Dibutyl fumarate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD guidelines for Testing of Chemicals, guideline No. 211: "Daphnia magna, Reproduction Test", Adopted: October 2012. In addition, the procedures were designed to meet the test methods prescribed by the following guidelines and guidance document: • Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.20. "Daphnia magna Reproduction Test". • ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30. • Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by these deviations.

Principles of method if other than guideline:

Deviations:1. During the flow-through tests daphnids were fed with ~0.4 mg organic carbon per daphnid per day. The protocol stated the amount provided during a semi-static test.Evaluation: This did not affect the study results.2. Final test: At day 6 and day 11 of the test oxygen concentrations in all but the blank-control vessels were low, i.e. ranging between 2.3 and 3.4 mg/l.Evaluation: Probably due to the presence of DMF. This however did not negatively affect mortality or reproduction of the daphnids.3. Final test: Temperature varied between 19.3 and 21.9°C during the study and was therefore not kept within 2°C.Evaluation: Temperature changes were gradual and did not affect the daphnids.The study integrity was not adversely affected by these deviations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the study, samples for analyses were taken at the start of exposure and on days 7, 14 and 21. Measured concentrations were always lower than target and not stable. It was therefore considered appropriate to calculate the mean measured concentrations for targets up to and including 1.6 mg/l. The highest target concentration of 5.0 mg/l was only used during the first two days of exposure, hence the initially measured concentration was used. The effect parameters were based on the following exposure concentrations: 0.013, 0.055, 0.093, 0.54 and 4.1 mg/l.

Vehicle:

no

Details on test solutions:

The effect parameters were based on the following exposure concentrations: 0.013, 0.055, 0.093, 0.54 and 4.1 mg/l.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.In-house laboratory culture with a known history.Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium.Maximum age of the cultures: 4 weeksFeeding: Daily, a suspension of fresh water algaeValidity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Total hardness varied between 161 and 232 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the protocol (>140 mg CaCO3 per liter).

Test temperature:

Temperature of medium: 18-22°C

pH:

The pH remained within the range of 7.2 to 8.1 throughout the test and thus was maintained within the limits prescribed by the protocol (6.0-9.0, constant within 1.5 units).

Dissolved oxygen:

The oxygen concentration in all test solutions remained within the range of 2.3 to 9.0 mg/l during the exposure period and thus did not comply to the requirements as laid down in the protocol (> 3 mg/l)

Nominal and measured concentrations:

Nominal concentrations: 0.050, 0.16, 0.50, 1.6 and 5.0 mg/lMeasured concentrations: 0.013, 0.055, 0.093, 0.54 and 4.1 mg/l.

Details on test conditions:

Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Reference substance (positive control):

yes

Remarks:

DMF

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.055 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

All parental daphnids exposed to 4.1 mg/l died during the test period, while no significant mortality was observed at any of the lower test concentrations. Fumaric acid di-n-butyl ester did not affect reproduction of Daphnia magna at 0.013 mg/l after 21 days of exposure (NOEC). Exposure to concentrations of 0.055 mg/l induced significant inhibition of the reproductive capacity of the parental daphnids.

Results with reference substance (positive control):

The average cumulative number of young per female in the DMF-control after 21 days was 224 with a coefficient of variation of 8%.

 Target and measured exposure concentrations

Target conc. Test substance^1 (mg/l)	Measured concentration (mg/l)
	Day 0	Day 7	Day 14	Day 21	Mean measured
0.050	0.019	0.015	0.010	0.0096	0.013
0.16	0.088	0.057	0.046	0.041	0.055
0.50	0.32	0.12	0.050	<LOD	0.093
1.6	1.1	0.67	0.33	0.30	0.54
5.0	4.1	-	-	-	4.1

¹The test substance is Fumaric acid di-n-butyl ester

-: All parental daphnids dead

Validity criteria fulfilled:

yes

Conclusions:

Fumaric acid di-n-butyl ester:NOEC reproduction of Daphnia magna (21 days) = 0.013 mg/l

Executive summary:

Daphnia magna, 21-day reproduction study withFumaric acid di-n-butyl ester.

 

The study procedures described in this report were based on theOECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition,the procedures were designed to meet the test methods and validity criteria of theISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

 

The reproduction test was performed using a flow-through system with target concentrations of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/l, which were based on the results of two preceding range-finding tests. A blank- and solvent-control (DMF) were also included.

 

In the flow-through system the dilution water was dosed separately from the test substance stock solutions into mixing vessels. Stock solutions in DMF were a factor of 10,000 higher than in the target concentrations. The dosed volumes and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The experimental set-up included stainless steel vessels with 4 stainless steel mesh containers per test group. Each container contained five neonate (<24 h old)Daphnia magna. The study duration was 21 days and the test solutions were renewed at a flow rate of 0.7 litres/hour. The daphnids were fed continuously withChlorella pyrenoidosa(~0.4 mg organic C per daphnid per day). The condition of the parental daphnids was recorded daily and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

 

During the study, samples for analyses were taken at the start of exposure and on days 7, 14 and 21. Measured concentrations were always lower than target and not stable. It was therefore considered appropriate to calculate the mean measured concentrations for targets up to and including 1.6 mg/l. The highest target concentration of 5.0 mg/l was only used during the first two days of exposure, hence the initially measured concentration was used. The effect parameters were based on the following exposure concentrations: 0.013, 0.055, 0.093, 0.54 and 4.1 mg/l.

  

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

All parental daphnids exposed to 4.1 mg/l died during the test period, while no significant mortality was observed at any of the lower test concentrations.

 

The average cumulative number of young per female in the DMF-control after 21 days was 224 with a coefficient of variation of 8%.

 

Fumaric acid di-n-butyl esterdid not affect reproduction of Daphnia magna at 0.013 mg/l after 21 days of exposure (NOEC). Exposure to concentrations of 0.055 mg/l induced significant inhibition of the reproductive capacity of the parental daphnids.

 

Effect parameters obtained in this study are summarized in the table below

 

Parameter	Concentration (mg/l)
NOEC for reproduction	0.013
EC10forreproduction	0.061
EC50for reproduction	0.11
NOEC for mortality	0.54
NOEC for growth	0.055

Description of key information

Fumaric acid di-n-butyl esterdid not affect reproduction of Daphnia magna at 0.013 mg/l after 21 days of exposure (NOEC). Exposure to concentrations of 0.055 mg/l induced significant inhibition of the reproductive capacity of the parental daphnids.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

13 µg/L