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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Solubility of test substance in medium differs significantly from solubility in water. Reasons could not be found. Micelle formation is suspected.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 15541457
- Expiration date of the lot/batch: September 28, 2017
- Purity test date: September 29, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in original sealed packaging in a cool and ventilated place.
Analytical monitoring:
yes
Details on sampling:
At test start and at each working day samples of each test solution were taken from the test solution preparation just before distributing it to the 10 replicates. A sample of the aged test solution was taken from a representative vessel per concentration at each working day and processed immediately. If processing was not possible immediately after sampling due to technical reasons, samples were stored under circumstances to maintain the test item stable. The processed samples were stored in a freezer until chemical analysis. Chemical analysis of the processed samples was applied after test end.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Column elution method
A column was filled with column matrix loaded with the test item. The top and the bottom of the fillings were covered with membrane filters to avoid the loss of matrix material. A constant flow of dilution water was pumped through the column from bottom to top. Over a pre-conditioning phase the concentration was controlled until a constant concentration level was reached over the whole test duration. The test solution was sampled in a pre-conditioned vessel for each renewal. Subsequently, the solution was distributed to the test vessels – either directly, or after a 1:10 dilution with dilution water. This procedure was repeated daily.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not visible, suspected micelle formation.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Justification for species other than prescribed by test guideline: Not applicable
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene
- Feeding during test
- Food type: Mixture of suspensions of unicellular alga Desmodesmus subspicatus and JBL ArtemioFluid in a ratio of 9 : 1 (vol/vol)
- Amount: 0.2 mg C/Daphnia/day

ACCLIMATION/METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). Algae growing in the log-phase were centrifuged and the pellet were re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium. The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

QUARANTINE (wild caught)
Not applicable

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
no
Hardness:
In spite of the passage through a lime-stone column, hardness was below the recommended (not required) range (140 – 250 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Historical data showed values of 70 – 120 mg/L as CaCO3. However, according to the guideline lower hardness may be also appropriate for other Daphnia species. Historical results from reference tests at the IME indicate that test conditions are also suitable for the Daphnia magna strain used at the IME.
Test temperature:
19.4 – 20.3 °C
pH:
7.4 – 8.37
Dissolved oxygen:
7.3 mg/L - 9.5 mg/L
Conductivity:
206 – 242 µS/cm
Nominal and measured concentrations:
Nominal: not applicable, as column elution method was applied for test medium preparation
Measured: 17.22 µg/L (eluate), 1.6 µg/L (10-fold dilution of eluate)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers covered with glass panes to minimize evaporation
- Material, size, fill volume: Glass, 50 mL, 50mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency): 1/d
- 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): Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water is used as holding- and dilution water, purification includes filtration with activated charcoal, passage through a limestone column, and aeration.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light - 8h dark
- Light intensity: 689 – 756 lx (corresponding to 9.2 – 10.1 µE/(m² *s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (daily), Newborn daphnids per beaker (daily), Abnormalities in in form of male sex or presence of winter eggs (daily), length of adults (at study termination).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: The hardly water soluble test item was applied as steady state column eluate with the most possible dissolved concentration and a 1:10 dilution of this eluate.
- Range finding study: No (Due to poor solubility of test item, see above)
Reference substance (positive control):
yes
Remarks:
Acute immobilization tests over 24 h with the reference substance K2Cr2O7 are performed in regular intervals. The results of the latest reference study (February 2016) are in agreement with historical 24 h EC50 -values [EC50(24h)=1.38 mg/L].
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.6 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
17.22 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight: None
- Other biological observations: None
- Mortality of control: One Daphnid on Day 10
- Other adverse effects control: None reported
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No visiual observations made; micelle formation was checked by particle scattering, but due to the low test item concentrations, the results were not valid.
- Effect concentrations exceeding solubility of substance in test medium: Undiluted eluate (16.94 µg/L TWA) exceeds water solubility of test item, presence of test material not in solution cannot be excluded.
Results with reference substance (positive control):
- Other: Historical reference data available.
Reported statistics and error estimates:
Shapiro-Wilk´s Test on Normal Distribution
Results: Number of residues = 26; Shapiro-Wilk´s W = 0.926; p(W) = 0.061; p(W) is greater than the selected significance level of 0.05; therefore, treatment data do not significantly deviate from normal distribution.

