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Long-term toxicity to aquatic invertebrates

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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
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

Key value for chemical safety assessment

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.