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

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

Study period:

2013-03-19 to 2013-04-11

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 211 (Daphnia magna Reproduction Test)

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 applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Sampling method: All concentration levels of the test item and the control were analysed by LC-MS/MS at the start of the exposure intervals on days 0, 2, 5, 14 (0 h) and at the end of the exposure intervals on days 2, 5, 7, 16 (48 or 72 h).
For the analyses at the end of the exposure intervals, separate replicates without algae and daphnids were prepared and stored under test conditions until sampling.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared in demineralised water one day prior to the start of each exposure interval. 100 mg/L of the test item were weighed out and transferred with an appropriate volume of demineralised water into a measuring flask. The measuring flask was not completely filled up with demineralised water, because the mineral salts of the dilution water had to be added immediately before the start of the exposure phase.
For the transfer of the weighed test item into the measuring flask, ultrasound was used for approximately 1 minute.
The stock solution was stirred with approximately 1100 rpm for 24 hours at room temperature.
After the stirring period, the mineral components as specified under 'Dilution water' were added to yield the highest test concentration.
- Eluate: Dilution water
- Differential loading:
6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: renewals 3 times per week


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2013-03-20 2013-03-22 2013-03-27 2013-03-29 2013-04-03 2013-04-05
100 144 142 148 141 142 146
Control 144 141 144 141 144 141

Test temperature:

Temperature [°C]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2013-03-20 2013-03-22 2013-03-27 2013-03-29 2013-04-03 2013-04-05
100 20.8 20.5 19.8 20.6 20.4 20.5
Control 19.8 20.6 19.0 20.4 19.6 20.5

pH:

pH-Values
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2013-03-20 2013-03-22 2013-03-27 2013-03-29 2013-04-03 2013-04-05
100 6.52 7.45 6.15 7.70 6.02 6.65
Control 8.32 7.77 8.89 7.73 7.91 7.35

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2013-03-20 2013-03-22 2013-03-27 2013-03-29 2013-04-03 2013-04-05
100 8.32 8.09 8.56 9.61 8.55 8.11
Control 8.99 7.73 10.51 8.59 8.74 5.91

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood: between days 8 and 11
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
Immobilisation Rates in the non GLP Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
test item concentration

[mg/L] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

24.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (14.0 - 42.0)

Details on results:

- Mortality of parent animals:
No significant adult mortality of > 20 % was observed during the test period of 21 days. In the concentration levels of 6.25 to 50.0 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. At the concentration level of 100 mg/L, the adult mortality was 20 %, which is not biologically significant. Therefore, the EC20 was assessed at 100 mg/L. Further EC-values were not calculated, because significant adult mortality was not observed.
- No. of offspring produced per day per female:
The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 83 in the control group. The reproductive output was statistically significant reduced in comparison to the control at the concentration levels of 25.0 to 100 mg/L (One Way Analysis of Variance, DUNNETT’S method, p = 0.05). At the concentration levels of 6.25 and 12.5 mg/L, the reproductive output was comparable to the reproductive output at the control.
The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 24.5 mg/L (95% confidence limits: 14.0 – 42.0 mg/L). The EC50 was calculated accordingly to be 66.8 mg/L (95 % confidence limits: 49.1 – 96.5 mg/L).
- Body length and weight of parent animals:
The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 6.25 to 100 mg/L were determined to be in the range of 3.84 to 5.30 mm per daphnid and 5.28 mm per daphnid in the control group.
The mean dry body weight was determined to be in the range of 0.30 to 0.89 mg per daphnid in the concentration levels 6.25 to 100 mg/L and 0.81 mg per daphnid in the control
- Type and number of morphological / behavioural abnormalities:
please refer to the respective table
- Time to first brood release or time to hatch:
The first brood was released between days 8 and 10 by all parental daphnids of the control and the concentration levels of 6.25 to 50.0 mg/L. At the concentration level of 100 mg/L, the first brood was released between days 11 and 15. Occurrence of the first brood after day 10 is regarded to be noticeably delayed.
At the tested concentration levels of 6.25 to 50.0 mg/L and in the control, 4 broods were produced by the surviving parental daphnids. At the concentration level of 100 mg/L, 2 – 3 broods were produced by the surviving parental daphnids.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.54 - 1.65) mg/L

Reported statistics and error estimates:

The NOEC and the LOEC based on the reduction of the
evaluation reproductive output were deduced directly from the results. Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test and analysis of variance (One Way Analysis of Variance, DUNNETT’S method).
Prior to running the analysis of variance, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests should be 0.05. For the determination of significant deviations of the intrinsic rates of natural increase, the P-value was < 0.05 and therefore the normality and the equal variance test failed.
The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.
The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Any other information on results incl. tables

