Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the analogue justification attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: source: source, RA-A, EC 946-056-8, Zschimmer & Schwarz, 2017, D. magna, 21 d, RL1

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: source: source, RA-A, EC 946-056-8, Zschimmer & Schwarz, 2017, D. magna, 21 d, RL1

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Jan 2017 - 09 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations, solvent-controls and stock solutions
- Sampling method: 2 mL samples were taken at the beginning and at the end of four intervals 24 hours (nominal days 0-1, 6-7, 13-14 and 20-21); Stock solutions: on nominal days 0, 6, 13 and 20
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15 °C) until analysis

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions were prepared in Dimethylformamide at the start of the test and weekly thereafter at a concentration of 100 mg/L. No special treatment other than careful mixing was needed to fully dissolve the test item in the solvent.
- Controls: only medium (test medium without test item)
- Chemical name of vehicle: DMF (99.8%, Merck, Darmstadt, Germany)
- Concentration of vehicle in test medium: 100 µL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history
- Age of parental stock: parental daphnids greater than two weeks old, maximum age of culture: 4 weeks
- Feeding during test: Twice daily an amount of 0.25 mL of a Chlorella sp. suspension. On weekend days an amount of 0.50 mL was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211
- Food type: a suspension of fresh water algae
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

161 - 214 mg/L CaCO3 (Control)
161 - 214 mg/L CaCO3 (Solvent control)
161 - 214 mg/L CaCO3 (56 µg/L)

Test temperature:

19 - 21 °C (Control)
20 - 21 °C (Solvent control)
20 - 21 °C (test item concentration)

pH:

7.6 - 8.3 (Control)
7.6 - 8.3 (Solvent control)
7.6 - 8.3 (test item concentration)

Dissolved oxygen:

8.0 - 10 mg/L (Control)
8.1 - 10 mg/L (Solvent control)
8.0 - 10 (test item concentration)

Nominal and measured concentrations:

Control, 1.0, 3.2, 10, 32 and 100 µg/L (nominal)
Control, 0.19, 0.19, 1.1, 12 and 56 µg/L (average measured)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with a Perspex plate)
- Material, size, headspace, fill volume: 60 mL-glass vessels, 50 mL volume
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7, as prescribed by Dr. Elendt-Schneider
- Hardness: 180 mg/L expressed as CaCO3
- pH: 7.7 ± 0.3
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, oxygen and pH was recorded at test start and just before and after each renewal of the test in one of the vessels; hardness was measured once a week

OTHER TEST CONDITIONS
- Photoperiod: 16 h photoperiod daily
- Light intensity: intensity at the start: 475-579 lux; at the end: 486-586 lux

EFFECT PARAMETERS MEASURED: The number of living, immobile and dead parental daphnids was daily recorded; body length was recorded at test end; number of newborn was recorded daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1
Range finding study
- Test concentrations: 0.0010, 0.010, 0.10 and 1.0 mg/L
- Results used to determine the conditions for the definitive study: Results of the range-finding test are summarized within table 1 in section " Any other information on results incl tables"

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 56 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

- Mortality of control: Two out of the twenty parental daphnids died during the test period in the DMF control, hence mortality did not exceed 20%
- Other: All biologicala nd analytical results are summarized within the tables 2-5 in section "Any other information on results incl. tables"

Reported statistics and error estimates:

Following statistical procedures were used to determine the NOEC for reproduction and growth:
Data distribution: Shapiro-Wilk´s Test ; Homogeneity of variance: Levene´s Test (with Residuals); Differences between two controls: Student-t test; Differencesbetween treatments and the (pooled) control: Williams Multiple Sequential t-test Procedure
Survival: Parental survival was not statistically analysed as the mortality did not exceed 20%.
EC-values for reproduction: No EC10 or EC50 values could be calculated as the observed effects were lower than 10%. All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Results of the range-finding test: Mortality and reproductive potency

Hexanedioic acid, di-C16-18 alkyl esters Concentration (mg/L)	Number Daphnia exposed	Cumulative number of dead/immobile parental daphnids
		Day 1-4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10
Blank-control	5	0	0 [1]	0 [5]	0 [5]	0 [5]	0 (13)	0[2]
	5	0	0 [2]	0 [4]	0 [5]	0 [5]	0 (12)	0 [4]
Acetone-control	5	0	0 [1]	0 [5]	0 [5]	0 [5]	0 (15)	0 [5]
	5	0	0 [1]	0 [5]	0 [5]	0 [5]	0 (6)	0 [5]
0.0010	5	0	0 [2]	0 [4]	0 [4]	0 [5]	0 (9)	0 [5]
	5	0	0 [1]	0 [4]	0 [4]	0 [5]	0 (8)	0 [5]
0.010	5	0	0 [1]	0 [4]	0 [5]	0 [5]	0 (12)	0 [5]
	5	0	0 [1]	0 [4]	0 [5]	0 [5]	0 (14)	0 [4]
0.10	5	0	0 [1]	0 [5]	0 [5]	0 [5]	0 (3)	0 [5]
	5	0	0 [2]	0 [5]	0 [5]	0 [5](1)1	0 (7)	0 [5]
1.0	5	0	0	0	0 [4]	0 [5]	0 [5]	0 [4]2
	5	0	0	0	0 [2]	0 [2]	0 [4]	0 [5]

