Diisodecyl azelate

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:

21 Aug 2016 - 22 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Analytical Method Study in Support of Daphnia magna Reproduction Test

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Analytical verification was not possible with the analytical method validation

GLP compliance:

yes

Specific details on test material used for the study:

The test substance is described as an unknown, variable composition, complex reaction product or biological material (UVCB) under REACH. Despite exhaustive attempts, an analytical method, based on the detection of a marker compound, capable of detecting the extremely low concentrations of this complex mixture in this project could not be developed. The study was consequently performed without analytical support and therefore no samples for analytical confirmation of actual exposure concentrations were taken during the main reproduction test.

Analytical monitoring:

no

Remarks:

An exhaustive and validated analytical method was not sensitive enough to detect the analyte due to the extremely low concentrations used.

Details on sampling:

The test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 512852. On the day of analysis, the test samples were defrosted at room temperature. The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 50/50 (v/v) acetonitrile/M7-medium to obtain concentrations within the calibration range.

Vehicle:

yes

Remarks:

Elendt M7 Media

Details on test solutions:

No test substance was observed in the range-finder test solutions used during the ecotoxicology test using the original method (TQD detection). To demonstrate proper preparation of the test solutions an aliquot of the filter residue was analysed. Based on the retention time of the compound the residues of the filters were identified as the test substance. This original methodology used TQD for detection and was validated with a low calibration standard of 0.02 mg/L. From the chromatograms it was concluded that the possible concentration in the ecotoxicology samples could be as low as 0.002 mg/L. In order to investigate whether the method could be improved to a level where test substance could be observed in the test solutions used during the ecotoxicity test, a more sensitive detector (Xevo TQ-S mass spectrometer (Waters)) was used for the analysis of reserve samples. The lowest analysed test concentration with the more sensitive detector was 0.002 mg/L (average detected area 739). No test substance was detected in samples taken from a water accommodated fraction (WAF) prepared at a loading rate of 1 mg/L. Some test item was visible in the samples taken from the WAF prepared at a loading rate of 10 mg/L (detected area 385). The detected response was smaller than the response of the lowest test calibration solution at the concentration of 0.002 mg/L and very close to the response of the detected carry over (detected area 240). Hence, it was concluded that the validated method could not be improved using available equipment. Additionally, according to the sponsor, test item has Log Koc value where solid phase extraction would not concentrate the sample and improve the sample preparation.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus,
1820), at least third generation, obtained by a
cyclical parthenogenesis under specified
breeding conditions.
Source: In-house laboratory culture with a known
history.
Reason for selection: This system has been selected as an
internationally accepted invertebrate species.
Validity of batch: 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.
Characteristics: To initiate the test, young daphnids < 24 hours
old were selected, from parental daphnids
greater than two weeks old.

Start of each batch: With new-born daphnids, i.e. less than 3 days
old, by placing them individually in 50 ml
M7-medium.
Maximum age of the cultures: 4 weeks
Monitoring of the individual cultures: Three times a week the young are counted and
the parental daphnids are transferred to new
media.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae
Validity 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.
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).

Test type:

semi-static

Water media type:

other: Elendt M7

Remarks:

OECD approved

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

N/A

Hardness:

161 - 196 mg/L CaCO3

Test temperature:

19.0 - 21.0 °C

pH:

7.5 - 8.4

Dissolved oxygen:

7.5 - 9.7 mg/L O2

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

The test substance is described as an unknown, variable composition, complex reaction product or biological material (UVCB) under REACH. Despite exhaustive attempts, an analytical method, based on the detection of a marker compound, capable of detecting the extremely low concentrations of this complex mixture in this project could not be developed. The study was consequently performed without analytical support and therefore no samples for analytical confirmation of actual exposure concentrations were taken during the main reproduction test.

Details on test conditions:

Parental daphnids
Condition: Every workday and upon renewal on non-workdays, the
number of living, immobile and dead parental daphnids
was recorded. Dead daphnids were removed when
observed.
Presence of eggs in Every workday and upon renewal on non-workdays.
the brood pouch:
Body length: At the end of the test.

Offspring
Appearance first brood: When observed.
New-born daphnids: Every workday and upon renewal on non-workdays, the
number of new-born young was counted and the
condition of the young recorded. Thereafter the young
were removed.
Presence of unhatched eggs: When observed
Incidence of immobility: When observed.