Normality check was passed (p > 0.05).
Levene´s test is chosen for variance homogeneity testing.

Levene´s Test on Variance Homogeneity (with Residuals)
Based on the pre-selected significance level of 0.05, the Levene test indicates variance homogeneity !
Variance homogeneity check was passed. Normal distribution and variance homogeneity requirements are fulfilled.
The preselected multiple test is performed.

Williams Multiple Sequential t-test Procedure
Comparison of treatments with "Control" by the t test procedure after Williams. Significance was Alpha = 0.05, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max. likelihood mean; %MDD: minimum detectable difference to Control (in percent of Control); t: sample t; t*: critical t for Ho: µ1 = µ2 = ... = µk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).
Treatm. [% eluate] Mean s df LhM %MDD t t* Sign.
Control 94 015
10%
of Eluate 92 015 26 92 -12 -0.17 -1.71 -
Eluate 69 015 26 69 -13 -3.64 -1.79 +
+: significant; -: non-significant
A NOEC of 10.0 % eluate is suggested by the program.

Mean Cumulative Offspring per Survivor in Daphnia magna after 21 d, as %Inhibition of mean cumulative offspring per survivor caused by the test item after 21.0 d.

                               Treatment                     Mean               Std. Dev.                             n             %Reduction

                                     Control                           94                       12.8                             9             0.0

                             1:10 dilution                          92                       14.8                           10             1.2

                                       eluate                           69                       16.7                           10             26.7

Validity criteria fulfilled:
yes
Conclusions:
The “No Observed Effect Concentration” based on mean cumulative offspring per survivor was equal to or greater than 0.00164 mg/L (based on the time weighted average (TWA) test concentration of the test medium), which is greater than the level of water solubility.
Executive summary:

The underlying study was performed to assess the long-term toxicity of diisotridecyl adipate to Daphnia magna. The test was conducted according to OECD guideline 211 under GLP.

Based on the available information prior to the test, indicating a water solubility of the test item of < 0.001 mg/L, the column elution method was chosen for preparing the test medium and obtaining the highest possible test concentration. For test medium preparation, a column was filled with column matrix loaded with the test item (1 mg/g loading rate, column material: silica gel 60). The top and the bottom of the fillings were covered with membrane filters to avoid the loss of matrix material. A constant flow of dilution water is pumped through the column from bottom to top with a flow rate of 10 ml/min. Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a limestone column, and aeration.

Young female Daphnia, aged less than 24 hours at the start of the test (10 replicates of a single daphnid per group) were exposed to a steady state column eluate in form of the 100% eluate and 10 % eluate for a period of 21 days. The test solutions were renewed on a daily basis (working days, latest after 3 days; semi-static exposure). The treatment groups were compared to the control group in regard to the total number of living offspring produced per parent animal alive at the end of the test. Other substance-related effects on parameters such as growth (length), and possibly intrinsic rate of increase, are also examined. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The daphnids were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

 Analysis of the fresh and old test media (Day 1, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 18, 19, 20, 21) column eluate and the 10%-eluate, resulted in time weighted average (TWA) test item concentrations of 17.22 µg/l and 1.64 µg/l, respectively. Based on the test medium preparation method, it was considered justifiable to base the results on the mean measured test concentrations, as no nominal concentration is available. Both of the measured concentrations are above the water solubility reported for DITA.

No significant differences between the control group and the test groups were found for the following endpoints: Mobility, age at first reproduction, length and intrinsic rate. In the undiluted eluate, a ~27% reduction of mean cumulative offspring per surviving daphnids after 21 days were seen, whereas the exposure to the 10%-dilution of the eluate resulted in no significant effects for this endpoint, as well.

