors per replicate were determined from the control and of two test groups where no significant mortality (0.316 mg/L) and mortality close to 50 % (1.00 mg/L) was observed. The fish were dried for at least 24 h at 60 °C. Dry biomass weight was measured to the nearest 0.001 mg



Chemical parameters

Water quality and Temperature, pH value and oxygen saturation were measured
light intensity daily in freshly prepared and old test media in one replicate per
measurements test concentration and the control, respectively. Temperature was recorded continuously in the dilution water by a datalogger. Total hardness was measured at the beginning of exposure from one replicate of the control and the highest test concentration, respectively. The light intensity on the surface of the test vessels was measured at start of the exposure and on study day 5.



VEHICLE CONTROL PERFORMED: no

Results and discussion

Effect concentrations

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatch, survival, length, weight

Details on results:

- Mortality/survival at embryo and larval stages:
The post hatch success in all control replicates met the guideline criteria. The post hatch success was calculated from the study day with the maximum hatch rate per test group (post hatch day 2: for the control and all tested concentrations) and the surviving larvae on post hatch day 5. The post hatch survival at the end of the study was 97 % in the control group and ranged from 97 to 100 % post hatch success in the tested concentration levels.
ANOVA, One Way Analysis of Variance was carried out for post hatch survival at the end of the study. No statistically significant differences were found for all tested concentrations when compared with the control.

- Overall mortality/survival:
Overall survival at the end of the study was 97 % in the control group and ranged from 90 to 100 % in the tested concentration levels.
ANOVA, One Way Analysis of Variance was carried out for the results of overall survival on post hatch day 5 (end of the study). No statistically significant differences were found for all tested concentrations when compared with the control.

- Days to hatch and numbers hatched:
Egg hatch began on study day 3 in the control and all tested concentrations and continued until study day 5. Study day 3 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 87 %.
Statistical procedures (One Way Analysis of Variance) of all test concentrations were applied for study days 3, 4 and 5 (post hatch days 0, 1 and 2). A statistically significant difference was found on post hatch day 0 for the concentration levels of 1.00 – 10.0 mg/L and 100 mg/L. However, this effect was transient and considered to be biologically not significant.
Swim-up was observed for a 3-day period on study days 5, 6 and 7. Newly hatched fry of the control began to swim up on study day 5 (post hatch day 2). On study day 6 (post hatch day 3) all surviving larvae of the control had swum up. In the test concentrations of 1.00 to 31.6 mg/L newly hatched fry began to swim up on study day 5 (post hatch day 2). In the test concentration of 100 mg/L newly hatched fry began to swim up on study day 6 (post hatch day 3). The completion of swim-up in the test concentration of 1.00 – 31.6 mg/L was on study 6 (post hatch day 3). In the test concentrations of 100 mg/L swim up was completed on study day 7 (post hatch day 4).


- Data for length and weight of surviving fish:
The fry growth, expressed as length and dry weight, was determined on study day 8 (post hatch day 5).
ANOVA, One Way Analysis of Variance was carried out on post hatch day 5 (end of the study) for the data of the control and the two highest test concentrations of 31.6 and 100 mg/L. No statistically significant differences were found for the tested concentrations of 31.6 and 100 mg/L when compared with the control.



- Type of and number with morphological abnormalities:
No biologically significant morphological and behavioural effects were observed in any tested replicate throughout exposure.

Reported statistics and error estimates:

One way analysis of variance (ANOVA) and DUNNETT’S test was used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first.
For the parameters hatch, post hatch survival and overall fry survival (mortality), growth (length and dry weight), the following statistical tests were conducted:
Hatching data of study day 3 (post hatch day 0) was analysed with ANOVA and DUNNETT’S test. Hatching data of study days 4 and 5 (post hatch days 1 and 2) were analysed with ANOVA. Equal variance test failed for hatch data on study day 3. No suitable transformation was found to pass the Equal variance test.
Post hatch survival and overall survival data (mortality), dry weight and length data, of study day 8 (post hatch day 5) were analysed with ANOVA. Normality test failed for post hatch survival and overall survival data. No transformations were carried out with these data because the data sets were not estimated to follow a normal distribution.
The statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The -value (acceptable probability of incorrectly concluding that there is a difference) was 0.05.