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal test item conc. [mg/L]												No. of parents prod. juv.	Mean no. of juveniles per parent producing juveniles			Comparison versus control
	Number of living juveniles in replicate no.										Total no.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
100	--	9	34	23	29	14	--	28	14	20	171	8	21 ±	9	41*	74	yes
50.0	60	72	37	65	54	66	69	62	50	61	596	10	60 ±	10	17	28	yes
25.0	63	60	74	87	70	47	71	60	68	76	676	10	68 ±	11	16	19	yes
12.5	77	71	89	87	84	95	69	87	87	75	821	10	82 ±	9	10	1	no
6.25	73	75	96	81	97	76	79	75	85	73	810	10	81 ±	9	11	2	no
Control	93	76	88	76	83	82	83	86	87	76	830	10	83 ±	6	7	—	—

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal test item concentration [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
100	1	171	172	1
50.0	1	596	597	1
25.0	17	676	693	2
12.5	21	821	842	2
6.25	3	810	813	1
Control	0	830	830	0

First Appearance of Living Juveniles in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles										First	Mean
	in replicate no.										appearance	number
	1	2	3	4	5	6	7	8	9	10	mean day	of broods
100	--	11*	15*	11*	11*	12*	--	11*	14*	11*	12.0*	2.5^#
50.0	8	9	8	9	8	8	9	8	9	8	8.4	4.0
25.0	9	9	9	9	9	8	9	8	9	9	8.8	4.0
12.5	9	9	9	9	9	9	9	9	9	9	9.0	4.0
6.25	10	10	8	8	8	8	8	8	8	8	8.4	4.0
Control	9	9	9	9	9	9	9	9	9	9	9.0	4.0

Total Body Length of the Parental Daphnids

Nominal test item concentration [mg/L]	Total length of the parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
100	--	4.00	3.50	4.00	4.00	4.25	--	4.25	3.00	3.75	3.84	8	2.4	0.30
50.0	4.75	5.00	5.25	5.00	5.00	5.00	5.75	4.50	4.50	4.75	4.95	10	4.7	0.47
25.0	5.25	5.25	5.25	5.50	5.50	5.50	5.50	5.25	5.00	5.00	5.30	10	8.6	0.86
12.5	5.50	5.00	5.00	5.25	5.50	5.25	5.25	5.25	5.50	5.50	5.30	10	8.9	0.89
6.25	5.75	5.00	5.75	5.25	5.25	5.00	5.00	5.00	5.25	5.00	5.23	10	8.7	0.87
Control	5.50	5.25	5.00	5.25	5.50	5.25	5.25	5.00	5.25	5.50	5.28	10	8.1	0.81

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
100	0	0	20
50.0	0	0	0
25.0	0	0	0
12.5	0	0	0
6.25	0	0	0
Control	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The significant reduction of the reproductive output at the concentration levels of 25.0 to 100 mg/L was the most sensitive effect in this study.
Therefore, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 12.5 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 25.0 mg/L.
The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 24.5 mg/L (95% confidence limits: 14.0 – 42.0 mg/L). The EC50 was calculated accordingly to be 66.8 mg/L (95 % confidence limits: 49.1 – 96.5 mg/L).
No significant adult mortality of > 20 % was observed during the test period of 21 days. In the concentration levels of 6.25 to 50.0 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. At the concentration level of 100 mg/L, the adult mortality was 20 %, which is not biologically significant. Therefore, the EC20 was assessed at 100 mg/L. Further EC-values were not calculated, because significant adult mortality was not observed.

Endpoints assessed Tripropenyl succinic acid
Nominal test item concentration
[mg/L]
EC20 Adult mortality 100 mg/L
EC10 Reproduction
(with 95 % confidence limits) 24.5 mg/L (14.0 – 42.0 mg/L)
EC50 Reproduction
(with 95 % confidence limits) 66.8 mg/L (49.1 – 96.5 mg/L)
NOECReproduction 12.5 mg/L
LOECReproduction 25.0 mg/L

Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 d) of the test item Tripropenyl succinic acid (batch number: 12-SK002) was conducted according to OECD 211 (2008) from   2013-03-19 to 2013-04-11, with the definitive exposure phase from 2013-03-20 to 2013-04-10, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157Sarstedt, Germany.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions with renewal of the test solutions three times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

The succinic acid derivate typically forms a calcium salt with very low solubility (ca. 40 ppm) removing most of the test item from the aqueous solution by precipitation. Therefore, the recovery of the test item at the end of an exposure interval (48 h) is expected to be very low and the guideline quality criteria for the analytical recovery are no longer fulfilled. To reduce the precipitation of calcium salts, the total hardness in the Elendt M4 medium (B.P. Elendt, 1990), was set to a total hardness of approximately 140 mg CaCO₃/L still acceptable to achieve valid results. Nominal concentrations of Tripropenyl succinic acid were selected as follows:         6.25 – 12.5 –25.0 – 50.0 –100 mg/L.

The concentrations of the test item Tripropenyl succinic acid were analytically verified by LC-MS/MS of samples taken at the start of the exposure intervals on days 0, 2, 5, 14 (0 h) and at the end of the exposure intervals on days 2, 5, 7, 16 (48 or 72 h) of all concentration levels and the control.

