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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August 2016- 22 December 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 oC
pH:
7.5 - 8.4
Dissolved oxygen:
7.5 - 9.7 mg/L dO2
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  15 
 0.26 10  10 
 0.64 10  20 
 1.6 10  10 
 4.0 10  80  20 
 10 10 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  0.3 
 0.64 4.27  0.085  0.9 
 1.6 4.25  0.079  1.3 
 4.0 4.20  0.112  2.4* 
 10 4.00  0.210  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).

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Feb - 25 Feb 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study with acceptable restrictions: The analytical purity is only 77.2%.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard
Analytical monitoring:
yes
Details on sampling:
- Sampling method: sampled directly from each vessel.
- Sample storage conditions before analysis: no storage before analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance was measured and mixed with vehicle. Dilution water was added bit by bit to prepare the stock solution (1000 mg/L). Stock solution was diluted with dilution water and stable within 9 days in the dark and cool. Stock solution was prepared every 6 or 7 days.
- Controls: blank control and vehicle control
- Chemical name of vehicle: HCO-40
- Concentration of vehicle in test medium: 1000 mg/L in a stock solution, 1 mg/L in final concentration
- Evidence of undissolved material: Test water was clear and clorless after preparation.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: Incorporated Administrative Agency, National Institute for Environmental Studies, Tsukuba, Japan
- Age of parental stock: < 24 hours
- Feeding during test
- Food type: Chlorella vulgaris
- Amount: 0.2 mgC/day/animal

ACCLIMATION
- Acclimation period: 13 Jan - 04 Feb 2004
- Acclimation conditions: same as test
- Type and amount of food: Chlorella vulgaris, 0.2 mgC/day/animal
- Health during acclimation: Mortality of parental animals were 0% during 2 weeks before exposure
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
240 - 250 mg/L as CaCO3
Test temperature:
19.7 - 20.2 (20 ± 1)°C
pH:
7.5 - 8.4
Dissolved oxygen:
7.1 - 8.8 mg/L
Nominal and measured concentrations:
Nominal concenration: 0.1 mg/L
Measured concentration: 0.0637 mg/L (time-weighted mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type: closed with a teflon sheet
- Material: glass; Size: 100 mL; Fill volume: 80 mL
- Aeration: no aeration
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: aerated purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, water hardness, pH and oxygen were measured at day 0, 8, 15 and 20 in freshly prepared test solution and at day 1, 9, 16 and 21 in old test solutuion before renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment
- Photoperiod: 16 hours in the light and 8 hours in the dark
- Light intensity: < 800 lux

EFFECT PARAMETERS MEASURED: Mortality of parental animal, cumulative number of produced juveniles per alive adult, time to first brood production

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- Test concentrations: 0.1 mg/L (nominal concentration)
- Results used to determine the conditions for the definitive study: Acute daphnia toxicity test was performed to determine EC 50 for 48 hours. EC50 was > 0.1 mg/L (nominal concentration).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (analytical grade)
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
> 0.064 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.064 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: mean cumulative number of juveniles produced per adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.064 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: mean cumulative number of juveniles produced per adult
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.064 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: mean cumulative number of juveniles produced per adult
Details on results:
- Mortality of parent animals: see Table 2
- No. of offspring produced per day per female: see Table 3
- Time to first brood release or time to hatch: 8 - 9 days in all groups
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 0.76 ± 0.16 mg/L
Reported statistics and error estimates:
Student's t paired comparison test for NOEC and LOEC

Table 1: Measured concentration

Nominal concentration

[mg/L]

 

Measured concentration [mg/L]

TWM (*1)

[% of nominal]

Date

1 (new)

2 (old)

8 (new)

9 (old)

15 (new)

16 (old)

Control

<0.0002

<0.0002

<0.0002

<0.0002

<0.0002

<0.0002

-

Solvent control

<0.0002

<0.0002

<0.0002

<0.0002

<0.0002

<0.0002

-

0.1

0.0814

0.0551

0.0785

0.0500

0.0818

0.0433

0.0637

(64)

New: Freshly prepared test solutuion

Old: Old test solution before renewal

(*1): Time-weighted mean measured concentration during 21 days

Table 2: Cumulative number of dead parental Daphnia

Nominal concentration [mg/L]

Mean measured concentration

[mg/L]

Cumulative Number of immobilised [Percent Mortality]

Day 1

2

4

7

14

21

Control

-

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

1 (10)

Solvent control

 

0 (0)

1 (10)

1 (10)

1 (10)

1 (10)

1 (10)

0.100

0.0637

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

Table 3: Cumulative number of juveniles produced per adult alive for 21 days

Nominal Concentration

(Measured Concentration) [mg/L]

Mean Cumulative Numbers of Living Young after 21 d (Mean ± S.D.)

Inhibition rate [% of solvent control]

Significant difference

Control

122.9 ± 9.2

 

Solvent control

118.3 ± 12.7

 

 

0.100 (0.0637)

122.2 ± 8.3

-3.3

-

-: Indicates no significant difference

Description of key information

In a daphnia reproduction study 21 days exposure to C9-11 branched alcohols, C10 rich diesters with nonanedioic acid 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). At 4 mg/L there were some slight statistically significant effects on growth (decrease by 2.4%), which were considered not to be of toxicological relevance. Based on the complex nature of the substance analytical determination of the actual test concentrations was not possible (Migchielsen, 2016).

In a daphnia reproduction study with bis(2-ethylhexyl) azelate no effects on mortality, reproduction and growth were seen at a loading rate of 0.1 mg/L (0.0637 mg/L TWA measured concentration). The tested concentration is well above the water solubility of bis(2-ethylhexyl) azelate. Therefore it is concluded that the NOEC is above the water solubility (MOE 2004)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.1 mg/L

Additional information

Although in one of the two tests on the analogues no analytical measurements were performed, the results are indicative for very low toxicity at the water solubility level. As both substances are considered close analogues to diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid, no toxicity to daphnia at the water solubility level is expected for this substance. Therefore the NOELR is set at 0.1 mg/L (see also document on read-across rationale)