As mentioned above, the concentrations measured in the undiluted eluate are far above the reported and experimentally determined water solubility for diisotridecyl adipate. Hence, micelle formation or other processes causing the inhibition cannot be excluded.

Based on the results of this test the 21d NOEC value for mean cumulative offspring per survivor was determined to be 1.64 µg/L (based on the time weighted average (TWA) test concentration of the test medium), which is greater than the level of water solubility.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study with analytical monitoring, read-across, non-GLP
Qualifier:
according to
Guideline:
other: ASTM (American Society for Testing and Materials)
Principles of method if other than guideline:
Proposed standard practice for conducting Daphnia magna chronic toxicity tests in a flow-through system.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
yes
Test organisms (species):
Daphnia magna
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
250 +- 25 mg CaCO3/L
Test temperature:
20 °C
pH:
8.1 - 8.3
Dissolved oxygen:
6.2 - 8.6 mg/L
Salinity:
alkalinity: 350 +- 25 mg/L
Nominal and measured concentrations:
effective concentrations of 0.014; 0.024; 0.052; 0.087; 0.18 mg/L Di(2-ethylhexyl) adipate, measured with 14C-Di(2-ethylhexyl)adipate
Details on test conditions:
specific conductance 700 µOhm-1/cm

TEST PROCEDURE
- A Mount and Brungs proportional diluter delivered water and chemical at a rate of four replacement volumes per day for five exposure levels and a solvent control.
- All treatments and controls were conducted in quadruplicate with 10 first-instar daphnids (less than 24 h old) in each of the test chambers.
- the test chambers were fed 15 to 30 mL of a S.capricornutum suspension three times daily and 2 mL of trout chow suspension once daily
- The test was terminated on day 21. The surviving adults were removed from the test chamber, isolated on microscope depression slides and measured using a binocular dissecting scope and calibrated eyepiece.
Duration:
21 d
Dose descriptor:
other: MATC maximum acceptable toxicant concentration
Effect conc.:
0.024 - 0.052 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: mean length, survival and reproduction
Details on results:
Significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L. MATC (maximum acceptable toxicant concentration) for chronic toxicity on Daphnia magna was derived to range between 0.024 and 0.052 mg/L based on statistical analyses of adult meand length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate.
Validity criteria fulfilled:
no
Conclusions:
The results of the 21d-chronic study indicate that the read-across substance bis(2-ethylhexyl) adipate is moderately toxic to Daphnia magna. The mean young per adult per reproduction day after 21 d was significantly afffected in the mean measured exposure levels of 0.087 and 0.18 mg/L. The maximum acceptable toxicant concentration (MATC) range is 0.024 to 0.052 mg/L., based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
July 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with analytical monitoring, read-across
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Stock solution: 1 g/L test substance + 1 g/L vehicle, the calculated test concentrations of 0.19, 0.39, 0.77 mg/L contain 2.0 mg/L vehicle (nominal)
- Chemical name of vehicle: 1 g/L MARLOWET R 40
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: own breeding
- Feeding during test
- Food type: green algae
- Amount: 0.5E7 cells/daphnid from day 0 to day 6; 1.0E7 cells/daphnid from day 7 to day 21
- Frequency: daily
- Age of test organisms: < 24 hours

HOUSING
- Housing medium: M4-medium (Elendt, 1990)
- Housing conditions: each test organism were kept separate in 60 ml test water
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
20 °C
pH:
7.8 - 8.6
Dissolved oxygen:
94 - 107 mg/L
Nominal and measured concentrations:
Nominal test concentrations: 0.5, 1.0, 2.0 mg/L
Measured concentrations: 0.19, 0.39, 0.77 mg/L (geometric mean, biological active)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, total volume, fill volume: glass beakers, 150 ml, 100 ml
- Renewal rate of test solution (frequency): after 2, 6, 7, 9, 12, 14, 16, 19 days
- 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
- Biomass loading rate: 1 daphnid/100 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater according to Elendt (M4-medium)
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value and oxygen content of the new test solutions at day 2, 12, 16 and of the old test solutions at day 6, 14, 19.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: approx. 450 and 95 Lux