Test medium
Temperature, oxygen and pH: At the start of the test and just before and after each
renewal in one of the vessels of each test group with
surviving daphnids.
Hardness: Once a week in fresh and old media from the control
and the highest test concentration
Light: At the start and the end of the test

Reference substance (positive control):

not required

Remarks:

OECD TG 211 (2012)

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

9.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LL50

Effect conc.:

8.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Age at first brood

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Under the conditions of the present study Diisodecyl azelate did not affect reproduction of Daphnia magna at a WAF prepared at a loading rate of 4.0 mg/L after 21 days of exposure (NOELR).

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

Parental Mortality: Fisher’s exact test (p<0.05)
Reproduction: Multiple sequentially-rejective U-test after Bonferroni-Holm (p<0.05)
Body length: Shapiro-Wilk's test (p<0.05)

Mortality (immobility) of parental daphnids at the end of the test

Test group Diisodecyl azelate WAF (mg/L)	Introduced	Mobile	Immobile	% Immobility
Control	20	17	3	15
0.26	10	9	1	10
0.64	10	8	2	20
1.6	10	9	1	10
4.0	10	80	2	20
10	10	3	7	70*

* Statistically significant; Fisher’s exact test (p<0.05)

Group mean cumulative number of juveniles per introduced parent and reduction of reproduction at the end of the test

Test group Diisodecyl azelate WAF (mg/L)	Mean	Std. Dev.	n	%Reduction
Control	118.4	20.29	20	-
0.26	127.0	7.76	10	-7.3
0.64	116.3	21.67	10	1.7
1.6	130.3	12.75	10	-10.1
4.0	122.4	43.25	10	-3.4
10	64.3	36.67	10	45.7*

* Statistically significant; Multiple sequentially-rejective U-test after Bonferroni-Holm (p<0.05)

Group mean body lengths (mm) and reduction of growth of parental daphnids at the end of the test.

Test group Diisodecyl azelate WAF (mg/L)	Mean (mm)	Std. Dev.	n	%Reduction
Control	4.31	0.162	17	-
0.26	4.29	0.129	9	0.3
0.64	4.27	0.085	8	0.9
1.6	4.25	0.079	9	1.3
4.0	4.20	0.112	8	2.4*
10	4.00	0.210	3	7.2*

* Statistically significant; Shapiro-Wilk's test (p<0.05)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Diisodecyl azelate did not affect reproduction of Daphnia magna at a WAF prepared at a loading rate of 4.0 mg/L after 21 days of exposure (NOELR).

Executive summary:

Under the conditions of the present study Diisodecyl azelate did not affect reproduction of Daphnia magna at a WAF prepared at a loading rate of 4.0 mg/L after 21 days of exposure (NOELR).

Description of key information

NOELR (21 d) = 4.0 mg/L (Daphnia magna, OECD 211, nominal)

Key value for chemical safety assessment

Marine water invertebrates

Marine water invertebrates

Effect concentration:

4 mg/L

Additional information

One study investigating the chronic toxicity of Diisodecyl azelate (CAS 28472-97-1) to Daphnia magna is available (Charles River, 2016). This test was conducted with a semi-static design according to OECD guideline 211 and under GLP conditions. The batch of Diisodecyl azelate tested is a liquid UVCB and the test item is not soluble in test medium at the loading rates initially prepared. Test solutions for the reproduction test were individually prepared by magnetic stirring of various loading rates for two days followed by a settling period of approximately 1 h. The Water Accommodated Fraction (WAF) was subsequently collected from the middle clear fraction using tubing filled with glass wool. The resulting clear and colourless WAFs were used for testing. WAFs prepared at nominal loading rates of 0.26, 0.64, 1.6, 4.0 and 10 mg/L were tested. The study duration was 21 days and the test solutions were renewed three times a week. The test substance is a UVCB. Despite exhaustive attempts, an analytical method, based on the detection of a marker compound, capable of detecting the extremely low concentrations of this complex mixture could not be developed. The study was consequently performed without analytical support and study endpoints are based on loading rates. It was concluded that the No Observable Effect Loading Rate (NOELR) for Diisodecyl azelate on the reproduction of Daphnia magna is 4.0 mg/L after 21 days of exposure based on reproduction and immobilisation. The NOELR is far above the water solubility which was determined to be < 0.05 mg/L and it is unclear if the effects resulted from intrinsic properties of the substance or by physical effects.