The measured concentrations of the test item at the start of the exposure intervals were in the range of 97 to 111 % of the nominal values. At the end of the exposure intervals (48 or 72 h), the measured concentrations of the test item were in the range of 92 to 114 % of the nominal values. This demonstrates, that the test item concentrations were successfully maintained for the duration of the test.   The nominal concentrations were used for assessment of the endpoints.

·          The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 83 in the control group. The reproductive output was statistically significant reduced in comparison to the control at the concentration levels of 25.0 to 100 mg/L (One Way Analysis of Variance, Dunnett’s method, p = 0.05). At the concentration levels of 6.25 and 12.5 mg/L, the reproductive output was comparable to the reproductive output at the control.
The EC₁₀ for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 24.5 mg/L (95% confidence limits: 14.0 – 42.0 mg/L). The EC₅₀ was calculated accordingly to be 66.8 mg/L (95 % confidence limits: 49.1 – 96.5 mg/L).

Based on the significant reduction of the reproductive output at the concentration levels of 25.0 to 100 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 12.5 mg/L andthe Lowest Observed Effect Concentration (LOEC) was assessed at 25.0 mg/L.

·   The coefficient of variation of the number of living offspring produced per parent was 7 % in the control. In the test groups the coefficient of variation was in the range of 10 to 41 %. The coefficient of variation of 41 % at the concentration level of 100 mg/L is regarded to be biologically noticeable

·   The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of natural increase determined for the surviving daphnids at the concentration levels of 6.25 to 50.0 mg/L were comparable to the IR at the control (One Way Analysis of Variance, Dunnett’s method, p = 0.05). At the concentration level of 100 mg/L, there was a statistical significance in comparison to the control.

·   No stillborn juveniles or aborted eggs were observed in the control group during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was in the range of 1 to 2 % in the tested concentration levels of 6.25 to 100 mg/L.

·   The first brood was released between days 8 and 10 by all parental daphnids of the control and the concentration levels of 6.25 to 50.0 mg/L. At the concentration level of 100 mg/L, the first brood was released between days 11 and 15. Occurrence of the first brood after day 10 is regarded to be noticeably delayed.
At the tested concentration levels of 6.25 to 50.0 mg/L and in the control, 4 broods were produced by the surviving parental daphnids. At the concentration level of 100 mg/L, 2 – 3 broods were produced by the surviving parental daphnids

·   The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 6.25 to 100 mg/L were determined to be in the range of 3.84 to 5.30 mm per daphnid and 5.28 mm per daphnid in the control group.

     The mean dry body weight was determined to be in the range of 0.30 to 0.89 mg per daphnid in the concentration levels 6.25 to 100 mg/L and 0.81 mg per daphnid in the control

·   No significant adult mortality of > 20 % was observed during the test period of 21 days. In the concentration levels of 6.25 to 50.0 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. At the concentration level of 100 mg/L, the adult mortality was 20 %, which is not biologically significant. Therefore, the EC₂₀ was assessed at 100 mg/L. Further EC-values were not calculated, because significant adult mortality was not observed.

·       No males or ephippia (winter eggs) were observed in the control or in the test groups.

 

·   Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.

A summary of all test item related effects and the assessed endpoints based on the nominal concentrations of the test item is given in Table 1.

Table 1:           Test Item Related Effects (NOEC, LOEC, EC₁₀, EC₂₀ and EC₅₀)

                         (based on the nominal concentrations of the test item Tripropenyl succinic acid)

Effects	Tripropenyl succinic acid Nominal concentrations of the test item [mg/L]
	Control	6.25	12.5	25.0	50.0	100
Mean Number of Living Juveniles per Producing Parent (Reproduction Rate ± SD)	83 ± 6	81 ± 9	82 ± 9	68 ± 11^1)	60 ± 10^1)	21 ± 9^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]	7	11	10	16	17	41^2)
Mean Intrinsic Rates of Natural Increase	0.44	0.45	0.44	0.44	0.45	0.29^5)
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]	0	1	2	2	1	1
Mean Number of Broods	4.00	4.00	4.00	4.00	4.00	2.50^3)
Appearance of First Brood [Mean Day]	9.0	8.4	9.0	8.8	8.4	12.0^4)
Parental Daphnids: Mean Dry Weight [mg]	0.81	0.87	0.89	0.86	0.47	0.30
Parental Daphnids: Mean Body Length [mm]	5.28	5.23	5.30	5.30	4.95	3.84
Adult Mortality after 21 Days [%]	0	0	0	0	0	20
EC20 Adult mortality	100 mg/L
EC10 Reproduction (with 95 % confidence limits)	24.5 mg/L (14.0 – 42.0 mg/L)
EC50 Reproduction (with 95 % confidence limits)	66.8 mg/L (49.1 – 96.5 mg/L)
NOECReproduction	12.5 mg/L
LOECReproduction	25.0 mg/L