Any other information on results incl. tables

Egg Hatch / Hatching Time: Study Day 1 - 5 (Post Hatch Day -2 - 2)

Nominal test item concentration [mg/L]	Replicate	Egg hatch [%]
		Post hatch day -2 (Study day 1)	Post hatch day -1 (Study day 2)	Post hatch day 0 (Study day 3)	Post hatch day 1  (Study day 4)	Post hatch day 2   (Study day 5)
100	1	0	0	40	100	100
	2	0	0	20	80	80
	3	0	0	20	80	90
	Mean	0	0	+      27	87	90
31.6	1	0	0	70	90	100
	2	0	0	70	90	90
	3	0	0	10	70	100
	Mean	0	0	50	83	97
10.0	1	0	0	20	90	100
	2	0	0	40	90	90
	3	0	0	60	90	90
	Mean	0	0	+      40	90	93
3.16	1	0	0	40	100	100
	2	0	0	50	90	100
	3	0	0	20	100	100
	Mean	0	0	+      37	97	100
1.00	1	0	0	30	90	100
	2	0	0	30	100	100
	3	0	0	10	90	90
	Mean	0	0	+      23	93	97
Control	1	0	0	80	90	100
	2	0	0	100	100	100
	3	0	0	80	80	100
	Mean	0	0	87	90	100

Post Hatch Survival on Study Day 8 (Post Hatch Day 5)

Nominal test item concentration [mg/L]	Study day with maximum hatch	Replicate	Hatched larvae on study day with maximum hatch n/10	Vital Larvae on study day 8 (PHD 5) n/10	Post Hatch survival [%]
100.0	5 (PHD 2)	1	10	10	100
		2	8	8	100
		3	9	9	100
		Mean	9.0	9.0	100
31.6	5 (PHD 2)	1	10	10	100
		2	9	9	100
		3	10	10	100
		Mean	9.7	9.7	100
10.0	5 (PHD 2)	1	10	10	100
		2	9	9	100
		3	9	9	100
		Mean	9.3	9.3	100
3.16	5 (PHD 2)	1	10	10	100
		2	10	9	90
		3	10	10	100
		Mean	10.0	9.7	97
1.00	5 (PHD 2)	1	10	10	100
		2	10	10	100
		3	9	9	100
		Mean	9.7	9.7	100
Control	5 (PHD 2)	1	10	10	100
		2	10	10	100
		3	10	9	90
		Mean	10.0	9.7	97

Overall Survival and Mortality on Study Day 8 (Post Hatch Day 5)

Nominal test item concentration [mg/L]	Replicate	Vital larvae on post hatch day 5 (Study day 8) n/10	Overall survival [%]	Mortality [%]
100	1	10	100	0
	2	8	80	20
	3	9	90	10
	Mean	9	90	10
31.6	1	10	100	0
	2	9	90	10
	3	10	100	0
	Mean	10	97	3
10.0	1	10	100	0
	2	9	90	10
	3	9	90	10
	Mean	9	93	7
3.16	1	10	100	0
	2	9	90	10
	3	10	100	0
	Mean	10	97	3
1.00	1	10	100	0
	2	10	100	0
	3	9	90	10
	Mean	10	97	3
Control	1	10	100	0
	2	10	100	0
	3	9	90	10
	Mean	10	97	3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Tripropenyl succinic acid caused no significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions up to the dosage level of 100 mg/L (nominal test item concentration). The LC50 on study day 8 (post hatch day 5) was determined to be > 100 mg/L. The NOEC for the parameters hatch, post hatch and overall survival (mortality), growth (expressed as length and dry weight) was 100 mg/L. For further LOEC values, please refer to Table 1. All effect values are given based on the nominal concentrations of the test item Tripropenyl succinic acid

Executive summary:

The effects of the test item Tripropenyl succinic acid to the embryo and sac-fry stages of the zebrafish (Danio rerio) were determined according to OECD Guideline 212 from 2013-03‑12 to 2013-03-22, with the definitive exposure phase from 2013-03-13 to 2013-03-21 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with renewal of the test media every 2 – 3 days was performed with the nominal test item concentrations of 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L (dilution factor 3.16).

The test was started by placing fertilized eggs in the test vessels and lasted 8 days (5 days post-hatch). 30 eggs of Danio rerio were exposed per test concentration and control (3 replicates with 10 eggs each), respectively.

On day three 87 % of the control larvae have hatched. Therefore, study day 3 was defined as post hatch day 0 (PHD 0).

Different toxic endpoints were determined: egg hatch, time to hatch, post hatch survival, overall fry survival and mortality, fry growth (expressed as length and weight), morphological and behavioural effects. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results. The revealing results are summarized in Table 1 and Table 2.

All concentrations of Tripropenyl succinic acid and the control were analytically verified by LC-MS/MS at the start and the end of three exposure intervals. The measured concentrations of Tripropenyl succinic acid in freshly prepared test concentrations were in the range of 102 to 112 % of the nominal values. In the corresponding 2 to 3 days aged test concentrations, the recoveries were in the range of 93 to 114 % of the nominal values. All effect values are given based on the nominal concentrations of the test item Tripropenyl succinic acid.

NOEC, LOEC: Hatch, Fry survival, Growth, Behaviour

                      based on nominal test item concentrations [mg/L]

Parameter	NOEC	LOEC
Hatch	100	> 100
Post hatch survival	100	> 100
Overall survival	100	> 100
Length	100	> 100
Weight	100	> 100

Table 2:       LC₅₀-Value with 95 % Confidence Interval on Study Day 8 (Post Hatch Day 5)

                      based on nominal test item concentrations [mg/L]

LC50	> 100
95 % confidence interval	Not applicable