EFFECT PARAMETERS MEASURED: mortality and reporduction after 2, 6, 7 9, 12, 14, 16, 19, 21 days

VEHICLE CONTROL PERFORMED: yes


Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Details on results:
Toxic effects on the reproduction and mortality of the adult daphnids, respectively, are above the water solubility of the test substance.
Validity criteria fulfilled:
yes
Executive summary:

In a valid 21 day reproduction test according to OECD test guideline 202 (1984, part 2), the water flea Daphnia magna was exposed to three different concentrations of the read-across substance di-2 -ethylhexyl adipate (nominal concentrations 0.5, 1.0, and 2.0 mg/L, measured concentrations 0.19, 0.39, and 0.77 mg/L). To solubilise the test substance, a vehicle was used (1g/L MARLOWET R 40).

During the test, no effects were observed. Based on reproduction, a NOEC of>= 0.77 mg/L was determined. Toxic effects on reproduction and mortality of adult daphnids are above the water solubility of the test substance (Huels AG, 1996a).

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study with analytical monitoring, read-across
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: ASTM Method E 1193-097
Deviations:
not specified
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Samples from the D. magna test were collected on test days 0, 2, 4, 7, 14 and 20 and analyzed for the DEHA. Samples were collected in 500-mL glass bottles with ground glass stoppers (the bottles and stoppers were rinsed three times with small amount of test or acetone control solution which was discarded previously). Samples of diluent water were collected on test days 1 and 8 and samples of solvent control solution were collected on test days 0 and 14 and also analyzed for DEHA. On test days 1, 14 and 21 samples of the test solutions were collected directly from the test chambers after having been in the the test chambers for 24 h (approx. 40 mL from each chamber and combined to provide 400 mL).
The measured initial concentration of DEHA in the test solution at the time of renewal varied from 1.65 to 8.32 µg/L. The majority of the measured values were within 3-6 µg/L. The DEHA concentration in duplicate samples on days 0, 7 and 20 suggest that the concentration of DEHA was homogeneous in the test solutions. The average initial DEHA concentration in all renewal solutions in this limit test was 5 ± 1µg/L.
Vehicle:
yes
Details on test solutions:
1 mL of a DEHA stock solution in acetone (50 mg/L) was added to 3 L of diluent water in a 4-L Erlenmeyer flask (nominal DEHA concentration = 16 µg/L, acetone concentration = 0.3 mL/L). A stir bar was added, the flask covered with sterilized foil, and the mixture vigorously stirred with a vortex height of 1/2 to 3/4 of total solution depth for 24 ± 2 h in the dark at 20 ± 2°C. The DEHA-saturated solution was removed by siphoning through a glass tube that extended from the bottom of the flask through the neck opening. The first several tube volumes of solution were siphoned off and discarded. A solvent control solution, acetone-diluent water, was prepared in the same manner as the DEHA-diluent water test solution.
Test chemical, solvent control and diluent water control solutions were prepared daily.
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna neonates, < 4-h old, were obtained from ABS (Aquatic BioSystems, Inc., Fort Collins, CO, USA) and used immediately. At ABS, adult organisms were cultured in moderately hard water (hardness of 102 mg/L (as CaCO3), alkanity of 65 mg/L (as CaCO3), and pH 7.9) at 22°C under a 16/8 (light/dark) cycle and fed a 2:3 mixture of yeast, trout chows and cerophyll (YTC) and green algae (Pseudokirchneriella subcapitata).
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
160 - 180 mg/L (as CaCO3)
Test temperature:
20 ± 1°C
pH:
7.8 - 8.6
Dissolved oxygen:
5.4 - 7.7 mg/L
Nominal and measured concentrations:
nominal: 16 µg/L
measured: 5 ± 1 µg/L (initial)
Details on test conditions:
TEST SYSTEM
The test was initiated by placing one young (<4 h) neonate Daphnia magna in each 100 mL glass beaker containing 80 mL of solution. Ten replicates each of diluent water control, solvent control and DEHA solution were used in teh test. Shortly after addition of the daphids, 0.4 mL of a 2:3 undigested YTC:algae mix was added to each chamber. The diluent water control, solvent control and DEHA test solution were renewed daily.

