Dibutyl adipate

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

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.
Range-finding test:
A loading rate of 100 mg/L was prepared by magnetic stirring for one hour to achieve maximum dissolution in test medium. The Water Soluble Fraction (WSF) was collected after one hour of stabilisation by siphoning the middle fraction. In addition, a concentration of 10 mg/L was prepared by magnetic stirring for one hour. Lower test concentrations were prepared by subsequent dilution of the 10 mg/L solution. All were clear and colourless solutions.
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

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals: Two out of the twenty parental daphnids died during the test period in the untreated control. Hence, parental mortality did not exceed 20% in the controls. On day 10 all parental daphnids exposed to a nominal concentration of 4.8 mg/l had died. Mortality at nominal concentrations between 0.06 and 1.6 mg/l ranged between 10 and 20% and was not considered to be relevant as 20% mortality is allowed as validity criterion for the control. Microscopic examinations of the dead daphnia revealed that no test substance particles were stuck to the body of the organisms thus excluding physical effects. It was however observed that some algae were stuck to the tail and/or tentacles. Generally, observed mortality effects did not follow a concentration-response pattern, and are considered inadvertent.
- No. of offspring produced per day per female: The reproduction curves recorded at 0.06 to 1.6 mg/l generally followed the curve of the control or were just above it. Reproduction in the control averaged 66.1 young, whereas the test group’s reproduction averaged 65.0 to 82.5 young.
From day 15 onwards most of the offspring were immobilised thus flattening the curves. It was observed in all test groups including the control. It was unknown what the cause of this phenomenon was. It was however not test substance related and the same was observed in the invalid first reproduction test attempt. It was thought that daily handlings might have affected the organisms.
Statistical analysis on the cumulative reproduction showed that the reproduction of the daphnids was not significantly reduced up to and including 1.6 mg/l nominally when compared to the control group.
- Body length and weight of parent animals: The mean parental body length in the Dibutyl Adipate treated groups was slightly higher (2 to 4%) than the mean body length in the control group. Statistical analyses confirmed that none of the groups was statistically different from the control.
- Time to first brood release or time to hatch: In the controls, the presence of eggs in the brood pouch was recorded for the first time on day 5 or 6 and the first brood generally appeared on day 8 or 9. The first recording of the presence of eggs in the brood pouch was similar to the control and treated solutions up to and including 1.6 mg/l. Hence, no delay in appearance of the first brood was observed in the treatments when compared to the control, except for the daphnids exposed to 4.8 mg/l that did not have any brood.

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.

Validity criteria fulfilled:

yes

Conclusions:

The long-term toxicity of dibutyl adipate to aquatic invertebrates was tested in accordance with GLP and OECD guideline 211. test concentrations between 0.0045 and 5.2 mg/L were tested. The 21d-NOEC value is determined to be 1.5 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. 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. Reproduction in the control averaged 66.1 viable young, whereas the test group’s reproduction averaged 65.0 to 82.5 young per surviving parent animal. No delay in appearance of the first brood was observed in the treatments when compared to the control, except for the daphnids exposed to 4.8 mg/l that did not have any brood due to the fact that all parents died before onset of reproduction. No significant numbers of aborted or unhatched eggs were observed at any of the concentrations tested. From day 15 onwards most of the offspring were immobilised in all test groups including the control. It was unknown what the cause of this phenomenon was. It was however not test substance related. Thus, the 21d-NOEC value is determined to be 1.5 mg/L based on the mean initial measured test concentration.

Description of key information

NOEC (21d) = 1.5 mg/L (measured) for Daphnia magna (OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 mg/L

Additional information

One study investigating the long-term toxicity of dibuty adipate to aquatic invertebrates is available. The semi-staic study was performed in accordance with GLP and OECD guideline 211 (Migchielsen 2014). 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. Reproduction in the control averaged 66.1 viable young, whereas the test group’s reproduction averaged 65.0 to 82.5 young per surviving parent animal. No delay in appearance of the first brood was observed in the treatments when compared to the control, except for the daphnids exposed to 4.8 mg/l that did not have any brood due to the fact that all parents died before onset of reproduction. No significant numbers of aborted or unhatched eggs were observed at any of the concentrations tested. From day 15 onwards most of the offspring were immobilised in all test groups including the control. It was unknown what the cause of this phenomenon was. It was however not test substance related. Thus, the 21d-NOEC value is determined to be 1.5 mg/L based on the mean initial measured test concentration.