Dibutyl adipate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

other: OECD 211

Deviations:

yes

Remarks:

Hardness of the test medium was below 140 mg/l (125 mg/l as CaCO3) on day 10.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Singular samples were taken from all test concentrations and the control.
- Sampling method: 2 mL samples were taken at the beginning and at the end of four daily renewal intervals (days 0-1, 6-7, 13-14, 20-21)
- Sample storage conditions before analysis: Samples not analyzed on the day of sampling were stared in a freezer.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of Dibutyl Adipate tested was a clear colourless liquid with a purity of 99.7% (corr. area %) and a water solubility of 26.5 mg/L at 20°C (Report Henkel AG & Co. KGaA; 28.08.2013). Dibutyl Adipate proved not soluble in test medium at 100 mg/L, but at 4.8 mg/L the test substance was fully soluble after one hour of stirring.
Final test:
A concentration of 4.8 mg/L was magnetically stirred for one hour and subsequently used as highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All were clear and colourless solutions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All treatments were clear and colourless solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house laboratory culture
- Age of parental stock: < 24 h
- Feeding during test
- Food type, Amount: 0.25 mL of Chlorella pyrenoidosa suspension (in the last week: 0.75 mL)
- Frequency: twice daily (except for weekends)

ACCLIMATION
- Acclimation period: none
- Health during acclimation (any mortality observed): no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

125 - 232 mg/L as CaCO3

Test temperature:

19.2 - 20.3 °C

pH:

7.9 - 8.6

Dissolved oxygen:

8.8 - 10.2 mg/L

Nominal and measured concentrations:

Nominal: 0.06, 0.18, 0.54, 1.6 and 4.8 mg/L
Measured (mean of initial concentrations): 0.045, 0.16, 0.48, 1.5 and 5.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass vessels covered with a Perspex plate
- Fill volume: 50 mL
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium
- Culture medium different from test medium:
- Intervals of water quality measurement: Temperature, oxygen and pH was measured at the start of the test and just before and after each renewal in one of the vessels of each test group. Hardness was measured once a week in fresh and old media from the control and the highest test concentration.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light
- Light intensity: at start: 634 - 692 lux; at the end: 642 - 684 lux

EFFECT PARAMETERS MEASURED: The number of living, immobile and dead parental daphnids, the presence of eggs in the brood pouch, the number of newborn daphnids were recorded daily. Bode length was recorded at the end of the test. Appearance of first brood, presence of unhatched eggs and incidence of immobility was recorded when observed.

RANGE-FINDING STUDY
- Test concentrations: The concentrations of Dibutyl Adipate tested were: 0.10, 1.0, 10 and a WSF prepared at 100 mg/L. A control group was also included.
- Results used to determine the conditions for the definitive study: No data were available from an acute toxicity test. Therefore, a preliminary test was performed prior to the reproduction test. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was seven days. Test conditions were held as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 1, 3 and 6 during the test. Samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 3 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae. No effects on survival or mobility of the parental daphnia were observed at concentrations up to and including 1.0 mg/L. Nine out of ten daphnids exposed to 10 mg/L died during the course of the 7-day test period, while all daphnids exposed to a WSF prepared at a loading rate of 100 mg/L had died after one day of exposure. Based on these observations it was concluded that Dibutyl Adipate did not affect survival or development during the range-finding test up to and including a nominal concentration of 1.0 mg/L.

Reference substance (positive control):

no

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

> 5.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Additional analytical experiments (WIL Research project 502605)

Experiment 1: check whether decrease is linear over time:

A nominal concentration of 10 mg/L (batch A) was prepared in M7 medium applying 1 hour of magnetic stirring, which resulted in a clear and colourless solution. A volume of 100 mL was collected and made up to 1 liter in M7 medium thus preparing a nominal concentration of 1 mg/L. This solution was divided over 7 daphnia vessels each containing ~50 mL. Samples for analytical determination of actual exposure concentrations were taken at various time points from the individual daphnia vessels. Results showed that the start concentration was in agreement with nominal and then a linear decrease was observed in time, i.e. decreases of 10, 20, 93 and >99% after 4, 6, 24 and 29 hours, respectively.