TEST MEDIUM / WATER PARAMETERS
The test diluent waster was city tap water that had been filtered through charcoal and a Millipore system (Milli-Q: Millipore, Billerica, MA, USA), then amended to a hardness of 160-180 mg/L (as CaCO3) with selected salts (USEPA 1994). After the addition and dissolution of the salts, the diluent water was sterilized by filtration (Gelman® pleated capsule with 0.2 µm Versapore® membrane)

OTHER TEST CONDITIONS
The test was performed in a temperature-controlled environmental chamber at 20±1°C. Lightning was controlled to provide a 16/8 light/dark cycle, with a mean illumination of 75±25 ft-c /(807±269 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Each day of the test survival and health (e.g., mobility, presence of ephippia) of the initial organisms were determined. Then, the test organisms were transferred into new test chambers. Neonates were counted each day after the first brood was observed (e.g., on day 7), then discarced. The test was terminated after 21 days of exposure and the cumulative number of neonates determined. The adult females were transferred to individual pre-tarred pans, dried overnight at 74°C and their dry weights measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
not performed, limit test
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Survival of Daphnia magna was highest in the DEHA exposure (100%) compared to both the control (90%) and solvent control (90%) exposures. Neonates were first observed in the control and solvent control on day 7, and in the DEHA treatment on day 8. By day 8, 50% of the females in both the control and solvent control and 60% of the females in the DEHA treatment had produced first broods. The average number of neonates per surviving female was 137 for the control and 148 for the solvent control. By comparison, each female exposed to the DEHA solution produced an average of 152 neonates (10% more). Consistent with survival and productivity, the mean dry weight per surviving female was highest in the DEHA exposure (0.804 mg) compared to 0.779 and 0.742 mg for the control and solvent control, respectively.
Three times during the course of the test, the DEHA solution after being in the test chambers for 24 h was collected for analysis. The data for all three samples showed that the concentration of DEHA had decreased during 24 h in the test chambers in the presence of daphnids and food (YTC:algae)
Validity criteria fulfilled:
yes
Remarks:
Survival and reproduction of the control organisms surpassed the minimum OECD (1998) and ASTM (2001) criteria for acceptability.
Conclusions:
DEHA had no observed detrimental effect on survival, growth or reproduction in Daphnia magna in the range of water solubility.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2013-01-14 until 2013-05-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with analytical monitoring; read-across
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(No lux reading observations on Day 9 with no impact on the study itself. Lux reading on all other days were within the acceptable range.)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples were taken from the solvent control and each surviving test group (replicates pooled) for quantitative analysis. Duplicate samples were taken of both, the fresh test preparations on Days 0, 5, 12 and 19, and of the expired test preparations on Day 2, 7, 14 and 21.
- Sample storage conditions before analysis: at approx. -20°C
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preliminary solution in vehicle (100 mg test item/ 10 mL vehicle)
- Final stock solution: 1 mg test item/L medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (final stock solution/final test solution(s) /solvent control(s)): <= 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): any undissolved material in the stock solution after stirring was removed via filtration through a preconditioned 0.2 µm Gelman Acrocap filter
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: waterflea
- Source: in-house laboratory culture
- Age of parental stock: not specified
- Feeding during test
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin(R) flake suspension
- Amount during test period: 2-7 µL algal suspension, 10-32 µL Tetramin(R) Flake suspension (corresponding to 0.1-0.2 mg carbon/daphnid/day)
- Frequency: daily