[ ] number of parents with eggs in their brood pouch, ( ) number of offspring, 1 one immobile offspring, 2 one

 

Table 2: Main test: Analytical Results: Nominal and mean measured exposure concentrations

Hexanedioic acid, di-C16-18 alkyl esters Nominal concentration (µg/L)	Mean exposure concentration (µg/L) at day				Average exposure concentration (µg/L)
	0-1	6-7	13-14	20-21
1.0	0.281	0.281	0.0932	0.0932	0.19
3.2	0.281	0.281	0.0932	0.0932	0.19
10	2.5	1.3	0.48	0.26	1.1
32	19	16	6.5	5.1	12
100	78	76	37	32	56

1 Half of the limit of detection of the analytical method (LOD= 0.568 µg/L)

2 Half of the limit of detection of the analytical method (LOD= 0.185 µg/L)

 

Table 3: Main test Biological Results: Mortality (immobility) of parental daphnids at the end of the test

Hexanedioic acid, di-C16-18 alkyl esters Average concentration (µg/L)	Introduced	Mobile	Immobile	% Immobility
Blank-control	10	10	0	0
DMF-control	10	8	2	20
Pooled control	20	18	2	10
0.19	10	10	0	0
0.19	10	10	0	0
1.1	10	8	2	20
12	10	8	2	20
56	10	9	1	10

 

Table 4: Main test Biological Results: Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at the end of the test

Hexanedioic acid, di-C16-18 alkyl esters Average concentration (µg/L)	Mean	Std. Dev.	n	%Change1
Pooled control	84.9	5.10	18
0.19	86.0	9.23	10	-1.3
0.19	85 (77.0)	9.37 (27.13)	9 (10)	-0.30 (9.3)
1.1	86.1	15.43	8	-1.5
12	86.6	6.44	8	-2.0
56	88.9	7.59	9	-4.7

1 Negative numbers indicate an increase, () – between brackets values before removal of the outlying replicate are given

 

Table 5: Main test Biological Results: Incidence of immobilised offspring, offspring trapped at the surface and aborted eggs

Abnormality	Hexanedioic acid, di-C16-18 alkyl esters; Average concentration (µg/L)
	Blank-control	DMF-control	0.19	0.19	1.1	12	56
Immobile offspring	5	1	2	2	3	1	5
Aborted eggs	4	3	2	1	15	14	15

 

Table 6: Main test Biological Results: Group mean body lengths (mm) and reduction of growth of parental daphnids at the end of the test.

Hexanedioic acid, di-C16-18 alkyl esters Average concentration (µg/L)	Mean	Std. Dev.	n	%Change1
Pooled control	4.02	0.137	18
0.19	3.95	0.092	10	1.8
0.19	3.90 (3.75)	0.165 (0.492)	9 (10)	3.0 (6.7)
1.1	3.93	0.079	8	2.1
12	4.06	0.164	8	-1.1
56	3.94	0.150	9	1.9

1 Negative numbers indicate an increase, () – between brackets values before removal of the outlying replicate are given

Description of key information

NOEC (21 d) ≥ 56 µg/L for Daphnia magna (OECD 211); read across

Additional information

Experimental studies investigating chronic toxic effects of the target substance Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester on Daphnia magna are not available. Thus, in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5 a read across to the structurally related source substance Hexanedioic acid, di-C16-18 alkyl esters (CAS 92969-90-9) was conducted to fulfill the data requirements according to Annex VII. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substance was used for the assessment. This read-across is justified in detail within the analogue justification in IUCLID Section 13.

The toxicity of the source substance Hexanedioic acid, di-C16-18 alkyl esters (CAS 92969-90-9) to Daphnia magna was tested under semi-static conditions according to OECD guideline 211 under GLP conditions. D. magna was exposed to nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L (0.19, 0.19, 1.1, 12 and 56 µg/L (mean measured concentrations)) for 21 days. Due to the low solubility of the test item dimethylformamide (DMF) was used as a solvent. Due to the decrease of test substance concentration during testing the results were based on average measured concentrations over the 21-day test period. Hexanedioic acid, di-C16-18 alkyl esters did not affect reproduction of Daphnia magna. Thus a NOEC (21 d) ≥ 56 µg/L was determined. Based on the available data for the read across substance and the very low water solubility (<0.57 µg/L at 20°C, pH 6.3) of the target substance a chronic toxicity of Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester is not expected.