Experiment 2: check slow stirring and analyses of test solution collected from the bottom of the vessel:

The test substance (batch A) was carefully exposed to the surface of the M7-medium in 2 liter flasks that contained a tap at the bottom for sample collection. The nominal concentration of the test solution was 10 mg/L. The substance was clearly present as a floating layer of ‘oily’ droplets. After a 5-minute stabilization period, stirring was started slowly (~100 rpm, no vortex).

After ~48 hours it was observed that the floating layer had slightly changed from big round oily droplets to a very thin film of smaller loose ‘aggregates’. In addition it was observed that the stirring rod had some brown material stuck to it. After 96 hours the solution was observed to have slightly changed in color. The solution was very slightly yellow and some brown precipitate was observed at the flaks bottom and on the stirring rod. It was further noticed that the floating material was still present but consisted of even smaller ‘aggregates’.

Samples were taken from the bottom of the flask at various time points. Results showed that the measured concentration increased from 4 to 5 mg/L during the first 6 hours, remained stable until 24 hours and then rapidly decreased by > 99% after 48 hours.

Experiment 3: check results batch A (as used for prior experiments) with newly derived test substance (batch B):

A new batch of test substance (batch B) was used for this experiment. The test procedure was identical to experiment 2. The results of this experiment were also identical, i.e. a slightly increasing concentration during the first 6 hours, then a relative stable concentration until 24 hours and thereafter a rapid decrease in concentration. It was therefore concluded that the decreasing concentrations were not related to a problem with the first batch of test substance.

As in both experiment 2 and 3 a typical yellow-brownish precipitate was observed it was suspected that the decreasing concentrations in water might perhaps be related to a reaction with ions present in the test medium rather than a degradation process like hydrolyses.

Starting from the assumption that undissolved or precipitated particles of the test item might physically hamper the Daphnia (e.g. by ingestion), thus potentially compromising the results of the reproduction test, the solution behaviour of Dibutyl Adipate in other media than M7 was to be checked.

Experiment 4: compare behaviour in purified water, natural water and M7-medium:

Preparation procedures were identical to the previous pre-tests performed. The test substance (batch A) was very carefully added to the surface of the different media (purified water, natural water and M7-medium) in 2 liter flasks that contained a tap at the bottom for sample collection. The nominal concentration of the test solutions was 10 mg/l. The substance was clearly present as a floating layer of droplets at the start in all solutions. After a 5-minute stabilization period, stirring was started slowly (~100 rpm, no vortex). The appearances of the three media were as follows:

Purified water: from 6 hours onwards it was observed that a thin film of test substance was observed just above the surface against the inside of the flask wall. No changes observed until end of test.

Natural water: at t=3 hours slightly yellow (water colour) with one big drop of test substance floating at surface and precipitate. From 24 hours onwards a change to a more thin film of smaller loose ‘flakes’(identical to prior tests) , precipitate and a minor film just above surface against wall. Unchanged until end of test.

M7 medium: identical to natural water (except for yellow colour). From 48 hours onwards the floating layer diminished/disappeared and a slight amount of precipitate was present at the stirring rod (yellow/brown) and at the flask bottom.

The results of analyses showed that the concentration in natural water and in M7-medium decreased according to the decreases observed in the prior experiments, however, the concentration in purified water was stable during the 96-hour test period and in agreement with nominal.

Results from the extra analytical experiments (performed and reported under WIL Research project 502605) indicated that Dibutyl Adipate was stable over 96 hours in purified water, but highly unstable in solutions containing salts, e.g. daphnia test medium (M7) or natural water. One possible explanation is that Dibutyl Adipate precipitates in the presence of salts and subsequently adsorbs to surfaces; however, other explanations, e.g. biodegradation, could be true as well.