ACCLIMATION
- Acclimation period: no

QUARANTINE (wild caught)
not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Gravid adult daphnids were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These yound were removed from the cultures and used for testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
220-250 mg CaCO3/L
Test temperature:
19-21°C
pH:
7.6 - 8.3
Dissolved oxygen:
>= 5.6 mg O2/L
Nominal and measured concentrations:
Given the overall decline in measured concentration between each media renewal period, it was considered justifiable to base the results on the mean measured test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass flask, coverd with plastic lid to reduce evaporation
- Fill volume: 100 mL test preparation
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency): 3 days
- 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
- Test medium: Elendt M7
- Preparation of dilution water: deionized reverse osmosis water
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: not mentioned
- Photoperiod: 16 h (8 h darkness with 20 min dawn and dusk transition periods)
- Light intensity: 733-781 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Parental: daily (dead/alive)
- Filial: daily (dead alive)
- Other: unhatched eggs

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY (48h)
- Test concentrations: 1.0, 10 and 100 % v/v
- Results used to determine the conditions for the definitive study: yes (no immobilization in any concentration)
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.001 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.001 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: 0
- Mean number of live offspring per surviving adult (solvent contro/100 % v/vl): 116/138
- Coefficient of variation (solvent control): 5.3%
- Time to first offspring (solvent control/100 % v/v): 8 d/9 d
- Other biological observations: no unhatched eggs, no dead young
Validity criteria fulfilled:
yes
Conclusions:
The 21-Day EC50 (immobilization) value based on the mean measured test concentrations of the test media, for the parental Daphnia generation (P1) was estimated to be > 0.00063 mg/L. Exposure of Daphnia magna to the test item resulted in no immobilization at all the concentrations employed during the test.
The 21-Day EC50 (reproduction) value based on the mean measured test concentrations was estimated to be 0.00063 mg/L. The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the mean measured test concentration of the test media were greater than and equal to 0.00063 mg/L respectively. The “Maximum Acceptable Toxicant Concentration” was calculated to be 0.00063 mg/L.
This study showed no toxic effects at the limit of water solubility.
Executive summary:

The study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2008) No 211, “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 “Daphnid Chronic Toxicity Test”.

Information provided by the Sponsor indicated the water solubility of the test item to be less than 0.050 mg/L. Additionally, pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing . A pre-study media preparation trial indicated that the most appropriate method of preparation was using a solvent spike followed by filtration.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of test concentrations of 10, 18, 32, 56 and 100% v/v solution for a period of 21 days. The test solutions were renewed 3 times per week. The test item solution was prepared by initially dissolving 100 mg of test item in a final volume of 10 mL of dimethylformamide. An aliquot of the solvent stock solution was dispersed in Elendt M7 media using a magnetic stirrer for approximately 10 minutes to give a 1.0 mg/L stock solution. After stirring, any undissolved test item was removed vial filtration to give the 100% v/v solution test concentration, from which dilutions were performed to give the remainder of the test series. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Analysis of the freshly prepared 100 % v/v solution preparation on days 0, 5, 12 and 19 showed measured concentrations to range from less than the limit of quantification to 0.0024 mg/L. Analysis of the old or expired media on days 2, 7, 14 and 21 showed measured concentrations to range from less than the limit of quantification to 0.00093 mg/L. Given the overall decline in measured concentration between each period of media renewal, it was considered justifiable to base the results on the mean measured test concentrations.

The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the mean measured test concentration of the test media were greater than and equal to 0.00063 mg/L respectively. The “Maximum Acceptable Toxicant Concentration” was calculated to be 0.00063 mg/L. This study showed no toxic effects at the limit of water solubility.

Description of key information

NOEC(21d) = 1.64 µg/L (measured, column elution method) for immobilisation of Daphnia magna (OECD 211)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
1.64 µg/L

Additional information

Chronic Daphnia data are available for DITA itself and the respective read across substances.