Based on these results, and in consult with the sponsor, it was decided to continue with a semi-static test applying daily renewals to minimise the loss of test substance from solution. In addition, it was decided to dip the parent daphnia before each transferal to fresh medium shortly in clean water to minimise biodegradation processes. It was further decided to check immobilised parents under a microscope to assess whether the cause was physical hampering by precipitated particles or not. The test range for the final reproduction test was set at 0.06, 0.18, 0.54, 1.6 and 4.8 mg/L.

Test substance analytics

The analytical support generally confirmed the repeatability of the preparation of the test concentrations with measured concentrations just below or close to nominal. Results of the 24-hour old solutions showed that measured concentrations had significantly decreased as was expected. During the first week of exposure concentrations after 24 hours could still be measured in the two or three highest test groups. However, from day 14 all were below the Limit Of Quantification (LOQ) of the analytical method. Consequently, no reliable mean measured concentrations could be calculated. The test range based on the mean of the initial measured concentrations of the freshly prepared solutions was 0.045, 0.16, 0.48, 1.5 and 5.2 mg/L.

Conclusions:

The long-term toxicity of dibutyl adipate to aquatic invertebrates was tested in accordance with GLP and OECD guideline 211. Mortality of parental daphnids was observed at the first observation date (after 5 days) in order to evaluate the chronic acute toxicity of the substance to Daphnia magna. Test concentrations between 0.0045 and 5.2 mg/L were tested. No mortality was observed in the control and any treatment after 5 days exposure time. Thus, the 5d-EC50 is determined to be > 5.2 mg/L based on the mean initial measured test concentration.

Executive summary:

The long-term toxicity of dibutyl adipate to aquatic invertebrates was tested in accordance with GLP and OECD guideline 211. Mortality of parental daphnids was observed at the first observation date (after 5 days) in order to evaluate the chronic acute toxicity of the substance to Daphnia magna.

The analytical support generally confirmed the repeatability of the preparation of the test concentrations with measured concentrations just below or close to nominal. Results of the 24-hour old solutions showed that measured concentrations had significantly decreased as was expected. During the first week of exposure concentrations after 24 hours could still be measured in the two or three highest test groups. However, from day 14 all were below the Limit Of Quantification (LOQ) of the analytical method. Thus, the test range is based on the mean of the initial measured concentrations of the freshly prepared solutions: 0.045, 0.16, 0.48, 1.5 and 5.2 mg/L. Thus, the 21d-NOEC value is determined to be 1.5 mg/L based on the mean initial measured test concentration.

No mortality was observed in the control and any treatment after 5 days exposure time. Thus, the 5d-EC50 is determined to be > 5.2 mg/L based on the mean initial measured test concentration.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Validated QSAR model

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Principles of method if other than guideline:

Calculation based on ECOSAR v1.00, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.

GLP compliance:

no

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

other: Daphnid

Test type:

other: not specified

Water media type:

freshwater

Total exposure duration:

48 h

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.614 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: calculation for neutral organics

Details on results:

For detailed description on the model and its applicability, see "Any other information on materials and methods incl. tables".

Although in the QSAR literature the daphnid endpoint is often denoted "LC50", the determination of lethality is based on immobilization, i.e. "EC50". Thus, the two endpoints can be considered identical.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Feb - 19 Feb 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: Water hardness lower than recommended in current guidelines. No analytical determination performed. Only 24 h test duration. The vehicle concentration was set at 100 mg/L.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Principles of method if other than guideline:

Test for acute immobilization with Daphnia sp. for 24 h.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: blank control and vehicle control
- Chemical name of vehicle: mixture of DMSO and HC40 (DMSO : HCO = 9:1)
- Concentration of vehicle in test medium (stock solution and final test solution): 100 mg/L