Key study / Diisotridecyl adipate

The available long-term toxicity study towards aquatic invertebrates for DITA (Simon, 2016) was conducted by using the column elution method for test medium preparation. Daphnids were exposed to a steady state column eluate in form of the 100% eluate and 10 % eluate for a period of 21 days in a semi-static test design. No significant differences between the control group and the test groups were found for the following endpoints: Mobility, age at first reproduction, length and intrinsic rate. In the undiluted eluate, a ~27% reduction of mean cumulative offspring per surviving daphnids after 21 days were seen, whereas the exposure to the 10%-dilution of the eluate resulted in no significant effects for this endpoint, as well.

The concentrations measured in both, the undiluted eluate and the 10%- eluate tests solution, are above the reported and experimentally determined (e.g. Kotthoff, 2016) water solubility for DITA of ~0.7 µg/L. Hence, micelle formation or other processes causing the inhibition cannot be excluded.

 

As seen for the read-across substances (details below) and based on the valid water solubility figure for DITA (0.7 µg/l), this study supports the fact that there is no toxicity in the range of water solubility.

Supporting information / Read-Across substances

In addition, there are three long-term toxicity tests with aquatic invertebrates available on the read-across substance bis(2-ethylhexyl) adipate (CAS 103-23-1; DEHA) and one study on the read-across substance diisotridecyl dodecanedioate (CAS 84731-63-5), which show no toxicity in the range of water solubility. All of these substances, DEHA, DITA and diisotridecyl dodecanedioate, are dialkylesters of dicarboxylic acids, either adipic acid (1,6-hexanedioic acid) or 1,12-dodecandioic acid. Both dicarboxylic acids are linear and have even-numbered carbon chains. The alcohol component of all three substances has branched alkyl groups (C8 or C13).

DEHA Chronic Daphnia studies showing non-toxicity

In a valid 21 day reproduction test according to OECD test guideline 202 (1984, part 2), the water flea Daphnia magna was exposed to three different concentrations of bis-(2-ethylhexyl) adipate (nominal concentrations 0.5, 1.0, and 2.0 mg/L, measured concentrations 0.19, 0.39, and 0.77 mg/L). To solubilise the test substance, a vehicle was used (1g/L MARLOWET R 40). During the test, no effects were observed. Based on the reproduction rate, a NOEC of >= 0.77 mg/L was determined (Huels AG, 1996a). This is considered a valid proof of the absence of long-term aquatic toxicity of bis(2-ethylhexyl) adipate to invertebrates in the range of water solubility. This result is confirmed in another study performed by Robillard et al. (2008) (key study). A chronic Daphnia magna limit test was conducted at an average exposure concentration of 4.4 μg/L (measured water solubility = 5.5 µg/L) in laboratory diluent water to avoid insoluble test material and physical entrapment. One hundred percent of the DEHA-treated organisms survived compared to 90% survival in both the controls and solvent controls. Mean neonate reproduction was 152, 137, and 148 and mean dry weight per surviving female was 0.804, 0.779, and 0.742 mg in the treatment, control and solvent control, respectively. No adverse effects were observed. In the third study (Felder et al. (1986), not conducted according to a current guideline and GLP), which is the only one with DEHA detailed discussed in the Substance Evaluation Draft Decision, a significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L were found. A MATC (maximum acceptable toxicant concentration) for long-term toxicity to Daphnia magna was calculated to be between 0.024 and 0.052 mg/L based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate. Acetone was used as a solvent in this study. No adverse effects to survival growth or reproduction were observed even at levels five times the water solubility of DEHA. Effects were reported at concentrations significantly higher than the water solubility of DEHA (0.005 mg/L). Under such conditions it is likely that the observed effects have been caused by physical entrapment rather than chemical interactions (Rhodes et al. 1995). Furthermore, information provided in the results section of the publication is rather scarce. Neither raw data nor details about the extent of impairment daphnids had to face at a given concentration are given, and no information about a dose-response relationship is included. In our opinion also the study of Felder et al. (1986) with the NOEC of 0.024 mg/L shows that no toxicity occurred within the range of water solubility of 0.005-0.0032 mg/L. The lowest measured water solubility value of DEHA is lower than the NOEC. The study supports the fact that there was no toxicity in the range of water solubility. Therefore this value cannot be used for the PNEC-derivation.

Diisotridecyl dodecanedioate Chronic Daphnia study showing non-toxicity

In addition to the presented long-term toxicity studies with Daphnia magna performed with DEHA, a recently (2013) conducted long-term toxicity test with Daphnia magna (OECD 211) on the read-across substance diisotridecyl dodecanedioate is available. The GLP study was performed under semi-static conditions using DMF as a solvent (key study). In the Substance Evaluation Draft Decision it is discussed by the MSCA that the study cannot be considered valid due to uncertainties in the preparation of samples which resulted in declining of 99 % of the test substance in 48 h to below the LOQ. We would like to defend the study and attached a letter with the complete rationale of the responsible test institute Harlan. "The decline in measured test concentrations in the inoculated test samples was due to adsorption of the test item to the algal cells that were present. In the Daphnia magna Reproduction Test, algal cells are added to the test solutions in order to provide a food source for the daphnids, it is therefore considered that the decline in measured concentrations over each test media renewal period in this test was due to adsorption of the test item to the algal cells and not due to instability and / or volatility. As the test item was adsorbed to the algal cells that the daphnids ingest as a food source it can be considered that the daphnids were exposed to the test item over the period of each test media renewal" (please see attached the complete wording of the rationale). Furthermore based on the structure of diisotridecyl dodecanedioate it is expected that this substance is even more water insoluble than DITA (< 0.001 mg/L).The analytics for such water insoluble UVCB-substances are very difficult. The measured concentrations in this study were extremely low. Therefore it is not unlikely that the measured concentrations were inconsistent in such a sensitive study. Analysis of the freshly prepared 100 % v/v solution preparation on days 0, 5, 12, 19 showed measured concentrations ranging from less than the LOQ (assessed as 0.00027 mg/L) to 0.0024 mg/L. Analyses of the old or expired media on days 2, 7, 14, 21 showed measured concentrations ranging from less than the limit of quantification to 0.00093 mg/L. Given to the apparent decline in measured concentration between each period of media renewal, it was considered justifiable to base the results on the mean measured test concentrations of the test media to give a "worst case" analysis of the data. The No Observed Effect Concentration based on the mean measured concentration of the test media was equal to 0.00063 mg/L respectively. The mean measured concentration of 0.00063 mg/L was estimated for the 100 % v/v solution. We provide additional information including a better justification for the choice of test solution preparation used for the diisotridecyl dodecanoate test (Harlan 2013), and why it would achieve saturation for a low solubility UVCB (please see attachment “issue1_OECD211comments2_diisotridecyldodecanoate_studyno41202662.pdf"). In the study of Robillard et al. (2008) the water solubility of 5.5 (± 0.22) μg/L for di(2- ethylhexyl) adipate (DEHA) was measured using the slow-stir method which is in the Registrants’ scientific opinion, one of the most suitable methods for substances with a very low water solubility. Compared to DEHA, the water solubility of DITA and of diisotridecyl dodecanoate can be expected to be lower because of longer C-chains. Therefore, the higher measured water solubility values for DITA and diisotridecyl dodecanoate must be interpreted with caution, since they were determined with the ASTM-E1148 Standard Test Method, a comparably less sensitive method. In general, the determination of a definite value for the water solubility of UVCB substances poses a very complex problem and is by far more complicated than the determination for single substances (monoconstituents). This study shows like the DEHA-studies no toxic effects at the limit of water solubility.

Overall assessment of Chronic Daphnia for DITA

Thus, on the basis of the available data for DITA and the supporting information, it can be concluded that DITA is neither acutely, nor chronically toxic to aquatic invertebrates up to its limit of water solubility.

Although the measured water solubility values for DITA, as mentioned above, are lower than the determined NOEC in this study, the obtained NOEC (Simon, 2016) will be used as basis for the PNEC-derivation and hence be taken into account for the environmental exposure assessment.