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

ACCLIMATION
- Type and amount of food: Chlorella vulgaris
- Feeding frequency: once a day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Hardness:

64

Test temperature:

22 ± 0.5 °C

pH:

7.5 - 7.7

Dissolved oxygen:

8.0 - 8.3 mg/L

Nominal and measured concentrations:

Nominal concentration: 5.6, 10, 18, 32 and 58 mg/L

Details on test conditions:

TEST SYSTEM
- Fill volume: 50 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Alkalinity: 26.5
- Ca/mg ratio: 8.2

OTHER TEST CONDITIONS
- Photoperiod: 16 hours in the light, 8 hours in the dark
- Light intensity: 3500 lux

EFFECT PARAMETERS MEASURED: Immobility after 24 d test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study was performed
- Test concentrations: 0.1, 1, 10 and 100 mg/L (vehicle concentration was 100 mg/L)
- Results used to determine the conditions for the definitive study: 15% and 100% immobility was observed in 10 mg/mL and 100 mg/L, respectively. In other groups including controls, immobility was 0%.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

16.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 12.9 - 21.1 mg/L

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0% mortality

Reported statistics and error estimates:

Probit test

Table 1. Cumulative immobilited daphnia during 24 hours

Nominal Concentration [mg/L]	Cumulative Immobilized Daphnia [% immobility]
Control	0 (0)
Solvent control	2 (10)
5.6	2 (10)
10	8 (40)
18	6 (30)
32	16 (80)
58	20 (100)

Description of key information

EC50 (48 h) = 1.6 mg/L (ECOSAR v1.00)

EC50 (24h) = 16.6 mg/L (similar to OECD 202)

EC50 (120h) > 5.6 mg/L (OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.6 mg/L

Additional information

One study investigating the acute toxicity to aquatic invertebrates of Dibutyl adipate (CAS 105-99-7) is available. The study was conducted by a Japanese authority equivalent to OECD guideline 202 using Daphnia magna as test organism (MOE, 1993). The 24 h-EC50 was determined to be 16.6 mg/L based on nominal test concentration. However, the test was only conducted for 24 hours which is considered as insufficient for assessing the acute toxicity to Daphnia magna. Therefore, results of a long-term toxicity test with dibutyl adipate and a QSAR calculation was additionally used to evaluate the acute toxicty of the substance to aquatic invertebrates in a weight-of-evidence approach.

The long-term toxicity of dibutyl adipate to aquatic invertebrates was tested in accordance with GLP and OECD guideline 211 (Migchielsen 2014). Mortality of parental daphnids was observed at the first observation date (after 5 days) in order to evaluate the chronic acute toxicity of the substance to Daphnia magna. The analytical support generally confirmed the repeatability of the preparation of the test concentrations with measured concentrations just below or close to nominal. Results of the 24-hour old solutions showed that measured concentrations had significantly decreased as was expected. During the first week of exposure concentrations after 24 hours could still be measured in the two or three highest test groups. However, from day 14 all were below the Limit Of Quantification (LOQ) of the analytical method. Thus, the test range is based on the mean of the initial measured concentrations of the freshly prepared solutions: 0.045, 0.16, 0.48, 1.5 and 5.2 mg/L. Thus, the 21d-NOEC value is determined to be 1.5 mg/L based on the mean initial measured test concentration. No mortality was observed in the control and any treatment after 5 days exposure time. Thus, the 5d-EC50 is determined to be > 5.2 mg/L based on the mean initial measured test concentration.

A (Q)SAR calculcation using ECOSAR v1.00 resulted in a lower LC50 for aquatic invertebrates (48h-LC50 = 1.6 mg/L, calculation for neutral organics). Based on the available data it is assumed that the 48h-EC50 value will be > 1 and < 10 mg/L. In a worst case approach the lowest effect value observed using the ECOSAR calculation is used as key information for the short term toxicity of dibutyl adipate to aquatic invertebrates.